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Abstract
This study used restriction fragment length polymorphism (RFLP) with Aeromonas-specific primers to identify species of Aeromonas and to investigate their distribution in a trout farm and stream.
In January, May, August and November 2000, presumptive Aeromonas species were recovered from a farm and a sedimentation pond in a fish farm and stream, and identified by PCR-RFLP analysis with Aeromonas-specific primers. The specificity of Aeromonas-specific primers and the suitability of PCR-RFLP analysis for identifying Aeromonas spp. were confirmed with fatty acid methyl esters (FAMEs) and 16S rDNA sequencing analyses, respectively. Levels of Aeromonas spp. sampled in May and August were higher than in January and November at all sampling sites. Aeromonas salmonicida was the dominant species in January and November, and the proportion of pathogenic species (Aer. hydrophila, Aer. caviae and Aer. veronii) increased in May and August.
PCR-RFLP analysis with Aeromonas-specific primers is a rapid and reliable method for identifying widely distributed Aeromonas spp. from environmental samples.
To minimize human health risk, monitoring the levels and species composition of Aeromonas in fish farm is advisable.
... Aeromonas hydrophila is a ubiquitous bacterium of freshwater environments where it can be found in water and sediments as well as in association with coldwater (Pettibone 1998;Zhang et al. 2000;Lee et al. 2002;Maalej et al. 2003;Silva et al. 2014;Patil et al. 2016). Many studies have tried to identify the environmental factors that affect abundance and distribution of A. hydrophila, primarily due to its potential as a human pathogen (Pettibone 1998;Lee et al. 2002;Maalej et al. 2003;VandeWalle et al. 2012;Odeyemi and Ahmad 2017). ...
... Aeromonas hydrophila is a ubiquitous bacterium of freshwater environments where it can be found in water and sediments as well as in association with coldwater (Pettibone 1998;Zhang et al. 2000;Lee et al. 2002;Maalej et al. 2003;Silva et al. 2014;Patil et al. 2016). Many studies have tried to identify the environmental factors that affect abundance and distribution of A. hydrophila, primarily due to its potential as a human pathogen (Pettibone 1998;Lee et al. 2002;Maalej et al. 2003;VandeWalle et al. 2012;Odeyemi and Ahmad 2017). Early studies on the ecology of Aeromonas showed a positive correlation between total aerobic counts (TAC) and Aeromonas counts (AC) (Kaper et al. 1981;Lee et al. 2002); however, we did not find a significant correlation (data not shown) between TAC and AC in any of the samples analyzed. ...
... Many studies have tried to identify the environmental factors that affect abundance and distribution of A. hydrophila, primarily due to its potential as a human pathogen (Pettibone 1998;Lee et al. 2002;Maalej et al. 2003;VandeWalle et al. 2012;Odeyemi and Ahmad 2017). Early studies on the ecology of Aeromonas showed a positive correlation between total aerobic counts (TAC) and Aeromonas counts (AC) (Kaper et al. 1981;Lee et al. 2002); however, we did not find a significant correlation (data not shown) between TAC and AC in any of the samples analyzed. In water samples, AC were at least one order of magnitude lower than TAC at any sampling point, which is in agreement with what has been previously reported from other aquaculture environments (Lee et al. 2002). ...
The genus Aeromonas comprises more than 60 recognized species that include many important fish pathogens such as the causative agents of furunculosis, and motile Aeromonas septicemia (MAS). Although MAS is typically considered a secondary infection, a new virulent A. hydrophila (vAh) strain has been causing devastating losses to the catfish industry in Alabama since 2009. The objective of this study was to characterize the spatiotemporal distribution of Aeromonas sp. and, specifically, vAh in a commercial catfish farm in West Alabama. We sampled biofilm, sediment and water from 3 ponds during 4 consecutive months during the growing season. Total aerobic counts were between 8.8 x 105 and 1.5 x 106 CFU/ml but were significantly higher in biofilm and sediment than in water throughout the sampling period. Total Aeromonas counts in water samples significantly increased in all three ponds after the month of August and ranged from 7.8 x 103 to 4.9 x 104 CFU/ml. A similar trend was observed in biofilm and sediment samples with total Aeromonas counts increasing in those samples in late summer to early fall. Overtime, the concentration of Aeromonas in water samples decreased by one order of magnitude, while there was a significant increase in sediments as temperature dropped. vAh was detected in 35.4% of biofilm samples and 22.9% of sediment samples, suggesting that both environments could serve as the major reservoir for this pathogen. Future monitoring efforts should focus on targeting sediment and biofilms since those two samples appear to naturally enrich for the presence of vAh and other Aeromonas. This article is protected by copyright. All rights reserved.
... Although recently Lee et al. (2002) reported a PCR-RFLP analysis for identification of Aeromonas spp., the amplified fragments could not dif ferentiate some species. ...
... Therefore, the method may lead misidentification if the bacterial strain subjected to be analysed is not confirmed as Aeromonas by bio chemical or other molecular method. Lee et al. (2002) proposed a method by using genus specific PCR prim ers and alteration of some endonucleases. ...
... These primers yielded a 953 bp PCR product. However, the subsequent RFLP was unable to differentiate A. bestiarum and A. salmonicida because the differentiable sections of these species were near the terminal region of the PCR-amplified products (Lee et al., 2002). In the present study, a new primer set was chosen from different positions representing differ ent sequences that yielded a 1206 bp PCR product from the DNA of type or reference strains of all recognized Aeromonas genospecies. ...
The study was conducted to develop a PCR and an improved PCR-RFLP analysis method for rapid species-identification of Aeromonas genospecies. The forward and reverse primers for PCR were designed from the complementary sequences of the 16S rDNA of all 15-recognized Aeromonas genospecies to amplify a 1206-bp PCR product. The PCR amplified the expected product from the DNA template of type or reference strains of all recognized Aeromonas genospecies as well as 106 Aeromonas strains, while no PCR product was obtained from any of the non-Aeromonas strains tested. The PCR-RFLP analysis using the restriction enzymes (AluI, MboI, PvuII, PstI and NarI) provided identification of almost all Aeromonas species. However, the Aeromonas sp. T8 group and A. caviae exhibited a similar RFLP pattern. Some selected biochemical tests were found to be helpful for differentiation of the two species.
... The study revealed that 80% of the examined samples were identified as Aeromonas spp. by the 16S rRNA gene amplification and identified as A. hydrophila by the RFLP pattern, which nearly agreed with the findings of Lee, Cho, Lee, Lee, and Kim (2002) who identified by the 16S rRNA gene-based PCR that 78% of the examined fish samples were infected with Aeromonas spp., whereas only 14% were identified by RFLP as A. hydrophilia. A Lower prevalence was observed by Onuk et al. (2013), who identified 23.33% as A. hydrophila using PCR-RFLP pattern analysis as well as by Hussain, Jeyasekaran, Shakila, Raj, and Jeevithan (2014) who confirmed that 56% of the fish samples contained Aeromonas spp. ...
... These differences may be attributed to the different species, sampling time, and geographical range. Lee et al. (2002) found that the distribution of Aeromonas spp. showed a close relationship between the sampling time and dominant species especially pathogenic species, such as A. hydrophila, A. caviae, and A. veronii, which were dominant at all sites during warmer months. ...
Aquaculture sector in Egypt has demonstrated a remarkable development; however, it has also faced challenges with respect to disease outbreaks. Nile tilapia is the most widely cultured species in Egypt. Under stress conditions, tilapia is vulnerable to a variety of bacterial diseases that tend to be ubiquitous in the freshwater environment. Semi-intensive Nile tilapia farms in Kafr El-Sheikh Governorate, Egypt are experiencing acute mortality especially during summer months. The samples from these farms were collected and subjected to investigations to identify the etiological agent(s) behind these mortalities. Bacteriological examination revealed that the dominant bacteria were Gram-negative rods and identified as Aeromonas spp. through biochemical tests. PCR, restriction fragment length polymorphism (RFLP) assays, and sequencing confirmed the presence of Aeromonas hydrophila. In addition, the aerA gene, a virulence factor in A. hydrophila, was detected by PCR in all identified A. hydrophila isolates. In order to confirm that the isolated A. hydrophila was the causative agent of tilapia mortality, healthy Nile tilapia were challenged with the isolated strains, which produced the same clinical picture of the collected samples. This study implicates that A. hydrophila could be the causative agent of the summer mortality in Nile tilapia farms in Kafr El-Sheikh Governorate, Egypt.
... The different thermal cycler settings are shown in Table 3. The method described by Lee et al. (2002) was used for the detection of Aeromonas species, and that described by Trakhna et al. (2009) with slight modification was used for A. hydrophila identification. ...
... The results obtained by PCR amplification of targeted 16S rDNA sequences proved the results obtained by initial isolation on R-S medium, as 22 isolates were identified by bands at 953 and 103 bp that are specific for the genus Aeromonas and A. hydrophila, respectively which is consistent with the results of many other studies as (Lee et al. 2002;Trakhna et al. 2009;Omar et al. 2016;Aboyadak et al. 2015Aboyadak et al. , 2017. The PCR technique was very helpful for the identification of Pseudomonas especially at the species level as the colonies grown on specific media did not have a distinct demarcation feature that allowed differentiation between the two recovered Pseudomonas species. ...
Bacterial diseases are the main cause of high economic loss in aquaculture, particularly gram-negative bacteria. This study was conducted for the isolation and identification of Aeromonas and Pseudomonas spp. from diseased fish. Twenty-two Aeromonas and sixteen Pseudomonas isolates were recovered from diseased Nile tilapia (Oreochromis niloticus) raised in eight earthen ponds in Elhox, Metoubes, Kafrelsheikh, Egypt. The recovered isolates were further identified using PCR as 22 Aeromonas hydrophila, 11 Pseudomonas aeruginosa, and 5 Pseudomonas fluorescens isolates. The 22 A. hydrophila isolates were screened for the presence of four virulence genes. Sixteen of the isolates (72.72%) were positive for the aerolysin gene (aer); 4 (18.18%) harbored the cytotoxic enterotoxin gene (act); and 2 (9.09%) carried the hemolysin A gene (hylA) while the cytotonic heat–stable enterotoxin gene (ast) was absent from all the tested isolates. The pathogenicity test indicated the direct relationship between the mortality percentage and the genotype of the tested A. hydrophila isolates as the mortality rates were 63.3 and 73.3% for isolates with two virulence genes (aer⁺ & act⁺, and aer⁺ and hylA⁺, respectively), followed by 40, 53.3, and 56.6% for isolates with only one virulence gene (hylA, act, and aer, respectively) and 20% for isolates lacking virulence genes. Based on the sensitivity test, the multi-antibiotic resistance profiles were as follows: 90.9% of the A. hydrophila isolates were sensitive to florfenicol and doxycycline; then 68.18% were susceptible to oxytetracycline, norfloxacin, and ciprofloxacin; and 63.63% were susceptible to sulfamethoxazole-trimethoprim, while only 27.27 and 4.5% were sensitive to erythromycin and cephradine, respectively, and all the isolates were resistant to amoxicillin and ampicillin.
... specific primer identified twelve Aeromonas spp. isolate as specific band appeared by electrophoresis at a molecular weight of 953 bp which is specific for Aeromonas spp. as showed in Figure 5 that was typically described by Lee et al. [9] and Aboelgalagel [17]. The PCR amplification using Aeromonas hydrophila specific primer (16S rRNA) identified twelve Aeromonas hydrophila strain as the PCR assay results in the amplification of 103 bp band specific Aeromonas hydrophila, as showed in Figure 6. ...
... b) Thermal cycle adjustment for:i. Amplification of Aeromonas species targeted DNA as described by Lee et al.[9]: Amplification was carried out in thermal cycler (Peltier thermal cycler Model: MG 96+ en-zyme® USA), with an initial denaturation at 94°C for 4 mins followed by 35 cycles of denaturation each at 94°C for 1 min, annealing at 68 °C for 30 sec and an extension step at 72°C for 45 sec. After the end of cycle's one final extension step at 72°C for 10 mins was performed. ...
An outbreak recorded in Tilapia farms in Kafrelsheikh governorate with high mortalities ranged between 30-70 % in the summer season of 2014. This study was conducted for isolation and identification of the causative agent responsible for mortalities in four fish farms. Twelve Aeromonas isolates were identified by PCR using Aeromonas species primer at the molecular weight of (953 bp) then all strains were also confirmed by PCR as Aeromonas hydrophila using Aeromonas hydrophila specific-16S rRNA gene primer at the molecular weight of (103 bp).
... Aeromonas and Aeromonas hydrophyla target DNA: Amplification of genus Aeromonas targeted DNA was performed according to the method described by Lee et al. [19], with an initial denaturation at 94°C for 4 min followed by 35 cycles of denaturation each at 94°C for 1 min, annealing at 68°C for 30 sec and extension steps at 72°C for 45 secs, then final extension step at 72°C for 10 min. ...
... Isolate that characterized by appearance of specific bands at 953 bp, all these isolates were further identified by PCR as Aeromonas hydrophyla as characteristic bands appeard at 103 bp ( Figure 2). Other researches indicating identification of such genus and species using the same primers design including Lee et al. [19] and Zakaria [23] for genus areomonas and Trankhan et al. [20] and Aboyadak et al. [4] for Aeromonas hydrophyla. ...
The current study was conducted to determine the bacterial pathogens incorporated in mass mortality observed in
cultured Oreochromis niloticus farms at Kafrelsheikh province, Egypt, during the summer season of 2015. Moribund
fish samples were collected from six infected farms. General signs of septicemia were dominant in the clinical and
gross internal examination of diseased fish. The pathogenic bacteria were isolated on specific media then confirmed
by polymerase chain reaction (PCR). Out of thirty isolates, nineteen Aeromonas hydrophila, seven Vibrio cholera and
three Vibrio alginolyticus isolates were recovered and identified using PCR. Current study indicated non-selectivity
of Rimler-Shotts media for selective isolation of Aeromonas hydrophila also non-selectivity of TCBS media for Vibrio
spp. isolated from diseased Oreochromis niloticus.
... specific primer identified twelve Aeromonas spp. isolate as specific band appeared by electrophoresis at a molecular weight of 953 bp which is specific for Aeromonas spp. as showed in Figure 5 that was typically described by Lee et al. [9] and Aboelgalagel [17]. The PCR amplification using Aeromonas hydrophila specific primer (16S rRNA) identified twelve Aeromonas hydrophila strain as the PCR assay results in the amplification of 103 bp band specific Aeromonas hydrophila, as showed in Figure 6. ...
... b) Thermal cycle adjustment for:i. Amplification of Aeromonas species targeted DNA as described by Lee et al.[9]: Amplification was carried out in thermal cycler (Peltier thermal cycler Model: MG 96+ en-zyme® USA), with an initial denaturation at 94°C for 4 mins followed by 35 cycles of denaturation each at 94°C for 1 min, annealing at 68 °C for 30 sec and an extension step at 72°C for 45 sec. After the end of cycle's one final extension step at 72°C for 10 mins was performed. ...
An outbreak recorded in Tilapia farms in Kafrelsheikh governorate with high
mortalities ranged between 30-70 % in the summer season of 2014. This study
was conducted for isolation and identification of the causative agent responsible
for mortalities in four fish farms. Twelve Aeromonas isolates were identified by
PCR using Aeromonas species primer at the molecular weight of (953 bp) then
all strains were also confirmed by PCR as Aeromonas hydrophila using Aeromonas
hydrophila specific-16S rRNA gene primer at the molecular w eight of (103 bp).
... New molecular techniques, such as the 16S rDNA polymerase chain reaction (PCR) assay have recently been applied to bacteria screening. This assay relies on the amplification of the gene coding for ribosomal RNA (16S rRNA), which is present in almost all bacteria (Lee et al., 2002;Vernon et al., 2002). This assay has several advantages over traditional microbiological methods. ...
... Some bacteria are difficult to identify with phenotypic identification schemes commonly used outside reference laboratories. 16S ribosomal DNA (rDNA)-based identification of bacteria potentially offers a useful alternative when phenotypic characterization methods fail are been used many researcher (Drancourt et al., 2000;Nakatsu et al., 2000;Lee et al., 2002). However, as yet, the usefulness of 16S rDNA sequence analysis in the identification of conventionally unidentifiable isolates has not been evaluated with a large collection of isolates. ...
Aeromonas spp infections are probably the most common bacterial disease diagnosed in cultured warm water fish. In the present study, six strains of Aeromonas spp bacteria were isolated from the gourami (Colisa lalia) by 16S rDNA sequencing analyses that are pathogenic to freshwater fish. Among them, three were under Aeromonas veronii species, two were Aeromonas sp ATCC and one was Aeromonas hydrophila. Colisa lalia usually imported in Korea from the South and Southeast Asian countries for recreational purposes. However, they are playing important role as a disease vector or carriers. The infected fish of this study frequently have hemorrhages at the base of the fins or on the skin, and gross ulcerative lesions. Internal signs include, fluid in the abdomen, swollen liver and spleen, and the intestine was distended and fluid-filled. In this study, the utility of 16S rDNA sequencing was employed to isolate Aeromonas bacteria from freshwater imported fish are important to environment, veterinary, and clinical purposes.
... Such additional PCR tools would complement existing primer sets capable of identifying presumptive Aeromonas spp. isolates [97][98][99][100], isolates that are either A. hydrophila or A. dhakensis [96], isolates suspected to be A. dhakensis [16], and A. hydrophila ST251 [16,76,101], which has caused significant disruption to channel catfish (Ictalurus punctatus) culture in the United States, carp production in China, and striped catfish farms in Vietnam [12,16,17,102]. ...
Aeromonas dhakensis is increasingly recognised to be an important pathogen responsible for disease losses in warm-water aquaculture and, similar to several other Aeromonas species, it can infect humans. Knowledge of A. dhakensis is accumulating, but this species remains relatively under-investigated compared to its close relative, Aeromonas hydrophila. The significance of A. dhakensis may have been overlooked in disease events of aquatic animals due to issues with reliable identification. Critical to appreciating the importance of this pathogen is the application of dependable molecular tools that enable accurate identification and discrimination from A. hydrophila and other motile aeromonads. This review aims to synthesise the key literature on A. dhakensis, particularly with relevance to aquaculture, including knowledge of the bacterium derived from disease case studies in aquatic hosts. Identification methods and strain phylogeny are discussed, with accurate detection important for prompt diagnosis and for distinguishing strains with heightened virulence. Increasing evidence suggests that A. dhakensis may be more virulent than A. hydrophila and correct identification is required to determine the zoonotic risks posed, which includes concerns for antibiotic-resistant strains. This review provides an impetus to improve species identification in the future and screen strain collections of presumptive Aeromonas spp. retrospectively to reveal the true prevalence and impact of A. dhakensis in aquaculture, the environment, and healthcare settings.
... isolate as specific band appeared by electrophoresis at a molecular weight of 953 bp that is specific for Aeromonas spp. [58][59]. ...
... For the 16s rDNA gene, forward and reverse primers were used as 16s rDNA-AERR and 16s rDNA-AERR, respectively, where the PCR product was 935 bp. The sequences of this primer set were followed by Lee et al. (2002) and Tasanapak et al. (2018). DNA extraction was performed on each isolate and used as a SIRIWAT ET AL S9 DNA template. ...
Nowadays, most people are more concerned with their personal health. Salads that are ready to eat are frequently a healthy eating option. It is consumed raw and unheated, which promotes the growth of numerous microorganisms. Furthermore, microbial contamination may occur because of the use of contaminated water for cleaning and packing. Aeromonas spp. are bacteria that grow on the water’s surface. They can survive in water that has been chlorinated to eliminate bacteria, which is critical for public health. Because bacteria can produce and secrete a variety of enzymes that are toxic to human tissue, there are a number of factors that contribute to violence. In people with low immunity, the majority of them can cause serious disease. As a result, the goal of this research is to look into and identify Aeromonas spp. isolated from ready-to-eat salad. The 16s rDNA gene was used to confirm the findings, and a PCR was used to look into the virulence factor genes. In this study, 9 isolates of Aeromonas spp. were found in 136 ready-to-eat salad samples, accounting for 6.6 percent of the total. Six virulence genes (ast, fla, lip, act, alt, and aphB) were used to identify each of 9 isolates where fla were found 4 isolates (44.44 %), and ahpB were also discovered 8 isolates (88.88 %). Therefore, there is the potential that ready to eat salad can be contaminated by Aeromonas spp. containing virulence factor which can cause a severe health risk such as diarrhea to consumers.
... Aeromonas 16S-specific primers AERF (5'-CTACTTTTGCC GGCGAGCGG-3') and AERR (5'-TGATTCCCGAAGGCACTCCC-3'), designed by LEE et al. (2002), were used to amplify 953 bp of the 16S gene from the selected bacterial DNA. The product was sequenced directly after purification with a PCR clean up column kit (QIAGEN). ...
The present study was undertaken to characterize Aeromonas spp. isolates from diseased Nile tilapia(Oreochromis niloticus) and humans using an Aeromonasspecific 16S rDNA primer, to determine their antibiotic multidrug resistance profile and to investigate the virulence of the strains in Nile tilapia. Aeromonas spp. were isolated from fish suffering from haemorrhagic septicaemia and from human stool and swab samples. Initially, all Aeromonas isolates were identified biochemically to the species level. Random Amplified Polymorphic DNA (RAPD)-PCR was applied for definitive identification and twelve bacterial isolates were confirmed as Aeromonas spp. Four representative isolates were selected for each host, subjected to 16S rDNA PCR amplification, sequenced and compared with reference sequences in GenBank. A phylogenetic tree constructed using the neighbour-joining method classified all isolates as motile Aeromonas species closely related to the Indian strain of A. hydrophila. All isolates showed multidrug resistance to ≥3 of the antimicrobials tested. Fish and human motile Aeromonas strains were able to infect Nile tilapia fish, inducing clinical signs and mortality. The results provide preliminary evidence that Aeromonas strains from fish show genetic similarity to human strains. This finding is consistent with the premise that infected fish could be a source of human contamination and vice versa.
... by using two endonucleases (AluI and MboI) simultaneously, Figueras et al., [40] completed the previous protocol of Borrell et al., [39] to differentiate A. salmonicida, A. bestiarum and A. popoffi using endonucleases AlwNI and PstI. Also Lee et al., [41] reported the identification of Aeromonasa species by sequence analysis corresponded to the identification by PCR-RFLP analysis. ...
In this study 60 broiler chicken, 7 layer and 11 duck farms of different ages suffering from diarrhea and stunted growth were investigated for Aeromonas species, 9 broiler chicken farms yielded 14 Aeromonas isolates (7 A. caviae, 2 A. hy-drophila, 3 A. schubertii and 2 A. trota), 1 layer farm yielded only one A. trota isolate and 2 duck farms produced 2 A. hydrophila isolates. Bacteriological examination of ration and water samples of 50 poultry farms revealed A. caviae (3 isolates), A. hydrophila (8 isolates) from ration and A caviae (4 isolates), A. hydrophila (2 isolates) from water. The previous Aeromonas isolates from poultry farms were compared with standard A. hydrophila and A. hydrophila, A. schubertii of fish source when sensitivity, PCR identification and RFLP. Sensitivity test of poultry A. hydrophila was similar to that of standard but differed from that of the fish isolates, A. hydrophila isolates were more resistant to antibiotics than the other Aeromonas species followed by A. caviae, A. trota then A. schubertii. Also A. hydrophila isolates from fish and poultry were more resistant to antibiotics than those from water. Elctrophoretic analysis of the PCR product (using specific primer 16S rRNA gene) revealed the specific amplification of 599 bp fragment for all selected Aeromonas isolates (identified poultry Aeromonas, standard and fish isolates). RFLP of 16S rRNA gene using Mbo1 and Alu1 restriction enzymes resulted in similarity among poultry, standard and fish isolates.16S rRNA gene of A. hydrophila, A. caviae and A. schubertii was digested with Mbo1 only, while that of A. trota was digested with both Mbo1 and Alu1. Pathogenicity test for A. hydrophila, A. trota, A. caviae and A. schubertii were applied, mortality was 13.3% in A. hydrophila, 20% in A. trota, 13.3% in A. caviae and 6.7% in A. schubertii infected groups. A. schubertii infection induced marked effect on body weight than A. caviae, A. trota and A. hydrophila. Small intestine, liver, lung, spleen and kidney were collected from fresh ailing sacrified infected chicks for histopathologi-cal examination.
... Poor water quality is often the origin of outbreaks in farmed species [47]. Additionally, mesophilic Aeromonas species' abundance is interconnected with environmental temperature and their levels are considered higher in Summer months when water temperature increases [48]. In modelling experiments regarding anthropogenic pressures, both S. pyrenaicus and I. lusitanicum are considered to display an intermediate tolerance to environmental alterations [49]. ...
Assessments regarding health aspects of Iberian leuciscids are limited. There is currently an information gap regarding effects of infectious diseases on these populations and their role as a possible conservation threat. Moreover, differences in susceptibility to particular agents, such as Aeromonas spp., by different species/populations is not clear. To understand potential differences in Aeromonas diversity and load, as well as in the prevalence and proportion of skin lesions, in fishes exposed to similar environmental conditions, an observational study was implemented. Using a set of 12 individuals belonging to two sympatric Iberian leuciscid species ( Squalius pyrenaicus and Iberochondrostoma lusitanicum ), the skin lesion score in each individual was analyzed. Furthermore, a bacterial collection of Aeromonas spp. isolated from each individual was created and isolates’ load was quantified by plate counting, identified at species level using a multiplex-PCR assay and virulence profiles established using classical phenotypic methods. The similarity relationships of the isolates were evaluated using a RAPD analysis. The skin lesion score was significantly higher in S . pyrenaicus , while the Aeromonas spp. load did not differ between species. When analyzing Aeromonas species diversity between fishes, different patterns were observed. A predominance of A . hydrophila was detected in S . pyrenaicus individuals, while I . lusitanicum individuals displayed a more diverse structure. Similarly, the virulence index of isolates from S . pyrenaicus was higher, mostly due to the isolated Aeromonas species. Genomic typing clustered the isolates mainly by fish species and skin lesion score. Specific Aeromonas clusters were associated with higher virulence indexes. Current results suggest potential differences in susceptibility to Aeromonas spp. at the fish species/individual level, and constitute important knowledge for proper wildlife management through the signalization of at-risk fish populations and hierarchization of conservation measures.
... For molecular identification, the genomic DNA of bacterial isolates was extracted using the InstaGene matrix (Bio-Rad laboratories, Hercules, CA, USA) according to the manufacturer's protocol. All the suspected isolates which were screened by the phenotypic tests were subjected to PCR examination using primer sets to detect specific Aeromonas genus (953 bp) and Aeromonas hydrophila species (AeroH; 625 bp) as described in previous studies [24,25] (Table 5). PCR reaction mixtures and conditions were modified from previous study [25]. ...
The study aims to evaluate the infection prevalence, virulence gene distribution and an-timicrobial resistance of Aeromonas hydrophila associated in diseased outbreaks of cultured freshwater fish in Northern Vietnam. The confirmed A. hydrophila were screened for the presence of the five pitutative-virulence genes including aerolysin (aerA), hemolysin (hlyA), cytotonic enterotoxin (act), heat-labile cytotonic enterotoxin (alt), and heat-stable enterotoxin (ast), and examined the susceptibility to 16 antibiotics. A total of 236 A. hydrophila isolates were recovered and confirmed from 506 diseased fish by phenotypic tests, PCR assays, and gyrB, rpoB sequenced analyses, corresponding to the infection prevalence at 46.4%. A total of 88.9% of A. hydrophila isolates harbored at least one of the tested virulence genes. The genes aerA and act were most frequently found (80.5% and 80.1%, respectively) while the ast gene was absent in all isolates. The resistance to oxa-cillin, amoxicillin and vancomycin exhibited the highest frequencies (>70%), followed by eryth-romycin, oxytetracycline, florfenicol, and sulfamethoxazole/trimethoprim (9.3-47.2%). The multiple antibiotic resistance (MAR) index ranged between 0.13-0.88 with 74.7% of the isolates having MAR values higher than 0.2. The results present a warning for aquaculture farmers and managers in preventing the spread of A. hydrophila and minimizing antibiotic resistance of this pathogen in fish farming systems.
... [16], virF gene for Shigella spp. [17], conserved regions of the 16S rDNA gene for Campylobacter [18], the Yersinia heat-stable enterotoxin (YST) gene [19], and conserved regions of 16S rDNA for Aeromonas [20] and the C. difficile toxin B (tcdB) gene [21], (Table 1). ...
Little information is available regarding the pathogens that cause diarrhea in hospitalized patients who also have various clinical problems. The purpose of this study was to determine the presence of pathogens in fecal samples of hospitalized patients all suffering diarrhea in addition to other problems in Mexico. Diarrheic stools from 240 patients were obtained in a third-level hospital in Monterrey, Mexico. PCR was used for the detection of Salmonella spp., Shigella spp., Campylobacter spp., Yersinia spp., Aeromonas spp., Clostridioides difficile, and norovirus GI and GII. The presence of trophozoites, cysts of protozoa, eggs, and/or helminth larvae was determined by microscopic observation. Of the 240 patients analyzed, 40.4% presented at least one of the pathogens analyzed. Norovirus was the pathogen most frequently found (28.6%), followed by bacteria (11.7%), and parasites (8.3%). The majority of co-infections were parasites + norovirus, and bacteria + norovirus. Norovirus was detected mainly in children aged 0 to 10 years (9/15, 60%). Patients aged 0–20 years did not present co-infections. Entamoeba coli and Entamoeba histolytica were the most common parasites, (8/240), and Salmonella was the most prevalent bacteria (10/240). This information can help design specific strategies useful for hospitalized people with a compromised status.
... The developed m-PCR involves amplifying the three multiple genes in single reaction based upon primers deduced from the regions carrying etfA of E. tarda (Sakai et al., 2007), 16S RNA of S. iniae (Zlotkin et al., 1998) and 16S rRNA of Aeromonas spp. (Lee et al., 2002). All primers used were synthesized by Invitrogen ® and Biomatik ®, Lithuania. ...
... Clarridge (2004), analisando a sequência 16S rRNA para identificação bacteriana afirma que o uso desse gene constitui uma forma robusta, precisa e de fácil reprodução na identificação bacteriana. Em nosso estudo, o uso do gene 16S rRNA como alvo para a identificação do gênero Aeromonas foi embasado nos trabalhos realizados por Figueras et al. (2000), Lee et al. (2002) e Morandi et al. (2005). ...
RESUMO: As infecções causadas por bactérias do gênero Aeromonas estão entre as doenças mais comuns em peixes cultivados em todo o mundo, com ocorrência de aeromoniose em todos os países que possuem cultivo de tilápia do Nilo (Oreochromis niloticus). O presente trabalho descreve o desenvolvimento de uma nova multiplex PCR (mPCR) para diagnóstico de Aeromonas spp. e identificação do gene aerolisina (aerA). Para padronização da mPCR foram utilizadas cepas de referência de várias espécies do gênero Aeromonas e de outros gêneros. Também foram usadas cepas de campo de A. hydrophila oriundas de cultivos de peixes pacamãs (Lophiosilurus alexandri) e Aeromonas spp. de tilápias do Nilo. Os primers foram desenhados com base na região 16S rRNA e aerA. Para verificar a melhor temperatura de anelamento foram utilizados gradientes entre 59°C a 61°C com 40ng de DNA molde. Os produtos da amplificação da região 16S rRNA e do gene aerA apresentaram 786 e 550pb, respectivamente. A mPCR apresentou melhor temperatura de anelamento a 57,6°C com limite de detecção das concentrações de DNA em ambos genes (16S rRNA and aerA) de 10-10g/μL. A mPCR padronizada é rápida, sensível e específica no diagnóstico de Aeromonas spp. e identificação do gene aerolisina. Esta metodologia apresenta vantagens quando comparada aos métodos de diagnóstico convencionais, podendo ser utilizada em cultivos comerciais de tilápias do Nilo ou outros peixes. A identificação do gene aerolisina é uma importante ferramenta na determinação do potencial patogênico dos isolados de Aeromonas spp. estudados.
... The PCR targeting conserved sequence of 16S ribosomal RNA gene was considered for the identification of Aeromonas genus and species. Amplification of 16S rRNA gene was performed with Aeromonas (AER)-F: (5'-CTACTTTTGCCGGCGAGCGG-3') and AER-R: (5'-TGATTCCCGAAGGCACTCCC -3') primers to amplify a 954 bp fragment [15]. PCR amplification was carried out in a thermal cycler (Eppendorf, Germany). ...
Introduction:
Aeromonas spp. have recently emerged as opportunistic pathogens and only few studies are available regarding the isolation of these bacteria from burn wound infections. This study aimed at isolating Aeromonas as an infrequent cause of infection in this group of immunocompromised patients.
Methods:
A total of 300 samples were collected from the wounds of burn patients hospitalized in Gotbodin Shirazi Burn Center in a year. The samples were cultured on Aeromonas specific media. The API-E20 kit was used for basic biochemical identification and 16S rRNA and gyrB genes amplification and sequencing was performed. The antibiotic susceptibility of the isolates was determined using the agar diffusion and broth microdilution methods.
Results:
Biochemical tests and the API-20E kit demonstrated five samples of Aeromonas, while, molecular testing confirmed only three. All isolates were resistant to ampicillin-sulbactam, erythromycin, oxacillin and vancomycin. However, they were susceptible to gentamicin, meropenem, nitrofurantoin, chloramphenicol, cephalexin and cefotaxime. Two A. veronii isolates were resistant to sulfamethoxazole.
... The first application that used PCR-RFLP analysis for the differentiation of Aeromonas species was developed by Borrell et al. [22] and was based on evaluation of the 16S rRNA gene. Since then, similar approaches have been reported by Graft et al. [23], Figueras et al. [24], Ghatak et al. [25] and Lee et al. [43]; however, the problem of discriminatory power was still present. This paper reports a new PCR-RFLP method that uses the rpoD gene and has performed well in discriminating between 27 Aeromonas species using the sequences of the species type strains. ...
Purpose:
The taxonomy of Aeromonas keeps expanding and their identification remains problematic due to their phenotypic and genotypic heterogeneity. In this study, we aimed to develop a rapid and reliable polymerase chain reaction-restriction fragment length polymorphism assay targeting the rpoD gene to enable the differentiation of aeromonads into 27 distinct species using microfluidic capillary electrophoresis.
Methodology:
A pair of degenerate primers (Aero F: 5'-YGARATCGAYATCGCCAARCGB-3' and Aero R: 5'-GRCCDATGCTCATRCGRCGGTT-3') was designed that amplified the rpoD gene of 27 Aeromonas species. Subsequently, in silico analysis enabled the differentiation of 25 species using the single restriction endonuclease AluI, while 2 species, A. sanarelli and A. taiwanensis, required an additional restriction endonuclease, HpyCH4IV. Twelve type strains (A. hydrophila ATCC7966T, A. caviae ATCC15468T, A. veronii ATCC9071T, A. media DSM4881T, A. allosaccharophila DSM11576T, A. dhakensis DSM17689T, A. enteropelogens DSM7312T, A. jandaei DSM7311T, A. rivuli DSM22539T, A. salmonicida ATCC33658T, A. taiwanensis DSM24096T and A. sanarelli DSM24094T) were randomly selected from the 27 Aeromonas species for experimental validation.Results/key findings. The twelve type strains demonstrated distinctive RFLP patterns and supported the in silico digestion. Subsequently, 60 clinical and environmental strains from our collection, comprising nine Aeromonas species, were used for screening examinations, and the results were in agreement.
Conclusion:
This method provides an alternative method for laboratory identification, surveillance and epidemiological investigations of clinical and environmental specimens.
... Each phenotypically identified isolate was incubated in alkaline peptone water at 378C for 24 hr and genomic DNA was purified using Exgene Cell SV (GeneAll, Seoul, Korea) DNA extraction kit. The isolates were identified at the genus level based on their 16S rRNA gene sequences (Lee, Cho, Lee, Lee, & Kim, 2002). Further species identification and the phylogenetic analyses of 44 Aeromonas spp. ...
We evaluated 44 strains of Aeromonas spp. consisting of five species; A. hydrophila, A. veronii, A. caviae, A. enteropelogenes, and A. media isolated from frozen white‐leg shrimp marketed in Korea. All the isolates were positive in DNase, protease, gelatinase, and lipase tests whereas 29 isolates (66%) were positive for slime production. Ten virulence genes were detected by PCR in which the prevalence of act, alt, ast, aerA, lip, ahyB, ser, fla, gcat, and ascV were 50%, 77%, 72%, 82%, 84%, 93%, 80%, 64%, 84%, and 23%, respectively. As observed in antibiogram, 100% resistance was detected in ampicillin and clindamycin while nalidixic acid, tetracycline, cephalothin, erythromycin, and trimethoprim‐sulfamethoxazole were resisted by 95%, 89%, 86%, 68%, and 66% of the isolates, respectively. Multiple antibiotic resistance index was >0.20 in 42/44 (95%) of the isolates. Overall, our results indicated that these aeromonads are indicative of a serious public health risk due to their virulence potential and the multiple‐antimicrobial resistance.
Practical applications
Similar to other seafood, shrimp have been identified as vehicles of Aeromonas spp. which are the etiological agents of many foodborne infections. The gyrB gene sequencing and phylogenetic analysis confirmed the occurrence of five species of Aeromonas in frozen white‐leg shrimp marketed in Korea. Putative virulence properties were examined by both phenotypic tests and PCR assays whereby majority of the isolates exhibited the virulence both phenotypically and genetically. Besides, the disc diffusion test was carried out to assess the antibiotic susceptibility of the isolates. Following the results, the MAR index was calculated for each strain thereby suggested their high risk owing to MAR indices higher than 0.20. With our results, frozen white‐leg shrimp associated Aeromonas spp., exhibiting potential virulence and antimicrobial resistance, can be considered as a potential public health risk. Moreover, it is noteworthy that freezing the shrimp may not reduce the incidence of pathogenic Aeromonas.
... The culture-based presumptive Aeromonas isolates were exposed to DNA extraction by boiling as described previously . To determine the genus of the bacteria, we screened all the presumptive culture-originated Aeromonas isolates using genus-specific primers AERF (5 0 -CTACTTTTGCCGGC GAGCGG-3 0 ) and AERR (5 0 -TGATTCCCGAAGG CACTCCC-3 0 ) that target Aeromonas 16S rDNA (Lee et al. 2002). To determine Aeromonas species, all isolates that were confirmed to belong to genus Aeromonas, were examined using a species-specifying primers A. hydrophila 16S rRNA forward (5 0 -GGCCTTGCGCGATTGTATAT-3 0 ) and reverse (5 0 -GTGGCGGATCATCTTCTCAGA-3 0 ) (Trakhna et al. 2009). ...
... Traditionally, the diagnosis of fish diseases is based on the isolation of the pathogen from the tissues of diseased fish and its identification by conventional microbiological methods or commercial phenotypic identification systems. However, these methods are cumbersome and time-consuming and do not always allow the differentiation between closely related bacteria (Austin et al. 1998;García-González et al. 2011;Keeling et al. 2013;Lee et al. 2002). Non-culture-based detection systems such as immunoassays (Hiney et al. 1994), loop-mediated isothermal amplification (LAMP) (Kulkarni et al. 2009), multiplex-PCR (Altinok et al. 2008), multiplex PCR coupled to DNA microarray (González et al. 2004) and conventional PCR have been developed for identification of A. salmonicida strains (Beaz-Hidalgo et al. 2008;Byers et al. 2002a, b;Gustafson et al. 1992;Hiney et al. 1992;Miyata et al. 1996). ...
A SYBR Green I real-time polymerase chain reaction protocol for specific detection of the fish pathogen Aeromonas salmonicida subsp. salmonicida was developed and validated for rapid diagnosis of typical furunculosis. The sequence of the aopO gene of A. salmonicida subsp. salmonicida, which encodes for a serine/threonine protein kinase linked to virulence, was chosen for primer design. The selected primers amplified a 119-bp internal fragment of the aopO gene. The specificity test proved that 100 % (40/40) of the A. salmonicida subsp. salmonicida strains tested showed a positive amplification with subspecies-specific melting temperatures (Tm) of 80.75 ± 0.35 °C. Atypical A. salmonicida subspecies and other non-related bacterial fish pathogens did not amplify or showed unspecific melting profiles, except for one strain of A. salmonicida subsp. achromogenes and one strain of A. salmonicida subsp. smithia. The detection sensitivity was 21 fg of purified bacterial DNA per reaction, corresponding to 1-2 bacterial cells and 6-60 bacteria per reaction for seeded kidney and blood. The assay was highly reproducible with low variation coefficient values for intra-run and inter-run assays. The assay also allowed the specific detection of A. salmonicida subsp. salmonicida in tissues of fish naturally and experimentally infected. No amplification was detected when tissues from healthy fish or fish affected by other diseases were tested. The SYBR Green real-time PCR and melt curve analysis developed in this study is a rapid and accurate method for the specific identification of A. salmonicida subsp. salmonicida isolates and its detection on tissues of fish affected by furunculosis.
... and, Kingombe et al. (2004) successfully employed RFLP technique on PCR amplicons for detection and speciation of aeromonads from foods. Moreover, this technique has also been utilized to detect and characterize Aeromonas isolates from chicken carcasses and in fish farm (Lee et al. 2002, Abdullah et al. 2003. One limitation that most of these studies suffered from is apparent similarity among RFLP fingerprints of various strains and isolates. ...
The members of the genus Aeromonas have emerged as an important food-borne pathogen that pose many challenges to the scientific community. Difficulties regarding aeromonads involve its debated taxonomy, true role as pathogen and detection, particularly from foods. Various conventional techniques involving culture media and broths have been devised for identification form foods and immunological techniques viz. ELISA protocols have been standardized. Due to shortcomings of these methodologies molecular techniques were explored. These included nucleic acid hybridization probes, various formats of PCR, restriction fragment length polymorphism, amplified fragment length polymorphism, pulsed field gel electrophoresis, real time PCR, sequence analysis, DNA microarray technique, etc. Many of these modern assays have been promising, indicating that future detection paradigm for detection of Aeromonas will predominantly be based on molecular techniques. In this communication we have attempted to review these available molecular tools for detection of aeromonads from foods.
... The multiplex PCR reactions were performed in a 20 µl reaction mixture consisting of 0.2 µl of Taq DNA polymerase (5U/µl) (iNtRON Biotechnology, USA), 2 µl of PCR buffer (100 mM Tris HCl, pH 8.3), 2 µl of 10 mmol dNTP mix (2.5 mmol each), 10.8 µl of sterile distilled water, 1 µl of each primer (10 µmol each), and 1 µl of DNA template (50 ng). The sequences of primers used in this study are listed in Table 2 (Lee et al., 2002;Meiri-Bendek et al., 2002). PCR amplifications were performed with a T-personal combi thermocycler ® (Biometra ® , Germany). ...
A multiplex Polymerase Chain Reaction (m-PCR) assay was developed for simultaneous detection of two major pathogens, Streptococcal and Aeromonad bacteria, in farmed tilapia. DNA fragments of Streptococcus spp. and Aeromonas spp. were amplified using genus specific primers, C1/C2 and AERF/AERR, which produced PCR of 207 bp and 953 bp, respectively. The lowest concentration of each Streptococcus and Aeromonas spp. extracted genomic DNA from a colony detected by m-PCR was 2 ng. The m-PCR assay was proven applicable for detection of bacterial genomic DNA in tissues (brain and posterior kidney) of infected fish. Specificity of the assay tested with other Gram-positive (Staphylococcus aureus) and Gram-negative water borne bacteria (Escherichia coli, Enterococcus faecalis, Pseudomonas aeruginosa, Salmonella enteritidis, Salmonella typhimurium, Vibrio harveyi and Vibrio parahemolyticus) showed no amplification. As Streptococcal and Aeromonad infections are common concurrent bacterial diseases, the m-PCR assay established in this study enabled effective simultaneous detection of these two major bacterial infections responsible for current economic losses in tilapia farming in Thailand.
... Molecular method for detecting of A. hydrophila was introduced and applied in numerous previous studies (Nielsen et al. 2001;Swaminathan et al. 2004;Yogananth et al. 2009). The genomic DNA was the coding region of ribosomal RNA, which was used for phylogenetic studies as it is highly conserved between different species of bacteria and archaea and as important taxonomic tools ( Weisburg et al. 1991;Coenye and Vandamme 2003) such as taxonomy of the genera Aeromonas ( Lee et al. 2002;Chen et al. 2012; Liu and Li 2012;Sarkar et al. 2012). Two universal primers, 27F and 1492R, were developed in amplification of genomic DNA genes and had allowed discriminating of identification up to the species level and typing of other bacteria ( Jiang et al. 2006;Sarkar et al. 2012). ...
This study was carried out to confirm whether Aeromonas hydrophila is a main haemorrhagic pathogen in blunt snout bream Megalobrama amblycephala (BSB). The fish was challenged with bacterial concentrations of 1.7×10 4 , 1.7×10 5 , 1.7×10 6 and 1.7×10 7 cfu/fish for 7 days post infection. The results showed that mortality was bacterial-dose dependent, with 100% mortality observed at 1 day at the highest dose level (1.7×10 7 cfu/fish). For comparison, control fish exhibited cumulative mortalities of 0%. The median lethal dose (LD 50) was 5×10 5 cfu/fish. A total of 15 bacterial strains of Aeromonas were re-isolated from challenged fish and re-identified based on morphological characteristics, biochemical tests and genomic DNA gene sequencing. No bacteria were isolated from the control group. This study results indicated that A. hydrophila is capable of causing haemorrhagic septicaemia in BSB. Antibiotic susceptibility test with two strains D4 and HU201301 was investigated; both strains showed sensitive to most of the tested drugs.
... rDNA RFLP is considered to be a simple and reliable method for discrimination of Aeromonas spp. without the need of sequencing and has been used to study the distribution of Aeromonas spp. in fish farms(Lee et al., 2002).Borrell et al. (1997) developed a method for identification of all known Aeromonas species on the basis of restriction patterns of the PCRamplified 16S rRNA gene, using AluI-MboI restriction enzymes. These enzymes produce species-specific profile for A. hydrophila, A. ...
A total of 50 strains of Aeromonas spp. were isolated from 50 water and fish samples, and
identified biochemically and genetically. Biochemical characterization indicated that 20 out of 50
aeromonads were A. veronii , 19 were A. caviae , 9 were A. hydrophila and 2 were A. trota . Molecular
identification of isolated aeromonads revealed restriction profile of four species i.e. A. sobria , A. media ,
A. allosaccharophila and A. schubertii in addition to A. veronii, A. caviae, A. hydrophila and A. trota in
16S rDNA RFLP. The 16S rDNA sequencing results for the 13 strains validated the results of PCR-RFLP
analysis for Aeromonas identification. Most of the isolates identified by 16S rDNA RFLP were positive for
virulence factors as revealed by phenotypic tests such as hemolysin production, casein hydrolysis,
gelatinase activity, lipase activity, DNase activity, lecithinase production and siderophore production.
Further, a variable number of Aeromonas spp. was positive for lipase and elastase genes indicating the
presence of other genes responsible for these activities observed phenotypically. The present study
highlights important incidence of motile Aeromonas spp. with virulence potential, from water and fish
samples.
... To determinate the genetic relatedness between bacterial strains by molecular methods are powerful and more commonly useful for bacterial typing [9] . Several PCR based protocols have been designed to identify and characterization of Aeromonas strains [10,11] . Moreover RAPD PCR with ERIC primer has been used for typing Aeromonas isolates [12,13] . ...
Phenotypic and molecular characterization of Aeromonas sobria (A. sobria) isolates by antibiotyping, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole cell proteins, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was aimed in this study. For this aim, thirty-six A. sobria isolates were analysed. Isolates were divided into 12 different antibiotypes and 4 proteotypes according to their antibiotic susceptibilities and SDS-PAGE patterns, respectively. Thirteen RAPD types were observed among all isolates. In conclusion, the use of double or triple combination of typing methods in this study was found to be more useful for discriminating the strains. The results obtained from this study may give information about phenotypic and genotypic variability of the A. sobria strains isolated from different regions of Turkey and can be helpful to control disease in fish through guiding the antibiotic therapy and giving information that will be useful to development vaccine.
... To determinate the genetic relatedness between bacterial strains by molecular methods are powerful and more commonly useful for bacterial typing [9] . Several PCR based protocols have been designed to identify and characterization of Aeromonas strains [10,11] . Moreover RAPD PCR with ERIC primer has been used for typing Aeromonas isolates [12,13] . ...
Phenotypic and molecular characterization of Aeromonas sobria (A. sobria) isolates by antibiotyping, sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of whole cell proteins, random amplified polymorphic DNA-polymerase chain reaction (RAPD-PCR) was aimed in this study. For this aim, thirty-six A. sobria isolates were analysed. Isolates were divided into 12 diferent antibiotypes and 4 proteotypes according to their antibiotic susceptibilities and SDS-PAGE patterns, respectively. Thirteen RAPD types were observed among all isolates. In conclusion, the use of double or triple combination of typing methods in this study was found to be more useful for discriminating the strains. The results obtained from this study may give information about phenotypic and genotypic variability of the A. sobria strains isolated from diferent regions of Turkey and can be helpful to control disease in fish through guiding the antibiotic therapy and giving information that will be useful to development vaccine.
... In the present study, sequence analysis ascribed to Aeromonas spp. some strains from the Tortoli gut samples; this taxonomic group was also found among the microflora of trout (Lee et al., 2002;Pond et al., 2006;Kim et al., 2007), the intestinal microbial community of carp (Namba et al., 2007) and the gut of salmon (Skrodenyte-Arbaciauskiene et al., 2008). Additionally, the occurrence of high GC Gram-positive bacteria belonging to Leucobacter spp. ...
1-The bacterial flora of the digestive tract of aquatic organisms reflects various factors, such as the aqueous environment (temperature, salinity, etc.), seasonal variation, diet, fish species and anatomy of gastrointestinal section. 2-In the present work, culturable bacteria isolated from intestinal samples of gilthead sea bream caught in two coastal lagoons of Sardinia, were quantified and identified in order to detect the effect of different habitats on the microbial ecology of fish gut.
... and Salmonella spp. Polymerase chain reaction (PCR) was done with a thermal cycler using the amplification programme followed by LEE et al. 2002 andAMIT-ROMACH et al. 2004. The PCR mixtures consisted of 2.5 µl of 10X buffer (with MgCl 2 ), 3 µl of dNTP mixtures (2.5 µM each), 1 µl of each primer and 0.5 µl of Taq DNA polymerase (3 U/µl) (GENEI, BANGALORE), in a final volume of 25 µl. ...
An investigation was conducted to identify the bacterial isolates and to study the antibiotic resistance patterns of Aeromonas spp. and Salmonella spp. from the skin, gills, and gut of the fresh water loach, Lepidocephalichthys guntea (HAMILTON BUCHANAN) and water sampled from four different sites along the River Lotchka in Darjeeling District, West Bengal, India. Isolated bacteria were identified by different biochemical procedures, and Polymerase Chain Reaction was performed using genus specific 16S rDNA primers for confirmation of identification of Aeromonas spp. and Salmonella spp. Antibiotic susceptibility test of bacterial isolates was also done by Disc Diffusion method. A total of 49 Aeromonas spp. and 24 Salmonella spp. were isolated from tested samples. Maximum resistance was exhibited for Penicillin-G, Ampicillin, and Cephalothin (>75% resistant Aeromonas spp. and >60% Salmonella spp. in the four different sites of river and body parts of fish). Ciprofloxacin and Tetracycline resistance was almost nil. The results indicated that the river water and resident fish were contaminated with multi-antibiotic resistant enteric pathogenic bacteria. This study, thus, provides valuable information for making policy decisions aimed at reducing microbial contamination of fish and the indiscriminate use of antibiotics.
... Aeromonas genus-specific polymerase chain reaction (PCR) detection was carried out as previously described using the primers AERF and AERR (Lee, Cho, Lee, Lee & Kim 2002) (Table 3). Strains with positive reactions were harvested as presumptive Aeromonas isolates and were confirmed as members of the genus Aeromonas in terms of motility, positive reactions for both oxidase and catalase, fermentation of glucose and resistance to O/129 on a 150-lg disc (Abbott et al. 2003). ...
Under experimental challenges, the gastrointestinal (GI) tract of fish has been proposed as an infectious route of several pathogenic bacteria. Is this also the case for diseased fishponds? A field research was conducted to verify this hypothesis. A crucian carp (Carassius carassius) reared fishpond with motile Aeromonas septicaemia outbreak was sampled in this study. A total of 62 strains of Aeromonas hydrophila were isolated and identified. The clonal relationship among these strains was determined by sequencing the gyrB gene, ERIC-PCR, RAPD-PCR, and the presence of seven virulence genes. Strains with identical genotypes were further confirmed as the same clone by multilocus sequence typing analysis. Experimental infection assays were also conducted in zebrafish (Danio rerio). The results show that the same clone strains identical to those in the blood of diseased fish existed in the intestinal digesta of diseased and uninfected fish. Regardless of their origins, all these strains were highly pathogenic to zebrafish. The result indicates that pathogenic strains of A. hydrophila had existed in the GI tract of fish before the infection occurred. This increases our knowledge on infectious route of A. hydrophila in crucian carp.
... Then, two to three single colonies were randomly selected and re-purified from each TSA plate. Aeromonas genus-specific polymerase chain reaction (PCR) detection was carried out as previously described (Lee et al., 2002). All isolates were positively amplified and were further confirmed as members of the genus Aeromonas in terms of motility, positive reactions for both oxidase and catalase, fermentation of glucose and resistance to O/129 on a 150 μg disk (Abbott et al., 2003). ...
... The PCR methods were performed according to published protocols (El Sayed and El-Adrosy, 2007;Gómez-Duarte et al., 2009;Iturriza Gómara et al., 2002;La Rosa et al., 2011;Leblanc-Maridor et al., 2011;Lee et al., 2002;Lemee et al., 2004;Miwa et al., 1998;Park et al., 2006;Vidal et al., 2005;Vinjé and Koopmans, 1996). No limit of detection was calculated for each singleplex PCR assay. ...
... could be a potential food-borne pathogen, in particular in RTE foods minimally processed and stored under refrigeration (Palumbo et al., 1985). In agreement with previous studies carried out in different geographical areas (Lee et al., 2002; Pianetti et al., 2008), this study shows that Aeromonas spp. are ubiquitous and may be isolated from a wide range of vegetal and animal origin food. ...
This study provides data on the prevalence of potentially pathogenic Aeromonas spp. in ready-to-eat (RTE) seafood products by evaluating the occurrence of Aeromonas spp. and the presence of virulence-associated genes. Aeromonas spp. was detected in 57 ⁄ 81 (70.3%) RTE seafood samples. Specifically, Aeromonas spp. was highlighted in 19 ⁄ 21 (90.5%) sushi, in 18 ⁄ 21 (85.7%) sea salad, 11 ⁄ 12 (91.7%) surimi and 9 ⁄ 12 (75%) peeled shrimp samples. Aeromonas spp. was not observed in marinated anchovies and octopus salad samples. Then, PCRs aimed at the hlyA, aerA, alt and ast genes, encoding, respectively, haemolysin A, aerolysin, aeromonas labile temperature cytotonic enterotoxin and aeromonas stable temperature cytotonic entero-toxin, demonstrated a widespread distribution of these genes among Aeromonas isolates. The results underline the need to implement an adequate control plan performing an intensive and continuous monitoring to guarantee the human health.
The study aims to examine the pathogenicity, the effects of environmental conditions on the growth of Aeromonas hydrophila in tilapia, and histopathological changes in infected fish. A. hydrophila isolates, which were recovered from diseased tilapia samples collected at farming cages/ponds in several northern Vietnam provinces, were identified by biochemical tests, PCR confirmation, and sequencing of 16S rRNA and housekeeping genes (gyrB). Three representative strains after identification were subjected to evaluate the pathogenicity via challenge experiments using the intraperitoneal injection method and to determine the impacts of environmental factors on their growth. The present study showed an average LD50 value (lethal dose 50%) of A. hydrophila to tilapia at 4.6×105 CFU/fish, the infected fish in the challenge tests presented the clinical and gross lesions similar to the natural diseased fish, including haemorrhaging at the base of fins, skin, anal opening and in visceral organs, especially in liver and intestine. Histopathological examination of the diseased fish showed the hyperplasia of epithelial cells and haemorrhage of the gill filaments. The tissues of the liver, kidney, and spleen exhibited the lesion of haemorrhage, congestion, and degeneration, while the brain tissue appeared the colonisation by the bacteria. The A. hydrophila strains from tilapia showed a high tolerance to environmental conditions with the capacity to survive and multiply at a temperature of 15-45°C, a salinity of 0-60‰ and a pH of 5-10. The present results provide useful information to establish the strategies in prevention of A. hydrophila infection in tilapia and other freshwater fish.
In this study, a total of 240Oreochromis niloticus species were collected randomly and monthly during an outbreak of disease mass mortalities from different fish farms in the areas of Bahr El-Baqar. Clinical signs and postmortem lesions of affected Oreochromis niloticus were recorded. The most isolated bacterial agent was Aeromonas veronii biovar sobria which identified using Vitek 2 system and traditional method as Aeromonas sobria. The identification was confirmed by detection of 16s rRNA gene by PCR then sequencing of this gene and by analysis of sequence was identified as Aeromonas veronii. The total prevalence was 86.25% in the four months. The highest prevalence was in July 95% followed by August 91.67% and June 86.67% then May 71.67%. The experimental infection was studied and the mortality was 70 and 35% of the I/P and I/M respectively.
Blunt snout bream (BSB), Megalobrama amblycephala, is a herbivorous freshwater fish native to China and a major aquaculture species in Chinese freshwater polyculture systems. Recently, the bacterium Aeromonas hydrophila has been reported to be its pathogen causing hemorrhagic septicemia clinical signs and great great ecomomic losses of farmed BSB. This study used next generation sequencing technology to better comprehensive the transcriptome profiles of response-related genes in the BSB which will facilitate further research into the resistance and susceptibility of this fish species to exogenously invasive pathogens. The data analysis of transcriptome profile and expression of immune-related genes from BSB upon A. hydrophila stimulation was also described. The main results are as follows:
1. As an initial step, the pathogenicity of A. hydrophila to BSB was carried out to confirm whether A. hydrophila is a main pathogen causing hemorrhagic septicemia in BSB. The fish was challenged with bacterial concentrations of 1.7×105, 1.7×106, 1.7×107 and 1.7×108 cfu/mL for 7 days post infection. The results showed that mortality was bacterial-dose dependent, with 100% mortality observed at day 1 at the highest dose (1.7×108 cfu/mL). Control fish exhibited cumulative mortalities of 0%. The median lethal dose (LD50) was 5×106 cfu/mL. A total of 15 Aeromonas strains were re-isolated from challenged fish and re-identified based on morphological characteristics, biochemical tests and genomic DNA gene sequencing. No bacteria were isolated from the control group. This study results indicated that A. hydrophila is capable of causing hemorrhagic septicemia in BSB. Moreover, antibiotic susceptibility test with two strains D4 and HU201301 was investigated; the results showed that both strains were sensitive to most of the tested antibiotic drugs.
2. To understand the immune response of the BSB to A. hydrophila infection, the RNA-Seq technology was utilized to analyze the transcriptomic profile after artificial bacterial infection. Two cDNA libraries synthesized from tissues collected from control BSB or those injected with A. hydrophila were sequenced by using Illumina HiSeq2000. After de novo assembly, 155,052 unigenes (average length 692.8 bp) were isolated. All unigenes were annotated using BLASTX relative to several public databases. The sequence similarity (86%) of the assembled unigenes was to zebrafish based on the Nr database. A number of unigenes (n = 30,482) were assigned to three GO categories: biological processes (25,242 unigenes), molecular functions (26,096 unigenes), and cellular components (22,778 unigenes). 20,909 unigenes were classified into 25 KOG categories and 28,744 unigenes were assigned to 315 specific signaling pathways. In total, 238 significantly differentially expressed unigenes (mapped to 125 genes) were identified: 101 upregulated genes and 24 downregulated genes. Another 303 unigenes were mapped to unknown or novel genes. Among the known expressed genes identified, 53 were immune-related genes and were distributed in 71 signaling pathways.
3. Microsatellites (n = 10,877), including di- to pentanucleotide repeat motifs, were also identified in the BSB transcriptome profiles. Dinucleotide repeats were most common (7,152, 65.8%), followed by tri- (3,172, 29.2%), tetra- (549, 5.05%), and pentanucleotide repeat motifs (4, 0.04%). The lengths of the SSRs ranged from 12 to 25 bp. PCR primers were successfully designed for 5077 (46.7%) of the identified microsatellites. Furthermore, a total of 36,326 putative SNPs were discovered from the transcripts. The frequency of SNP was one SNP in 29.2 bp sequence length. The transition and transversion mutation was 21,445 and 12,553 SNPs, respectively. A ratio of transition to transversion was 1.71. Within those yielded SNPs, 10,812 SNPs identified from 2,421 unigenes could be annotated to differential functionality in comparing to public database using BLASTX for GO, KOG and KEGG. A number of SNPs (n = 7,727, 71.5%) found from 1,628 unigenes were assigned to three main GO categories: ‘cellular components’, ‘molecular function’ and ‘biology process’. In total, 5,812 (53.8%) SNPs identified from 1,324 unigenes were classified into 25 KOG categories. 4,589 (42.4%) SNPs detected from 975 unigenes were assigned to 278 KEGG pathways. Furthermore, a number of 600 SNPs found from 111 unigenes were successfully annotated to the term ‘immune system’ via KEGG classification. The database could be useful for further genetic studies in BSB.
4. In silico characterization and homology modeling of encoded proteins, including MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaCTSL, MaMMP-9, MaIL-8 and MaIL-10 in BSB were performed. Physicochemical and functional characteristics of proteins were analyzed. The secondary and tertiary of the protein’s domains were constructed applying the comparative modeling method.
5. Reverse transcription–quantitative PCR (RT–qPCR) was used to analyze the expression of immune-related genes (MaTLR5, MaNFKBIA, MaMyD88, MaTRAF6, MaC3, MaC7, MaCTSL, MaMMP-9, MaIL-8, and MaIL-10) in response to A. hydrophila. The results showed that all investigated genes were highly up- or and downregulated in the liver, spleen and kidney during the challenge time from 4 to 120 hours post injection. This research provides the important roles of these genes in the BSB’s innate immune system.
The results provide significant valuable information of molecular data, which are useful for further study of the immunogenetics of BSB.
The intestinal microbiota play an important role in the maintenance of the fish health. In this study, 150 Oreochromis niloticus were divided into five groups (G1–G5) and were subjected to alternate weekly exchange of feeding rate and frequency (3, 3, 1.5, 1.5 and 0%) and (2, 1, 2, 1 and 0 time), respectively, for 8 weeks. Enumeration of total intestinal bacteria revealed that the abundance of Lactobacillus was negatively correlated with those of Aeromonas, Pseudomonas and Edwardsiella. The abundance and proportion of Lactobacillus were affected by the change in feeding frequency. When fish were infected with Aeromonas hydrophila, survival rate significantly decreased in G5 (starved fish) compared with G1 (control group). The pro‐inflammatory cytokines, interleukin (IL)‐1β and tumour necrosis factor‐α, and anti‐inflammatory cytokine IL‐10 as well as serum antibacterial activity and respiratory burst activity were positively correlated with the proportion of Lactobacillus. The alternate weekly exchange of feeding regime did not alter the parametric morphology of the intestine, height and width of intestinal villi, number of goblet cells and width of muscle layer, except in G5. In conclusion, the alternate weekly exchange of feeding regime to starvation (G5), decrease in the feeding frequency to 1 time/day (G3) and decrease in feeding rate to 1.5% (G4) suppressed the immune status of fish, which became vulnerable to bacterial infection. Immune suppression was positively correlated with a decreased proportion of Lactobacillus spp.
Bu çalışmada, 2015-2019 yılları arasında Veteriner Kontrol Merkez Araştırma Enstitüsü Su Ürünleri Hastalıkları Araştırma ve Teşhis Laboratuvarı’na gönderilen balık örneklerinden izole edilen bakteriyel etkenlerin dağılımı ve bu etkenlerin de bölgesel ve aylara göre dağılımı incelendi. Bu süreçte laboratuvara gönderilen balık örneklerinden etken izolasyonu ve identifikasyonu, konvansiyonel yöntemler ve hızlı teşhis kitleri (Vitek 2) kullanılarak gerçekleştirildi. Çalışmada 60 adet balık örneği incelendi ve bunların 27’sinde bakteriyel etken izole edildi. İzole edilen etkenlerin dağılımının; Aeromonas sobria (%23,3), Aeromonas hydrophila (%6,7), Shewanella putrefaciens (%5), Aeromonas veroni (%3,3), Serratia rubidaea (%1,7), Kocuria rhizophila (%1,7 ), Streptococcus iniae (%1,7 ), Pseudomonas aeruginosa (%1,7 ) ve Proteus spp. (%1,7 ) oranında olduğu belirlendi. Elde edilen sonuçlar göre; 2015-2019 yılları arasında balıklardan en fazla izole edilen etkenin Aeromonas sobria olduğu görüldü. Yine balıklardan izole edilen bakteriyel etkenlerin bölgesel ve aylara göre dağılımı incelendiğinde; bu etkenlerin en yaygın Haziran, Ağustos, Eylül, Ekim aylarında ve Ankara, Bolu, Kastamonu illerinde görüldüğü belirlendi.
The diversity and distribution of Aeromonas spp. associated with virulence profiles from the Rodrigo de Freitas Lagoon were investigated using phylogenetic analysis of gyrB/rpoB gene sequences for speciation. The concatenated gyrB/rpoB gene sequences clustered into five species: Aeromonas punctata/caviae (n ¼ 37), A. hydrophila (n ¼ 10), A. dhakensis (n ¼ 16), A. jandaei (n ¼ 1) and A. enteropelogenes/trota (n ¼ 3). The virulence genes (atc/aerA/hlyA/asp/amp) resulted in 19 virulence profiles, distributed heterogeneously among the five Aeromonas species. Out of the 67 isolates, 16% presented five distinct profiles carrying four virulence genes and 7% showed all genes investigated. The hemolytic genes were detected as follows: act 54% (37/67), aerA 36% (24/67), hlyA 26% (18/67) and proteolytic genes such as asp 36% (24/57) and amp in 85% (57/67) were widely distributed in lagoon sampling stations. Meanwhile, 88% (59/67) and 92% (62/67) of the isolates showed hemolytic and proteolytic activity, respectively. Our results demonstrated that concatenated sequences of the gyrB and rpoB genes showed to be an adequate approach for the Aeromonas speciation and prevalence. The high heterogeneity of virulence genes among the species resulted in several virulence profiles, as well as high percentages of hemolytic and proteolytic activity, demonstrating the necessity of further epidemiological surveys of Aeromonas species pathogenicity in an aquatic recreational lagoon.
Aeromonas hydrophila is a Gram-negative, bacterial pathogen of humans and other vertebrates. To delineate pathotypes, the genomes of twenty-eight Aeromonas isolates were screened by PCR to determine the presence of virulence factors including: aerolysin (aerA), cytotoxic enterotoxin (act), hemolysin (ahh1), elastase (ahyB), enolase (eno), S-layer protein (ahsA), serine protease (ser), Type IV Aeromonas pilus (tapA), lipase (lip), and Type Three Secretion System (T3SS) components (aopB, ascV). Genes for ahh1, lip, ser, and ahyB were present in all 28 strains tested, others genes varied. The tapA gene encoding a type IV pilus was absent in all 28 isolates screened. After the presence or absence of these genes was determined, corresponding activity was determined using phenotypic assays. Analysis of the data defined 11 different pathotypes based on the genotypic and phenotypic profiles with the largest cluster being ahh1+, ahyB+, lip+, ser+, act+, aerA+, eno-, aopB-, ascV-, ahsA-, and tapA-. Representatives of the pathotype groups were used in a nematode challenge assay and a cell culture assay to assess the importance of virulence factors in their pathogenicity and their cytotoxicity, respectively. All Aeromonas strains tested, with the exception of A. hydrophila ML09-119, showed significantly greater lethality compared to the E. coli negative control in the nematode challenge, yet only A. hydrophila 1127 was significantly more lethal than the positive control Pseudomonas aeruginosa PAO1. Variation in lethality between A. hydrophila strains suggests that C. elegans is a suitable model for studying pathogenic mechanisms and elucidating the combination of factors that define highly virulent strains.
The genus Aeromonas is ubiquitous in aquatic environments encompassing a broad range of fish and human pathogens. Aeromonas strains are known for their enhanced capacity to acquire and exchange antibiotic resistance genes and therefore, are frequently targeted as indicator bacteria for monitoring antimicrobial resistance in aquatic environments. This study evaluated temporal trends in Aeromonas diversity and antibiotic resistance in two adjacent semi-intensive aquaculture facilities to ascertain the effects of antibiotic treatment on antimicrobial resistance. In the first facility, sulfadiazine-trimethoprim was added prophylactically to fingerling stocks and water column-associated Aeromonas were monitored periodically over an 11-month fish fattening cycle to assess temporal dynamics in taxonomy and antibiotic resistance. In the second facility, Aeromonas were isolated from fish skin ulcers sampled over a 3-year period and from pond water samples to assess associations between pathogenic strains to those in the water column. A total of 1200 Aeromonas isolates were initially screened for sulfadiazine resistance and further screened against five additional antimicrobials. In both facilities, strong correlations were observed between sulfadiazine resistance and trimethoprim and tetracycline resistances, whereas correlations between sulfadiazine resistance and ceftriaxone, gentamicin, and chloramphenicol resistances were low. Multidrug resistant strains as well as sul1, tetA, and intI1 gene-harboring strains were significantly higher in profiles sampled during the fish cycle than those isolated prior to stocking and these genes were extremely abundant in the pathogenic strains. Five phylogenetically distinct Aeromonas clusters were identified using partial rpoD gene sequence analysis. Interestingly, prior to fingerling stocking the diversity of water column strains was high, and representatives from all five clusters were identified, including an A. salmonicida cluster that harbored all characterized fish skin ulcer samples. Subsequent to stocking, diversity was much lower and most water column isolates in both facilities segregated into an A. veronii-associated cluster. This study demonstrated a strong correlation between aquaculture, Aeromonas diversity and antibiotic resistance. It provides strong evidence for linkage between prophylactic and systemic use of antibiotics in aquaculture and the propagation of antibiotic resistance.
Epizootic ulcerative syndrome (EUS) is an international problem that has been studied independently and collaboratively by many different workers. In some quarters, EUS is considered a fungal disease of freshwater and brackish-water fish. The disease has been given several colloquial names: mycotic granulomatosis in Japan, red spot disease in Australia, and EUS in Asia. In addition to the foregoing, significant well-documented epizootics have occurred in Thailand, Cambodia (Kampuchea), Lao PDR, Myanmar, Vietnam, China, Hong Kong, the Philippines, Bangladesh, and India.
The present study determined the inhibitory effect of tannin from sweet chestnut wood against tilapia Oreochromis niloticus bacterial pathogens, Aeromonas hydrophila (20 isolates) and Streptococcus agalactiae (20 isolates). Minimum Inhibitory Concentrations (MICs) corresponding to tannin and oxytetracycline (OTC) for A. hydrophila were 18.75-300 μg/ml and 0.125-128 μg/ml; and for S. agalactiae were 600 μg/ml and 0.5-8 μg/ml, respectively. The antibacterial potency of tannin from sweet chestnut wood was found in the test system of pH: 6.5-7.0, suggesting that the inhibitory effect of tannins is unrelated to their acidity property. The OTC-resistant A. hydrophila and S. agalactiae (MIC of OTC > 4 μg/ml) responded to lower MICs of OTC in the test system with sub-inhibitory concentration of tannins (150 μg/ml). Concerning the trend towards the evolution of antimicrobial resistant strains, the antimicrobial activity of tannins may be a possible additive compound against fish pathogens treated with antibiotics.
Aeromonas spp. are opportunistic pathogens causing a broad spectrum of human illnesses like gastroenteritis, chronic diarrhea, wound infections, peritonitis, urinary tract infections, and septicemia. Their ability to grow in foods stored in a refrigerator poses a substantial threat for human consumption. We investigated the prevalence of Aeromonas from commercial food products across Riyadh, Saudi Arabia. A total of 250 samples were randomly collected and processed for the isolation and identification of Aeromonas by morphological and biochemical means and for their identification by PCR. A total of 102 strains of Aeromonas were isolated, including 47% from raw meat samples, 34% from raw fish samples, and 18.6% from milk and dairy products; 56.8% were identified as A. hydrophila and 43.1% as A. sobria. Antibiotic susceptibility tests done revealed 100% sensitivity to chloramphenicol, colistin, ciprofloxacin, and nitrofurantoin. 16S rDNA PCR revealed the presence of the 953 bp fragment in all the strains. The present investigation suggested the occurrences of A. sobria and A. hydrophila in human consumable stored and refrigerated foods.
This study examined the antimicrobial susceptibility and mutation(s) in quinolone resistance-determining regions (QRDRs) in streptococcal pathogens isolated from farmed Nile tilapia Oreochromis niloticus in Thailand. Surveillance of antimicrobial susceptibility in tilapia streptococcal pathogens reveals that Streptococcus agalactiae (n = 97) and Streptococcus iniae (n = 3) from diseased tilapia were susceptible to amoxicillin, florfenicol, sulfamethoxazole/trimethoprim and sulfadimethoxine/ormetoprim, however, only 78 isolates were susceptible to enrofloxacin. Twenty-two enrofloxacin-resistant S. agalactiae isolates were further examined for mutations in the QRDRs of gyrA, gyrB, parC and parE genes. Twenty isolates had single base pair changed in the gyrA sequence, C-242-T. Point mutations in gyrB, GC-1135, 1136-AA and T-1466-G, were identified in one isolate. All resistant isolates harboured a mutation in the parC gene, C-236-A, while no mutations were observed in the parE gene. The study represented mutations of gyrA and parC genes as marked modification of the enrofloxacin-resistant S. agalactiae from farmed tilapia. This study is a primary report of the QRDRs mutations associated with fluoroquinolone resistance from streptococcal pathogen in the cultured fish. The phenotypic and genotypic characterization of enrofloxacin resistance S. agalactiae evident in this study has led to an improved regulation of antimicrobial use in Thai aquaculture.
This study aimed to isolate and characterize an indigenous algicidal bacterium named LTH-1 and its algae-lysing compounds active against three Microcystis aeruginosa strains (toxic TH1, nontoxic TH2 and standard FACHB 905). The LTH-1 isolated from Lake Taihu, near Wuxi City in China, was identified as Aeromonas sp. based on its morphological characteristic features and phylogenetic analysis by sequencing of 16S rDNA. Extracellular compounds produced by LTH-1 showed strong algaelysing activity, and they were water-soluble and heat-tolerant, with a molecular mass lower than 2 kDa. Two algae-lysing compounds were isolated and purified from extracellular filtrate using silica gel column chromatography. One of these was identified as phenylalanine (C9H11NO2, m/z 166.0862) and the other (C8H16N2O3, m/z 189.1232) was unidentified by hybrid ion trap/time-of-flight mass spectrometry coupled with a high-performance liquid chromatography (LC/MS-IT-TOF) system. The half maximal effective concentration (EC50) of phenylalanine produced by LTH-1 against FACHB 905 was 68.2 +/- 8.2 microg mL(-1) in 48h. These results suggest that the algicidal Aeromonas sp. LTH-1 could play a role in controlling Microcystis blooms, and its extracellular compounds are also potentially useful for regulating blooms of the harmful M. aeruginosa.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons, a variety of definitional, algorithmic, and statistical refinements permits the execution time of the BLAST programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program that runs at approximately three times the speed of the original. In addition, a method is described for automatically combining statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using this matrix. The resulting Position Specific Iterated BLAST (PSLBLAST) program runs at approximately the same speed per iteration as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities.
The Ribosomal Database Project (RDP-II), previously described by Maidak et al., continued during the past year to add new rRNA sequences to the aligned data and to improve the analysis commands. Release 7.1 (September 17, 1999) included more than 10 700 small subunit rRNA sequences. More than 850 type strain sequences were identified and added to the prokaryotic alignment, bringing the total number of type sequences to 3324 representing 2460 different species. Availability of an RDP-II mirror site in Japan is also near completion. RDP-II provides aligned and annotated rRNA sequences, derived phylogenetic trees and taxonomic hierarchies, and analysis services through its WWW server (http://rdp.cme.msu.edu/ ). Analysis services include rRNA probe checking, approximate phylogenetic placement of user sequences, screening user sequences for possible chimeric rRNA sequences, automated alignment, production of similarity matrices and services to plan and analyze terminal restriction fragment length polymorphism (T-RFLP) experiments.
Phenotypic and genetic studies were performed on some atypical aeromonas strains of uncertain taxonomic position. 16S rRNA gene sequence analysis revealed that these strains represent a hitherto unknown genetic line within the genus Aeromonas, for which the name Aeromonas allosaccharophila sp. nov. is proposed. The type strain is CECT 4199.
The spatiotemporal distributions of Aeromonas spp. and fecal coliforms in a cove receiving sewage treatment effluent and draining into a brackish lagoon were studied for 34 months with sampling at six stations. A total of 452 strains of Aeromonas spp. were isolated and identified at the outflow of the treatment system and at stations in the cove. Hemolytic activity of 289 Aeromonas strains was determined. The Aeromonas spp. and fecal coliform distributions showed seasonal cycles in the pond effluent. These seasonal bacterial cycles were persistent in effluent, at the discharge point, and in the cove. However, the abundance levels of these bacterial distributions decreased gradually from the coast to the open lagoon. A dilution model showed that the Aeromonas spp. and fecal coliform distributions in the cove water were subject not only to dilution effect but also to other environmental factors, such as salinity. A. sobria is the most common species identified in the Aeromonas population present in the cove water. Survival studies confirmed that Aeromonas spp., especially A. sobria, were more sensitive to saline and/or marine stress than fecal coliforms. Among the Aeromonas hydrophila and A. sobria strains, 96 and 97%, respectively, produced hemolysin, whereas among the Aeromonas caviae strains, 95% were nonhemolytic.
The spatiotemporal dynamics of Aeromonas spp. and fecal coliforms in the sewage treatment ponds of an urban wastewater center were studied after 20 months of sampling from five stations in these ponds. Isolation and identification of 247 Aeromonas strains were undertaken over four seasons at the inflow and outflow of this pond system. The hemolytic activity of these strains was determined. The Aeromonas spp. and the fecal coliform distributions showed seasonal cycles, the amplitude of which increased at distances further from the wastewater source, so that in the last pond there was an inversion of the Aeromonas spp. cycle in comparison with that of fecal coliforms. The main patterns in these cycles occurred simultaneously at all stations, indicating control of these bacterial populations by seasonal factors (temperature, solar radiation, phytoplankton), the effects of which were different on each bacterial group. The analysis of the Aeromonas spp. population structure showed that, regardless of the season, Aeromonas caviae was the dominant species at the pond system inflow. However at the outflow the Aeromonas spp. population was dominated by A. caviae in winter, whereas Aeromonas sobria was the dominant species in the treated effluent from spring to fall. Among the Aeromonas hydrophila and A. sobria strains, 100% produced hemolysin; whereas among the A. caviae strains, 96% were nonhemolytic.
The 16S rRNA gene sequences of the type strains of Aeromonas enteropelogenes and Aeromonas ichthiosmia were determined by polymerase chain reaction direct sequencing in order to clarify their interrelationships with other aeromonad species. On the basis of 16S rRNA gene sequence analysis, A. enteropelogenes and A. ichthiosmia were found to be identical to Aeromonas trota and Aeromonas veronii, respectively.
We investigated the usefulness of a novel DNA fingerprinting technique, AFLP, which is based on the selective amplification of genomic restriction fragments by PCR, to differentiate bacterial strains at the subgeneric level. In totals, 147 bacterial strains were subjected to AFLP fingerprinting: 36 Xanthomonas strains, including 23 pathovars of Xanthomonas axonopodis and six pathovars of Xanthomonas vasicola, one strain of Stenotrophomonas, 90 genotypically characterized strains comprising all 14 hybridization groups currently described in the genus Aeromonas, and four strains of each of the genera Clostridium, Bacillus, Acinetobacter, Pseudomonas and Vibrio. Depending on the genus, total genomic DNA of each bacterium was digested with a particular combination of two restriction endonucleases and the resulting fragments were ligated to restriction halfsite-specific adaptors. These adaptors served as primer-binding sites allowing the fragments to be amplified by selective PCR primers that extend beyond the adaptor and restriction site sequences. Following electrophoretic separation on 5% (w/v) polyacrylamide/8.3 M urea, amplified products could be visualized by autoradiography because one of the selective primers was radioactively labelled. The resulting banding patterns, containing approximately 30-50 visualized PCR products in the size range 80-550 bp, were captured by a high-resolution densitoscanner and further processed for computer-assisted analysis to determine band-based similarity coefficients. This study reveals extensive evidence for the applicability of AFLP in bacterial taxonomy through comparison of the newly obtained data with results previously obtained by well-established genotypic and chemotaxonomic methods such as DNA-DNA hybridization and cellular fatty acid analysis. In addition, this study clearly demonstrates the superior discriminative power of AFLP towards the differentiation of highly related bacterial strains that belong to the same species or even biovar (i.e. to characterize strains at the infrasubspecific level), highlighting the potential of this novel fingerprinting method in epidemiological and evolutionary studies.
We describe a new method for studying the structure and diversity of bacterial communities in the natural ecosystem. Our approach is based on single-strand-conformation polymorphism (SSCP) analysis of PCR products of 16S rRNA genes from complex bacterial populations. A pair of eubacterial universal primers for amplification of the variable V3 region were designed from the 16S rRNA sequences of 1,262 bacterial strains. The PCR conditions were optimized by using genomic DNAs from five gram-positive and seven gram-negative strains. The SSCP analysis of the PCR products demonstrated that a bacterial strain generated its characteristic band pattern and that other strains generated other band patterns, so that the relative diversity in bacterial communities could be measured. In addition, this method was sensitive enough to detect a bacterial population that made up less than 1.5% of a bacterial community. The distinctive differences between bacterial populations were observed in an oligotrophic lake and a eutrophic pond in a field study. The method presented here, using combined PCR amplification and SSCP pattern analyses of 16S rRNA genes, provides a useful tool to study bacterial community structures in various ecosystems.
Identification of Aeromonas species, emergent pathogens for humans, has long been controversial due to their phenotypic and genomic heterogeneities. Computer analysis of the published 16S rRNA gene sequences revealed that restriction fragment length polymorphism of the PCR-amplified 16S rRNA gene is a good and rapid way of assessing the identities of all known species of Aeromonas. The method was evaluated with the reference strains of all species (or DNA homology groups) and 76 clinical isolates of diverse origin. Most results from the two approaches were in agreement, but some discrepancies were discerned. Advantages over previous phenotypic and genetic methods are discussed.
The BLAST programs are widely used tools for searching protein and DNA databases for sequence similarities. For protein comparisons,
a variety of definitional, algorithmic and statistical refinements described here permits the execution time of the BLAST
programs to be decreased substantially while enhancing their sensitivity to weak similarities. A new criterion for triggering
the extension of word hits, combined with a new heuristic for generating gapped alignments, yields a gapped BLAST program
that runs at approximately three times the speed of the original. In addition, a method is introduced for automatically combining
statistically significant alignments produced by BLAST into a position-specific score matrix, and searching the database using
this matrix. The resulting Position-Specific Iterated BLAST (PSIBLAST) program runs at approximately the same speed per iteration
as gapped BLAST, but in many cases is much more sensitive to weak but biologically relevant sequence similarities. PSI-BLAST
is used to uncover several new and interesting members of the BRCT superfamily.
We examined the taxonomic position of seven Aeromonas isolates, recovered from Flemish and Scottish drinking water production plants and reservoirs, which were previously recognized by numerical analysis of genomic AFLP fingerprints as members of an unknown Aeromonas taxon that most closely resembled the species Aeromonas bestiarum (DNA hybridization group [HG] 2). The new phenotypic and DNA-DNA hybridization data obtained in this study show that the A. bestiarum-like strains constitute a separate Aeromonas species, for which the name Aeromonas popoffii sp. nov. is being proposed. The new species exhibited an internal DNA relatedness ranging from 79 to 100% and was 22 to 63% related to the type or reference strains of other Aeromonas spp. The highest DNA binding values were determined with A. bestiarum (51 to 63%), followed by Aeromonas hydrophila sensu stricto (HG1; 50 to 60%) and Aeromonas salmonicida (HG3; 39 to 55%). Although fingerprints generated by ribotyping and cellular fatty acid analysis often were highly similar, minor differences between the respective fingerprints were of significance for the differentiation of A. popoffii from its closest taxonomic neighbors, HG1, HG2, and HG3. Phenotypically, all seven strains of A. popoffii were positive for acid and gas production from D-glucose and glycerol, growth in KCN broth, arginine dihydrolase, DNase, Voges-Proskauer reaction, and resistance to vibriostatic agent O/129 and ampicillin but displayed negative reactions for production of urease, tryptophan deaminase, ornithine decarboxylase, and lysine decarboxylase (LDC). None of the strains displayed strong hemolytic activity. The lack of D-sucrose fermentation and LDC production and the ability to utilize DL-lactate as the sole energy and carbon source were useful characteristics for the biochemical separation of A. popoffii from A. bestiarum. Other Aeromonas spp. could be differentiated phenotypically from the new species by at least two features. The chromosomal G+C content of A. popoffii ranges from 57.7 to 59.6 mol%. Strain LMG 17541 is proposed as the type strain.
The recently reported chemotaxonomic and genotypic description of two well-separated subgroups (I and II) in Aeromonas eucrenophila and their affiliation to Aeromonas encheleia and the unnamed Aeromonas DNA hybridization group (HG) 11 (G. Huys, M. Altwegg, M.-L. Hänninen, M. Vancanneyt, L. Vauterin, R. Coopman, U. Torck, J. Lüthy-Hottenstein, P. Janssen, and K. Kersters, Syst. Appl. Microbiol. 19:616-623, 1996) has questioned the original species descriptions of A. eucrenophila and A. encheleia. In order to elucidate the unclear taxonomic status of these taxa in the genus Aeromonas, we have further investigated a collection of 14 reference strains and 14 related isolates encompassing the taxa A. eucrenophila subgroups I and II, A. encheleia, and HG11 by DNA-DNA hybridization (on 17 of the 28 strains) and phenotypic characterization (on all 28 strains). Genotypically, the investigated strains could be grouped into two DNA hybridization groups that exhibited between-group homologies ranging from 42 to 52%. The members of DNA homology group I (DNA binding, 76 to 100%) were strains of A. eucrenophila subgroup I, including the type strain LMG 3774, and two A. eucrenophila-like isolates, leading to the conclusion that these strains should be considered true representatives of the species A. eucrenophila. The strains of A. eucrenophila subgroup II, HG11, and A. encheleia, on the other hand, were closely joined in DNA homology group II (DNA binding, 74 to 105%) together with two presumptive A. encheleia isolates. The fact that strain LMG 16330T of A. encheleia was the only type strain residing in DNA homology group II implies that HG11 and A. eucrenophila subgroup II should be classified in the species A. encheleia. Except for the somewhat aberrant phenotypic positions of HG11 strains LMG 13075 and LMG 13076, the establishment of DNA homology groups I and II was supported by the delineation of phena 1 and 2 (level of correlation, 90%), respectively, as revealed by numerical analysis of 136 phenotypic test results. These data indicate that A. eucrenophila and A. encheleia are phenotypically highly related but can be easily separated by testing the production of acid from D-cellobiose and lactose and the assimilation of D-cellobiose. Extended descriptions of both species are given.
Aeromonas strains which phenotypically and genetically belong to the Aeromonas salmonicida species but that according to their phenotypic properties constitute a new subspecies have been isolated from the water of a heavily polluted river, the Matanza river, situated near the central district of Buenos Aires city. These strains were ascribed to the A. salmonicida species by using 65 biochemical tests and by DNA-DNA hybridization. They produce acid from -sorbitol, an unusual biochemical property found in a few members of the A. salmonicida species. They also utilize urocanic acid and do not ferment L-rhamnose or utilize LD-lactate, and are elastase- and gluconate-negative. The DNA relatedness was over 70%, the current limit accepted for the phylogenetic definition of a species, to the described A. salmonicida subspecies and nearly 100% within the new group of Aeromonas strains. Phenotypic differentiation from other A. salmonicida subspecies was readily achieved using the following characteristics: growth at 37 degrees C, melanin production, indole and Voges-Proskauer assays, growth on KCN broth, mannitol and sucrose fermentation and gas from glucose. A remarkable property of the strains of the new group was their ability to degrade polypectate, an unusual feature among Aeromonas species in general. The complete 16S rRNA gene of one strain of the new group was sequenced. Comparison with rDNA sequences of Aeromonas members available in databases revealed a close relationship between this strain and strains belonging to A. salmonicida subsp. salmonicida, masoucida and achromogenes, in agreement with the biochemical data. Since the new A. salmonicida strains constitute a tight genomic group that can be identified by phenotypic properties it was concluded that they represent a new subspecies for which the name Aeromonas salmonicida subsp. pectinolytica is proposed. The type strain of A. salmonicida subsp. pectinolytica is 34melT (= DSM 12609T).
A previously described molecular method, based on 16S rDNA RFLP analysis, for the identification of Aeromonas spp. was unable to separate the species Aeromonas salmonicida, Aeromonas bestiarum and the recently described Aeromonas popoffii. In this study, the method has been extended with endonucleases AIwNI and PstI for the identification of these species. A molecular frame for the identification of all known Aeromonas spp. is presented.
A novel DNA fingerprinting method, named AFLPTM, was used to determine the genotypic diversity among 168 Aeromonas isolates originating from five drinking water production plants in Flanders, Belgium. The AFLPTM technique determines the genomic similarity between bacterial strains through numerical analysis of banding patterns generated by the electrophoretic separation of selectively amplified restriction fragments. Using an identification library (AER094) comprising AFLP fingerprints of 107 well-characterized Aeromonas strains, a total of 144 isolates (86%) could be allocated to one of the 14 DNA hybridization groups (HGs) so far recognized in the genus Aeromonas. The majority of these strains belonged to Aeromonas hydropbila HGs 2 and 3, Aeromonas caviae HGs 5A and 5B, Aeromonas sobria HG 7, and Aeromonas veronii HG8/10. Cluster analysis of individual banding patterns revealed that eight isolates identified as Aeromonas eucrenophila HG6 were dispersed over two well-separated AFLP clusters, suggesting the existence of a genotypic subdivision within this species. The remaining 24 unidentified isolates constituted a homogeneous AFLP cluster which was found to be most closely related to HG2. Possibly, these strains may represent a currently unknown HG within the A. bydropbila complex. In conclusion, this study clearly elucidates the taxonomic value of the AER094 database for the identification and classification of unknown aeromonads and further demonstrates the general applicability of AFLP-based libraries to determine genotypic relationships in other bacterial genera.
The culturability of the intestinal microflora of 48 rainbow trout was detected by comparing direct microscopic counts (4′,6-diamidino-2-phenylindole, DAPI) with plate counts (tryptone soya agar, TSA). In general, a high percentage (average 50%) of the microflora could be cultured. The counts of the intestinal microflora varied 3–5 log units between fish within the same sampling point. A total of 504 bacteria were identified by physiologic criteria and 153 strains also by partial sequencing of the 16S rRNA gene. High agreement was found between classical and molecular identification. The dominant intestinal microflora was identified as bacteria belonging to the gamma subclass of Proteobacteria (of the genera Citrobacter, Aeromonas and Pseudomonas), to the Gram-positive bacteria with low G+C-content (of the genus Carnobacterium) and as bacteria belonging to the beta subclass of Proteobacteria. However, the composition of the intestinal microflora showed high variation among three investigated fish farms and also at different time points within one fish farm.
The fish pathogen, Aeromonas salmonicida, has been the focus of a number of molecular genetic studies designed to characterize the microorganism and its pathogenesis. The paracrystalline surface protein layer (A-layer) of A. salmonicida has been studied in considerable detail. The A-layer gene, vapA, has been cloned and sequenced and studies have been performed on its regulation. The secretion pathway specific for the A-layer subunits has also been partially characterized as has the general protein secretion pathway. Other genes involved in the biogenesis of the A. salmonicida surface include abcA, asoA and asoB. The abcA gene encodes a protein which is involved in lipopolysacharide O-chain synthesis and secretion and may have a role in the regulation of vapA gene expression. A. salmonicida also possesses plasmids of various sizes which exhibit a high degree of conservation and can encode antibiotic resistance elements. Insertion sequence elements have been identified in two strains of A. salmonicida and are capable of transposing within a cell to cause mutations that affect virulence. Molecular biology techniques have also been applied to the problem of detection of low levels of A. salmonicida in natural environments and carrier fish. The development of tools such as specific DNA probes and PCR primer pairs allows the detection of extremely low numbers of A. salmonicida even in the presence of high numbers of other bacteria. The development of vaccines against A. salmonicida has incorporated some new techniques such as the generation of specific mutations in the chromosome or the production of large quantities of particular proteins, such as the outer membrane porins, in expression systems. Another approach involves the use of an avirulent A. salmonicida strain as a shuttle system to express fragments of genes from viral pathogens with a view to providing protective immunity against multiple diseases with a single vaccine.
The phylogenetic interrelationships of members of the genera Aeromonas and Plesiomonas were investigated by using small-subunit ribosomal DNA (rDNA) sequencing. Members of the genus Aeromonas formed a distinct line within the gamma subclass of the Proteobacteria. Plesiomonas shigelloides also clustered within the confines of the gamma subclass of the Proteobacteria but exhibited a closer association with members of the family Enterobacteriaceae than with members of the family Aeromonadaceae. Species of the genus Aeromonas exhibited very high levels of overall sequence similarity (ca. 98 to 100%) with each other. Several of the relationships derived from an analysis of the rDNA sequence data were in marked disagreement with the results of chromosomal DNA-DNA pairing experiments. Diagnostic rDNA signatures that have possible value for differentiating most Aeromonas species were discerned.
. The aerobic flora of the skin of 56 Atlantic salmon from coastal, estuarine and river waters was analysed quantitatively; 50 skin and 33 gill samples were analysed qualitatively. The water at each sampling station was also analysed. The principal genera on the skin and gills were Moraxella, Flavobacterium, Cytophaga and Pseudomonas; members of Acinetobacter, Bacillus, Aeromonas, Vibrio, the Entrobacteriaceae, Micro-coccaceae and some coryneforms were also present. The gill flora was similar to that of the skin, which reflected that of the environment.
The occurrence of Aeromonas spp. in the metropolitan water supply of Perth, Western Australia, Australia, was monitored at several sampling points during a period of 1 year. Water within the distribution system conformed to international standards for drinking water but contained Aeromonas spp. in numbers comparable to those in raw surface water, although this water was free of Escherichia coli. Coliforms and E. coli were found in raw surface waters, and Aeromonas spp. were found in raw water from surface and underground sources. Chemical treatment, followed by chlorination at service reservoirs, resulted in water free of E. coli and a decrease in the number of Aeromonas spp. Aeromonas spp. were found in the greatest numbers in summer. Multiple regression analysis showed that growth of Aeromonas spp. in chlorinated water was related to water temperature, residual chlorine, and interaction between these variables. The incidence of Aeromonas-associated gastroenteritis, determined from isolates referred to us for enterotoxin testing, paralleled the pattern of isolation of Aeromonas spp. in water within the distribution systems. We suggest that the presence of Aeromonas spp. in drinking water needs public health appraisal and that further work should be undertaken to permit reevaluation of standards for the quality of drinking water.
Gas-liquid chromatography of cellular fatty acid methyl esters (FAMEs) was used to determine the phenotypic and genotypic diversity among 489 presumptive Aeromonas strains isolated from five Flemish drinking water production plants. FAME profiles were compared with the predetermined library profiles of a representative database, AER48C, which contains the mean FAME data of all 14 currently established hybridization groups (HGs) or genospecies within Aeromonas. Using AER48C, more than 93% (457 strains) of all presumptive aeromonads isolated on ampicillin-dextrin agar were unequivocally identified as belonging to this genus. Moreover, 85.5% and 73.5% of these strains could be assigned to a particular phenospecies or HG, respectively. Raw and treated surface water samples were dominated by members of the Aer. hydrophila complex (38.8%, comprising HGs 1-3), followed by the Aer. caviae complex (22.7%, comprising HGs 4-6) and the Aer. sobria complex (16.7%, comprising HGs 7-9). HGs 3, 5A/B and 8 were the most prominent genospecies in this type of water. On the other hand, it was found that raw and treated phreatic groundwater samples displayed a much more limited species diversity since these were almost entirely dominated (95.8%) by strains belonging to HGs 2 and 3 of the Aer. hydrophila complex. In general, flocculation-decantation and sand filtration were not shown to influence the overall species distribution in any of the plants examined.
A chemotaxonomic study was carried out on representative strains of 13 Aeromonas genomic species. Quinone, polyamine, and fatty acid patterns were found to be very useful for an improved characterization of the genus and an improved differentiation from members of the families Enterobacteriaceae and Vibrionaceae. The Q-8-benzoquinone was the predominant ubiquinone, and putrescine and diaminopropane were the major polyamines of the genus. The fatty acid patterns of 181 strains, all characterized by DNA-DNA hybridization, showed a great homogeneity within the genus, with major amounts of hexadecanoic acid (16:0), hexadecenoic acid (16:1), and octadecenoic acid (18:1), and minor amounts of the hydroxylated fatty acids (3-OH 13:0, 2-OH 14:0, 3-OH 14:0) in addition to some iso and anteiso branched fatty acids (i-13:0, i-17:1, i-17:0, and a-17:0). Although some differences in fatty acid profiles between the genomic species could be observed, a clearcut differentiation of all species was not possible.
A case of food poisoning outbreak involving Aeromonas hydrophila is reported in this study. A group of 27 people consumed a typical Swedish food "landgång" which is a type of smörgåsbord containing shrimps with mayonnaise, liver paté, ham, sausage, and legume salad which was purchased from a food store. Twenty-two of the 27 persons became ill within 20-34 hr of consumption of the food and reported the symptoms ranging from severe acute diarrhea, abdominal pain, headache, fever and vomiting. One person also fainted. The symptoms lasted for a couple of days. Of the remaining 5 healthy persons who consumed the left-over food the next day, 2 became ill with similar symptoms. The bacteriological examination of left-over food samples resulted in the isolation of A. hydrophila from shrimps with mayonnaise, smoked sausage, liver paté and boiled ham. The total number of A. hydrophila in these foods were log 10(6) to log > 10(7) organisms per gram of food sample. A. hydrophila was however, not isolated from legume/mayonnaise salad samples. All the food samples tested showed low numbers of other expected food contaminating organisms such as coliforms at 37 C and 44 C, fecal streptococci, Staphylococcus aureus, fungi and yeast etc., while Bacillus cereus, Clostridium perfringens and Salmonella spp. were not detected in the food samples. Investigations of the virulence profiles of the A. hydrophila isolates showed their capacity to produce beta-hemolysin, cytotoxins, cytotonic toxins, enterotoxins, and adhesion to and invasion of human intestinal (Henle 407) cells in culture.
Aeromonas spp. are Gram-negative rods of the family Vibrionaceae. They are normal water inhabitants and are part of the regular flora of poiquilotherm and homeotherm animals. They can be isolated from many foodstuffs (green vegetables, raw milk, ice cream, meat and seafood). Mesophilic Aeromonas spp. have been classified following the AeroKey II system (Altwegg et al., 1990; Carnahan et al., 1991). The major human diseases caused by Aeromonas spp. can be classified in two major groups: septicemia (mainly by strains of A. veronii subsp. sobria and A. hydrophila), and gastroenteritis (any mesophilic Aeromonas spp. but principally A. hydrophila and A. veronii). Most epidemiological studies have shown Aeromonas spp. in stools to be more often associated with diarrhea than with the carrier state; an association with the consumption of untreated water was also conspicuous. Acute self-limited diarrhea is more frequent in young children, in older patients chronic enterocolitis may also be observed. Fever, vomiting, and fecal leukocytes or erythrocytes (colitis) may be present (Janda, 1991). The main putative virulence factors are: exotoxins, endotoxin (LPS), presence of S-layers, fimbriae or adhesins and the capacity to form capsules.
We characterized a collection of 268 Aeromonas isolates from diverse sources (clinical, animal, and environmental sources) for their species and serogroup designations. Overall, 97% of these strains could be identified to the genomospecies level by using an expanded battery of biochemical tests. Members of the Aeromonas hydrophila complex (A. hydrophila, HG2, and A. salmonicida), a group that has previously been difficult to separate biochemically, could easily be distinguished from one another by using a number of recently described phenotypic properties which included utilization of DL-lactate and urocanic acid. Differences in species distributions on the basis of the source of isolation were noted. Serogroup analysis of these 268 isolates plus a number of reference cultures indicated that (i) each genomospecies is serologically heterogeneous and individual serogroups can be found in more than one species, (ii) most type or reference strains for each hybridization group are not serologically representative of the genomospecies at large, (iii) serogroups O:11, O:34, and O:16 predominate clinically (48%), supporting previous studies indicating their importance in human infections, and (iv) most A. trota strains do not express the O139 antigen of Vibrio cholerae. The collective results suggest that both species and serogroup designations are important factors in establishing which isolates can cause human infections when they are acquired from nonclinical sources (foods, animals, and the environment).
This study investigates the role of water quality parameters as possible risk or protection factors in Aeromonas spp-affected rainbow trout hatcheries in northeastern Spain. The results revealed an association between oxygen concentration, ammonia concentration and total dissolved solids (TDS) with the prevalence of Aeromonas spp cases on the affected fish farms. The oxygen and ammonia concentrations acted as risk factors when their values were lower than 7.06 mg/L or higher than 0.05 mg/L, respectively, generating odds ratio (OR) values of 2.0 and 2.3. TDS levels, however, acted as a protective factor (OR = 0.39), from an epidemiological point of view but not statistically, when the values were outside the normal range, 199-261 mg/L. On a multivariate level, fish farms exposed to all the associated factors at the same time, had a risk of being affected by Aeromonas spp that was 1.74 times higher than farms with normal quality parameters; the risk increased when TDS had a normal value and it decreased when oxygen and/or ammonia concentrations had normal values.
Aeromonas spp. are common contaminants of fish and seafood. They also are ubiquitous in the water environment. Aeromonas spp. were identified in 27 (93%) of 29 fish, in 17 (100%) fish-egg, in two (16%) of 12 shrimp samples and in 23 (100%) freshwater samples. In total, 117 Aeromonas strains were isolated from 69 positive samples, several samples having had two or three Aeromonas species. Included in this were also 26 mesophilic Aeromonas strains isolated in association with the study on fish diseases. The distribution of the species into 13 known hybridization groups (HGs) were studied by phenotypic and molecular methods. Ribopattern analysis of SmaI digested DNA was used for the identification of HGs. The predominant HG in fish, fish-eggs and freshwater samples was A. hydrophila HG 3 because 63% (22/37), 28% (16/57) or 80% (16/20) of the strains, respectively, were in HG 3. A. hydrophila HG 2 was also common in fresh fish samples but was not identified in fish-egg samples. HG 7 was common in fish samples studied for fish diseases and in freshwater samples. Strains which were not allotted to any HGs were common (19 of 143 strains). A. hydrophila HG 1, A. caviae HG 4, A. veronii subspecies sobria or subspecies veronii HG 8/10 known to be associated with human diarrhea were uncommon in all samples. The three strains isolated from frozen shrimp during two suspected food-borne outbreaks were A. hydrophila HG 2 and HG 3.
It has been almost 10 years since a major review on the association of Aeromonas with human disease has been published. During that period the number of valid species in the genus has grown to 14, with
a new family (Aeromonadaceae) established to house this genus. Despite this explosion in the number of new genomospecies,
only five (Aeromonas hydrophila, A. caviae, A. veronii, A. jandaei, and A. schubertii) are currently recognized as human pathogens. New syndromes attributed to this genus include hemolytic uremic syndrome, burn-associated
sepsis, and a variety of respiratory tract infections, including epiglottitis. Convincing evidence suggests that some aeromonads
do cause gastroenteritis, but it is presently unclear whether many of the strains isolated from feces are involved in diarrheal
disease. Many questions regarding this genus remain unanswered.
Density of Aeromonas spp. at one site in the Buffalo River and at four sites on its upstream tributaries was followed from June 1992-June 1993. Membrane filtration counts of Aeromonas during the summer ranged between 18 and 4000 ml-1, which were one to two logs higher than faecal coliform and faecal streptococci densities. Aeromonas spp. in the Buffalo River, and faecal coliforms, faecal streptococci, and the heterotrophic plate count throughout the watershed, increased by approximately one log during summer rainstorms. However, Aeromonas spp. increased only by a factor of two during rainstorms at the upstream sites. Aeromonas spp. showed a strong positive correlation with both indicator bacteria and total suspended solids at the upstream sites during the summer but not the winter. Correlations between Aeromonas and indicator bacteria remained strong in the Buffalo River during the winter, signifying that different conditions exist in the Buffalo River and its upstream tributaries. The strong correlation between Aeromonas spp. and indicator bacteria in the Buffalo River suggest that, in the absence of media capable of the quantitative recovery of potentially pathogenic aeromonads, standard faecal coliform analyses may adequately assess public health risks from Aeromonas spp. in an urban river used for recreational purposes.
To identify a group of eight Aeromonas strains of our collection showing ribotyping patterns similar to those described for the species Aeromonas popoffii, 16S rRNA gene sequence analysis was performed. Results were in agreement with the DNA binding values, and allowed the identification of a 'signature region' differentiating the A. popoffii strains from all other members of the genus Aeromonas.
Microbiological surveys, to determine the quality and safety, were conducted on 45 sorghum samples comprising dry powders (n = 15) and corresponding fermented (n = 15) and cooked fermented porridge (n = 15) samples collected from households in an informal settlement of the Gauteng Province of South Africa. Mean aerobic plate counts, Gram-negative counts and bacterial spore counts of sorghum powder samples decreased in fermented and cooked fermented porridge samples. However, mean lactic acid bacteria counts increased in fermented porridge samples, but decreased slightly in cooked fermented porridge samples. The mean pH value of sorghum powder samples decreased in fermented and cooked fermented porridge, respectively. Bacillus (B.) cereus was detected in all 15 sorghum powder samples, while Escherichia (E.) coli was detected in 53%, Clostridium perfringens in 27%, Listeria monocytogenes in 13% and Aeromonas spp., Salmonella spp., Staphylococcus aureus, Shigella spp. and Yersinia spp., each in 7% of sorghum powder samples. Of the fermented porridge samples, 40% contained B. cereus and 7% contained E. coli. None of the pathogens tested for were detected in cooked fermented porridge samples. B. cereus (53%), B. subtilis (21%), B. thuringiensis (13%), B. licheniformis (10%) and B. coagulans (3%) were identified from 120 isolates randomly selected from spore count plates of the highest dilution showing growth.
The 16S rDNA sequences of the recently described Aeromonas encheleia and Aeromonas popoffii, were determined and compared with data from all known Aeromonas sp. Diagnostic 16S rDNA regions were also sequenced for some strains previously considered as an extension of A. encheleia and a strain of Aeromonas Group 501 (formerly Enteric Group 501). Results indicated that A. encheleia and A. popoffii are phylogenetically separated species as originally described. A conclusion about HG11 taxonomic status is not recommended until previous discrepancies are clarified by further DNA-DNA hybridization and sequencing studies.
More than 900 culturable, heterotrophic aerobic isolates were obtained from the sediments of a forested, pristine stream and analyzed using three classical microbiological tests: API 20E, amplified ribosomal DNA restriction analysis (ARDRA), and fatty acid analysis. Gram-negative bacteria comprised most of the heterotrophic aerobic isolates (66.7%), similar to other oligotrophic environments. The isolates were assigned to the genus level as Pseudomonas, Flavobacterium, Micrococcus, Bacillus, Chromobacterium, Acinetobacter, Alcaligenes, Aeromonas, Methylobacterium, Enterobacter, Corynebacterium, and Sporolactobacillus. Genotypic analysis by ARDRA facilitated the comparison among strains within Pseudomonas, Bacillus, and Enterobacter groups. Temperature and predation may influence the survival of bacteria during seasons, as shown previously by others. Our results showed that the number of heterotrophic aerobic bacteria, especially Enterobacter, Alcaligenes, and Aeromonas, and Gram-positive bacteria, decreased in winter compared to summer conditions.
Aeromonas bacteraemia is not a common infectious disease, but can cause a grave outcome in infected cases. In this study, clinical presentations and prognostic factors of cases of monomicrobial Aeromonas bacteraemia were analysed. Also, the impact of beta-lactam and aminoglycoside in combination and of emerging cephalosporin-resistance during therapy was discussed.
From 1989 to 1998 in a medical centre in southern Taiwan, those cases with monomicrobial Aeromonas bacteraemia were included for study.
A total of 104 episodes of monomicrobial Aeromonas bacteraemia, accounting for 74% of all Aeromonas bacteraemia, were encountered. The infections usually occurred in the patients with hepatic cirrhosis (54%) or malignancy (21%) and were community-acquired (74%). Cases of community-acquired bacteraemia were more likely to have cirrhosis, a high severity score at onset, and a worse prognosis than those of nosocomial bacteraemia did and nosocomial isolates were less susceptible to cefoxitin and cefotaxime. Forty-three percent of cases had a concomitant infection focus, such as primary peritonitis, invasive cellulitis or necrotizing fasciitis, biliary tract or burn wound infections. Crude fatality rate within 2 weeks after the onset was 30%. Secondary bacteraemia and a higher severity score ( > or = 4) for illness at the first presentation were independently associated with a fatal outcome. The therapeutic superiority of beta-lactam and aminoglycoside in combination cannot be demonstrated in patients with Aeromonas bacteraemia. Cefotaxime resistance emerged in 3.4% of 58 patients treated with a cephalosporin for at least 72 h. None of the community-acquired isolates, but one-quarter of the nosocomial isolates, were resistant to cefotaxime.
Aeromonas bacteraemia usually occurred in patients with liver cirrhosis or malignancy, and heralded a poor prognosis, especially while associated with a relevant infectious source or with a higher severity score at presentation. The superiority of aminoglycoside and beta-lactam in combination cannot be demonstrated while treating those patients, and the emergence of antimicrobial resistance to cephalosporin was a rare event during cephalosporin therapy. Thus, a broad-spectrum cephalosporin remains one of the antimicrobial alternatives for invasive community-acquired Aeromonas infections.
To study the phenotypic and chemotaxonomic (i.e. phospholipid and cellular fatty acid composition) characteristics of environmental Aeromonas spp. and Vibrio spp. isolated from a drinking water reservoir near Vladivostok City, and the application of some chemotaxonomic markers for discrimination of the two genera and species.
Presumptive Aeromonas species were dominant in surface water samples (up to 25% of the total number of bacteria recovered). These strains were consistent with respect to the cultural and biochemical properties used to define the species Aeromonas sobria (seven strains) and Aer. popoffii (three strains). Vibrio mimicus (two strains) and Vibrio metschnikovii (one strain) were identified according to phenotypic features and cellular fatty acid composition.
Environmental Aer. sobria isolates were atypical in their ability to grow at 42 degrees C, and were haemolytic, proteolytic and cytotoxic. Although it was present in a high proportion in the water samples, atypical Aer. sobria is not an indicator of polluted water.
The incidence of Aeromonas in the drinking water reservoirs in the Far East of Russia is reported for the first time.
Environmental Impacts of inland based trout-farms on the water quality of a stream
- J.-C Cho
- S.-H Lee
- S.-J Park
- S.-J Kim
Cho, J.-C., Lee, S.-H., Park, S.-J. and Kim, S.-J. (1995) Environmental Impacts of inland based trout-farms on the water quality of a
stream. Korean Journal of Sanitation 10, 56-66.
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MIDI (1999). Microbial Identification System Operating Manual,
Version 3.0, September, 1999. Newark, DE, USA: MIDI Inc.
Microbial Identification System Operating Manual
- Midi
MIDI (1999). Microbial Identification System Operating Manual,
Version 3.0, September, 1999. Newark, DE, USA: MIDI Inc.
An epidemic of food poisoning by Aeromonas hydrophila
- L Zeng-Shan
- C Guilian
- F Shumei
Zeng-Shan, L., Guilian, C. and Shumei, F. (1988) An epidemic of food
poisoning by Aeromonas hydrophila. Chinese Journal of Preventive
Medicine 22, 333-334.
- I D E N T I F I C A T I O N A N D D I S T R I B U T I O N O F A E R O M O N A S
I D E N T I F I C A T I O N A N D D I S T R I B U T I O N O F A E R O M O N A S 985
ª 2002 The Society for Applied Microbiology, Journal of Applied Microbiology, 93, 976-985