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Aggregative phenotype of Shiga toxin–producing Escherichia coli cells grown to stationary phase in LB-half salt broth glass tubes at 28°C for 24 h in a rotary shaker. Top panel, left to right: Photograph of cultures of environmental (RM9131), sporadic illness (M10001248), outbreak (SD134209), and enteroaggregative Escherichia coli (EAEC) (EAEC 042) (Chaudhuri et al., 2010) strains. Note the aggregates at the bottom of the tube (arrowheads) and the resulting clear suspension in cultures of strain RM9131 and SD134209, whereas cells of strain M10001248 and EAEC 042 remain in suspension. Long arrows point to biofilm rings that formed during rotary agitation at the liquid–air interface. Lower panel: Phase-contrast images of the cells at the bottom of each culture tube are shown immediately above in the top panel. Large dense clusters of cells are visible for strain RM9131 and SD134209, but only nonaggregated individual cells are present for strain M10001248 and EAEC 042. (Magnification, 40 · ).
Source publication
Escherichia coli serogroup O111 is among the six most commonly reported non-O157:H7 Shiga toxin-producing E. coli (STEC), which are emerging as important foodborne pathogens. We have assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of...
Contexts in source publication
Context 1
... were observed at the bottom of LB-HS cultures of a subset of O111 strains when incubated to a stationary phase of growth in glass tubes with agitation (Fig. 2, upper panel). Over time during incubation, the planktonic cells were depleted in the cultures and the number and size of aggregates increased. The formation of a very dense biofilm ring at the culture liquid-air interface during rotary agitation was visible. This aggregation phenotype was observed also in M9 glucose minimal medium (data not shown). ...
Context 2
... genomic DNA patterns. Genotypic relatedness and percent similarity are shown on the left side. The number of strains of environmental, sporadic clinical, and outbreak origin are marked in black, light gray, and dark gray text, respectively. Pulsogroups based on at least 90% similarity are delineated with brackets on the right. culture conditions (Fig. 2, upper panel) but did form a biofilm coating the inner surface of the tube (not shown). Preliminary data indicate that the EAEC pAA-encoded aggregative deter- minants were not present in two of the aggregative O111 strains that we sequenced (unpublished data). Observation of the O111 aggregates by phase contrast microscopy revealed the presence of ...
Context 3
... of the tube (not shown). Preliminary data indicate that the EAEC pAA-encoded aggregative deter- minants were not present in two of the aggregative O111 strains that we sequenced (unpublished data). Observation of the O111 aggregates by phase contrast microscopy revealed the presence of numerous large and dense cell clusters of various sizes (Fig. 2, lower panel), whereas only single cells were detected in the nonaggregative strains. All O111 environmental and outbreak strains in our study (24/38 strains) had the ability to aggregate, whereas none of the strains causing sporadic illness displayed this unique behavior (Table 3). Aggregation did not correlate with H-type (Tables 2 and ...
Similar publications
An important challenge relating to clinical diagnostics of the foodborne pathogen Shiga toxin-producing E. coli (STEC), is that PCR-detection of the shiga-toxin gene (stx) in DNA from stool samples can be accompanied by a failure to identify an STEC isolate in pure culture on agar. In this study, we have explored the use of MinION long-read sequenc...
Citations
... Like Ag43, other types of adhesins, such as the Va-autotransported adhesins like TibA or AIDA-I (self-associating autotransporters [SAATs]) (65), trimeric autotransporters (TAAs), conjugation pili (66), or fimbriae, including curli or type IV pilus, can mediate aggregate formation through homotypic interactions and thus participate in kin recognition (67)(68)(69)(70)(71)(72)(73)(74)(75). Although all these cell surface appendages participate in kin recognition, they might trigger different transcriptional responses upon aggregation. ...
Bacteria can rapidly tune their physiology and metabolism to adapt to environmental fluctuations. In particular, they can adapt their lifestyle to the close proximity of other bacteria or the presence of different surfaces. However, whether these interactions trigger transcriptomic responses is poorly understood. We used a specific setup of E. coli strains expressing native or synthetic adhesins mediating bacterial aggregation to study the transcriptomic changes of aggregated compared to nonaggregated bacteria. Our results show that, following aggregation, bacteria exhibit a core response independent of the adhesin type, with differential expression of 56.9% of the coding genome, including genes involved in stress response and anaerobic lifestyle. Moreover, when aggregates were formed via a naturally expressed E. coli adhesin (antigen 43), the transcriptomic response of the bacteria was more exaggerated than that of aggregates formed via a synthetic adhesin. This suggests that the response to aggregation induced by native E. coli adhesins could have been finely tuned during bacterial evolution. Our study therefore provides insights into the effect of self-interaction in bacteria and allows a better understanding of why bacterial aggregates exhibit increased stress tolerance. IMPORTANCE The formation of bacterial aggregates has an important role in both clinical and ecological contexts. Although these structures have been previously shown to be more resistant to stressful conditions, the genetic basis of this stress tolerance associated with the aggregate lifestyle is poorly understood. Surface sensing mediated by different adhesins can result in various changes in bacterial physiology. However, whether adhesin-adhesin interactions, as well as the type of adhesin mediating aggregation, affect bacterial cell physiology is unknown. By sequencing the transcriptomes of aggregated and nonaggregated cells expressing native or synthetic adhesins, we characterized the effects of aggregation and adhesin type on E. coli physiology.
... In our study, we found only one strain belonging to the addressed serogroups, corresponding to an O111 strain isolated from a pig. The detection of O111 strains in cattle and swine feces, as well as their association with severe illness in humans, is not surprising and was previously reported [70][71][72][73]. However, O111 STEC strains isolated from humans with moderate to severe illness are mainly associated with the presence of stx1 type, together with eae and ehxA genes [72][73][74][75]. ...
... The detection of O111 strains in cattle and swine feces, as well as their association with severe illness in humans, is not surprising and was previously reported [70][71][72][73]. However, O111 STEC strains isolated from humans with moderate to severe illness are mainly associated with the presence of stx1 type, together with eae and ehxA genes [72][73][74][75]. ...
Shiga toxin-producing Escherichia coli (STEC) is a zoonotic pathogen that causes severe illness in humans and is an important cause of foodborne disease. In Chile, there is limited information on the virulence characteristics of this pathogen in livestock, and none in companion animals. The aim of this study was to characterize STEC strains isolated from cattle, swine, dogs, and cats, in Chile, in terms of the presence of Shiga toxin types and subtypes, virulence genes, serogroups, and clonality. One-thousand two-hundred samples were collected, isolating 54 strains (4.5%), where stx1a (68.5%) and ehxA (74.1%) were the most frequently detected virulence genes. Only one strain belonging to the most clinically relevant serogroups was identified. Pulsed field gel electrophoresis analysis showed high clonal diversity among strains isolated from cattle, while those from swine showed the same pattern. This study provides further evidence regarding cattle and swine in Chile as a potential source of a wide variety of STEC strains that could potentially cause severe illness in humans, and that companion animals do not seem to represent a relevant reservoir. It also argues that preventive and control strategies should not be focused on detecting serogroups, but instead, on detecting their determinants of virulence.
... The Enterobacteriaceae family represents the main bacterial species involved with food contamination, mainly in products of animal origin. In this family, the Shiga-toxin producing Escherichia coli (STEC) have had several large-scale outbreaks cases (Diodati et al. 2015;Jenkins et al. 2015;Launders et al. 2016;Sinclair et al. 2017). The pathogenesis of the strains present in this bacterial group is due to the acquisition of the virulence gene of the bacterium Shigella dysenteriae named stx1 and stx2, with stx2 toxin being responsible for the most severe cases of foodborne diseases (Ogura et al. 2015;Castro et al. 2017). ...
Aims:
First, two inactivation models were compared for different phenotypic profiles of Escherichia coli O26 using UV-C light (UV-C) and thermal treatment (T), by means of Central Composite Rotatable Design of Experiment (CCRD). Second, we aimed to evaluate subsequent survival and persistence of cells in simulated gastric fluid (SGF).
Methods and results:
Two strains of E. coli O26, a wild-type strain and a clinical ATCC strain were used in both steps. A CCRD was used in a 22 arrangement in random order. The goodness-of-fit of the models were determined. The lack of Fit, and the normality of residual data were checked with the Shapiro-Wilk's test, and the model accuracy factor, bias factor, and the model mean square error (MSE) were measured. Subsequently, the resistance capacity of the strains was evaluated after exposure to simulated gastric acid. The CCRD results obtained indicate that the mild heat (< 70 °C) has a recovery effect. In addition, for the clinical strain, the UV-C and heat (above 70 °C) has an additive inactivation effect. Moreover, temperature (65 °C) induced SGF resistance by the wild and clinical strain. For the clinical strain, cells exposed to UV-C were more sensitive to SGF. In contrast to clinical strain, exposing cells of the wild strain to UV-C increased the survival capacity in the SGF.
Conclusion:
Response surface analyses showed that the wild-type O26 strain has higher persistence under unfavorable conditions than the clinical strain, and the stresses caused by applied microbial control technologies can increase the survival capacity in the SGF.
Significance and impact:
The present study shed light on different phenotypic responses in the same bacterium serogroup. Moreover, the impact of the study was that strain selection criteria must be adequate to develop effective models of inactivation. This article is protected by copyright. All rights reserved.
... Strain SD134209 was isolated from a patient with bloody diarrhea during an outbreak at a daycare facility in South Dakota (10). These three strains exhibited a strong aggregative phenotype characterized by the settlement of large bacterial assemblages in broth culture with agitation, a behavior commonly exhibited by environmental and outbreak STEC O111 isolates (11). ...
Non-O157 Shiga toxin-producing Escherichia coli (STEC) strains are a common source of foodborne illness. STEC O111 is among the most prevalent non-O157 STEC serogroups. Few completed genomes of STEC O111 strains have been reported to date. We report here the complete genomic sequences of three O111:H8 strains that display a distinct aggregation phenotype.
... Inactivation of RcsB or RpoS in E. coli O157:H7 was suggested to be beneficial to the bacterial population as a whole, since two subpopulations with an array of distinct phenotypes emerged. Remarkably, the polymorphic nature of autoaggregation observed in a collection of STEC O111 strains was attributed to loss-of-function mutations in RpoS (29,48), suggesting that adaptive mutation is a common mechanism in STEC to fine-tune its colonization and survival under changing habitat conditions. ...
Cah is a calcium-binding autotransporter protein involved in autoaggregation and biofilm formation. Although cah is widespread in Shiga toxin-producing Escherichia coli (STEC), we detected mutations in cah at a frequency of 31.3% in this pathogen. In STEC O157:H7 supershedder strain SS17, a large deletion results in a smaller coding sequence, encoding a protein lacking the C-terminal 71 amino acids compared with Cah in STEC O157:H7 strain EDL933. We examined the function of Cah in biofilm formation and host colonization to better understand the selective pressures for cah mutations. EDL933-Cah played a conditional role in biofilm formation in vitro: it enhanced E. coli DH5α biofilm formation on glass surfaces under agitated culture conditions that prevented autoaggregation but inhibited biofilm formation under hydrostatic conditions that facilitated autoaggregation. This function appeared to be strain dependent since Cah-mediated biofilm formation was diminished when an EDL933 cah gene was expressed in SS17. Deletion of cah in EDL933 enhanced bacterial attachment to spinach leaves and altered the adherence pattern of EDL933 to bovine recto-anal junction squamous epithelial (RSE) cells. In contrast, in trans expression of EDL933 cah in SS17 increased its attachment to leaf surfaces, and in DH5α, it enhanced its adherence to RSE cells. Hence, the ecological function of Cah appears to be modulated by environmental conditions and other bacterial strain-specific properties. Considering the prevalence of cah in STEC and its role in attachment and biofilm formation, cah mutations might be selected in ecological niches in which inactivation of Cah would result in an increased fitness in STEC during colonization of plants or animal hosts.
IMPORTANCE
Shiga toxin-producing Escherichia coli (STEC) harbors genes encoding diverse adhesins, and many of these are known to play an important role in bacterial attachment and host colonization. We demonstrated here that the autotransporter protein Cah confers on E. coli DH5α cells a strong autoaggregative phenotype that is inversely correlated with its ability to form biofilms and plays a strain-specific role in plant and animal colonization by STEC. Although cah is widespread in the STEC population, we detected a mutation rate of 31.3% in cah, which is similar to that reported for rpoS and fimH. The formation of cell aggregates due to increased bacterium-to-bacterium interactions may be disadvantageous to bacterial populations under conditions that favor a planktonic state in STEC. Therefore, a loss-of-function mutation in cah is likely a selective trait in STEC when autoaggregative properties become detrimental to bacterial cells and may contribute to the adaptability of STEC to fluctuating environments.
... Our findings agree with previous research that determined that E. coli does not express curli in static broth (LBG) or agar plates at 37 • C ( Barnhart and Chapman, 2006). The flocculation phenotype described in our work has been seen previously under environmental stress, including LB supplemented with NaCl, for the Shigatoxin producing E. coli O111 serogroup (Diodati et al., 2015). ...
Shiga toxin producing Escherichia coli (STEC) strains vary in acid resistance; however, little is known about the underlying mechanisms that result in strain specific differences. Among 25 STEC O157:H7 strains tested, 7 strains flocculated when grown statically for 18 h in minimal salts medium at 37°C, while 18 strains did not. Interestingly, the flocculation phenotype (cells came out of suspension) was found to correlate with degree of acid sensitivity in an assay with 400 mM acetic acid solution at pH 3.3 targeting acidified foods. Strains exhibiting flocculation were more acid sensitive and were designated FAS, for flocculation acid sensitive, while the acid resistant strain designated PAR for planktonic acid resistant. Flocculation was not observed for any strains during growth in complex medium (Luria Bertani broth). STEC strains B201 and B241 were chosen as representative FAS (2.4 log reduction) and PAR (0.15 log reduction) strains, respectively, due to differences in acid resistance and flocculation phenotype. Results from electron microscopy showed evidence of fimbriae production in B201, whereas fimbriae were not observed in B241.Curli fimbriae production was identified through plating on Congo red differential medium, and all FAS strains showed curli fimbriae production. Surprisingly, 5 PAR strains also had evidence of curli production. Transcriptomic and targeted gene expression data for B201 and B241indicated that csg and hde (curli and acid induced chaperone genes, respectively) expression positively correlated with the phenotypic differences observed for these strains. These data suggest that FAS strains grown in minimal medium express curli, resulting in a flocculation phenotype. This may be regulated by GcvB, which positively regulates curli fimbriae production and represses acid chaperone proteins. RpoS and other regulatory mechanisms may impact curli fimbriae production, as well. These findings may help elucidate mechanisms underlying differences among STEC strains in relating acid resistance and biofilm formation.
... Ultimately, there are no comprehensive prospective studies directly linking microbial contamination of fresh produce to that of hands, soil, water, or surfaces in the natural setting of the agricultural production environment. This may be due to logistical constraints in setting up a matched study design (6,17), resource constraints in acquiring large sample sizes (18), application of the appropriate statistical analyses to account for multivariable factors affecting microbial contamination (6,(19)(20)(21), and data variability from diverse microbial distributions on produce and environmental samples (22)(23)(24). Because of the low rate of detection of pathogen contamination (25)(26)(27)(28), populations of microbial-indicator organisms, including organisms that indicate filth (coliforms) and/or fecal contamination (Escherichia coli, Enterococcus spp., and somatic coliphage), can be monitored (reviewed in references 29 to 31). ...
To improve food safety on farms, it is critical to quantify the impact of environmental microbial contamination sources on fresh produce. However, studies are hampered by difficulties achieving study designs with powered sample sizes to elucidate relationships between environmental and produce contamination. Our goal was to quantify, in the agricultural production environment, the relationship between microbial contamination on hands, soil, and water and contamination on fresh produce. In 11 farms and packing facilities in northern Mexico, we applied a matched study design: composite samples (n = 636, equivalent to 11,046 units) of produce rinses were matched to water, soil, and worker hand rinses during two growing seasons. Microbial indicators (coliforms, Escherichia coli, Enterococcus spp., and somatic coliphage) were quantified from composite samples. Statistical measures of association and correlations were calculated through Spearman's correlation, linear regression, and logistic regression models. The concentrations of all microbial indicators were positively correlated between produce and hands (ρ range, 0.41 to 0.75; P < 0.01). When E. coli was present on hands, the handled produce was nine times more likely to contain E. coli (P < 0.05). Similarly, when coliphage was present on hands, the handled produce was eight times more likely to contain coliphage (P < 0.05). There were relatively low concentrations of indicators in soil and water samples, and a few sporadic significant associations were observed between contamination of soil and water and contamination of produce. This methodology provides a foundation for future field studies, and results highlight the need for interventions surrounding farmworker hygiene and sanitation to reduce microbial contamination of farmworkers' hands.
... Similarly, Bai et al. [19] and Cernicchiaro et al. [16] found IMS beads for O111 often yielded colonies of O103 due to non-specific binding of antibodies, while Cooley et al. [20] concluded that using specific O-type IMS kits did not significantly improve isolation of the target serogroup. Isolates of O111 obtained in the present study were similar to environmental and outbreak strains of this serogroup surveyed by Diodati et al. [21] in that all harbored stx 1 and only a subset carried stx 2 . That all O111 colonies isolated were STEC was unexpected, but perhaps reflects the stability of the cryptic prophage carrying stx 1 associated with this serogroup [22]. ...
Pooled feces collected over two years from 1749 transport trailers hauling western-Canadian slaughter cattle were analysed by PCR for detection of Escherichia coli serogroups O26, O45, O103, O111, O121, O145, and O157. Sequential immunomagnetic separation was then used to collect bacterial isolates (n = 1035) from feces positive for target serogroups. Isolated bacteria were tested by PCR to confirm serogroup and the presence of eae, ehxA, stx1, and stx2 virulence genes. Based on PCR screening, serogroup prevalence in feces ranged from 7.0% (O145) to 94.4% (O103) with at least 3 serogroups present in 79.5% of samples. Origin of cattle affected serogroup PCR prevalence and O157 was most prevalent in feces from south-west Alberta (P < 0.001). All serogroups demonstrated seasonal variations in PCR prevalence, with O26, O45, O103, O121, and O157 least prevalent (P < 0.001) in cooler winter months, while uncommon serogroups O111 and O145 increased in prevalence during winter (P < 0.001). However, isolates collected during winter were predominantly from serogroups O103 and O45. No seasonal variation was noted in proportion of isolates which were Shiga toxin containing E. coli (STEC; P = 0.18) or positive for Shiga toxin and eae (enterohemorrhagic E. coli; EHEC; P = 0.29). Isolates of serogroups O111, O145, and O157 were more frequently EHEC than were others, although 37.6–54.3% of isolates from other serogroups were also EHEC. Shiga-toxin genes present also varied by geographic origin of cattle (P < 0.05) in all serogroups except O157. As cattle within feedlots are sourced from multiple regions, locational differences in serogroup prevalence and virulence genes imply existence of selection pressures for E. coli and their virulence in western-Canadian cattle. Factors which reduce carriage or expression of virulence genes, particularly in non-O157 serogroups, should be investigated.
... We have previously assembled a collection of environmental and clinical strains of E. coli O111 from diverse sources and investigated various genotypic and phenotypic characteristics of these strains to gain a better understanding of the epidemiology and biology of this serogroup. Our study revealed that many environmental STEC O111 strains isolated from diverse sources over time and from various geographical locations are genotypically identical, suggesting that these strains circulate in the environment and may contaminate the food chain (11). The environmental and outbreak strains in this collection were similar in their virulence determinants, and all displayed a strong aggregative phenotype characterized by the settlement of large bacterial assemblages in broth culture with agitation; in contrast, the sporadic case strains, which are strains isolated from single patients that were not part of a recognized outbreak, were nonaggregative (11). ...
... Our study revealed that many environmental STEC O111 strains isolated from diverse sources over time and from various geographical locations are genotypically identical, suggesting that these strains circulate in the environment and may contaminate the food chain (11). The environmental and outbreak strains in this collection were similar in their virulence determinants, and all displayed a strong aggregative phenotype characterized by the settlement of large bacterial assemblages in broth culture with agitation; in contrast, the sporadic case strains, which are strains isolated from single patients that were not part of a recognized outbreak, were nonaggregative (11). ...
... Given the role of aggregation in the pathogenicity and environmental fitness of E. coli and our previous observation that STEC O111 outbreak and environmental strains aggregate in culture, whereas sporadic strains are nonaggregative (11), we sought to identify the factors involved in this aggregative behavior in order to further our understanding of STEC O111 epidemiology. Because STEC O111 aggregation strongly correlated with high levels of curli fimbrial production and RpoS function in our previous study (11), we assessed the role of these fimbriae and sigma RpoSmediated regulation in the aggregation of this important enteric pathogen. ...
Escherichia coli
O111 is an emerging non-O157:H7 Shiga toxin-producing
E. coli
(STEC). We previously reported that outbreak and environmental, but not sporadic case, strains of STEC O111 share a distinct aggregation phenotype (M.E. Diodati, A.H. Bates, M.B. Cooley, S. Walker, R.E. Mandrell, and M.T. Brandl, Foodborne Pathog. Dis. 2015, 12: 235-243, doi:10.1089/fpd.2014.1887). We show here, the natural occurrence of non-aggregative variants in single STEC O111 strains. These variants do not produce curli fimbriae, lack RpoS function, but synthesize cellulose. Deletion of
csgBAC
or
rpoS
in an aggregative outbreak strain abolished aggregate formation, which was rescued when curli biogenesis or RpoS function, respectively, was restored. Complementation of a non-aggregative variant with RpoS also conferred curli production and aggregation. These observations were supported by Western blot analysis with an anti-CsgA antibody. Immunomicroscopy revealed that curli was undetectable on the cells of the non-aggregative variant and the RpoS mutant, but present in large quantities in the intercellular matrix of the assemblages formed by aggregative strains. Sequence analysis of
rpoS
in the aggregative strain and its variant showed a single substitution of threonine for asparagine at aa124. Our results indicate that the multicellular behavior of STEC O111 is RpoS-dependent via positive regulation of curli production. Aggregation may confer a fitness advantage in O111 outbreak strains under stressful conditions in hydrodynamic environments along the food production chain and in the host, while the occurrence of non-aggregative variants may allow for the cell population to adapt to conditions benefiting a planktonic life style.
... However, in the equally Stx-producing O111:HϪ serotype 11128 strain, mlrA is fully intact. Moreover, a distinct aggregative behavior was recently reported for O111 strains that was positively correlated with the production of curli fibers and RpoS function (59). Our finding that the O111:HϪ strain 11128 is a PdeR-deficient mutant, but wild-type with respect to mlrA, suggests that this strain overproduces CsgD and curli fibers in comparison to most other E. coli strains (i.e., pdeR ϩ mlrA ϩ strains). ...
... Due to a mutation in pdeR (yciR) and an intact mlrA gene, this strain can be expected to even overproduce CsgD, curli, and cellulose. Indeed, it shows curli-dependent cellular aggregation (59). With respect to Stx and high curli production this strain thus resembles the O104:H4 strains rather than classical STEC (see below). ...
Importance:
Our analysis reveals interesting trends in pathogenic E. coli that could reflect different host cell adherence mechanisms. These may either benefit from or be counteracted by the c-di-GMP-stimulated production of amyloid curli fibres and cellulose. Thus, EAEC - which adhere in a 'stacked brick' biofilm mode - have a potential for high c-di-GMP accumulation due to DgcX, a strongly expressed additional DGC. By contrast, EHEC and UPEC - which use alternative adherence mechanisms - tend to have extra PDEs, suggesting that low cellular c-di-GMP levels are crucial for these strains under some specific conditions. Overall, our study also indicates that GGDEF/EAL domain proteins evolve rapidly and thereby contribute to adaptation to host-specific and environmental niches of various types of E. coli.
