Article

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry

March 2010
Applied Microbiology and Biotechnology 87(1):331-41

March 2010
87(1):331-41

DOI:10.1007/s00253-010-2526-2

Source
PubMed

Authors:

Yingying Wang

Nankai University

Lieve Claeys

David Van Der Ha

Willy Verstraete

Ghent University

Show all 5 authorsHide

The present study reports the disinfection effects of chemically and electrochemically dosed chlorine on two models for typical water-borne bacteria (Escherichia coli and Legionella beliardensis) by plating and flow cytometry (FCM) in combination with different fluorescence dyes. The residual effect on various cell functions, including cultivability, esterase activity, membrane polarization, and integrity, was tested at different free chlorine concentrations. In comparison, chemical disinfection yielded on average 60% more E. coli cells entering the viable but nonculturable (VBNC) state than electrochemical disinfection. Here, VBNC is defined as those cells with intact cell membrane but which cannot be cultured on solid nutrient agar plates. L. beliardensis was about five times more resistant to chlorine disinfection than E. coli. The results also suggested the two methods result in different disinfection mechanisms on L. beliardensis, i.e., chemically dosed chlorine targeted cell membrane integrity before enzyme activity, while electrochemically dosed chlorine acted the other way round. In addition, both bacteria lost the integrity of their cell membranes at three times lower chlorine concentration over a longer contact time (i.e., 40 vs. 10 min) by the chemical method. Our results showed that FCM is an appropriate tool to evaluate the effects of water disinfection and the percentage of cells in VBNC in a matter of hours. Electrochemical disinfection is suggested to be a favorable alternative for chemical disinfection.

Differential development of Legionella sub-populations during short- and long-term starvation

Article

Full-text available

Apr 2018
WATER RES

Legionellae are among the most important waterborne pathogens in industrialized countries. Monitoring and surveillance of Legionella in engineered water systems is usually performed with culture-based methods. Since the advent of culture-independent techniques, it has become clear that Legionella concentrations are often several orders of magnitude higher than those measured by culture-based techniques and that a variable proportion of these non-culturable cells are viable. In engineered water systems, the formation of these viable but non-culturable (VBNC) cells can be caused by different kinds of stress, such as, and most importantly, nutrient starvation, oxidative stress and heat. In this study, the formation of VBNC cells of six Legionella strains under conditions of starvation was monitored in mono-species microcosms for up to one year using a combination of different viability indicators. Depending on the strain, complete loss of culturability was observed from 11 days to 8 weeks. During the starvation process, three distinct phases and different sub-populations of VBNC cells were identified. Until complete loss of culturability, the number of membrane-intact cells decreased rapidly to 5.5-69% of the initial cell concentration. The concentration of the sub-population with low esterase activity dropped to 0.03-55%, and the concentration of the highly esterase-active sub-population dropped to 0.01-1.2% of the initial concentration; these sub-populations remained stable for several weeks to months. Only after approximately 200 days of starvation, the number of VBNC cells started to decrease below detection limits. The most abundant VBNC sub-populations were characterized by partially damaged membranes and low esterase-activity. With this study, we showed that upon starvation, a stable VBNC Legionella community may be present over several months in a strain-dependent manner even under harsh conditions. Even after one year of starvation, a small proportion of L. pneumophila cells with high esterase-activity was detected. We speculate that this highly active VBNC subpopulation is able to infect amoebae and human macrophages.

Enhanced killing of Escherichia coli using a combination of polyhexamethylene biguanide hydrochloride and 1-bromo-3-chloro-5,5- dimethylimidazolidine-2,4-dione

Article

Oct 2017
FEMS MICROBIOL LETT

The bactericidal activities of polyhexamethylene biguanide hydrochloride (PHMB), 1-bromo-3-chloro-5,5-dimethylimidazolidine-2,4-dione (BCDMH), and the combination of the two (designated as PB) were compared using Escherichia coli as the test organism. PB exhibited strong bactericidal activity: 10 mg/L PHMB combined with 8 mg/L BCDMH resulted in approximately 5.74 log10 reduction (LR), whereby 320 mg/L PHMB or 20 mg/L BCDMH was about 5.53 and 6.56 LR, respectively. Analyses using scanning electron microscopy, flow cytometry, and atomic absorption spectroscopy indicated that PB, PHMB, and BCDMH disrupted cell membranes and changed membrane structure and permeability, resulting in the leakage of intracellular soluble proteins and ions. PB exerted stronger effects on potassium and magnesium leakage, membrane potential, and permeability than BCDMH did. PB caused less protein leakage than PHMB did. These results suggest that at a relatively low concentration, PB exhibited good bactericidal activity and physiological effect on E. coli.

Determination of viable legionellae in engineered water systems: Do we find what we are looking for?

Article

Full-text available

Feb 2016
WATER RES

Alexander Kirschner

In developed countries, legionellae are one of the most important water-based bacterial pathogens caused by management failure of engineered water systems. For routine surveillance of legionellae in engineered water systems and outbreak investigations, cultivation-based standard techniques are currently applied. However, in many cases culture-negative results are obtained despite the presence of viable legionellae, and clinical cases of legionellosis cannot be traced back to their respective contaminated water source. Among the various explanations for these discrepancies, the presence of viable but non-culturable (VBNC) Legionella cells has received increased attention in recent discussions and scientific literature. Alternative culture-independent methods to detect and quantify legionellae are proposed in order to complement or even substitute the culture method in the future. Such methods should detect VBNC Legionella cells and provide a more comprehensive picture of the presence of legionellae in engineered water systems. However, it is still unclear whether and to what extent these VBNC legionellae are hazardous to human health. Current risk assessment models to predict the risk of legionellosis from Legionella concentrations in the investigated water systems contain many uncertainties and are mainly based on culture-based enumeration. If VBNC legionellae should be considered in future standard analysis, quantitative risk assessment models including VBNC legionellae must be proven to result in better estimates of human health risk than models based on cultivation alone. This review critically evaluates current methods to determine legionellae in the VBNC state, their potential to complement the standard culture-based method in the near future, and summarizes current knowledge on the threat that VBNC legionellae may pose to human health.

A simple and rapid method for detecting fecal pollution in urban rivers by measuring the intrinsic β-D-glucuronidase activity of Escherichia coli

Article

Oct 2023
WATER RES

As urban rivers are domestic, industrial, and agricultural water resources, fecal pollution poses human health and environmental risks. In this study, we developed a simple and rapid method to detect fecal pollution in urban rivers. Water samples were mixed with liquid medium, including a fluorescent substrate and fluorescence intensity (F.I.) was measured using a microplate reader to determine Escherichia coli (E. coli) β-D-glucuronidase (GUS) activity instead of E. coli concentration. GUS activities measurements in pure E. coli cultures revealed that E. coli incubated with a GUS substrate accumulated GUS enzymes in their cells, whereas those incubated without a GUS substrate did not. The increase in GUS activity corresponded to the proliferation of E. coli and the GUS activity increased linearly even during the lag growth phase of E. coli, indicating the presence of intrinsic GUS (iGUS) in E. coli cells before incubation. iGUS activity persisted at 81 % in the chlorinated samples, even though the E. coli concentration was reduced by a factor of 106. The iGUS activity persisted for approximately three days. Therefore, we assumed that E. coli present in fecal contaminants, in which GUS substrates are present, could be distinguished from those surviving in the natural environment for three days or longer by measuring iGUS activity. River water samples were collected upstream and downstream of the discharge outlets of municipal wastewater treatment plants and a combined sewer outlet. The iGUS activities were <0.24 mMFU/mL for the upstream samples and >0.21 mMFU/mL for the downstream samples. Interestingly, E. coli concentrations were not necessarily associated with fecal pollution. This indicates that by setting a threshold for iGUS activity, our method can be used as a simple and rapid method for detecting fecal pollution in urban rivers. Because the limit of detection for our method is 20 CFU/mL, our method is applicable to detecting high fecal pollution in a small river.

A Novel Flow Cytometric Approach for the Quantification and Quality Control of Chlamydia trachomatis Preparations

Article

Full-text available

Dec 2021

Chlamydia trachomatis is an obligate intracellular pathogenic bacterium with a biphasic developmental cycle manifesting two distinct morphological forms: infectious elementary bodies (EBs) and replicative intracellular reticulate bodies (RBs). Current standard protocols for quantification of the isolates assess infectious particles by titering inclusion-forming units, using permissive cell lines, and analyzing via immunofluorescence. Enumeration of total particle counts is achieved by counting labeled EBs/RBs using a fluorescence microscope. Both methods are time-consuming with a high risk of observer bias. For a better assessment of C. trachomatis preparations, we developed a simple and time-saving flow cytometry-based workflow for quantifying small particles, such as EBs with a size of 300 nm. This included optimization of gain and threshold settings with the addition of a neutral density filter for small-particle discrimination. The nucleic acid dye SYBR® Green I (SGI) was used together with propidium iodide and 5(6)-carboxyfluorescein diacetate to enumerate and discriminate between live and dead bacteria. We found no significant differences between the direct particle count of SGI-stained C. trachomatis preparations measured by microscopy or flow cytometry (p > 0.05). Furthermore, we completed our results by introducing a cell culture-independent viability assay. Our measurements showed very good reproducibility and comparability to the existing state-of-the-art methods, indicating that the evaluation of C. trachomatis preparations by flow cytometry is a fast and reliable method. Thus, our method facilitates an improved assessment of the quality of C. trachomatis preparations for downstream applications.

Sanitising efficacy of lactic acid combined with low-concentration sodium hypochlorite on Listeria innocua in organic broccoli sprouts

Article

Jan 2020
INT J FOOD MICROBIOL

The sanitising eﬀects of combined lactic acid (LA, 2%, v/v) and low-concentration sodium hypochlorite (SH, 4mg/L) on Listeria innocua were investigated. The disinfectant kinetics and percentage of injured cells showed that the combined treatment resulted in dramatic inactivation of L. innocua after 60s. The Weibull model with a high R2 (1.00) and low root mean squared error (RMSE) (0.19) and Akaike Information Criterion (AIC) (−7.58) was veriﬁedasthemostsuperiormodeltodescribeeﬀectsofthecombination treatment.Proteinleakageandthe ﬂuorescence intensity ratio revealed that LA might disrupt the cytoplasmic membrane rapidly and that SH could synergistically promote the sanitising eﬀect. Atomic force microscopy (AFM) images showed notable morphological changes in the combination treated cells, which presented lower width (0.73μm) and height (0.29μm), and increased roughness (10.37nm). Moreover, massive leakage of intracellular components indicated severe cell collapse. The antimicrobial eﬀects on organic broccoli sprouts showed that the combined treatment resulted in reductions of 0.82, 1.51, and 1.77 log colony forming units (CFU)/g fresh weight (FW) for aerobic bacteria, yeasts and moulds, and inoculated L. innocua at day 0. In addition, there were no signiﬁcant diﬀerences of sensory quality results between treatment and control groups and they were all greater than the acceptance limit. Thus, the disinfection treatments had no negative eﬀects on the storage quality of the organic broccoli sprouts. The combined treatment could be a potential sanitising strategy for organic sprouts.

Development of fungal spore staining methods for flow cytometric quantification and their application in chlorine-based disinfection

Article

Nov 2019
CHEMOSPHERE

The Risk To Human Health From Free-Living Amoebae Interaction With Legionella In Drinking And Recycled Water Systems

Thesis

Full-text available

Sep 2012

Jacqueline Thomas

Treated drinking water in developed countries still causes disease despite meeting current regulations and compliance monitoring needs that focus on faecal contamination. Of growing concern are the increasing rates of community acquired pneumonia (CAP) infections which are in part due to inhalation of treated drinking water aerosols containing opportunistic pathogens including Legionella and Mycobacterium. CAP infections due to Legionella presently are estimated to account for one third of all reported drinking water outbreaks in the USA. If the number of Legionella infections are to be reduced then the means of replication in drinking water need to be better understood as it is apparent that current drinking water disinfection and control mechanism are ineffective. Free-living amoebae (FLA) are eukaryotic microorganisms which are known to feed on bacteria but some microorganisms, such as pathogenic Legionella, effectively avoid digestion and infect the FLA. Microorganisms which can infect FLA are collectively known as amoebae resistant microorganisms (ARM). As hosts FLA effectively facilitate the survival, growth and infectivity of their pathogenic ARM. Despite FLA being an accepted host for pathogenic Legionella and other ARM, very little is known about their density and diversity nor their interactions with Legionella in drinking and recycled water systems and end use applications. The aim of this research was to address the present knowledge gap about FLA by analysing FLA and their interactions with Legionella in a drinking and recycled water systems and applications and then use that information in a novel quantitative microbial risk assessment for FLA and Legionella. Through literature analysis of 29 published studies reporting on FLA in drinking water systems and applications it appears that FLA are ubiquitous. FLA were detected in drinking water tap samples at a mean frequency of 46 % (n = 18, = 28). Overall, the density of FLA was poorly reported but there was a large diversity of FLA with 14 different genera of FLA identified. Already, six different genera of FLA have been isolated from drinking water systems and applications infected with 14 different species of pathogenic ARM, thus highlighting the variety of FLA and ARM interaction occurring in drinking water. From water and biofilm samples FLA were isolated using traditional culture methods utilising non-nutrient agar plates with Escherichia coli overlays. More sensitive molecular techniques were also applied with qPCR targeting Acanthamoeba spp., H. vermiformis and Naegleria spp. Detected FLA were identified by cloning and partial 18S rRNA gene sequencing. Legionella spp. were also isolated using standard culture methods and detected in samples and FLA using PCR and qPCR targeting the Legionella genus. Identification of detected Legionella was achieved by cloning and partial 16S rRNA gene sequencing. Water quality characteristics including biofilm quantity, heterotrophic plate counts and free and combined chlorine were also recorded during sampling and the course of the experiments to ascertain possible trends. To partly address the knowledge gap about FLA in recycled water systems a water recycling plant (WRP) and its recycled water distribution system were sampled for FLA and Legionella. By qPCR H. vermiformis was detected breaking through the WRP at densities of 2.7 amoebae.mL-1 and entering the distribution system. The water and biofilm for both the parallel recycled and drinking systems within the dual distribution system was sampled using a modified Robbins Device (MRD). Acanthamoeba spp. and H. vermiformis were detected by culture and qPCR in drinking and recycled water and biofilm samples with the highest mean densities (4.6 amoebae.cm-2 ) detected in drinking water biofilm. Pathogenic Legionella anisa were detected in low densities in one recycled water sample (40 cells.mL-1) and biofilm samples (14 cells.cm-2) but no Legionella was detected in the drinking water samples. Two of the 16 FLA isolated from drinking and recycled water were shown to be infected with Legionella sp. illuminating how common FLA infections with ARM were. Garden hoses were identified as an exposure pathway for Legionella infection that had been neglected in the literature to date. To evaluate FLA and Legionella density and diversity two garden hoses types (standard green and lilac) supplied with drinking water were sampled over 18 months. Over the three sampling periods the lilac hose type water consistently had higher mean density of Acanthamoeba sp. (324 amoebae.mL-1) and H. vermiformis (300 amoebae.mL-1) compared to the green hose water Acanthamoeba sp. (18 amoebae.mL-1) and H. vermiformis (31 amoebae.mL-1). Similarly, Legionella detection was significantly higher (1 way ANOVA, p < 0.0001) for the lilac hose type water (6.5 103 cells.mL-1) which were higher than all the other Legionella densities reported in the literature. Two thirds of the Legionella detected were identified as unclassified Legionella species with unknown pathogenicity that needs to be explored further. To replicate the conditions in heated applications of drinking water, such as showering, annular reactors were incubated at 42 C for 13.5 months. Despite having comparable biofilm quantities to the ambient reactors there were consistently less FLA detected in the heated reactor biofilm by both culture and qPCR. A greater diversity of FLA were identified in the reactors including Echinamoeba exudans and Vahlkampfia sp. compared to the MRD drinking water which supplied the reactors. The highest mean detection of Legionella was for heated reactor water (80 cells.mL-1) while heated reactor biofilm had the lowest mean detection (13 cells.mL-1). During heated conditions biofilm appears to be less important in the proliferation of FLA and Legionella than under ambient conditions which was incorporated into the risk assessment. Acanthamoeba sp. and H. vermiformis isolates from the MRD, garden hoses and annular reactors were incubated with fluorescently labeled L. pneumophila at 22 and 37 C and tracked by fluorescent microscopy over seven days to determine if the FLA would be infected. After seven days at 22 C all of the Acanthamoeba sp. trophozoites were infected with L. pneumophila but only a maximum mean of 25 % of the H. vermiformis were. Accordingly, the numbers of L. pneumophila increased in the presence of Acanthamoeba sp. by 4.4 103 - 7.3 103 times compared to only 58 - 108 times when incubated with H. vermiformis. The FLA isolated from the drinking water systems and applications were infected with L. pneumophila at different rates which was used in the risk assessment calculations. FLA density and diversities in water and biofilm were incorporated to form a quantitative microbial risk assessment (QMRA) for Legionella infection. The QMRA was unique in that the input data for FLA and Legionella came from real drinking water end use applications, showering and garden hose use. When the model was run with estimated mean densities of pathogenic L. pneumophila and FLA the probability of human infection was greater than the recommended risk benchmark of 1 10-4 for both garden hose use and showering. Hence, based on these novel probabilities of infection it is recommended that those people who are at risk of Legionella infection, such as the elderly, diabetics and people with suppressed immune systems, should make behavioral changes to reduce exposure. Innovative changes recommended include draining and hanging garden hoses and avoiding using them on spray settings. Furthermore, it is advised to take baths over showers or if that it not possible to use a point of use filter system. It must be remembered that FLA and Legionella are only two groups of microorganism capable of interaction in a drinking water system. There is a range of hosts and pathogenic ARM in drinking water whose density, diversity and interactions need to be further explored. New next generation sequencing platforms and fluorescent assisted cell sorting technologies present excellent opportunities to make advancement in this area of research. Combined with improved epidemiological surveying then the true health burden of pathogenic ARM in drinking water can be established and in turn controlled.

Sanitising efficacy of lactic acid combined with low-concentration sodium hypochlorite on Listeria innocua in organic broccoli sprouts

Article

Feb 2019
INT J FOOD MICROBIOL

The sanitising effects of combined lactic acid (LA, 2%, v/v) and low-concentration sodium hypochlorite (SH, 4 mg/L) on Listeria innocua were investigated. The disinfectant kinetics and percentage of injured cells showed that the combined treatment resulted in dramatic inactivation of L. innocua after 60 s. The Weibull model with a high R ⁠ 2 (1.00) and low root mean squared error (RMSE) (0.19) and Akaike Information Criterion (AIC) (−7.58) was verified as the most superior model to describe effects of the combination treatment. Protein leakage and the fluorescence intensity ratio revealed that LA might disrupt the cytoplasmic membrane rapidly and that SH could synergistically promote the sanitising effect. Atomic force microscopy (AFM) images showed notable morphological changes in the combination treated cells, which presented lower width (0.73 μm) and height (0.29 μm), and increased roughness (10.37 nm). Moreover, massive leakage of intracellular components indicated severe cell collapse. The antimicrobial effects on organic broccoli sprouts showed that the combined treatment resulted in reductions of 0.82, 1.51, and 1.77 log colony forming units (CFU)/g fresh weight (FW) for aerobic bacteria, yeasts and moulds, and inoculated L. innocua at day 0. In addition, there were no significant differences of sensory quality results between treatment and control groups and they were all greater than the acceptance limit. Thus, the disinfection treatments had no negative effects on the storage quality of the organic broccoli sprouts. The combined treatment could be a potential sanitising strategy for organic sprouts.

Flow cytometry applications in water treatment, distribution, and reuse: A review

Article

Dec 2018
WATER RES

Ensuring safe and effective water treatment, distribution, and reuse requires robust methods for characterizing and monitoring waterborne microbes. Methods widely used today can be limited by low sensitivity, high labor and time requirements, susceptibility to interference from inhibitory compounds, and difficulties in distinguishing between viable and non-viable cells. Flow cytometry (FCM) has recently gained attention as an alternative approach that can overcome many of these challenges. This article critically and systematically reviews for the first time recent literature on applications of FCM in water treatment, distribution, and reuse. In the review, we identify and examine nearly 300 studies published from 2000 to 2018 that illustrate the benefits and challenges of using FCM for assessing source-water quality and impacts of treatment-plant discharge on receiving waters, wastewater treatment, drinking water treatment, and drinking water distribution. We then discuss options for combining FCM with other indicators of water quality and address several topics that cut across nearly all applications reviewed. Finally, we identify priority areas in which more work is needed to realize the full potential of this approach. These include optimizing protocols for FCM-based analysis of waterborne viruses, optimizing protocols for specifically detecting target pathogens, automating sample handling and preparation to enable real-time FCM, developing computational tools to assist data analysis, and improving standards for instrumentation, methods, and reporting requirements. We conclude that while more work is needed to realize the full potential of FCM in water treatment, distribution, and reuse, substantial progress has been made over the past two decades. There is now a sufficiently large body of research documenting successful applications of FCM that the approach could reasonably and realistically see widespread adoption as a routine method for water quality assessment.

Long-Term Effects of Residual Chlorine on Pseudomonas aeruginosa in Simulated Drinking Water Fed With Low AOC Medium

Article

Full-text available

May 2018

Residual chlorine is often required to remain present in public drinking water supplies during distribution to ensure water quality. It is essential to understand how bacteria respond to long-term chlorine exposure, especially with the presence of assimilable organic carbon (AOC). This study aimed to investigate the effects of chlorination on Pseudomonas aeruginosa in low AOC medium by both conventional plating and culture-independent methods including flow cytometry (FCM) and quantitative PCR (qPCR). In a simulated chlorinated system using a bioreactor, membrane damage and DNA damage were measured by FCM fluorescence fingerprint. The results indicated membrane permeability occurred prior to DNA damage in response to chlorination. A regrowth of P. aeruginosa was observed when the free chlorine concentration was below 0.3 mg/L. The bacterial response to long-term exposure to a constant low level of free chlorine (0.3 mg/L) was subsequently studied in detail. Both FCM and qPCR data showed a substantial reduction during initial exposure (0-16 h), followed by a plateau where the cell concentration remained stable (16-76 h), until finally all bacteria were inactivated with subsequent continuous chlorine exposure (76-124 h). The results showed three-stage inactivation kinetics for P. aeruginosa at a low chlorine level with extended exposure time: an initial fast inactivation stage, a relatively stable middle stage, and a final stage with a slower rate than the initial stage. A series of antibiotic resistance tests suggested long-term exposure to low chlorine level led to the selection of antibiotic-resistant P. aeruginosa. The combined results suggest that depletion of residual chlorine in low AOC medium systems could reactivate P. aeruginosa, leading to a possible threat to drinking water safety.

Monitoring the live to dead transition of bacteria during thermal stress by a multi-method approach

Article

Feb 2016

Rapid microbiological methods to assess cell physiological properties of microorganisms are gaining interest in the elucidation of the effect of antimicrobial agents or physical inactivation. Fluorescent probes combined with flow cytometry or microplate assays provide information about cellular targets of chemical or physical stressors and help to clarify the underlying mode of action. In this work we exemplarily monitored the bacterial response of Listeria innocua, Staphylococcus aureus, Salmonella enterica and Escherichia coli to a mild thermal treatment by applying various methods to illustrate bacterial vital functions like the redox activity, membrane potential, esterase activity, efflux activity, glucose uptake, membrane integrity and plate counts. It was observed that some cellular properties are affected earlier than others. Respiration, glucose-uptake and pump activity were the most sensitive parameters, followed by the loss of membrane potential and membrane integrity. Unspecific esterase was found to be relatively resistant to mild heat exposure. This study shows that such a multi-method approach is a suitable tool to monitor the impact of inactivation treatments on bacteria, providing information about the mode of action, the heterogeneity of populations, species-specific differences to stressors and valuable insight in vital functions beyond pure culturability.

Sediment bioelectrochemical system with potential application against vibriosis in aquaculture

Article

Dec 2023

Emerging Contaminants in the Water Environment: Disinfection-induced Viable but Non-culturable Waterborne Pathogens

Article

Sep 2023
J HAZARD MATER

Role of Resuscitation Promoting Factor-like Protein from Nocardiopsis halophila

Article

Full-text available

Feb 2023

Resuscitation promoting factors (Rpf), a class of proteins secreted by gram-positive bacteria including actinobacteria, promote the resuscitation of dormant bacteria and spore germination. Here, we describe the reconstitution of the resuscitation promoting activity of the Rpf protein from Nocardiopsis halophila CGMCC 4.1195T in vitro and in vivo. The Rpf protein was expressed in the host Escherichia coli BL21 codon plus (DE3) and was confirmed to have a significant resuscitation effect on the viable but non-culturable (VBNC) N. halophila. Subsequently, the rpf gene of N. halophila was knocked out. We found that the growth rate of the mutant strain (Δrpf) was slower than that of the wild strain, and the former produced significantly shorter spores than the wild-type strain. Our results confirmed the activity of the Rpf protein in N. halophila to promote dormant bacteria resuscitation. This study will lay the foundation for the application of the Rpf protein from N. halophila to exploit actinomycetes resources.

Possible Processes and Mechanisms of Hexachlorobenzene Decomposition by the Selected Comamonas testosteroni Bacterial Strains

Article

Full-text available

Oct 2022

Background: The bacterial destructing activity toward pesticides has been the focus of research in the last few decades. Hexachlorobenzene is included in the organochlorine pesticides group that are prohibited for use. However, large hexachlorobenzene amounts are still concentrated in the soil, stressing the relevance of research on hexachlorobenzene-destroying bacteria. Methods: The ability to destroy hexachlorobenzene by Comamonas testosteroni UCM B-400, B-401, B-213 strains was investigated and established. Bacteria were cultivated (7 days at 28 °C) in mineral Luria-Bertrani (LB) medium with three hexachlorobenzene doses: 10, 20, 50 mg/L. The hexachlorobenzene concentrations were recorded by a gas chromatography method. Results: The results showed that C. testosteroni UCM B-400, B-401 have high destructive activity toward hexachlorobenzene. The highest (50 mg/L) initial concentration decreased to 41.5 and 43.8%, respectively, for C. testosteroni UCM B-400, B-401. The unadapted C. testosteroni UCM B-213 was tolerant to hexachlorobenzene (cell titers after cultivating with 10.0, 20.0, 50.0 mg/mL were higher compared to initial titer), but had a low-destructing activity level (two times less than B-400 and B-401). Conclusions: Bacterial strains C. testosteroni UCM B-400, B-401 can be seen as a potential soil bioremediation from hexachlorobenzene pollution.

Possible Processes and Mechanisms of Hexachlorobenzene Decomposition by the Selected Comamonas testosteroni Bacterial Strains

Article

Full-text available

Oct 2022

Nadiya Yamborko

The bacterial destructing activity toward pesticides has been the focus of research in the last few decades. Hexachlorobenzene is included in the organochlorine pesticides group that are prohib-ited for use. However, large hexachlorobenzene amounts are still concentrated in the soil, stress-ing the relevance of research on hexachlorobenzene-destroying bacteria. Methods: The ability to destroy hexachlorobenzene by Comamonas testosteroni UCM B-400, B-401, B-213 strains was in-vestigated and established. Bacteria were cultivated (7 days at 28 °C) in mineral Luria-Bertrani (LB) medium with three hexachlorobenzene doses: 10, 20, 50 mg/L. The hexachlorobenzene con-centrations were recorded by a gas chromatography method. Results: The results showed that C. testosteroni UCM B-400, B-401 have high destructive activity toward hexachlorobenzene. The highest (50 mg/L) initial concentration decreased to 41.5 and 43.8%, respectively, for C. testos-teroni UCM B-400, B-401. The unadapted C. testosteroni UCM B-213 was tolerant to hexachloro-benzene (cell titers after cultivating with 10.0, 20.0, 50.0 mg/mL were higher compared to initial titer), but had a low-destructing activity level (two times less than B-400 and B-401). Conclu-sions: Bacterial strains C. testosteroni UCM B-400, B-401 can be seen as a potential soil bioremedi-ation from hexachlorobenzene pollution. Keywords: hexachlorobenzene; toxicity; microbial metabolism; destroying activity; bacterial decomposition; destruction potential

Exploring the use of flow cytometry for understanding the efficacy of disinfection in chlorine contact tanks

Article

Apr 2022
WATER RES

A pilot scale chlorine contact tank (CCT) with flexible baffling was installed at an operational water treatment plant (WTP), taking a direct feed from the outlet of the rapid gravity filters (RGF). For the first time, disinfection efficacy was established by direct microbial monitoring in a continuous reactor using flow cytometry (FCM). Disinfection variables of dose, time, and hydraulic efficiency (short circuiting and dispersion) were explored following characterisation of the reactor's residence time distributions (RTD) by tracer testing. FCM enabled distinction to be made between changes in disinfection reactor design where standard culture-based methods could not. The product of chlorine concentration (C) and residence time (t) correlated well with inactivation of microbes, organisms, with the highest cell reductions (N/N0) reaching <0.025 at Ctx̄ of 20 mg.min/L and above. The influence of reactor geometry on disinfection was best shown from the Ct10. This identified that the initial level of microbial inactivation was higher in unbaffled reactors for low Ct10 values, although the highest levels of inactivation of 0.015 could only be achieved in the baffled reactors, because these conditions enabled the highest Ct10 values to be achieved. Increased levels of disinfection were closely associated with increased formation of the trihalomethane disinfection by-products. The results highlight the importance of well-designed and operated CCT. The improved resolution afforded by FCM provides a tool that can dynamically quantify disinfection processes, enabling options for much better process control.

Ancient Oriental Wisdom still Works: Removing ARGs in Drinking Water by Boiling as compared to Chlorination

Article

Nov 2021
WATER RES

Antibiotic resistance genes (ARGs) in municipal drinking water may not be effectively removed during centralized treatment. To reduce potential health risks, water disinfection at the point-of-use scale is warranted. This study investigated the performance of boiling, a prevalent household water disinfection means, in response to ARGs contamination. We found that boiling was more efficient in inactivating both Escherichia coli and environmental bacteria compared to chlorination and pasteurization. Boiling of environmental bacteria suspension removed a much broader spectrum of ARGs and mobile genetic elements (up to 141 genes) than chlorination (up to 13 genes), such better performance was largely attributed to a stronger inactivation of chlorine-tolerant bacteria including Acinetobacter and Bacillus. Accumulation of extracellular ARGs was found during low-temperature heating (≤ 80°C) of E. coli and at lower chlorination dosages (≤3 min). These extracellular ARGs as well as the intracellular ARGs got removed as the heating temperature increased or the chlorination time prolonged. Under the same treatment time (30 min), high-temperature heating (≥ 90.1°C) damaged the DNA structure more thoroughly than chlorination (5 mg/L). Taking into account the low transferability of ARGs after DNA melting, boiling may provide an effective point-of-use approach to attenuating bacterial ARGs in drinking water and is still worth promoting in the future.

Phenolic-rich extracts from acerola, cashew apple and mango by-products cause diverse inhibitory effects and cell damages on enterotoxigenic Escherichia coli

Article

Full-text available

Oct 2021
LETT APPL MICROBIOL

This study aimed to evaluate the inhibitory effects of phenolic-rich extracts from acerola (Malpighia emarginata D.C., PEA), cashew apple (Anacardium occidentale L., PEC) and mango (Mangifera indica L., PEM) by-products on distinct enterotoxigenic E. coli (ETEC) strains. The capability of PEA and PEC of impairing various physiological functions of ETEC strains was investigated with multiparametric flow cytometry. Procyanidin B2 , myricetin and p-coumaric acid were the major phenolic compounds in PEA, PEC and PEM, respectively. PEA and PEC had lower minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) (MIC: 31.25 mg ml-1 ; MBC: 62.5 mg ml-1 ) on ETEC strains than PEM (MIC and MIC: > 1000 mg ml-1 ). PEA and PEC (15.6, 31.2, 62.5 mg ml-1 ) caused viable count reductions (P < 0.05) on ETEC strains after 24h of exposure, notably the ≥3 log reductions caused by 62.5 mg ml-1 . The 24h exposure of ETEC strains to PEA and PEC (31.2, 62.5 mg ml-1 ) led to high sizes of cell subpopulations with concomitant impairments in cell membrane polarization and permeability, as well as in enzymatic, respiratory and efflux activities. PEA and PEC are effective in inhibiting ETEC through a multi-target action mode with disturbance in different physiological functions.

Inactivation efficacies of lactic acid and mild heat treatments against Escherichia coli strains in organic broccoli sprouts

Article

Mar 2022
FOOD CONTROL

The kinetics of the inactivation of Escherichia coli in organic broccoli sprouts by mild heat (MH) and lactic acid (LA) were investigated. In vitro survival kinetics showed that the cell number decreased dramatically and was not detectable after 120–135 s under combination treatment. Protein leakage and atomic force microscopy analyses showed that inactivation of E. coli cells resulted from disruption of the outer membrane by the sanitisers. LA almost destroyed the outer membrane and induced high protein leakage (3.78–10.75 μg/mL). MH enhanced the membrane damage caused by LA. The in vivo inactivation and following recovery during storage suggested that the combination treatment presented a potential inactivation process for organic vegetables or sprouts. Weibull model with higher R² values (0.66–0.99) best described the in vitro antibacterial kinetics and in vivo recovery dynamics. Moreover, the metabolic analysis revealed that citrate cycle (TCA) and amino acid metabolism contributed to the adaptive responses.

Evaluation of DNA extraction yield from a chlorinated drinking water distribution system

Article

Full-text available

Jun 2021
PLOS ONE

Desalination technology based on Reverse Osmosis (RO) membrane filtration has been resorted to provide high-quality drinking water. RO produced drinking water is characterized by a low bacterial cell concentration. Monitoring microbial quality and ensuring membrane-treated water safety has taken advantage of the rapid development of DNA-based techniques. However, the DNA extraction process from RO-based drinking water samples needs to be evaluated regarding the biomass amount (filtration volume) and residual disinfectant such as chlorine, as it can affect the DNA yield. We assessed the DNA recovery applied in drinking water microbiome studies as a function of (i) different filtration volumes, (ii) presence and absence of residual chlorine, and (iii) the addition of a known Escherichia coli concentration into the (sterile and non-sterile, chlorinated and dechlorinated) tap water prior filtration, and directly onto the (0.2 μm pore size, 47 mm diameter) mixed ester cellulose membrane filters without and after tap water filtration. Our findings demonstrated that the co-occurrence of residual chlorine and low biomass/cell density water samples (RO-treated water with a total cell concentration ranging between 2.47 × 10 ² –1.5 × 10 ³ cells/mL) failed to provide sufficient DNA quantity (below the threshold concentration required for sequencing-based procedures) irrespective of filtration volumes used (4, 20, 40, 60 L) and even after performing dechlorination. After exposure to tap water containing residual chlorine (0.2 mg/L), we observed a significant reduction of E . coli cell concentration and the degradation of its DNA (DNA yield was below detection limit) at a lower disinfectant level compared to what was previously reported, indicating that free-living bacteria and their DNA present in the drinking water are subject to the same conditions. The membrane spiking experiment confirmed no significant impact from any potential inhibitors (e.g. organic/inorganic components) present in the drinking water matrix on DNA extraction yield. We found that very low DNA content is likely to be the norm in chlorinated drinking water that gives hindsight to its limitation in providing robust results for any downstream molecular analyses for microbiome surveys. We advise that measurement of DNA yield is a necessary first step in chlorinated drinking water distribution systems (DWDSs) before conducting any downstream omics analyses such as amplicon sequencing to avoid inaccurate interpretations of results based on very low DNA content. This study expands a substantial source of bias in using DNA-based methods for low biomass samples typical in chlorinated DWDSs. Suggestions are provided for DNA-based research in drinking water with residual disinfectant.

Environmental Management of Legionella in Domestic Water Systems: Consolidated and Innovative Approaches for Disinfection Methods and Risk Assessment

Article

Full-text available

Mar 2021

Legionella is able to remain in water as free-living planktonic bacteria or to grow within biofilms that adhere to the pipes. It is also able to enter amoebas or to switch into a viable but not culturable (VBNC) state, which contributes to its resistance to harsh conditions and hinders its detection in water. Factors regulating Legionella growth, such as environmental conditions, type and concentration of available organic and inorganic nutrients, presence of protozoa, spatial location of microorganisms, metal plumbing components, and associated corrosion products are important for Legionella survival and growth. Finally, water treatment and distribution conditions may affect each of these factors. A deeper comprehension of Legionella interactions in water distribution systems with the environmental conditions is needed for better control of the coloniza-tion. To this purpose, the implementation of water management plans is the main prevention measure against Legionella. A water management program requires coordination among building managers, health care providers, and Public Health professionals. The review reports a comprehensive view of the state of the art and the promising perspectives of both monitoring and disin-fection methods against Legionella in water, focusing on the main current challenges concerning the Public Health sector.

Water Stagnation and Flow Obstruction Reduces the Quality of Potable Water and Increases the Risk of Legionelloses

Article

Full-text available

Dec 2020

Legionella is an opportunistic waterborne pathogen associated with Legionnaires' disease and Pontiac fever. Despite improved public awareness, the incidence of Legionella associated infections has been increasing. Aerosols generated from engineered potable water systems are a demonstrated cause of both nosocomial and community-acquired legionellosis. The ecology of Legionella in these systems is complex with multiple factors impacting their colonization and persistence. Flow dynamics has been identified as an important factor and stagnation in cooling towers is an accepted risk for increased Legionella growth; however, less is known about the impact of flow dynamic on Legionella in potable water systems. This is especially complex due to the inherent intermittent and variable usage observed within outlets of a potable water system. This systematic literature review examines the role of fluid dynamics and stagnation on the colonization and growth of Legionella in potable water systems. Twenty two of 24 identified studies show a positive association between stagnation zones and increased colonization of Legionella. These zones included dead legs, dead ends, storage tanks, and obstructed water flow (such as intermittent usage or flow restriction). Prolonged stagnation in building plumbing systems also deteriorates the quality of thermally or chemically treated potable water. This stimulates the colonization of Legionella established biofilms. Such biofilms are intrinsically resistant to disinfection procedures and accelerate the rate of decay of chemical disinfectants. Sub-lethal doses of disinfectants and the presence of protozoan hosts in stationary water promote generation of viable but non-culturable Legionella cells. This results in false negatives in surveillance methods that use culture methodology. In conclusion, elimination of temporal and permanent stagnation points can improve the quality of potable water, efficacy of disinfectants, and reduce the risk of legionellosis. Current guidelines and water safety plans recognize the risks associated with permanent stagnation point (dead ends and dead legs); however, there is a need for greater emphasis on controlling temporal stagnation arising from intermittent usage.

Anion-exchange resin adsorption followed by electrolysis: A new disinfection approach to control halogenated disinfection byproducts in drinking water

Article

Sep 2019
WATER RES

Bromide and natural organic matter (NOM) are both precursors of halogenated disinfection byproducts (DBPs) in drinking water. During drinking water treatment process, chloride-form anion-exchange resin adsorption is expected to be capable of removing these DBP precursors and in the meantime releasing chloride ions. The released chloride as well as the chloride initially present in source water could be oxidized through electrolysis to generate free chlorine for disinfection. Based on the above assumptions, we developed a new disinfection approach using chloride-form anion-exchange resin adsorption followed by electrolysis to control halogenated DBPs. Parameter setup and optimization were performed for resin adsorption and electrolysis processes. Results showed that 93.7% of NOM and 90% of bromide could be removed at a resin dose of 20 mL per 2 L of simulated source water sample with a contact time of 1 h. Meanwhile, 49.5 mg/L of chloride was released from the resin to the water sample via anion-exchange, and the released chloride was further oxidized by electrolysis (Ti/RuO2-IrO2 anode and graphite cathode, current intensity of 0.4 A) to generate free chlorine (5 mg/L as Cl2) within 192 s. With this new approach, formation of total organic halogen, four trihalomethanes, and five haloacetic acids was reduced by 86.4%, 98.5%, and 93.2%, respectively, compared with chemical chlorination alone. Although the new approach might enhance the formation of some phenolic DBPs by decreasing bromide levels in source water, the overall cytotoxicity of the water samples treated with the new approach was significantly decreased by 68.8% according to a human hepatoma cell cytotoxicity assay. Notably, disinfection ability evaluation showed that the new approach achieved 3.36-log10 reductions of three seeded bacteria (Escherichia coli, Pseudomonas aeruginosa, and Staphylococcus aureus) in 19 s, suggesting that it was not only effective to E. coli but also effective to the chlorine-resistant bacteria (P. aeruginosa and S. aureus).

NOVEL SPECTROPHOTOMETRIC METHOD FOR ESTIMATION OF HYPOCHLORITE IN TAP WATER AND BLEACHING PRODUCTS SAMPLES

Article

Full-text available

Jan 2019

A simple, rapid and accurate spectrophotometric method has been developed for estimation of hypochlorite by trifluoperazine. The proposed method reports the reaction of hypochlorite with trifluoperazine in acetic acid medium to form a red colored product with an absorption maximum at 448 nm. Beer's Law was obeyed in the range of 0.2-3.6 µg/ml with molar absorptivity of 0.365×10 4 L.mol.-1 .cm-1. The relative standard deviation(RSD) of the method was less than 2% and average recovery (accuracy) was 100±1.2%. The optimum conditions for all color development are described and this method successfully applied for estimation of hypochlorite in drinking tap water and household bleaching samples.

Detection and Evaluation of Viable but Non-culturable Escherichia coli O157:H7 Induced by Low Temperature with a BCAC-EMA-Rti-LAMP Assay in Chicken Without Enrichment

Article

Full-text available

Feb 2019
FOOD ANAL METHOD

In this study, a rapid and sensitive method that combines ethidium bromide monoazide (EMA) staining with real-time loop-mediated isothermal amplification (Rti-LAMP) assay was tested in detecting and evaluating viable but non-culturable state (VBNC) Escherichia coli (E. coli) O157:H7 induced by low temperature. When 1.7 × 10⁴ CFU/ml of E. coli O157:H7 was 3 cycles of freeze-thaw, the cells were all dead. However, E. coli O157:H7 in 1.7 × 10⁶ CFU/ml and 1.7 × 10⁸ CFU/ml gradually transferred into VBNC state reaching 6.8 × 10² CFU/ml (0.04%) and 4.1 × 10⁵ CFU/ml (0.24%) after 6 cycles of freeze-thaw, respectively. Keeping E. coli O157:H7 (1.7 × 10⁸ CFU/ml) at 4 °C and − 20 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells increased from 0 to 1.1 × 10⁶ CFU/ml and 5.5 × 10⁶ CFU/ml detected by both EMA-Rti-LAMP and direct epifluorescence method (DEM) up to 258-day storage at 4 °C and − 20 °C, respectively. The EMA-Rti-LAMP had similar accuracy with DEM in detecting viable including VBNC cells, the former had specificity but not the later. Furthermore, the EMA-Rti-LAMP could detect as low as 25 CFU/g of VBNC E. coli O157:H7 derived from contaminated chicken combined with bentonite-coated activated carbon (BCAC) treatment to remove DNA amplification inhibitors, and the entire assay could be completed in 5 h. In addition, one same sample for E. coli O157:H7 positive was detected by both BCAC-EMA-Rti-LAMP and plate count method from 24 retail chicken samples, but four positive samples by BCAC-Rti-LAMP assay. The results obviously suggested that the BCAC-EMA-Rti-LAMP assay might be a rapid and sensitive method for detection of viable including VBNC E. coli O157:H7 cells in food without enrichment.

Searching for Activity Markers that Approximate (VBNC) Legionella pneumophila Infectivity in Amoeba after Ultraviolet (UV) Irradiation

Article

Full-text available

Sep 2018

Legionella pneumophila is an increasingly recognized threat to public health via aerosol exposures; with a variety of control measures including: water temperature/flow management and free chlorine used to reduce the risk of infection within healthcare centers. Despite these efforts, L. pneumophila often recolonizes plumbing systems after specific treatments, which prompted us to examine ultraviolet (UV) irradiation for a point-of-use, secondary control measure. Currently, there is no data on the efficacy of high (>254 nm) wavelength UV-C (100-280 nm) light inactivation of L. pneumophila with resuscitation of viable but non-culturable (VBNC) cells. We report for the first time L. pneumophila dose-responses for 268.6 nm and 288.6 nm UV-C, as compared to 256 nm, and demonstrate UV induced VBNC L. pneumophila remaining infectious to Acanthamoeba polyphaga during co-culture experiments. Findings were correlated to molecular-based activity assays to identify additional measures of L. pneumophila viability following UV disinfection compared to culture. A collection of viability markers may provide a more representative measure of risk compared to current culture-based detection, since UV-C irradiated L. pneumophila lose culturability, yet retain activity, increased ATP production, and the ability to be resuscitated by amoeba co-culture. This finding is significant as it identifies potential concern from VBNC cells following UV-C disinfection and the need for further research into the efficacy of UV inactivation as a point-of-use application for L. pneumophila control and management.

Application of flow cytometry in ballast water analysis — biological aspects

Article

Full-text available

Nov 2017

Ballast water may, when discharged, cause the spread of nonindigenous and potentially invasive species. International ballast water treatment regulations have accelerated the development of new methods to detect, enumerate and assess the status of organisms in the water to be discharged. Flow cytometry (FCM) is a powerful technique with a broad range of applications with the possibility for multi-parametric analysis and the potential of combining it with other techniques being two strong advantages. This review will discuss whether FCM is suitable for ballast water analysis according to international ballast water regulations, and sum up the advantages and disadvantages. It will also give an overview of available labeling techniques. Finally, a discussion on the knowledge gaps and future potential for FCM within ballast water analysis is presented.

Synergistic Effects of Micro-electrolysis-Photocatalysis on Water Treatment and Fish Performance in Saline Recirculating Aquaculture System

Article

Full-text available

Mar 2017

A new physico-chemical process for TAN (total ammonia nitrogen) removal and disinfection is introduced in saline recirculating aquaculture system (RAS), in which the biofilter is replaced with an integrated electrolysis cell and an activated carbon filter. The electrolysis cell which is based on micro current electrolysis combined with UV-light was self-designed. After the fundamental research, a small pilot scale RAS was operated for 30 days to verify the technical feasibility. The system was stocked by 42 GIFT tilapia (Oreochromis niloticus) fish with the rearing density of 13 kg/m3. During the experiments, the TAN concentration remained below 1.0 mg/L. The nitrite concentration was lower than 0.2 mg/L and the nitrate concentration had increased continuously to 12.79 mg/L at the end. Furthermore, the concentration of residual chlorine in culture ponds remained below 0.3 mg/L, ORP maintained slight fluctuations in the range of 190~240 mV, and pH of the water showed the downtrend. Tilapia weight increased constantly to 339.3 ? 10 g. For disinfection, the active chlorine generated by electrochemical treatment caused Escherichia coli inactivation. Enzyme activity assay indicated that the activity of glutamate dehydrogenase, carbonic anhydrase and glutamic pyruvic transaminase increased within the normal range. The preliminary feasibility was verified by using this physico-chemical technology in the RAS.

ELECTROCHEMICAL TREATMENT OF RECIRCULATING AQUACULTURE WASTEWATER USING A TI/RUO 2 -IRO 2 ANODE FOR SYNERGETIC TOTAL AMMONIA NITROGEN AND NITRITE REMOVAL AND DISINFECTION

Article

Aug 2016

Wastewater treatment and biosecurity are essential for intensive recirculating aquaculture system (RAS) production. In this study, the viability of the electrochemical process using a Ti/RuO 2-IrO 2 anode for synergetic total ammonia nitrogen (TAN) and nitrite removal and disinfection of semi-commercial RAS wastewater was evaluated. During the elec-trochemical oxidation process, the effects of the applied current density, sodium chloride concentration, and initial pH on the removal of TAN and nitrite were investigated. Experiment results indicated that under the conditions of 1.7 g L-1 sodium chloride concentration and 60 min electrolysis time, TAN removal efficiencies reached 78% at a current density of 60 mA cm-2 , while nitrite removal efficiencies reached more than 95% at a current density of 30 mA cm-2. TAN removal due to an indirect oxidation mechanism followed second-order kinetics, while nitrite removal was described by pseudo-first-order kinetics in low-salt water (1.7 g L-1). The kinetics for electro-oxidation of TAN and nitrite affected by the current density were expressed as k TAN = 1.0 × 10-5 J-0.0002 and k NO2-= 1.9 × 10-3 J-0.0041, respectively. For disinfection, the active chlorine in situ generated by electrochemical treatment caused Escherichia coli and Vibrio parahaemolyticus inactivation. The results showed that the two objective pathogens can be sterilized rapidly, which indicated that no extra treatment for disinfection was needed. Finally, a comparison was made of the energy consumption in this study with those in the literature. This study showed that the Ti/RuO 2-IrO 2 anodic oxidation process has a potential for sustainable RAS wastewater treatment.

Bactericidal Activity and Physiological Effects of Combined Application of Poly-Hexamethylene Biguanide Hydrochloride and 1-bromo-3-chloro-5, 5-dimethylimidazolidine-2, 4-dione on Staphylococcus aureus

Conference Paper

Full-text available

Jan 2017

Impact of pulsed light on cellular activity of Salmonella enterica

Article

Jul 2016
J APPL MICROBIOL

Aims: The objective of this study was a comprehensive characterization of physiological changes of Salmonella enterica induced by intense broad spectrum pulsed light (PL). After exposing the bacteria to this non-thermal decontamination technology on a gel surface, multiple viability parameters beyond culturability were assessed. Methods and results: By applying flow cytometry, a luciferin-luciferase bioluminescence assay and a microplate assay to measure the current redox activity, the impact of pulsed light on the membrane potential, membrane integrity, esterase activity, efflux pump activity, expression of the green fluorescent protein (GFP), respiration activity and ATP-content of Salmonella enterica ATCC BAA-1045 was determined. These culture independent methods for assessing the bacterial activity were compared to the ability to grow on tryptic soy agar. It is shown that this strain is rather sensitive to PL considering colony count reductions, while on the other hand unculturable bacteria still exhibit significant cellular energetic functions. However, this residual activity after PL exposure significantly decreases during sample storage in buffer for 24 h. This study also shows that the GFP expression of PL treated cells which have rendered unculturable is severely reduced. Conclusions: This study reveals that although not all cellular functions of Salmonella enterica are immediately shut down after PL exposure, the synthesis of new GFP is strongly reduced and affected to a similar extent as the culturability. Significance and impact of the study: It is shown for the first time, that even there is significant bacterial activity measurable after PL exposure, it is likely that non-growing pathogenic bacteria like Salmonella enterica are unable to express proteins, which is of great importance regarding their pathogenicity. This article is protected by copyright. All rights reserved.

Household Scale Clean Water Disinfection Technique with Chlorination Method

Article

Full-text available

Dec 2014

Disinfection is preventive efforts against the entry of pathogenic bacteria to the human body. Chlorination is one effort to give prevention with chlorine. The research objective was to determine of diffusion and mass transfer coefficients and then to develop of a chlorinated tool model. Effect of water flow rate on chlorine transport and granule size was studied to develop their relationship. The flow rates discharge used were 8 liters/minute, 14 liters/minute, and 20 liters/minute, whereas the granule sizes were 2.36 – 4.75 ml, 4.75-9.5 ml, and 9.5-16 ml. Diffusion coefficients and mass transfer determined by least summed of square of error. Diffusion coefficient and mass transfer used for disinfection technique was 0.4371 cm 2 minute-1 and 0.0039-minute-1 , as well as flow rate and granule size used was 9.5 ml-16 ml and 8 liters/minute. Experiment testing of chlorination performed to ensure of them was potentially for chlorination. Raw water chlorinated then was found of free chlorine residual and the total coliform met the quality standards based on regulation the Ministry of Health of Republic of Indonesia's number 416 of 1990 about the terms of supervision and the quality of water.

UV disinfection induces a VBNC state in Escherichia coli and Pseudomonas aeruginosa

Article

Full-text available

Jan 2015

UV Disinfection Induces a VBNC State in Escherichia coli and Pseudomonas aeruginosa

Article

Full-text available

Jan 2015

Xin Yu

New mechanistic insight into the microbial responses to on-line chemical cleaning of UF membranes with NaClO

Article

May 2024
SEP PURIF TECHNOL

Identification of significant live bacterial community shifts in different reclaimed waters during ozone and chlorine disinfection

Article

Jun 2023

Ozone and chlorine are the most widely used disinfectants for water and wastewater disinfection. They play important role in microbial inactivation but could also pose a considerable selection effect on the microbial community of reclaimed water. Classical culture-based methods that rely on the assessment of conventional bacterial indicators (e.g., coliform bacteria) could hardly reflect the survival of disinfection residual bacteria (DRB) and hidden microbial risks in disinfected effluents. Hence, this study investigated the shifts of live bacterial community during ozone and chlorine disinfection in three reclaimed waters (i.e., two secondary effluents and one tertiary effluent), adopting Illumina Miseq sequencing technology in combination with a viability assay, propidium monoazide (PMA) pretreatment. Notably, statistical analyses of Wilcoxon rank-sum test confirm the existance of distinct differences in bacterial community structure between samples with or without PMA pretreatment. On the phylum level, Proteobacteria commonly dominated in three undisinfected reclaimed waters, while ozone and chlorine disinfection pose varied effects on its relative abundance among different influents. On the genus level, ozone and chlorine disinfection significantly changed the bacterial composition and dominant species in reclaimed waters. Specifically, the typical DRB identified in ozone disinfected effluents were Pseudomonas, Nitrospira and Dechloromonas, while for chlorine disinfected effluents, Pseudomonas, Legionella, Clostridium, Mycobacterium and Romboutsia were recognized as typical DRB, which call for much attention. The Alpha and Beta diversity analysis results also suggested that different influent compositions greatly affected the bacterial community structure during disinfection processes. Since the experiments in present study were conducted in a short period and the dataset was relatively limited, prolonged experiment under different operational conditions are needed in future to illustrate the potential long-term effects of disinfection on the microbial community structure. The findings of this study could provide insights into microbial safety concern and control after disinfection for sustainable water reclamation and reuse.

Electro-Chlorination Technology for Disinfection of Drinking Water: A Patent Landscape

Article

Aug 2023

The Sustainable Development Goals (SDG) 2015, which are defined to achieve improved and more sustainable future, promotes safe and affordable drinking water facilities for all, till 2030. The rural and remotely placed population worldwide face the problem of shortage of pathogen-free drinking water due to the huge capital and maintenance costs involved in water purification. The current chemical disinfection treatment which is widely used for water disinfection has several disadvantages including the formation of Disinfection By-Products (DBPs). Electro-chlorination is one of the best alternatives as per the literature that can be installed as a decentralized system in a remote location and can overcome most of the issues related to chemical disinfection. The present paper provides an overview of the innovations in the area of electro-chlorination as a disinfection technique through a detailed patent analysis. The patenting activity and publications are considered as an indicator of research and innovation in the field. The patent analysis is also supported by literature analysis for understanding the research trends and the extent of research in the area. The patent data from the year 2000 to 2021 is analyzed country-wise and year-wise. The paper also discusses the IPC, CPC codes, assignees, investors, US class codes, patents types, and citations analysis for the patents in the field of electro-chlorination and DBPs. The keywords used for patent analysis are ‘Electro- chlorination’ and ‘Water’ and ‘Disinfection’ and ‘Disinfection by Products’.

Differentiation of DNA or membrane damage of the cells in disinfection by flow cytometry

Article

Apr 2022
J HAZARD MATER

Recently, the viabilities changes of fungal spores in the water supply system during different disinfection processes have been revealed. SYBR Green I (SG), a nucleic acid stain, its fluorescence intensity is correlated with the amount of double-stranded DNA. This study established a new method through successive SG-SG-PI staining (PI: Propidium Iodide) with flow cytometry (FCM). It could successfully distinguish DNA damage and membrane damage of fungal spores, clearly elucidating the intrinsic disinfection mechanism during the chemical disinfection. This method was briefly described as follows: firstly, (1) the fungal spores were stained with SG and washed by centrifugation; and then, (2) the washed spores were treated with disinfectants and terminated; after that, (3) the disinfected spores were re-stained with SG and analyzed by FCM; finally, (4) the SG re-stained spores were stained with PI and analyzed by FCM. The percentages of spores with DNA damage and membrane damage were determined by the fluorescence intensity obtained from steps (3) and (4), respectively. The repeatability and applicability of this developed method were confirmed. It was further applied to explore the inactivation mechanism during chlorine-based disinfection, and results demonstrated that chloramine attacked the DNA more seriously than the membrane, while chlorine and chlorine dioxide worked in a reverse way.

Mechanism and potential risk of antibiotic resistant bacteria carrying last resort antibiotic resistance genes under electrochemical treatment

Article

Jan 2022
SCI TOTAL ENVIRON

The significant rise in the number of antibiotic resistance genes (ARGs) that resulted from our abuse of antibiotics could do severe harm to public health as well as to the environment. We investigated removal efficiency and removal mechanism of electrochemical (EC) treatment based on 6 different bacteria isolated from hospital wastewater carrying 3 last resort ARGs including NDM-1, mcr-1 and tetX respectively. We found that the removal efficiency of ARGs increased with the increase of both voltage and electrolysis time while the maximum removal efficiency can reach 90%. The optimal treatment voltage and treatment time were 3 V and 120 min, respectively. Temperature, pH and other factors had little influence on the EC treatment process. The mechanism of EC treatment was explored from the macroscopic and microscopic levels by scanning electron microscopy (SEM) and flow cytometry. Our results showed that EC treatment significantly changed the permeability of cell membrane and caused cells successively experience early cell apoptosis, late cell apoptosis and cell necrosis. Moreover, compared with traditional disinfection methods, EC treatment had less potential risks. The conjugative transfer frequencies of cells were significantly reduced after treatment. Less than 1% of bacteria entered the viable but nonculturable (VBNC) state and less than 5% of intracellular ARGs (iARGs) turned into extracellular ARGs (eARGs). Our findings provide new insights into as well as important reference for future electrochemical treatment in removing ARB from hospital wastewater.

Disinfection of constructed wetland effluent by: In situ electrochemical chlorine production for water reuse

Article

Nov 2021

Constructed wetlands (CW) are globally used for the treatment of wastewater. Due to various causes, often the water is not fully treated in terms of pathogen removal requiring additional treatment. Here we evaluated electrochemical disinfection (ED) of CW effluents to guarantee safe wastewater reclamation in decentralized settings. We used a two-chamber electrochemical cell to produce chlorine at a Ti/RuO2 anode with a synthetic electrolyte containing 18.3 mol Cl− m−3 and subsequently tested with CW effluents from two locations (Ecuador and Belgium). The effluents ran first to the anode for disinfection by chlorine and then to the cathode for recovering a circumneutral pH. Different flow rates, current densities, and membrane types combinations were tested with the synthetic electrolyte to optimize chlorine production and later to disinfect CW effluents. The system produced about twice as much free chlorine when selecting an anion exchange membrane rather than a cation exchange membrane because of chloride electromigration to the anolyte. A 5-log removal of fecal indicators was observed without pathogen regrowth within 7-days after treatment when residual chlorine remained, allowing for non-potable water reuse. Lower residence times (15 s) and current densities (50 A m−2) induced the most energy-efficient operation with a charge density of 10.4 Ah.m−3 and energy consumption of <0.1 kWh.m−3. These results encourage CW+ED use, especially in low-income countries.

Evaluation of the vital viability and their application in fungal spores’ disinfection with flow cytometry

Article

Oct 2020
CHEMOSPHERE

More attention was focused on fungi contamination in drinking water. Most researches about the inactivation of fungal spores has been conducted on disinfection efficiency and the leakage of intracellular substances. However, the specific structural damage of fungal spores treated by different disinfectants is poorly studied. In this study, the viability assessment methods of esterase activities and intracellular reactive oxygen species (ROS) were optimized, and the effects of chlorine-based disinfectants on fungal spores were evaluated by flow cytometry (FCM) and plating. The optimal staining conditions for esterase activity detection were as follows: fungal spores (10⁶ cells/mL) were stained with 10 μM carboxyfluorescein diacetate and 50 mM ethylene diamine tetraacetic acid at 33 °C for 10 min (in dark). The optimal staining conditions for intracellular ROS detection were as follows: dihydroethidium (the final concentration of 2 μg/mL) was added into fungal suspensions (10⁶ cells/mL), and then samples were incubated at 35 °C for 20 min (in dark). The cell culturability, membrane integrity, esterase activities, and intracellular ROS were examined to reveal the structural damage of fungal spores and underlying inactivation mechanisms. Disinfectants would cause the loss of the cell viability via five main steps: altered the morphology of fungal spores; increased the intracellular ROS levels; decreased the culturability, esterase activities and membrane integrity, thus leading to the irreversible death. It is appropriate to assess the effects of disinfectants on fungal spores and investigate their inactivation mechanisms using FCM.

Biocidal potential of electrochemically activated solutions (ECAS) against Aeromonas sp. Enterobacter sp. and Escherichia coli in tap water

Article

Aug 2020

The biocidal efficiency of electrochemically activated solutions (ECAS) against isolated bacterial species from the water distribution network was analyzed. Predominant microbial consortia (identified using 16S rRNA gene sequencing as Aeromonas sp. Enterobacter sp. and Escherichia. coli) with a mean count of 1.15 × 10⁶ CFU/mL was inoculated in 100 L tap water in a prototype network to simulate real conditions. The physico-chemical quality of tap water of subsurface origin was assessed before experimentation. The contact times of 10 s, 10 min and 1 h were selected against the ECAS dosages of 1%, 5% and 10% (v/v), respectively. Each test organism showed an independent response at varying dosages. A 10% disinfectant dosage corresponding to a contact time of 1 h showed the highest Log reduction as 4.11 > 3.50 > 2.74 for Enterobacter sp. > E. coli > Aeromonas sp., respectively. The results revealed that due to varying physico-chemical composition and carbon content of test water, complete inhibition of test organism (1.15 × 10⁶ CFU/mL) was not achieved at 10% ECAS dosing for 1 h. The study concludes that adequate pretreatment could further enhance the removal efficacy and reduce the minimum inhibitory concentration of ECAS.

Identification and enumeration of microbial organisms in grey water in the Khayelitsha informal settlement, Western Cape, South Africa

Article

Mar 2020

Rapid and Sensitive Detection of Viable but Non-culturable Salmonella Induced by Low Temperature from Chicken Using EMA-Rti-LAMP Combined with BCAC

Article

Full-text available

Feb 2020
FOOD ANAL METHOD

In this study, a rapid and sensitive real-time loop-mediated isothermal amplification (Rti-LAMP) assay was developed for quantitative and evaluation of viable but non-culturable (VBNC) Salmonella. Four micrograms per milliliter of ethidium bromide monoazide (EMA) could significantly inhibit DNA amplification derived from dead cells in Rti-LAMP assays. The EMA-Rti-LAMP was used to monitor the culturable and VBNC Salmonella cells induced by 4 °C and − 20 °C, as direct fluorescence method (DEM) and plate counting method as controls. When 1.3 × 10⁴ CFU/mL Salmonella was 5 cycles of freeze-thaw, the cells were all dead. However, Salmonella in 1.3 × 10⁶ CFU/mL gradually transferred into VBNC state reaching 6.0 × 10² CFU/mL (0.05%) after 5 cycles of freeze-thaw. Keeping Salmonella 1.3 × 10⁴ CFU/mL and 1.3 × 10⁶ CFU/mL in 0.85% NaCl at 4 °C, the culturable cells persistently decreased in plate counting. Meanwhile, the VBNC cells generated gradually from 0 to 4.2 × 10³ CFU/mL and 1.3 × 10⁵ CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage, respectively. While in − 20 °C, 1.3 × 10⁴ CFU/mL Salmonella sharply inactivated during 20 days, but 1.3 × 10⁶ CFU/mL increasingly transferred into VBNC state reaching 3.5 × 10⁴ CFU/mL detected by both EMA-Rti-LAMP and DEM up to 110-day storage. The results indicated that the EMA-Rti-LAMP had similar accuracy with DEM in rapidly detecting viable including VBNC cells, and the former had specificity but the latter did not. The EMA-Rti-LAMP combined with bentonite-coated activated carbon (BCAC) treatment could detect as low as 35 CFU/g VBNC Salmonella derived from contaminated chicken, and the entire assay completed in 5 h. Furthermore, four identical samples were Salmonella positive from 24 retail frozen chicken samples detected by plate culture (GB4789.4-2016), BCAC-Rti-LAMP, and BCAC-EMA-Rti-LAMP. The BCAC-EMA-Rti-LAMP had one more sample for Salmonella positive than that of plate culture, but less two samples than that of BCAC-Rti-LAMP. Noticeably, the BCAC-EMA-Rti-LAMP had much more accuracy as plate counting than that of BCAC-Rti-LAMP in detection of viable Salmonella derived from chicken. These results strongly suggested that the BCAC-EMA-Rti-LAMP assay could be a rapid and sensitive method for detection of viable Salmonella including VBNC cells in chicken without enrichment.

Detection and Verification of the Viable but Nonculturable (VBNC) State of Escherichia coli O157:H7 and Listeria monocytogenes Using Flow Cytometry and Standard Plating

Article

Jun 2018

The use of electrolyzed oxidizing (EO) water to inactivate microorganisms on foods has been extensively studied and shown to be effective. However, the prospect of the formation of “viable but nonculturable” (VBNC) cells in pathogens after low free chlorine concentration (FCC) treatments under high organic loads presents safety concerns. This study investigated the effect of EO water FCC on inducing Escherichia coli O157:H7 and Listeria monocytogenes into the VBNC state and studied possible resuscitation triggering procedures of the VBNC cells. A 5‐strain cocktail of each pathogen (10⁶ colony forming units [CFU]/mL) was exposed to EO water (FCC of 20, 10, 5, 2.5, 1.25, 0.625 mg/L) and allowed to stand for 1 and 5 min, followed by the addition of neutralizing broth. Treated samples were plated on nonselective agar and analyzed using flow cytometry. For resuscitation, samples treated with identified VBNC induction conditions were exposed to elevated temperatures (37 °C) as well as addition of sodium pyruvate (SP) and Tween® 20 (T20) solutions. The initial culturing procedures suggested complete inactivation of both pathogens at 2.5 and 1.25 mg/L FCC in the growth medium. However, flow cytometry profiles showed VBNC cells were present. Subjecting samples to the recovery procedures further proved that VBNC E. coli O157:H7 can be resuscitated after exposure to SP and T20 at 37 °C, while L. monocytogenes did not resuscitate. These findings show that treating pathogens at low FCC can induce the VBNC state, and culturability of E. coli O157:H7 can be restored under appropriate conditions. Practical Application VBNC induction conditions for foodborne pathogens during chlorine washing treatment were determined in a broth system and the information can serve as a basis for future studies that address the prevention of VBNC formation during produce wash treatments.

In-situ features of LNA and HNA bacteria in branch ends of drinking water distribution systems

Article

May 2017

Preservation of Microbial Pure Cultures and Mixed Communities

Chapter

Feb 2015

Microorganisms are a valuable and irreplaceable resources for scientific research and biotechnological innovation and should be safeguarded. Therefore, systematic preservation of isolated pure cultures, enriched mixed cultures or environmental samples should become an integral part of good research practice. Cryopreservation of biological material is a low-tech, widely applicable way of long-term and stable storage. Its success is mostly dependent on the cryoprotective agent, used to protect cells from mechanical injuries due to ice formation, the stability of the freezing temperature, and the correct manipulations before and after storage. Although cryopreservation success can be organism-dependent, the protocol described here proved successful for various fastidious pure and mixed cultures when frozen at -80°C using 5% (v/v) dimethyl sulfoxide as cryoprotective agent. Numerous parameters of the protocol can be changed or optimized and guidelines are given to develop a custom-made cryopreservation protocol.

Assessment of sanitation efficacy against Escherichia coli O157:H7 by rapid measurement of intracellular oxidative stress, membrane damage or glucose active uptake

Article

Jul 2016
FOOD CONTROL

To reduce cross contamination with foodborne pathogens during washing procedures, sodium hypochlorite (NaOCl) and hydrogen peroxide (H2O2) are widely used in the fresh produce industry. To ensure food safety and minimize excessive use of sanitizers and water during sanitation, it is critical to develop a rapid method to assess sanitation efficacy. This study examines the potential for employing oxidative stress, membrane damage and glucose uptake measurements to assess the antimicrobial efficacy of NaOCl (0–70.4 ppm) and H2O2 (0–1.6% v/v) toward Escherichia coli O157:H7. The relative amount of intracellular reactive oxygen species (ROS) was measured using Aminophenyl fluorescein (APF) and 2′,7′- dichlorodihydrofluorescein diacetate (H2DCFDA) for cells treated with NaOCl and H2O2 respectively. Results from these ROS sensitive probes revealed a limited correlation between these oxidative stress measurements and inactivation of bacteria measured using the plate counting method. Sanitation experiments, conducted with contaminated pre-cut lettuce leaves in water, were also carried out. Measurement of the bacterial membrane integrity was assessed using the membrane permeable probe propidium iodide (PI) and by evaluating effects on active transport by the glucose transport system using 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2-Deoxyglucose (2-NBDG). The results demonstrate that the relative increase in membrane permeability using PI correlated with NaOCl, but did not correlate with H2O2 induced reduction of bacterial survival quantified by the plate counting method. In contrast to the other approaches, monitoring the loss of glucose transport system function by measuring the uptake of 2-NBDG displayed strong correlation with the reduction of bacterial survival for both NaOCl and H2O2 treatment. Overall, the study demonstrated potential of glucose uptake measurements with 2-NBDG to serve as surrogate method for the traditional plate counting, which is the current gold standard used by the Food and Drugs Administration, to provide a faster analysis of sanitation processes.

Measurement of In Situ Activities of Nonphotosynthetic Microorganisms in Aquatic and Terrestrial Habitats

Article

Full-text available

Jan 1985

James T Staley

A novel endovascular clip system for the treatment of intracranial aneurysms: Technology, concept, and initial experimental results: Laboratory investigation

Article

Full-text available

Jun 2008
J NEUROSURG

The authors describe a novel device for the endovascular treatment of intracranial aneurysms, the endovascular clip system (eCLIPs). Descriptions of the device and its delivery system as well as the results of flow model tests and the treatment of experimental aneurysms are provided. The eCLIPs comprises a flexible hybrid implantable device (an anchor and a covered leaf) and a balloon catheter delivery system, designed to be positioned and activated in the parent vessel in such a way that the covered portion will abut the aneurysm neck. The eCLIPs was subjected to testing in glass, elastomeric, and cadaveric flow models to determine its navigability, orientation, and activation compared with commercially available stents. In a second experiment, 8 carotid artery sidewall aneurysms in swine were treated using eCLIPs. The degree of occlusion was observed on angiography immediately following and 30 days after device activation, and a histological analysis was performed at 30 days. The device could navigate tortuous glass models and human cadaveric vessels. Compared with commercially available stents, the eCLIPs performed well. It could be navigated, oriented, and activated easily and reliably. With regard to the 8 porcine experimental aneurysms, immediate postactivation angiograms confirmed complete occlusion of 4 lesions and near occlusion of the other 4. Angiographic follow-up at 30 days postactivation showed occlusion of all 8 aneurysms and patency of all parent vessels. Histopathological analysis revealed aneurysm healing, with smooth-muscle cells growing across the lesion neck to allow reendothelialization. Aneurysm occlusion with a single extrasaccular endovascular device has potential advantages. The authors believe that eCLIPs may prove to be a useful tool in the endovascular treatment of cerebral aneurysms. The system should reduce risks associated with coiling, procedure time, costs, and radiation exposure. The device satisfactorily occluded 8 experimental sidewall aneurysms. The observed healing pattern is similar to that seen after microsurgical clipping.

Solar Disinfection (SODIS) and Subsequent Dark Storage of Salmonella Typhimurium and Shigella Flexneri Monitored by Flow Cytometry

Article

Full-text available

May 2009

Pathogenic enteric bacteria are a major cause of drinking water related morbidity and mortality in developing countries. Solar disinfection (SODIS) is an effective means to fight this problem. In the present study, SODIS of two important enteric pathogens, Shigella flexneri and Salmonella typhimurium, was investigated with a variety of viability indicators including cellular ATP levels, efflux pump activity, glucose uptake ability, and polarization and integrity of the cytoplasmic membrane. The respiratory chain of enteric bacteria was identified to be a likely target of sunlight and UVA irradiation. Furthermore, during dark storage after irradiation, the physiological state of the bacterial cells continued to deteriorate even in the absence of irradiation: apparently the cells were unable to repair damage. This strongly suggests that for S. typhimurium and Sh. flexneri, a relatively small light dose is enough to irreversibly damage the cells and that storage of bottles after irradiation does not allow regrowth of inactivated bacterial cells. In addition, we show that light dose reciprocity is an important issue when using simulated sunlight. At high irradiation intensities (>700 W m(-2)) light dose reciprocity failed and resulted in an overestimation of the effect, whereas reciprocity applied well around natural sunlight intensity (<400 W m(-2)).

Validation of SYTO 9/Propidium Iodide Uptake for Rapid Detection of Viable but Noncultivable Legionella pneumophila

Article

Full-text available

Dec 2008
MICROB ECOL

Legionella pneumophila is an ubiquitous environmental microorganism that can cause Legionnaires' disease or Pontiac fever. As a waterborne pathogen, it has been found to be resistant to chlorine disinfection and survive in drinking water systems, leading to potential outbreaks of waterborne disease. In this work, the effect of different concentrations of free chlorine was studied (0.2, 0.7, and 1.2 mg l(-1)), the cultivability of cells assessed by standard culture techniques (buffered charcoal yeast extract agar plates) and viability using the SYTO 9/propidium iodide fluorochrome uptake assay (LIVE/DEAD BacLight). Results demonstrate that L. pneumophila loses cultivability after exposure for 30 min to 0.7 mg l(-1) of free chlorine and in 10 min when the concentration is increased to 1.2 mg l(-1). However, the viability of the cells was only slightly affected even after 30 min exposure to the highest concentration of chlorine; good correlation was obtained between the rapid SYTO 9/propidium iodide fluorochrome uptake assay and a longer cocultivation with Acanthamoeba polyphaga assay, confirming that these cells could still recover their cultivability. These results raise new concerns about the assessment of drinking water disinfection efficiency and indicate the necessity of further developing new validated rapid methods, such as the SYTO 9/propidium iodide uptake assay, to assess viable but noncultivable L. pneumophila cells in the environment.

Use of Flow Cytometry To Monitor Legionella Viability

Article

Full-text available

Jan 2008
APPL ENVIRON MICROB

Legionella viability was monitored during heat shock treatment at 70°C by a flow cytometric assay (FCA). After 30 min of treatment, for 6 of the 12 strains tested, the FCA still detected 10 to 25% of cells that were viable but nonculturable (VBNC). These VBNC cells were able to produce ATP and to be resuscitated after culture on amoebae.

Disinfection of drinking water by using a novel electrochemical reactor employing carbon-cloth electrodes

Article

Full-text available

Mar 1992

A novel electrochemical reactor employing carbon-cloth electrodes was constructed for disinfection of drinking water. Escherichia coli K-12 (10(2) cells per cm3) was sterilized when a cell suspension was passed through the reactor at a dilution rate of 6.0 h-1, and a potential of 0.7 V versus a saturated calomel electrode was applied to an electrode. The survival ratio increased with increasing dilution rate but was less than 0.1% at dilution rates of less than 6.0 h-1. Although the survival ratio increased with increasing cell concentration above 10(3) cells per cm3, the disinfection rate also increased. The disinfection rate was 6.0 x 10(2) cells per cm3 per h at a cell concentration of 10(2) cells per cm3. Continuous sterilization of E. coli cells was carried out for 24 h. Sterilization is based on an electrochemical reaction between the electrode and the cell which is mediated by intracellular coenzyme A. Sterilization of drinking water by using this reactor was successfully performed, demonstrating the potential of such a reactor for clean and efficient water purification.

Comparative assessment of chlorine, heat, ozone, and UV light for killing Legionella pneumophila within a model plumbing system

Article

Full-text available

Mar 1987

Nosocomial Legionnaires disease can be acquired by exposure to the organism from the hospital water distribution system. As a result, many hospitals have instituted eradication procedures, including hypercholorination and thermal eradication. We compared the efficacy of ozonation, UV light, hyperchlorination, and heat eradication using a model plumbing system constructed of copper piping, brass spigots, Plexiglas reservoir, electric hot water tank, and a pump. Legionella pneumophila was added to the system at 10(7) CFU/ml. Each method was tested under three conditions; (i) nonturbid water at 25 degrees C, (ii) turbid water at 25 degrees C, and (iii) nonturbid water at 43 degrees C. UV light and heat killed L. pneumophila most rapidly and required minimal maintenance. Both UV light and heat (60 degrees C) produced a 5 log kill in less than 1 h. In contrast, both chlorine and ozone required 5 h of exposure to produce a 5 log decrease. Neither turbidity nor the higher temperature of 43 degrees C impaired the efficacy of any of the disinfectant methods. Surprisingly, higher temperature enhanced the disinfecting efficacy of chlorine. However, higher temperature accelerated the decomposition of the chlorine residual such that an additional 120% volume of chlorine was required. All four methods proved efficacious in eradicating L. pneumophila from a model plumbing system.

Recovery of culturability of an HOCl-stressed population of

Article

Full-text available

Dec 1997

An Escherichia coli population harvested in exponential phase at about 10(8) cells/ml was treated in phosphate buffer with HOCl at concentrations ranging from 0.4 to 1 mg/liter (7.7 to 19 microM). The HOCl stress resulted in the appearance of three cell subpopulations: a majority of dead (nonrespiring) cells, a few culturable cells (10(2) to 10(4)), and about 10(7) viable but nonculturable cells. In the absence of any added exogenous nutrient, a culturable population could be recovered after 1 day of incubation in phosphate buffer, and such a population would reach a cell density close to 10% of the initial density of the stressed population, whatever the initial number of survivors. When a small number of untreated cells were mixed with the stressed population, growth of the untreated cells was observed, demonstrating that damaged cells provided nutrients. Similarly, a filtrate and a disrupted-cell filtrate of the stressed population supported growth of untreated cells with the same efficiency. The number of CFU (untreated or stressed) at plateau phase depended on the initial density of the stressed cells. Taken together, these results suggest that recovery in phosphate buffer of an HOCl-stressed population is in large part due to growth of a few culturable cells at the expense of damaged cells. However, comparison of the growth rates of the stressed culturable population and of untreated bacteria growing in filtrate showed significantly faster growth of the stressed cells, a fact not fully compatible with the hypothesis that recovery is only the simple growth of survivors. We suggest, therefore, that in addition to growth of the few culturable stressed cells, there is repair and growth of some mildly injured viable but nonculturable cells.

Determination of the Biomasses of Small Bacteria at Low Concentrations in a Mixture of Species with Forward Light Scatter Measurements by Flow Cytometry

Article

Full-text available

Nov 1998
APPL ENVIRON MICROB

The forward light scatter intensity of bacteria analyzed by flow cytometry varied with their dry mass, in accordance with theory. A standard curve was formulated with Rayleigh-Gans theory to accommodate cell shape and alignment. It was calibrated with an extinction-culture isolate of the small marine organism Cycloclasticus oligotrophus, for which dry weight was determined by CHN analysis and 14C-acetate incorporation. Increased light scatter intensity due to formaldehyde accumulation in preserved cells was included in the standard curve. When differences in the refractive indices of culture media and interspecies differences in the effects of preservation were taken into account, there was agreement between cell mass obtained by flow cytometry for various bacterial species and cell mass computed from Coulter Counter volume and buoyant density. This agreement validated the standard curve and supported the assumption that cells were aligned in the flow stream. Several subpopulations were resolved in a mixture of three species analyzed according to forward light scatter and DNA-bound DAPI (4', 6-diamidino-2-phenylindole) fluorescence intensity. The total biomass of the mixture was 340 &mgr;g/liter. The lowest value for mean dry mass, 0.027 +/- 0.008 pg/cell, was for the subpopulation of C. oligotrophus containing cells with a single chromosome. Calculations from measurements of dry mass, Coulter Counter volume, and buoyant density revealed that the dry weight of the isolate was 14 to 18% of its wet weight, compared to 30% for Escherichia coli. The method is suitable for cells with 0.005 to about 1.2 pg of dry weight at concentrations of as low as 10(3) cells/ml and offers a unique capability for determining biomass distributions in mixed bacterial populations.

The use of multiple indices of physiological activity to access viability in chlorine disinfected Escherichia coli O157:H7

Article

Full-text available

Aug 1999

A suite of fluorescent intracellular stains and probes was used, in conjunction with viable plate counts, to assess the effect of chlorine disinfection on membrane potential (rhodamine 123; Rh123 and bis-(1,3-dibutylbarbituric acid) trimethine oxonol; DiBAC4(3)), membrane integrity (LIVE/DEAD BacLight kit), respiratory activity (5-cyano-2,3-ditolyl tetrazolium chloride; CTC) and substrate responsiveness (direct viable counts; DVC) in the commensal pathogen Escherichia coli O157:H7. After a 5 min exposure to the disinfectant, physiological indices were affected in the following order: viable plate counts > substrate responsiveness > membrane potential > respiratory activity > membrane integrity. In situ assessment of physiological activity by examining multiple targets, as demonstrated in this study, permits a more comprehensive determination of the site and extent of injury in bacterial cells following sublethal disinfection with chlorine. This approach to assessing altered bacterial physiology has application in various fields where detection of stressed bacteria is of interest.

Resolution of Viable and Membrane-Compromised Bacteria in Freshwater and Marine Waters Based on Analytical Flow Cytometry and Nucleic Acid Double Staining

Article

Full-text available

Oct 2001
APPL ENVIRON MICROB

The membrane integrity of a cell is a well-accepted criterion for characterizing viable (active or inactive) cells and distinguishing them from damaged and membrane-compromised cells. This information is of major importance in studies of the function of microbial assemblages in natural environments, in order to assign bulk activities measured by various methods to the very active cells that are effectively responsible for the observations. To achieve this task for bacteria in freshwater and marine waters, we propose a nucleic acid double-staining assay based on analytical flow cytometry, which allows us to distinguish viable from damaged and membrane-compromised bacteria and to sort out noise and detritus. This method is derived from the work of S. Barbesti et al. (Cytometry 40:214-218, 2000) which was conducted on cultured bacteria. The principle of this approach is to use simultaneously a permeant (SYBR Green; Molecular Probes) and an impermeant (propidium iodide) probe and to take advantage of the energy transfer which occurs between them when both probes are staining nucleic acids. A full quenching of the permeant probe fluorescence by the impermeant probe will point to cells with a compromised membrane, a partial quenching will indicate cells with a slightly damaged membrane, and a lack of quenching will characterize intact membrane cells identified as viable. In the present study, this approach has been adapted to bacteria in freshwater and marine waters of the Mediterranean region. It is fast and easy to use and shows that a large fraction of bacteria with low DNA content can be composed of viable cells. Admittedly, limitations stem from the unknown behavior of unidentified species present in natural environments which may depart from the established permeability properties with respect to the fluorescing dyes.

Legionella gresilensis sp. nov. and Legionella beliardensis sp. nov., isolated from water in France

Article

Full-text available

Dec 2001

Novel Legionella-like isolates, strains Montbéliard A1T and Gréoux 11 D13T, isolated from two different French water sources, were studied taxonomically and phylogenetically. Morphological and biochemical characterization revealed that they were Gram-negative, aerobic, non-spore-forming bacilli with a cut-glass appearance that grew only on L-cysteine-supplemented buffered charcoal yeast extract agar. Phenotypic characterization using fatty acid and ubiquinone profiles and SDS-PAGE analysis confirmed that they were closely related, but distinct from, other species of the genus Legionella, since serotyping could not relate them to any existing serogroup. Genotypic profiles generated by randomly amplified polymorphic DNA and 16S-23S rDNA spacer region PCR analyses were unique for each of these isolates. DNA-DNA relatedness values of strains Montbéliard A1T and Gréoux 11 D13T to each other and to other Legionella type strains were less than 25%. Phylogenetic affiliation of these organisms obtained by 16S rDNA sequence comparisons confirmed that they were distinct from any other known Legionella species. All the above results confirm that these strains constitute two novel species for which the names Legionella gresilensis sp. nov. (type strain Gréoux 11 D13T = ATCC 700509T = CIP 106631T) and Legionella beliardensis sp. nov. (type strain Montbéliard A1T = ATCC 700512T = CIP 106632T) are proposed.

Bare stent-graft technique: A new method of endoluminal vascular reconstruction for the treatment of giant and fusiform aneurysms

Article

Full-text available

Nov 2002

We present our initial experience with a newly developed endovascular stent graft technique in the treatment of two patients with giant aneurysms. In both of these patients, surgery and conventional endovascular techniques were likely to fail. The technique resulted in the successful management of the aneurysms in both cases. Our technique is described, and related experiences in the literature are discussed.

Stent-Graft Placement for Wide-Neck Aneurysm of the Vertebrobasilar Junction

Article

Full-text available

May 2004

We present a case in which a stent-graft was used to treat an aneurysm of the vertebrobasilar junction. According to our literature search, this is one of the first cases involving the intracranial placement of a stent-graft and the first case in which an aneurysm of the vertebrobasilar junction was treated in this manner. A stent-graft can be useful device for the neuroendovascular treatment of aneurysms in select patients.

Heterotrophic Plate Count Bacteria – What is Their Significance in Drinking Water?

Article

Full-text available

Jun 2004
INT J FOOD MICROBIOL

While the literature documents the universal occurrence of heterotrophic plate count (HPC) bacteria in soils, foods, air, and all sources of water, there is a lingering question as to whether this group of organisms may signal an increased health risk when elevated populations are present in drinking water. This paper reviews the relevant literature on HPC bacteria in drinking water, the lack of clinical evidence that elevated populations or specific genera within the HPC flora pose an increased health risk to any segment of the population, and the appropriate uses of HPC data as a tool to monitor drinking water quality changes following treatment. It finds no evidence to support health-based regulations of HPC concentrations.

Recovery of culturability of an HOCl-stressed population of Escherichia coli after incubation in phosphate buffer: resuscitation or regrowth?

Article

Nov 1997

Current and future applications of flow cytometry in aquatic microbiology

Article

Oct 2000

José Vives-Rego

Inactivation of Cryptosporidium parvum oocysts and Clostridium perfringes spores by a mixed-oxidant disinfectant and by free chlorine

Article

Jul 1997

Cryptosporidium parvum oocysts and Clostridium perfringens spores are very resistant to chlorine and other drinking-water disinfectants. Clostridium perfringens spores have been suggested as a surrogate indicator of disinfectant activity against Cryptosporidium parvum and other hardy pathogens in water. In this study, an alternative disinfectant system consisting of an electrochemically produced mixed-oxidant solution (MIOX; LATA Inc.) was evaluated for inactivation of both Cryptosporidium parvum oocysts and Clostridium perfringens spores. The disinfection efficacy of the mixed-oxidant solution was compared to that of free chlorine on the basis of equal weight per volume concentrations of total oxidants. Batch inactivation experiments were done on purified oocysts and spores in buffered, oxidant demand-free water at pH 7 an 25 degrees C by using a disinfectant dose of 5 mg/liter and contact times of up to 24 h. The mixed-oxidant solution was considerably more effective than free chlorine in activating both microorganisms. A 5-mg/liter dose of mixed oxidants produced a > 3-log10-unit (> 99.9%) inactivation of Cryptosporidium parvum oocysts and Clostridium perfringens spores in 4 h. Free chlorine produce no measurable inactivation of Cryptosporidium parvum oocysts by 4 or 24 h, although Clostridium perfringens spores were inactivated by 1.4 log10 units after 4 h. The on-site generation of mixed oxidants may be a practical and cost-effective system of drinking water disinfection protecting against even the most resistant pathogens, including Cryptosporidium oocysts.

Microbiological safety of natural mineral water

Article

Jun 2002

H Leclerc

Electrochemical Disinfection, an Environmentally Acceptable Method of Drinking Water Disinfection?

Article

Sep 2005
ELECTROCHIM ACTA

In general, chlorination is the method of drinking water disinfection most favoured by the water industry. Occasional outbreaks of water transmitted disease, the identification of chlorine as a source of potentially harmful disinfection by-products, and the emergence of recalcitrant pathogens has led to heightened regulation for the removal of microbial pathogens and disinfection by-products from drinking water. As a result, research and development of alternative disinfection technologies has intensified. Electrochemical disinfection has emerged as one of the more feasible alternatives to chlorination. Research using a range of cell configurations has shown electrochemical disinfection to be effective against a range of pathogens. However, in many of the systems, disinfection efficacy appears to be related to the generation of chlorine species. The apparent prevalence of chlorine as the mechanism of disinfection begs the question as to whether electrochemical disinfection has an advantage over chlorination in terms of its inactivation efficacy and potential to form disinfection by-products. This paper reports on a series of experiments evaluating the disinfection efficacy of an electrochemical disinfection technology against Escherichia coli and bacteriophage MS2. The results of these experiments conclude that electrochemical disinfection can be effective without the generation of chlorine species.

Electrochemical water disinfection Part I: Hypochlorite production from very dilute chloride solutions

Article

Jun 1999

Electrolytic production of hypochlorite in very dilute chloride solutions is investigated using platinum and iridium oxide coated titanium expanded metal electrodes as anodes. The dependence of the hypochlorite production rate on temperature, chloride concentration and current density was determined. It was found that the hypochlorite production rate is consistently higher on iridium oxide coated titanium compared to platinum coated titanium electrodes.

Legionella pneumophila in the Environment: The Occurrence of a Fastidious Bacterium in Oligotrophic Conditions

Article

May 2005

Legionella pneumophila, a micro-organism encountered in aquatic environments, can cause serious intracellular infections among humans. Since the bacterium is ubiquitous in aquatic habitats, it appears to be impossible to prevent L. pneumophila from entering man-made water systems. However, many questions concerning the survival and/or growth in the environment, the partners and opponents of L. pneumophila remain unanswered. This review focuses on the factors governing the ecology of L. pneumophila, since there is considerable divergence and even contradiction in literature on its environmental requirements. A key question to be resolved is the discrepancy between the fastidious nature of L. pneumophila in axenic cultures (e.g. 400 mg l−1 L-cysteine and 250 mg l-1 ferric iron) and the nutritionally poor environments in which it is commonly detected. It is assumed that dense microbial communities, as occurring in sediments and biofilms – but not likely in surface and drinking water, – can provide the necessary growth requirements for L. pneumophila. However, most of the studies concerning L. pneumophila have led to the general opinion that the organism can only multiply in the aquatic environment as a parasite in certain protozoa. The discovery of the non-classical siderophore legiobactin also indicates that the iron requirement for survival and autonomous growth is not as high as has been assumed. It thus appears that in order to control Legionella in the environment, focus should be on the eradication of microbial hotspots in which L. pneumophila resides.

Chlorine demand of biofilms in water distribution systems

Article

Feb 1999
WATER RES

Fixed biomass (biofilms) chlorine demand, produced from 3 test waters, is studied by the means of two methods: directly on colonized plastic beads and indirectly on biomass extracted after sonication. The first method results show that a steady state of biomass formation can be reached after an incubation period depending on test water quality. At steady state biofilms chlorine demand increases linearly with BDOC of water and increases according to the ratio surface/volume. The second method results show that extracted biomass chlorine demand is related with protein concentration and heterotrophic plate counts level in the extracts.

Approaches to prevention, removal and killing of biofilms

Article

Jun 2003
INT BIODETER BIODEGR

Bernhard Meyer

Biofilms contribute to hygiene problems in the food industry and in the medical field. Biofilms are diverse and due to the development of special phenotypes, biofilm organisms are not as susceptible to biocides as planktonic microorganisms. Biofilms may be prevented by regular disinfection. Since the attachment of microbes to surfaces and the development of biofilm phenotypes is a very fast process, it is, however, almost impossible, to prevent biofilm formation completely. The removal and killing of established biofilms requires harsh treatments, mostly using oxidising biocides. Depending on the nature of the biofilms, different biocides may be useful and the best biocide for a specific biofilm still has to be determined under practical conditions. Another approach is the prevention of biofilm formation by selection of materials that do not support the attachment of microorganisms. Some materials like glass and stainless steel show less biofilm formation than others. The ranking of materials, however, depends on the conditions, under which they are tested. A novel approach is biofilm inhibition by supplementation of systems with nutrients, to inhibit attachment. First results on inhibition of biofouling in reversed osmosis systems are presented.

Inactivation of Mycobacterium avium by copper and silver ions

Article

Jul 1998
WATER RES

Mycobacteriumavium and Legionella pneumophila are opportunistic pathogens that are found in hot water systems of hospitals. Hospital water supplies have also been suggested as sources for M. avium in AIDS patients. Copper/silver ionization has been shown to be an effective method for controlling Legionella in hospital hot water systems. The effect of copper/silver ions on M. avium is unknown. The susceptibility of M. avium to copper/silver ions was tested by performing kill-curve experiments in 100 ml buffered water at the following concentrations of copper/silver ions: (1) 0.1/0.01; (2) 0.2/0.02; (3) 0.4/0.04; (4) 0.8/0.08 mg/l. Initial M. avium concentration was ca. 3×106 CFU/ml. Viability was assessed daily for 1 week by plate count on Middlebrook 7H10 agar, and by the BACTEC system. Culture plates and BACTEC bottles were incubated at 37°C for two weeks. Contact times (days) required for 99% and 99.9% inactivation of M. avium by copper/silver ion concentrations of 0.1/0.01; 0.2/0.02; 0.4/0.04; 0.8/0.08 were 3 d and 5 d, 3 d and 5 d, 2 d and 4 d, 1 d and 2 d, respectively. The data indicate that M. avium is susceptible to these metal ions. However, M. avium are more resistant to the bactericidal effects of copper/silver ions than Legionella, requiring 100 times longer exposure to achieve comparable killing in vitro. Nevertheless, this suggests that the copper/silver ionization system may rid hospital hot water systems of both of these potential pathogens.

Water disinfection for developing countries and potential for solar thermal pasteurization

Article

Sep 1998
SOL ENERGY

Water-borne disease in developing countries leads to millions of deaths and billions of illnesses annually. Water disinfection is one of several interventions that can improve public health, especially if part of a broad program that considers all disease transmission routes and sustainably involves the community. Considering water volumes ≲30 m3/day, appropriate disinfection methods include chlorination, slow sand filtration, ultraviolet (UV) radiation and pasteurization. Pretreatment with a coarse roughing filtration is generally used with the first three of these technologies to reduce turbidity and maintain high effectiveness. Cysts and worm eggs are resistant to chlorination and UV but can be filtered relatively easily. Chlorination is widely used and inexpensive but requires a continual supply of chemicals. Slow sand filtration is lowest in cost but requires high investment in labor. Household filtration using indigenous devices requires little capital investment but is relatively ineffective and difficult to properly maintain. Batch treatment with solar UV is very easy to implement but effectiveness in practice is uncertain since temperatures above 50°C should be attained. UV lamp devices are inexpensive and easy to use but require power and access to maintenance infrastructure. Boiling of water requires no initial expense but fuel and labor costs are very high. Solar pasteurization devices (batch and flow-through) are effective and relatively maintenance-free, but existing products yield high treatment cost. Flow-through systems with selective flat plate collectors become cost-competitive with UV technology at costs of about $380/m2 and $80/m2 for home-scale and village-scale use, respectively. These cost goals might be attained with polymer thin film designs if durability issues are adequately resolved.

Uncertainty Characterization Approaches for Risk Assessment of DBPs in Drinking Water: A Review

Article

Feb 2009
J ENVIRON MANAGE

The management of risk from disinfection by-products (DBPs) in drinking water has become a critical issue over the last three decades. The areas of concern for risk management studies include (i) human health risk from DBPs, (ii) disinfection performance, (iii) technical feasibility (maintenance, management and operation) of treatment and disinfection approaches, and (iv) cost. Human health risk assessment is typically considered to be the most important phase of the risk-based decision-making or risk management studies. The factors associated with health risk assessment and other attributes are generally prone to considerable uncertainty. Probabilistic and non-probabilistic approaches have both been employed to characterize uncertainties associated with risk assessment. The probabilistic approaches include sampling-based methods (typically Monte Carlo simulation and stratified sampling) and asymptotic (approximate) reliability analysis (first- and second-order reliability methods). Non-probabilistic approaches include interval analysis, fuzzy set theory and possibility theory. However, it is generally accepted that no single method is suitable for the entire spectrum of problems encountered in uncertainty analyses for risk assessment. Each method has its own set of advantages and limitations. In this paper, the feasibility and limitations of different uncertainty analysis approaches are outlined for risk management studies of drinking water supply systems. The findings assist in the selection of suitable approaches for uncertainty analysis in risk management studies associated with DBPs and human health risk.

Long-Term Survival of Legionella pneumophila in the Viable But Nonculturable State After Monochloramine Treatment

Article

Oct 2008

Legionella pneumophila, a facultative intracellular human pathogen, can persist for long periods in natural and artificial aquatic environments. Eradication of this bacterium from plumbing systems is often difficult. We tested L. pneumophila survival after monochloramine treatment. Survival was monitored using the BacLight Bacterial Viability Kit (Molecular Probes), ChemChrome V6 Kit (Chemunex), quantitative polymerase chain reaction and culturability on buffered charcoal-yeast extract agar. In nonculturable samples, regain of culturability was obtained after addition of the amoeba Acanthamoeba castellanii, and esterase activity and membrane integrity were observed after >4 months after treatment. These results demonstrate for the first time that L. pneumophila could persist for long periods in biofilms into the viable but nonculturable (VBNC) state. Monitoring L. pneumophila in water networks is generally done by enumeration on standard solid medium. This method does not take into account VBNC bacteria. VBNC L. pneumophila could persist for long periods and should be resuscitated by amoeba. These cells constitute potential sources of contamination and should be taken into account in monitoring water networks.

Rapid, cultivation-independent assessment of microbial viability in drinking water

Article

Sep 2008
WATER RES

Fast and accurate monitoring of chemical and microbiological parameters in drinking water is essential to safeguard the consumer and to improve the understanding of treatment and distribution systems. However, most water utilities and drinking water guidelines still rely solely on time-requiring heterotrophic plate counts (HPC) and plating for faecal indicator bacteria as regular microbiological control parameters. The recent development of relative simple bench-top flow cytometers has made rapid and quantitative analysis of cultivation-independent microbial parameters more feasible than ever before. Here we present a study using a combination of cultivation-independent methods including fluorescence staining (for membrane integrity, membrane potential and esterase activity) combined with flow cytometry and total adenosine tri-phosphate (ATP) measurements, to assess microbial viability in drinking water. We have applied the methods to different drinking water samples including non-chlorinated household tap water, untreated natural spring water, and commercially available bottled water. We conclude that the esterase-positive cell fraction, the total ATP values and the high nucleic acid (HNA) bacterial fraction (from SYBR Green I staining) were most representative of the active/viable population in all of the water samples. These rapid methods present an alternative way to assess the general microbial quality of drinking water as well as specific events that can occur during treatment and distribution, with equal application possibilities in research and routine analysis.

Measurement of in Situ Activities of Nonphotosynthetic Microorganisms in Aquatic and Terrestial Habitats

Article

Feb 1985

Covered Stent Placement for Neurovascular Disease

Article

Apr 1997

We describe an endovascular technique in which covered stents were used to occlude a parent vessel. In one patient, with a giant paraclinoid aneurysm, a Gortex-covered Palmaz stent was used to occlude the cervical internal carotid artery and to create thrombosis in the aneurysm. In the second patient, with a high-flow vertebrojugular fistula, a hooded stent provided definitive treatment after an attempt to close the fistula by detachable balloon therapy failed. Follow-up of these patients revealed stable stent position and no untoward effects of permanent vessel occlusion.

Autologous Vein-covered Stent Repair of a Cervical Internal Carotid Artery Pseudoaneurysm: Technical Case Report

Article

Mar 1998

Stenting of a cervical internal carotid pseudoaneurysm is presented using a stent covered with saphenous vein. This procedure resulted in immediate exclusion of the aneurysm and maintained patency of the carotid artery. A gunshot to the neck resulted in airway obstruction and respiratory arrest with neurological injury in a male patient. Angiographic investigation revealed an enlarging cervical internal carotid pseudoaneurysm, possibly the source of a cerebral embolism. Anticoagulation had to be stopped because of bleeding complications. A decision was made to conduct definitive percutaneous treatment of the pseudoaneurysm. A Palmaz stent (JJIS, Warren, NJ) was covered with saphenous vein harvested from the patient's leg. Using standard technique and a simple innovation for stent construct introduction into the sheath, the vein-covered stent was placed across the opening of the aneurysm and dilated into position with a balloon. Immediate exclusion of a cervical internal carotid pseudoaneurysm was performed using an autologous vein-covered stent. One-month follow-up confirmed continued exclusion of the pseudoaneurysm and carotid patency without stenosis.

Two and three-color fluorescence flow cytometric analysis of immunoidentified viable bacteria

Article

Jul 2000

Traditional culture methods well established in the past and still in use are not able to detect the environmental microorganisms that exist in a viable but not culturable state. A number of different fluorescence-based assays have been developed over the past decade to detect and identify viable bacteria in the environment. We have developed a simple and rapid method for measuring the number and viability of immunolabeled bacteria by means of a two/three color fluorescence flow cytometric analysis. After washing, cultured bacteria in suspension were labeled with a rabbit polyclonal antibody recognizing the wall lipopolysaccharide complex. A secondary biotinylated anti-rabbit polyclonal antibody was added allowing the cells to be labeled with the streptavidin R-phycoerythrin-Cyanine 5 (RPE-Cy5) fluorochrome. Before flow cytometric analysis, bacterial suspensions were stained with SYBR Green I and propidium iodide which stain all of the cells and the non viable ones, respectively. With the appropriate filter sets of both Bryte-HS (Bio-Rad, Hercules, CA) and FACScan (Becton Dickinson, San Jose, CA) flow cytometers, the measurement of separated green (SYBR Green I), orange-red (propidium iodide), and far red (RPE-Cy5) fluorescence was possible, allowing the enumeration of viable immunodetected bacteria. The entire protocol is completed in less than 3 h, offering numerous possibilities for rapid and precise analyses in sanitary, industrial, and environmental microbiology.

Current and future applications of flow cytometry in aquatic microbiology

Article

Nov 2000

Flow cytometry has become a valuable tool in aquatic and environmental microbiology that combines direct and rapid assays to determine numbers, cell size distribution and additional biochemical and physiological characteristics of individual cells, revealing the heterogeneity present in a population or community. Flow cytometry exhibits three unique technical properties of high potential to study the microbiology of aquatic systems: (i) its tremendous velocity to obtain and process data; (ii) the sorting capacity of some cytometers, which allows the transfer of specific populations or even single cells to a determined location, thus allowing further physical, chemical, biological or molecular analysis; and (iii) high-speed multiparametric data acquisition and multivariate data analysis. Flow cytometry is now commonly used in aquatic microbiology, although the application of cell sorting to microbial ecology and quantification of heterotrophic nanoflagellates and viruses is still under development. The recent development of laser scanning cytometry also provides a new way to further analyse sorted cells or cells recovered on filter membranes or slides. The main infrastructure limitations of flow cytometry are: cost, need for skilled and well-trained operators, and adequate refrigeration systems for high-powered lasers and cell sorters. The selection and obtaining of the optimal fluorochromes, control microorganisms and validations for a specific application may sometimes be difficult to accomplish.

Analysis of bacterial function by multi-colour and single cell sorting

Article

Oct 2000
J MICROBIOL METH

With the increased awareness of the problems associated with the growth dependent analysis of bacterial populations, direct optical detection methods such as flow cytometry have enjoyed increased popularity over the last few years. Among the analyses discussed here are: (1) Bacterial discrimination from other particles on the basis of nucleic acid staining, using sample disaggregation to provide fast reliable enumeration while minimizing data artefacts due to post sampling growth; (2) Determination of basic cell functions such as reproductive ability, metabolic activity and membrane integrity, to characterise the physiological state or degree of viability of bacteria; and (3) The use of single cell sorting onto agar plates, microscope slides or into multi-well plates to correlate viability as determined by cell growth with fluorescent labelling techniques. Simultaneous staining with different fluorochromes provides an extremely powerful way to demonstrate culture heterogeneity, and also to understand the functional differences revealed by each stain in practical applications. Analysis of bacterial fermentations showed a considerable drop (20%) in membrane potential and integrity during the latter stages of small scale (5L), well mixed fed-batch fermentations. These changes, not found in either batch or continuous culture fermentations, are probably due to the severe, steadily increasing stress associated with glucose limitation during the fed-batch process, suggesting 'on-line' flow cytometry could improve process control. Heat injured cells can already show up to 4 log of differences in recovery in different pre-enrichment media, thus contributing to the problem of viable but non-culturable cells (VBNC's). Cytometric cell sorting demonstrated decreasing recovery with increasing loss of membrane function. However, a new medium protecting the cells from intracellular and extracellular causes of oxidative stress improved recovery considerably. Actively respiring cells showed much higher recovery improvement than the other populations, demonstrating for the first time the contribution of oxidative respiration to intracellular causes of damage as a key part of the VBNC problem. Finally, absolute and relative frequencies of one species in a complex population were determined using immunofluorescent labelling in combination with the analysis of cell function. The detail and precision of multiparameter flow cytometric measurements of cell function at the single cell level now raise questions regarding the validity of classical, growth dependent viability assessment methods.

Aberrant internal carotid artery in the middle-ear space

Article

Nov 2000

The incidence of an aberrant internal carotid artery in the middle ear is approximately one per cent and most patients are asymptomatic. We present two patients with an aberrant internal carotid artery who presented with pulsatile tinnitus and an intra-tympanic mass. Here we discuss the clinical presentation, relevant radiographic investigations and further management of these patients.

Exploring the frontier between life and death in Escherichia coli: Evaluation of different viability markers in live and heat- or UV-killed cells

Article

Dec 2000
RES MICROBIOL

A number of methods have been proposed to assess the viability of cells without culture. Each method is based on criteria that reflect different levels of cellular integrity or functionality. As a consequence, the interpretation of viability is often ambiguous. The purposes of this work were to evaluate the capacity of current viability markers to distinguish between live and dead Escherichia coli K-12 cells. Methods that assess 'viability' by the demonstration of metabolic activities (esterase activity, active electron transport chain, transport of glucose), cellular integrity (membrane integrity, presence of nucleic acids) or the building up of cellular material (cell elongation) have been evaluated in live and UV- or heat-killed cells. With live cells, viability markers detected cells in counts similar to the colony count. However, these so-called viability markers could stain dead cells for some time after the lethal treatment. For the UV-killed cells, residual activities were detected even after 48 h of storage at 20 degrees C. However, for heat-treated cells, these activities disappeared within hours after heat treatment. Only a combination of fluorescence in situ hybridization with rRNA probes and cell elongation in response to nutrients (in the presence of an inhibitor of cell division) had the ability to differentiate live from dead cells. Problems in the definition of a viable but nonculturable state are in part due to the lack of a clear definition of bacterial death. We consider death as an irreversible state where no growth, cell elongation or protein synthesis may occur.

Advances in the Bacteriology of the Coliform Group: Their Suitability as Markers of Microbial Water Safety

Article

Feb 2001

Advances in the elaboration of novel genomic types of beta-galactosidase-positive Enterobacteriaceae and comprehensive studies of their habitats have resulted in an innovative approach to the assessment of the merits and shortcomings of the thermotrophic and fecal species Escherichia coli and all other coliforms as markers of the microbiological safety of water. As one of the consequences, it is recommended to abolish the "technical" designation fecal coliforms because their current method of detection will result in the isolation of thermotrophic organisms that have been demonstrated, beyond a doubt, to be of environmental, rather than uniquely enteric origin. Additional population studies have demonstrated that none of the coliforms can function as reliable markers for all enteric pathogens (index organisms sensu Ingram), nor be of use in validating adequate processing for safety of raw water, which represents the indicator function of markers, as defined by Ingram. Future studies along these lines will have to provide the data required to assess the suitability of additional markers for the reliable monitoring of drinking water for microbiological safety.

Role of biofilms in the survival of Legionella pneumophila in a model potable-water system

Article

Dec 2001

Legionellae can infect and multiply intracellularly in both human phagocytic cells and protozoa. Growth of legionellae in the absence of protozoa has been documented only on complex laboratory media. The hypothesis upon which this study was based was that biofilm matrices, known to provide a habitat and a gradient of nutrients, might allow the survival and multiplication of legionellae outside a host cell. This study determined whether Legionella pneumophila can colonize and grow in biofilms with and without an association with Hartmannella vermiformis. The laboratory model used a rotating disc reactor at a retention time of 6.7 h to grow biofilms on stainless steel coupons. The biofilm was composed of Pseudomonas aeruginosa, Klebsiella pneumoniae and a Flavobacterium sp. The levels of L. pneumophila cells present in the biofilm were monitored for 15 d, with and without the presence of H. vermiformis, and it was found that, although unable to replicate in the absence of H. vermiformis, L. pneumophila was able to persist.

Bactericidal activity of electrolyzed acid water from solution containing sodium chloride at low concentration, in comparison with that at high concentration

Article

Jun 2002
J MICROBIOL METH

Electrolyzed strong acid water (ESW) containing free chlorine at various concentrations is becoming to be available in clinical settings as a disinfectant. ESW is prepared by electrolysis of a NaCl solution, and has a corrosive activity against medical instruments. Although lower concentrations of NaCl and free chlorine are desired to eliminate corrosion, the germicidal effect of ESW with low NaCl and free-chlorine concentrations (ESW-L) has not been fully clarified. In this study, we demonstrated that ESW-L possesses bactericidal activity against Mycobacteria and spores of Bacillus subtilis. The effect was slightly weaker than that of ESW containing higher NaCl and free-chlorine concentrations (ESW-H), but acceptable as a disinfectant. To clarify the mechanism of the bactericidal activity, we investigated ESW-L-treated Pseudomonas aeruginosa by transmission electron microscopy, a bacterial enzyme assay and restriction fragment length polymorphism pattern (RFLP) assay. Since the bacterium, whose growth was completely inhibited by ESW-L, revealed the inactivation of cytoplasmic enzyme, blebs and breaks in its outer membrane and remained complete RFLP of DNA, damage of the outer membrane and inactivation of cytoplasmic enzyme are the important determinants of the bactericidal activity.

Microbiological safety of natural mineral water

Article

Jul 2002

Natural mineral water originates from groundwater, an oligotrophic ecosystem where the level of organic matter is low and of a very limited bioavailability. The bacterial populations that evolve in these ecosystems are heterotrophic and in starvation-survival state resulting from an insufficient amount of nutrients; for this reason they enter a viable but non-culturable state. After bottling, the number of viable counts increases rapidly, attaining 10(4)-10(5) colony-forming units ml(-1) within 3-7 days. These bacterial communities, identified by culture or with specific probes, are primarily aerobic, saprophytic, Gram-negative rods. Groundwater sources for natural mineral waters are selected such that they are not vulnerable to fecal contamination. Ecological data, especially the diversity and physiological properties of bacterial communities, are essential together with epidemiological studies in order to perform a risk analysis for natural mineral waters. On a continuing basis, the management of microbial risks has to rely on assessment of the heterotrophic plate count and, more specially, on detection of marker organisms, i.e. the classic fecal contamination indicators that have to be absent, and vulnerability indicators for which the occurrence should be as low as possible. It is also recommended to search regularly, but not routinely, for viral and protozoan pathogens.

Keer JT, Birch L.. Molecular methods for the assessment of bacterial viability. J Microbiol Methods 53: 175-183

Article

Jun 2003
J MICROBIOL METH

A significant number of pathogenic microorganisms can be found in environmental reservoirs (air, water, soil). It is important to assess the viability status of these organisms to determine whether they pose a threat to public health. Classical methods for determining viability are time consuming. Hence, molecular methods have been developed to address this problem. Molecular methods offer speed, sensitivity and specificity. Both DNA and RNA have been analysed using molecular amplification methods such as polymerase chain reaction (PCR), reverse transcriptase PCR (RT-PCR) and nucleic acid sequence-based amplification (NASBA). However, due to the variable persistence of nucleic acids in cells post-death, the correlation between presence of DNA and RNA and viability is not clear-cut. Similarly, the choice of target and sensitivity of the method can significantly affect the validity of the viability assay. This review assesses the molecular methods currently available and evaluates their ability to assess cell viability with emphasis on environmental pathogens.

Elective Placement of Covered Stents in Native Coronary Arteries

Article

Jun 2003

To study the feasibility of placing a polytetrafluoroethylene (PTFE)-covered stent graft into native coronary arteries and assess the complications and the restenosis rate. Fifty consecutive patients with stable angina pectoris were included and the stent graft was placed into native coronary arteries. Clinical and angiographic follow-up were performed after 6 months. The stent grafts were successfully placed in all patients. The mean reference diameter was 3.3 +/- 0.6 mm. During follow-up the stent grafts occluded in patients after 1, 2 and 2.5 months and one more was occluded at 6 months. Three patients experienced myocardial infarction, 2 Q wave and one non-Q wave. After 6 months 42 (84%) patients had angina NYHA class 0 or 1. Target vessel revascularization was done in 11 cases for restenosis in the graft (n = 4), outside the graft (n = 3) and both (n = 4), giving a restenosis rate of 24%. The total major adverse coronary events at 6 months was 24%. The stent graft was deployed with a high success rate. The restenosis rate was not higher than expected for bare stents. However, this study showed that subacute occlusion may occur more frequently and we therefore recommend that ticlopidine or clopidogrel treatment should be prolonged to at least 3 months.

Are UV-induced nonculturable Escherichia coli K-12 cells alive or dead?

Article

Jul 2003

Cells that have lost the ability to grow in culture could be defined operationally as either alive or dead depending on the method used to determine cell viability. As a consequence, the interpretation of the state of 'nonculturable' cells is often ambiguous. Escherichia coli K12 cells inactivated by UV-irradiation with a low (UV1) and a high (UV2) dose were used as a model of nonculturable cells. Cells inactivated by the UV1 dose lost 'culturability' but they were not lysed and maintained the capacity to respond to nutrient addition by protein synthesis and cell wall synthesis. The cells also retained both a high level of glucose transport and the capacity for metabolizing glucose. Moreover, during glucose incorporation, UV1-treated cells showed the capacity to respond to aeration conditions modifying their metabolic flux through the Embden-Meyerhof and pentose-phosphate pathways. However, nonculturable cells obtained by irradiation with the high UV2 dose showed several levels of metabolic imbalance and retained only residual metabolic activities. Nonculturable cells obtained by irradiation with UV1 and UV2 doses were diagnosed as active and inactive (dying) cells, respectively.

The effect of free chlorine on Burkholderia pseudomallei in potable water

Article

Dec 2003
WATER RES

Chlorine is widely used in public water supplies to provide a disinfection barrier. The effect of chlorine disinfection on the water-borne pathogen Burkholderia pseudomallei was assessed using multiple techniques. After exposure to chlorine viable bacteria were undetectable by conventional plate count techniques; however, persistence of B. pseudomallei was verified by flow cytometry and bacteria were recoverable following a simple one-step broth procedure. The minimum residual chlorine concentration and contact time as prescribed by potable water providers in Australia was insufficient to reduce a B. pseudomallei population by more than 2 log(10). Chlorine had a bacteriostatic effect only on B. pseudomallei; viable bacteria were recovered from water containing up to 1000 ppm free chlorine. This finding has practical implications for water treatment in regions where B. pseudomallei is endemic. Future work to assess the effect of alternative water disinfection processes either singly or in sequence is necessary.

Enumeration of water-borne bacteria using viability assays and flow cytometry: A comparison to culture-based techniques

Article

Jan 2004
J MICROBIOL METH

Maintaining optimal conditions in catchments or distribution systems relies heavily on water authorities having access to rapid and accurate water quality data, including an indication of bacteriological quality. In this study, the BacLight bacterial viability kit and carboxyfluorescein diacetate (CFDA) were coupled with flow cytometry (FCM) for rapid detection of physiologically active bacteria from raw and potable waters taken from various locations around South Australia. Results were compared to the direct viable count (DVC) and quantitative DVC (qDVC), in addition to the culture-based methods of the heterotrophic plate count (HPC) and a commercial SimPlate technique. Raw and potable water analysis revealed that DVC and culture-based techniques reported significantly fewer viable bacteria compared to the number of physiologically active bacteria detected using the rapid FCM assays, where this difference appeared to be nonlinear across different samples. Inconclusive results were obtained using qDVC as a viability assay. In particular, HPC results were 2-4 log orders of magnitude below that reported by the FCM assays for raw waters. Few bacteria in potable waters examined were culturable by HPC, even though FCM assays reported between 5.56 x 10(2) and 3.94 x 10(4) active bacteria ml(-1). These differences may be attributed to the presence of nonheterotrophic bacteria, sublethal injury or the adoption of an active but nonculturable (ABNC) state.

Covered Stent Placement in a Traumatically Injured Vertebral Artery [1]

Article

Mar 2004
J VASC INTERV RADIOL

Evaluation of disinfective potential of reactivated free chlorine in pooled tap water by electrolysis

Article

Jun 2004
J MICROBIOL METH

Tap water is one of the causative factors of hospital infections. We examined the disinfective potential of electrolysis and mechanism of disinfection, and clarified the disinfective effect of electrolysis on tap water contaminated with bacteria, and discussed its clinical applications. Tap waters artificially contaminated with Pseudomonas aeruginosa, Escherichia coli, Legionella pneumophila, and Staphylococcus aureus could be sterilized by electrolysis at 20-30 mA for 5 min. A high-density suspension (10(6) CFU/ml) of a spore forming bacterium, Bacillus subtilis was not completely sterilized by electrolysis at 50 mA up to 30 min, but a low-density suspension (10(5) CFU/ml) was totally sterilized by electrolysis at 50 mA for 5 min. Electrolyzed P. aeruginosa changed morphologically, that is, there was bleb formation on the cell wall and irregular aggregation of cytoplasmic small granules. Moreover, cytoplasmic enzyme, nitrate reductase, was inactivated by the electrolysis. On the other hand, genomic DNA of the electrolyzed bacteria was not degenerated, therefore, their DNA polymerase activity was not completely inactivated. Consequently, the major agent in electrolysis for bactericidal action was considered to be free chlorine, and the possible bactericidal mechanism was by destruction of bacterial membranes, followed by the aggregation of peripheral cytoplasmic proteins. Electrolysis of tap water for both disinfecting contaminating bacteria and increasing the disinfectant capacity was considered effective with some limitations, particularly against high-density contamination by spore-forming bacteria. In clinical settings, electrolysis of tap water is considered effective to disinfect water for hand washing in operation theatres, and bathing water for immunocompromised hosts.

Water disinfection by electrochemical treatment

Article

Sep 2004
BIORESOURCE TECHNOL

The electrochemical disinfection of germinated brown rice (GBR) circulating water and cooling tower water containing Legionella bacteria was investigated. Results showed the total aerobic plate counts (APC) in the treated GBR circulating water decreased significantly and the turbidity was largely improved at a pulse voltage of 1.0 kV; Legionella bacteria were also disinfected effectively at 1.0 kV. The disinfection was attributed to the synergistic effects of the oxide anode, the electric field, and the radicals formed during the electrochemical treatment. This suggests that electrochemical treatment could be applicable to the disinfection of water from other sources.

The Viable but Nonculturable State in Bacteria

Article

Mar 2005

James D Oliver

It had long been assumed that a bacterial cell was dead when it was no longer able to grow on routine culture media. We now know that this assumption is simplistic, and that there are many situations where a cell loses culturability but remains viable and potentially able to regrow. This mini-review defines what the "viable but nonculturable" (VBNC) state is, and illustrates the methods that can be used to show that a bacterial cell is in this physiological state. The diverse environmental factors which induce this state, and the variety of bacteria which have been shown to enter into the VBNC state, are listed. In recent years, a great amount of research has revealed what occurs in cells as they enter and exist in this state, and these studies are also detailed. The ability of cells to resuscitate from the VBNC state and return to an actively metabolizing and culturable form is described, as well as the ability of these cells to retain virulence. Finally, the question of why cells become nonculturable is addressed. It is hoped that this mini-review will encourage researchers to consider this survival state in their studies as an alternative to the conclusion that a lack of culturability indicates the cells they are examining are dead.

Effects of chemically and electrochemically dosed chlorine on Escherichia coli and Legionella beliardensis assessed by flow cytometry

Abstract

No full-text available

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