Project description:In the absence of pipe-borne water, many people in Africa, especially in rural communities, depend on alternative water sources such as wells, boreholes and rivers for household and personal hygiene. Poor maintenance and nearby pit latrines, however, lead to microbial pollution of these sources. We evaluated the abundance of <i>Escherichia coli</i> and the prevalence of pathogenic <i>E. coli</i> virulence genes in water from wells, boreholes and a river in a South African peri-urban community. Monthly samples were collected between August 2015 and November 2016. In all, 144 water samples were analysed for <i>E. coli</i> using the Colilert 18 system. Virulence genes (<i>eagg</i>, <i>eaeA</i>, <i>stx1</i>, <i>stx2</i>, <i>flichH7</i>, <i>ST</i>, <i>ipaH</i>, <i>ibeA</i>) were investigated using real-time polymerase chain reaction. Mean <i>E. coli</i> counts ranged between 0 and 443.1 Most Probable Number (MPN)/100 mL of water sample. Overall, 99.3% of samples were positive for at least one virulence gene studied, with <i>flicH7</i> being the most detected gene (81/140; 57.6%) and the <i>stx2</i> gene the least detected gene (8/140; 5.7%). Both intestinal and extraintestinal pathogenic <i>E. coli</i> genes were detected. The detection of virulence genes in these water sources suggests the presence of potentially pathogenic <i>E. coli</i> strains and is a public health concern.
Project description:The Little Bighorn River is the primary source of water for water treatment plants serving the local Crow Agency population, and has special significance in the spiritual and ceremonial life of the Crow tribe. Unfortunately, the watershed suffers from impaired water quality, with high counts of fecal coliform bacteria routinely measured during run-off events. A metagenomic analysis was carried out to identify potential pathogens in the river water. The Oxford Nanopore MinION platform was used to sequence DNA in near real time to identify both uncultured and a coliform-enriched culture of microbes collected from a popular summer swimming area of the Little Bighorn River. Sequences were analyzed using CosmosID bioinformatics and, in agreement with previous studies, enterohemorrhagic and enteropathogenic Escherichia coli and other E. coli pathotypes were identified. Noteworthy was detection and identification of enteroaggregative E. coli O104:H4 and Vibrio cholerae serotype O1 El Tor, however, cholera toxin genes were not identified. Other pathogenic microbes, as well as virulence genes and antimicrobial resistance markers, were also identified and characterized by metagenomic analyses. It is concluded that metagenomics provides a useful and potentially routine tool for identifying in an in-depth manner microbial contamination of waterways and, thereby, protecting public health.
Project description:The aim of this study was to investigate the diversity of the Escherichia coli population, focusing on the occurrence of pathogenic E. coli, in surface water draining a rural catchment. Two sampling campaigns were carried out in similar hydrological conditions (wet period, low flow) along a river continuum, characterized by two opposite density gradients of animals (cattle and wild animals) and human populations. While the abundance of E. coli slightly increased along the river continuum, the abundance of both human and ruminant-associated Bacteroidales markers, as well as the number of E. coli multi-resistant to antibiotics, evidenced a fecal contamination originating from animals at upstream rural sites, and from humans at downstream urban sites. A strong spatial modification of the structure of the E. coli population was observed. At the upstream site close to a forest, a higher abundance of the B2 phylogroup and Escherichia clade strains were observed. At the pasture upstream site, a greater proportion of both E and B1 phylogroups was detected, therefore suggesting a fecal contamination of mainly bovine origin. Conversely, in downstream urban sites, A, D, and F phylogroups were more abundant. To assess the occurrence of intestinal pathogenic strains, virulence factors [afaD, stx1, stx2, eltB (LT), estA (ST), ipaH, bfpA, eae, aaiC and aatA] were screened among 651 E. coli isolates. Intestinal pathogenic strains STEC O174:H21 (stx2) and EHEC O26:H11 (eae, stx1) were isolated in water and sediments close to the pasture site. In contrast, in the downstream urban site aEPEC/EAEC and DAEC of human origin, as well as extra-intestinal pathogenic E. coli belonging to clonal group A of D phylogroup, were sampled. Even if the estimated input of STEC (Shiga toxin-producing E. coli) - released in water at the upstream pasture site - at the downstream site was low, we show that STEC could persist in sediment. These results show that, the run-off of small cattle farms contributed, as much as the wastewater effluent, in the dissemination of pathogenic E. coli in both water and sediments, even if the microbiological quality of the water was good or to average quality according to the French water index.
Project description:Hybridization with oligonucleotide microchips (microarrays) was used for discrimination among strains of Escherichia coli and other pathogenic enteric bacteria harboring various virulence factors. Oligonucleotide microchips are miniature arrays of gene-specific oligonucleotide probes immobilized on a glass surface. The combination of this technique with the amplification of genetic material by PCR is a powerful tool for the detection of and simultaneous discrimination among food-borne human pathogens. The presence of six genes (eaeA, slt-I, slt-II, fliC, rfbE, and ipaH) encoding bacterial antigenic determinants and virulence factors of bacterial strains was monitored by multiplex PCR followed by hybridization of the denatured PCR product to the gene-specific oligonucleotides on the microchip. The assay was able to detect these virulence factors in 15 Salmonella, Shigella, and E. coli strains. The results of the chip analysis were confirmed by hybridization of radiolabeled gene-specific probes to genomic DNA from bacterial colonies. In contrast, gel electrophoretic analysis of the multiplex PCR products used for the microarray analysis produced ambiguous results due to the presence of unexpected and uncharacterized bands. Our results suggest that microarray analysis of microbial virulence factors might be very useful for automated identification and characterization of bacterial pathogens.
Project description:The detection and abundance of Escherichia coli in water is used to monitor and mandate the quality of drinking and recreational water. Distinguishing commensal waterborne E. coli isolates from those that cause diarrhea or extraintestinal disease in humans is important for quantifying human health risk. A DNA microarray was used to evaluate the distribution of virulence genes in 148 E. coli environmental isolates from a watershed in eastern Ontario, Canada, and in eight clinical isolates. Their pathogenic potential was evaluated with Caenorhabditis elegans, and the concordance between the bioassay result and the pathotype deduced by genotyping was explored. Isolates identified as potentially pathogenic on the basis of their complement of virulence genes were significantly more likely to be pathogenic to C. elegans than those determined to be potentially nonpathogenic. A number of isolates that were identified as nonpathogenic on the basis of genotyping were pathogenic in the infection assay, suggesting that genotyping did not capture all potentially pathogenic types. The detection of the adhesin-encoding genes sfaD, focA, and focG, which encode adhesins; of iroN2, which encodes a siderophore receptor; of pic, which encodes an autotransporter protein; and of b1432, which encodes a putative transposase, was significantly associated with pathogenicity in the infection assay. Overall, E. coli isolates predicted to be pathogenic on the basis of genotyping were indeed so in the C. elegans infection assay. Furthermore, the detection of C. elegans-infective environmental isolates predicted to be nonpathogenic on the basis of genotyping suggests that there are hitherto-unrecognized virulence factors or combinations thereof that are important in the establishment of infection.
Project description:In Egypt as in many other countries, river water buffalo (Bubalus bubalis) is considered an important source of high-quality milk and meat supply. The objective of this study was to investigate serotypes, virulence genes, and antibiotic resistance determinants profiles of Escherichia coli isolated from buffalo at some places in Egypt; noticibly, this issue was not discussed in the country yet.A number of 58 rectal samples were collected from diarrheic buffalo calves in different regions in Egypt, and bacteriological investigated for E. coli existence. The E. coli isolates were biochemically, serologicaly identified, tested for antibiotic susceptibility, and polymerase chain reaction (PCR) analyzed for the presence of antibiotic resistance determinants and virulence genes.Overall 14 isolates typed as E. coli (24.1%); 6 were belonged to serogroup O78 (10.3%), followed by O125 (4 isolates, 6.9%), then O158 (3 isolates, 5.2%) and one isolate O8 (1.7%), among them, there were 5 E. coli isolates showed a picture of hemolysis (35.7%). The isolates exhibited a high resistance to β lactams over 60%, followed by sulfa (50%) and aminoglucoside (42.8%) group, in the same time the isolates were sensitive to quinolone, trimethoprim-sulfamethoxazole, tetracycline (100%), and cephalosporine groups (71.4%). A multiplex PCR was applied to the 14 E. coli isolates revealed that all were carrying at least one gene, as 10 carried blaTEM (71.4%), 8 Sul1 (57.1%), and 6 aadB (42.8%), and 9 isolates could be considered multidrug resistant (MDR) by an incidence of 64.3%. A PCR survey was stratified for the most important E. coli virulence genes, and showed the presence of Shiga toxins in 9 isolates carried either one or the two Stx genes (64.3%), 5 isolates carried hylA gene (35.7%), and eae in 2 isolates only (14.3%), all isolates carried at least one virulence gene except two (85.7%).The obtained data displayed that in Egypt, buffalo as well as other ruminants could be a potential source of MDR pathogenic E. coli variants which have a public health importance.
Project description:During a 2-year study, the presence of human pathogenic bacteria and noroviruses was investigated in shellfish, seawater and/or surface sediments collected from three French coastal shellfish-harvesting areas as well as in freshwaters from the corresponding upstream catchments. Bacteria isolated from these samples were further analyzed. Escherichia coli isolates classified into the phylogenetic groups B2, or D and enterococci from Enterococcus faecalis and E. faecium species were tested for the presence of virulence genes and for antimicrobial susceptibility. Salmonella members were serotyped and the most abundant serovars (Typhimurium and its monophasic variants and Mbandaka) were genetically characterized by high discriminative subtyping methods. Campylobacter and Vibrio were identified at the species level, and haemolysin-producing Vibrio parahaemolyticus were searched by tdh- and trh- gene detection. Main results showed a low prevalence of Salmonella in shellfish samples where only members of S. Mbandaka were found. Campylobacter were more frequently isolated than Salmonella and a different distribution of Campylobacter species was observed in shellfish compared to rivers, strongly suggesting possible additional inputs of bacteria. Statistical associations between enteric bacteria, human noroviruses (HuNoVs) and concentration of fecal indicator bacteria revealed that the presence of Salmonella was correlated with that of Campylobacter jejuni and/or C. coli as well as to E. coli concentration. A positive correlation was also found between the presence of C. lari and the detection of HuNoVs. This study highlights the importance of simultaneous detection and characterization of enteric and marine pathogenic bacteria and human noroviruses not only in shellfish but also in catchment waters for a hazard assessment associated with microbial contamination of shellfish.
Project description:Introduction: Community-acquired infections due to extended-spectrum beta-lactamase (ESBL) producing Escherichia coli are rising worldwide, resulting in increased morbidity, mortality, and healthcare costs, especially where poor sanitation and inadequate hygienic practices are very common. Objective: This study was conducted to investigate the prevalence and characterization of multidrug-resistant (MDR) and ESBL-producing E. coli in drinking water samples collected from Rohingya camps, Bangladesh. Methods: A total of 384 E. coli isolates were analyzed in this study, of which 203 were from household or point-of-use (POU) water samples, and 181 were from source water samples. The isolates were tested for virulence genes, ESBL-producing genes, antimicrobial susceptibility by VITEK 2 assay, plasmid profiling, and conjugal transfer of AMR genes. Results: Of the 384 E. coli isolates tested, 17% (66/384) were found to be ESBL producers. The abundance of ESBL-producers in source water contaminated with E. coli was observed to be 14% (27/181), whereas, 19% (39/203) ESBL producers was found in household POU water samples contaminated with E. coli. We detected 71% (47/66) ESBL-E. coli to be MDR. Among these 47 MDR isolates, 20 were resistant to three classes, and 27 were resistant to four different classes of antibiotics. Sixty-four percent (42/66) of the ESBL producing E. coli carried 1 to 7 plasmids ranging from 1 to 103 MDa. Only large plasmids with antibiotic resistance properties were found transferrable via conjugation. Moreover, around 7% (29/384) of E. coli isolates harbored at least one of 10 virulence factors belonging to different E. coli pathotypes. Conclusions: The findings of this study suggest that the drinking water samples analyzed herein could serve as an important source for exposure and dissemination of MDR, ESBL-producing and pathogenic E. coli lineages, which therewith pose a health risk to the displaced Rohingya people residing in the densely populated camps of Bangladesh.
Project description:BACKGROUND:Access to safe water for drinking and domestic activities remains a challenge in emerging economies like South Africa, forcing resource-limited communities to use microbiologically polluted river water for personal and household purposes, posing a public health risk. This study quantified bacterial contamination and the potential health hazards that wastewater treatment plant (WWTP) workers and communities may face after exposure to waterborne pathogenic bacteria in a WWTP and its associated surface water, respectively. RESULTS:Escherichia coli (Colilert®-18/ Quanti-Tray® 2000) and enterococci (Enterolert®/ Quanti-Tray® 2000) were quantified and definitively identified by real-time polymerase chain reaction targeting the uidA and tuf genes, respectively. An approximate beta-Poisson dose-response model was used to estimate the probability of infection (Pi) with pathogenic E. coli. Mean E. coli concentration ranged from 2.60E+?02/100?mL to 4.84E+?06/100?mL; enterococci ranged from 2.60E+?02/100?mL to 3.19E+?06/100?mL across all sampled sites. Of the 580 E. coli isolates obtained from this study, 89.1% were intestinal, and 7.6% were extraintestinal pathogenic E. coli. The 579 enterococci obtained were 50.4% E. faecalis (50.4%), 31.4% E. faecium, 3.5%, E. casseliflavus and 0.7% E. gallinarum. The community health risk stemming from the use of the water for recreational and domestic purposes revealed a greater health risk (Pi) from the ingestion of 1?mL of river water from upstream (range, 55.1-92.9%) than downstream (range, 26.8-65.3%) sites. The occupational risk of infection with pathogenic E. coli for workers resulting from a once-off unintentional consumption of 1?mL of water was 0% (effluent) and 23.8% (raw influent). Multiple weekly exposures of 1?mL over a year could result in a Pi of 1.2 and 100% for the effluent and influent, respectively. CONCLUSION:Our findings reveal that there is a potentially high risk of infection for WWTP workers and communities that use river water upstream and downstream of the investigated WWTP.
Project description:The detection of virulence determinants harbored by pathogenic Escherichia coli is important for establishing the pathotype responsible for infection. A sensitive and specific miniaturized virulence microarray containing 60 oligonucleotide probes was developed. It detected six E. coli pathotypes and will be suitable in the future for high-throughput use.