Development of a procedure for discriminating among Escherichia coli isolates from animal and human sources

Counts of Escherichia coli cells in water indicate the potential presence of pathogenic microbes of intestinal origin but give no indication of the sources of the microbial pollution. The objective of this research was to evaluate methods for differentiating E. coli isolates of livestock, wildlife, or human origin that might be used to predict the sources of fecal pollution of water. A collection of 319 E. coli isolates from the feces of cattle, poultry, swine, deer, goose, and moose, as well as from human sewage, and clinical samples was used to evaluate three methods. One method was the multiple-antibiotic-resistance (MAR) profile using 14 antibiotics. Discriminant analysis revealed that 46% of the livestock isolates, 95% of the wildlife isolates, and 55% of the human isolates were assigned to the correct source groups by the MAR method. Amplified fragment length polymorphism (AFLP) analysis, the second test, was applied to 105 of the E. coli isolates. The AFLP results showed that 94% of the livestock isolates, 97% of the wildlife isolates, and 97% of the human isolates were correctly classified. The third method was analysis of the sequences of the 16S rRNA genes of the E. coli isolates. Discriminant analysis of 105 E. coli isolates indicated that 78% of the livestock isolates, 74% of the wildlife isolates, and 80% of the human isolates could be correctly classified into their host groups by this method. The results indicate that AFLP analysis was the most effective of the three methods that were evaluated.     

Geographical variation in ribotype profiles of Escherichia coli isolates from humans,swine, poultry,beef, and dairy cattle in Florida

Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.     

Differentiation of faecal Escherichia coli from humans and animals by multiple antibiotic resistance analysis

AIMS: Multiple antibiotic resistance (MAR) was performed on 128 Escherichia coli isolates, recovered from faecal samples of humans and animals (cattle, goat, sheep) to determine and compare their antibiotic resistance patterns and to evaluate them statistically in order to specify the source of the faecal material. METHODS AND RESULTS: Disk diffusion method was applied with a selection of antibiotics. Statistical approach was performed with hierarchical cluster analysis (CA), discriminant analysis (DA) and principal component analysis. Comparing human and animal isolates there was significant difference in levels of resistance to all antibiotics tested (P<0.05) with 46 and 24 distinct resistance patterns for human and animal isolates respectively. CA and DA separated human and animal isolates with a high average rate of correct classification (99.2%), when all animal isolates were pooled together. CONCLUSIONS: MAR analysis compared with appropriate statistical evaluation may provide a useful tool for differentiating the human or animal origin of E. coli isolates derived from environmental samples. Subsequently, determination of the source of faecal pollution becomes possible. SIGNIFICANCE AND IMPACT OF THE STUDY: Determining the source of faecal pollution enables the prediction of possible risk for public health and the application of appropriate management plans for prevention of further contamination.     

Direct comparison of four bacterial source tracking methods and use of composite data sets

AIMS: Four bacterial source tracking (BST) methods, enterobacterial repetitive intergenic consensus sequence polymerase chain reaction (ERIC-PCR), automated ribotyping using HindIII, Kirby-Bauer antibiotic resistance analysis (KB-ARA) and pulsed-field gel electrophoresis (PFGE) were directly compared using the same collection of Escherichia coli isolates. The data sets from each BST method and from composite methods were compared for library accuracy and their ability to identify water isolates. METHODS AND RESULTS: Potential sources of faecal pollution were identified by watershed sanitary surveys. Domestic sewage and faecal samples from pets, cattle, avian livestock, other nonavian livestock, avian wildlife and nonavian wildlife sources were collected for isolation of E. coli. A total of 2275 E. coli isolates from 813 source samples were screened using ERIC-PCR to exclude clones and to maximize library diversity, resulting in 883 isolates from 745 samples selected for the library. The selected isolates were further analysed using automated ribotyping with HindIII, KB-ARA and PFGE. A total of 555 E. coli isolates obtained from 412 water samples were analysed by the four BST methods. A composite data set of the four BST methods gave the highest rates of correct classification (RCCs) with the fewest unidentified isolates than any single method alone. RCCs for the four-method composite data set and a seven-way split of source classes ranged from 22% for avian livestock to 83% for domestic sewage. Two-method composite data sets were also found to be better than individual methods, having RCCs similar to the four-method composite and identification of the same major sources of faecal pollution. CONCLUSIONS: The use of BST composite data sets may be more beneficial than the use of single methods. SIGNIFICANCE AND IMPACT OF THE STUDY: This is one of the first comprehensive comparisons using composite data from several BST methods. While the four-method approach provided the most desirable BST results, the use of two-method composite data sets may yield comparable BST results while providing for cost, labour and time savings.     

Association of multiple-antibiotic-resistance profiles with point and nonpoint sources of Escherichia coli in Apalachicola Bay.

A total of 765 Escherichia coli isolates from point and nonpoint sources were collected from the Apalachicola National Estuarine Research Reserve, and their multiple-antibiotic-resistance (MAR) profiles were determined with 10 antibiotics. E. coli isolates from point sources showed significantly greater resistance (P < 0.05) to antibiotics and higher MAR indices than isolates from nonpoint sources. Specifically, 65 different resistance patterns were observed among point source isolates, compared to 32 among nonpoint source isolates. Examples of this contrast in MAR profiles included percentages of isolates with resistance to chlortetracycline-sulfathiazole of 33.7% and to chlortetracycline-penicillin G-sulfathiazole of 14.5% for point source isolates versus 15.4 and 1.7%, respectively, for nonpoint source isolates. MAR profile homology, based on coefficient similarity, showed that isolates from point sources were markedly more diverse than isolates from nonpoint sources. Seven clusters were observed among point source isolates, with a coefficient value of approximately 1.8. In contrast, only four clusters were observed among nonpoint source isolates. Covariance matrices of data displayed six very distinct foci representing nonpoint source E. coli isolates. Importantly, E. coli isolates obtained directly from human and animal feces also clustered among point and nonpoint sources, respectively. We conclude that E. coli MAR profiles were associated with point and nonpoint sources of pollution within Apalachicola Bay and that this method may be useful in facilitating management of other estuaries.     

Prevalence and strain diversity of thermophilic campylobacters in cattle, sheep and swine farms

AIMS: To determine prevalence and strain diversity of thermophilic campylobacters in healthy ruminants and swine. METHODS AND RESULTS: Faecal samples collected from 343 herds (120 sheep, 124 beef cattle, 82 dairy cattle and 17 swine) in the Basque Country were screened in pools for thermophilic campylobacters. Two hundred and three herds were positive (67.1% dairy cattle, 58.9% beef cattle, 55.0% sheep and 52.9% pig), and species-specific PCR identified Campylobacter jejuni in 20.7% of the herds and Campylobacter coli in 6.4%. Campylobacter coli was isolated from the four production systems and was the most prevalent species in swine, where C. jejuni was not found. Other thermophilic campylobacters were found in all production systems. Four hundred and ninety-three animals from 11 positive herds were individually analysed, detecting significantly higher within-herd prevalences in dairy cattle (66.7%) and swine (57.8%) than in sheep (8.8%) or beef cattle (5.4%). flaA PCR-RFLP and pulsed-field gel electrophoresis analysis of a selection of isolates showed high genetic diversity. CONCLUSIONS: Healthy swine, cattle and sheep are important reservoirs of thermophilic campylobacters of different species and high genetic diversity. SIGNIFICANCE AND IMPACT OF THE STUDY: Efficient farm-based intervention measures are needed to reduce risk of infection. Non-C. jejuni/C. coli species should be monitored to investigate their significance for infection.     

Carbon source utilization profiles as a method to identify sources of faecal pollution in water

AIMS: Carbon source utilization profiles as a phenotypic fingerprinting methodology for determining sources of faecal pollution in water were evaluated. METHODS AND RESULTS: Three hundred and sixty-five Enterococcus isolates were collected from known faecal sources in four different geographical regions and were identified to species with the commercial Biolog system. Discriminant analysis (DA) was used to identify the substrate-containing wells that best classified the 365 isolates by source. By using 30 of the 95 wells for the analysis, the average rate of correct classification (ARCC) by source was 92.7% for a human vs non-human two-way classification when isolates from all regions were combined into one library. Corresponding ARCCs for other classification schemes were 81.9% for a four-way classification of human vs livestock vs wildlife vs domestic pets, and 85.7% for a three-way classification without human isolates. When three individual libraries were made based on classification of sources within Enterococcus species, the ARCC was 95.3% for the Ent. faecalis library, 95.8% for the Ent. gallinarum library and 94.7% for the Ent. mundtii library. Thirty Enterococcus isolates (unknown sources) were obtained from each of three stream sites where a specific source of pollution was apparent; 90.0% of the isolates from a human-suspected source were classified as human, 86.6% were classified as livestock from a livestock-suspected site, and 93.3% were classified as wildlife from a wildlife-suspected site. CONCLUSIONS: Phenotypic fingerprinting with carbon source utilization profiles provided levels of correct classification by sources from an Enterococcus library that were in the upper range of those reported in the literature. ARCCs for three Enterococcus species-specific libraries were very high and may be the best approach for further developing this concept and methodology. SIGNIFICANCE ANC IMPACT OF THE STUDY: The results, based on a modest Enterococcus library and a preliminary field validation test, demonstrated the potential for carbon source utilization profiles to be employed as a phenotypic method for determining sources of faecal pollution in water.     

Evaluation of antibiotic resistance analysis and ribotyping for identification of faecal pollution sources in an urban watershed

AIMS: The accuracy of ribotyping and antibiotic resistance analysis (ARA) for prediction of sources of faecal bacterial pollution in an urban southern California watershed was determined using blinded proficiency samples. METHODS AND RESULTS: Antibiotic resistance patterns and HindIII ribotypes of Escherichia coli (n = 997), and antibiotic resistance patterns of Enterococcus spp. (n = 3657) were used to construct libraries from sewage samples and from faeces of seagulls, dogs, cats, horses and humans within the watershed. The three libraries were analysed to determine the accuracy of host source prediction. The internal accuracy of the libraries (average rate of correct classification, ARCC) with six source categories was 44% for E. coli ARA, 69% for E. coli ribotyping and 48% for Enterococcus ARA. Each library's predictive ability towards isolates that were not part of the library was determined using a blinded proficiency panel of 97 E. coli and 99 Enterococcus isolates. Twenty-eight per cent (by ARA) and 27% (by ribotyping) of the E. coli proficiency isolates were assigned to the correct source category. Sixteen per cent were assigned to the same source category by both methods, and 6% were assigned to the correct category. Addition of 2480 E. coli isolates to the ARA library did not improve the ARCC or proficiency accuracy. In contrast, 45% of Enterococcus proficiency isolates were correctly identified by ARA. CONCLUSIONS: None of the methods performed well enough on the proficiency panel to be judged ready for application to environmental samples. SIGNIFICANCE AND IMPACT OF THE STUDY: Most microbial source tracking (MST) studies published have demonstrated library accuracy solely by the internal ARCC measurement. Low rates of correct classification for E. coli proficiency isolates compared with the ARCCs of the libraries indicate that testing of bacteria from samples that are not represented in the library, such as blinded proficiency samples, is necessary to accurately measure predictive ability. The library-based MST methods used in this study may not be suited for determination of the source(s) of faecal pollution in large, urban watersheds.     

Sourcing faecal pollution from onsite wastewater treatment systems in surface waters using antibiotic resistance analysis

AIMS: To identify the sources of faecal contamination in investigated surface waters and to determine the significance of onsite wastewater treatment systems (OWTS) as a major contributor to faecal contamination. METHODS AND RESULTS: Antibiotic resistance patterns (ARP) were established for a library of 717 known Escherichia coli source isolates obtained from human, domesticated animals, livestock and wild sources. Eight commonly used antibiotics, including amoxicillin, cephalothin, erythromycin, gentamicin, ofloxacin, chlortetracycline, tetracycline and moxalactam, at four different concentrations were used to obtain ARPs for E. coli isolates. Discriminant analysis (DA) was used to differentiate between the ARP of sources isolates. The developed ARP library was found to be adequate for discriminating human from nonhuman isolates, and was used to classify 256 enumerated E. coli isolates collected from monitored surface water locations. CONCLUSIONS: The resulting ARP DA indicated that a majority of the faecal contamination in more rural areas was nonhuman; however, the percentage of human isolates increased significantly in urbanized areas using OWTS for wastewater treatment. SIGNIFICANCE AND IMPACT OF THE STUDY: This study signifies the feasibility of using ARP for source tracking faecal contamination in surface waters, and linking faecal contamination to OWTS. The information will enable regulatory authorities to implement appropriate management practices to reduce the contamination of water resources caused by high densities and failing OWTS.     

Geographical Variation in Ribotype Profiles of Escherichia coli Isolates from Humans, Swine, Poultry, Beef, and Dairy Cattle in Florida

Waters impacted by fecal pollution can exact high risks to human health and can result in financial losses due to closures of water systems used for recreation and for harvesting seafood. Identifying the sources of fecal pollution in water is paramount in assessing the potential human health risks involved as well as in assessing necessary remedial action. Recently, various researchers have used the ribotyping method to identify sources of bacterial indicators (Escherichia coli and enterococci) in environmental waters. While these studies have identified genotypic differences between human- and animal-derived indicators that are capable of differentiating organisms isolated from humans and various animal hosts, most have focused on organisms collected from a confined geographic area and have not addressed the question of whether these ribotype profiles are watershed specific or if they can be applied universally to organisms from other geographic locations. In this study, E. coli isolates were obtained from humans, beef cattle, dairy cattle, swine, and poultry from locations in northern, central, and southern Florida and were subjected to ribotyping analysis. The intent was to determine (i) if ribotype profiles are capable of discriminating the source of E. coli at the host species level and (ii) if the resulting fingerprints are uniform over an extended geographic area or if they can be applied only to a specific watershed. Our research indicated that, using a single restriction enzyme (HindIII), the ribotyping procedure is not capable of differentiating E. coli isolates from the different animal species sampled in this study. Results indicate, however, that this procedure can still be used effectively to differentiate E. coli as being either human or animal derived when applied to organisms isolated from a large geographic region.     

Abundance and characteristics of the recreational water quality indicator bacteria Escherichia coli and enterococci in gull faeces

AIMS: To evaluate the numbers and selected phenotypic and genotypic characteristics of the faecal indicator bacteria Escherichia coli and enterococci in gull faeces at representative Great Lakes swimming beaches in the United States. METHODS AND RESULTS: E. coli and enterococci were enumerated in gull faeces by membrane filtration. E. coli genotypes (rep-PCR genomic profiles) and E. coli (Vitek GNI+) and enterococci (API rapid ID 32 Strep and resistance to streptomycin, gentamicin, vancomycin, tetracycline and ampicillin) phenotypes were determined for isolates obtained from gull faeces both early and late in the swimming season. Identical E. coli genotypes were obtained only from single gull faecal samples but most faecal samples yielded more than one genotype (median of eight genotypes for samples with 10 isolates). E. coli isolates from the same site that clustered at >/=85% similarity were from the same sampling date and shared phenotypic characteristics, and at this similarity level there was population overlap between the two geographically isolated beach sites. Enterococcus API(R) profiles varied with sampling date. Gull enterococci displayed wide variation in antibiotic resistance patterns, and high-level resistance to some antibiotics. CONCLUSIONS: Gull faeces could be a major contributor of E. coli (10(5)-10(9) CFU g(-1)) and enterococci (10(4)-10(8) CFU g(-)1) to Great Lakes recreational waters. E. coli and enterococci in gull faeces are highly variable with respect to their genotypic and phenotypic characteristics and may exhibit temporal or geographic trends in these features. SIGNIFICANCE AND IMPACT OF THE STUDY: The high degree of variation in genotypic or phenotypic characteristics of E. coli or enterococci populations within gull hosts will require extensive sampling for adequate characterization, and will influence methods that use these characteristics to determine faecal contamination sources for recreational waters.     

Antibiotic resistant Escherichia coli and Salmonella in Russian rooks (Corvus frugilegus) wintering in the Czech Republic

AIMS: To characterize antibiotic resistant Escherichia coli and Salmonella isolates in rooks wintering in the Czech Republic. METHODS AND RESULTS: Three hundred and sixty-three faeces samples from rooks were examined for antibiotic resistant Escherichia coli and Salmonella. Altogether 13.7%E. coli isolates were resistant to antimicrobial agents tested. The dominant type of resistance was to tetracycline. Resistant E. coli isolates were examined for antibiotic resistance genes and class 1 integrons. Five of 29 antibiotic resistant isolates possessed the int1 gene. Nine Salmonella isolates (2.5%) were found in rook faeces. All the isolates belonged to serotype Salmonella enterica serovar Enteritidis phage type PT8 and PT23. CONCLUSIONS: The study suggests that rooks can be infected by antibiotic resistant E. coli and Salmonella isolates, probably reflecting the presence of such isolates in their sources of food and/or water in the environment. SIGNIFICANCE AND IMPACT OF THE STUDY: Rooks can serve as reservoirs and vectors of antibiotic resistant E. coli and Salmonella isolates and potentially transmit these isolates over long distances.     

Expression of AmpC beta-lactamase in Enterobacter cloacae isolated from retail ground beef, cattle farm and processing facilities

AIMS: To better understand antibiotic resistance of Enterobacter cloacae isolates originated from food animals, the phenotypic and genotypic resistance of Ent. cloacae isolates from retail ground beef, cattle farm, processing facilities and clinical settings were investigated. METHODS AND RESULTS: The ampC, ampD and ampR genes in the isolates were sequenced and analysed. beta-Lactamase activities and beta-lactamase profiles of the isolates were analysed by the enzymatic hydrolysis of nitrocefin and isoelectric focussing, respectively. The ampC gene of the Ent. cloacae isolate was cloned and transformed into Escherichia coli strains. The genomic DNA profiles of Ent. cloacae isolates were analysed by using pulse field gel electrophoresis (PFGE). Mutation at one residue (Val-54-->Ile) in the AmpR amino acid sequence was consistently found in Ent. cloacae isolates that were resistant to a broadspectrum of beta-lactam agents. The enzyme activity in the isolates was induced by cefoxitin. The pI (isoelectric point) of the enzymes produced by the test strains ranged from 8.4 to 8.9. Cloning of ampC gene of the Ent. cloacae isolate conferred the resistance to ampicillin, cephalothin and amoxicillin in recipient E. coli strains. One recipient of E. coli O157:H7 strain additionally acquired resistance to ceftiofur. The genomic analysis of Ent. cloacae isolates by PFGE showed that the isolates from various sources were genetically unrelated. CONCLUSIONS: The spread of diverse clones of AmpC-producing Ent. cloacae occurred in the ecosystem and retail products. SIGNIFICANCE AND IMPACT OF THE STUDY: Our findings suggested that AmpC-producing Ent. cloacae could be a contributor in spreading beta-lactamase genes in farm environments and food processing environments.     

Class 1 integrons, selected virulence genes, and antibiotic resistance in Escherichia coli isolates from the Minjiang River, Fujian Province, China

Widespread fecal pollution of surface waters in developing countries is a threat to public health and may represent a significant pathway for the global dissemination of antibiotic resistance. The Minjiang River drainage basin in Fujian Province is one of China's most intensive livestock and poultry production areas and is home to several million people. In the study reported here, Escherichia coli isolates (n = 2,788) were sampled (2007 and 2008) from seven surface water locations in the basin and evaluated by PCR for carriage of selected genes encoding virulence factors, primarily for swine disease. A subset of isolates (n = 500) were evaluated by PCR for the distribution and characteristics of class 1 integrons, and a subset of these (n = 200) were evaluated phenotypically for resistance to a range of antibiotics. A total of 666 (24%) E. coli isolates carried at least one of the virulence genes elt, fedA, astA, fasA, estA, stx(2e), paa, and sepA. Forty-one percent of the isolates harbored class 1 integrons, and these isolates had a significantly higher probability of resistance to tobramycin, cefoperazone, cefazolin, ciprofloxacin, norfloxacin, azitromycin, and rifampin than isolates with no class 1 integron detected. Frequencies of resistance to selected antibiotics were as high as or higher than those in fecal, wastewater, and clinical isolates in published surveys undertaken in China, North America, and Europe. Overall, E. coli in the Minjiang River drainage basin carry attributes with public health significance at very high frequency, and these data provide a powerful rationale for investment in source water protection strategies in this important agricultural and urban setting in China.     

Genotypic analysis of Escherichia coli recovered from product and equipment at a beef-packing plant

AIMS: To identify sources of Escherichia coli on beef by characterizing strains of the organism on animals, equipment and product at beef-packing plant. METHODS AND RESULTS: Generic E. coli were recovered from hides, carcasses, beef trimmings, conveyers and ground beef during the summer of 2001 (750 isolates) and winter of 2002 (500 isolates). The isolates were characterized by Random Amplification of Polymorphic DNA (RAPD). The numbers of E. coli recovered from dressed carcasses were less than the numbers recovered from hides. The numbers recovered from chilled carcasses were too few for meaningful analysis of the strains present on them but the numbers recovered from trimmings and ground beef were larger. The RAPD patterns showed that the majority of isolates from hides, carcasses, beef trimmings, conveyers and ground beef were of similar RAPD types, but a few unique RAPD types were recovered from only one of those sources. The E. coli populations present on the hides of incoming animals and in the beef-processing environment were highly diverse. Randomly selected E. coli isolates from each of the five sources were further characterized by pulsed-field gel electrophoresis (PFGE). Most genotypes of E. coli defined by PFGE corresponded to the E. coli types defined by RAPD. CONCLUSIONS: The hides of the incoming animals appeared to be only one of the sources of the E. coli on trimmings and in ground beef, as additional sources were apparently present in equipment used for carcass breaking. SIGNIFICANCE AND IMPACT OF THE STUDY: This study indicates that hazardous microbiological contamination of meat may occur after the dressing of carcasses at commercial beef-packing plants, which suggests that attention should be given to the control of the contamination of meat during carcass breaking as well as during the dressing of carcasses.     

Farm disinfectants select for cyclohexane resistance, a marker of multiple antibiotic resistance, in Escherichia coli

AIMS: The aim of this study was to determine if three classes of farm disinfectants were able to select for ciprofloxacin or cyclohexane tolerant [indicative of a multiple antibiotic resistance (MAR) phenotype] Escherichia coli and if cyclohexane-tolerant E. coli could be isolated from farms. METHODS AND RESULTS: Chicken slurry containing ca 1 : 99 ratio ciprofloxacin resistant : susceptible E. coli (10 different resistant strains examined) was treated for 24 h with each of the disinfectants and examined for survival of resistant : susceptible strains. Ciprofloxacin-sensitive (n=5) and -resistant (n=5) E. coli were grown with sublethal concentrations of the disinfectants and then plated to agar containing ciprofloxacin or overlaid with cyclohexane. Escherichia coli (n=389) isolated from farms were tested for cyclohexane tolerance. Minimum inhibitory concentrations (MIC) were determined against representative isolates and mutants. The disinfectants did not select for the ciprofloxacin-resistant E. coli in poultry slurry but following growth with each of the three disinfectants, higher numbers (P < or = 0.023) of cyclohexane-tolerant E. coli were isolated and these had a MAR phenotype. Of the 389 farm E. coli tested, only one was cyclohexane tolerant. CONCLUSIONS: It is possible that in a farm environment, E. coli could be exposed to similar concentrations of the disinfectants that are selected for MAR type organisms under these laboratory conditions. SIGNIFICANCE AND IMPACT OF THE STUDY: Data from this study suggest that cyclohexane-resistant E. coli are not common on farms, but in view of the ease of isolating them in the laboratory with farm disinfectants, further investigations on farms are warranted.     

Fidelity of bacterial source tracking: Escherichia coli vs Enterococcus spp and minimizing assignment of isolates from nonlibrary sources

AIMS: The goal of the study was to improve the fidelity of library-dependent bacterial source tracking efforts in determining sources of faecal pollution. The first objective was to compare the fidelity of source assignments using Escherichia coli vs Enterococcus spp. The second objective was to determine the efficacy of using thresholds during source assignments to reduce the rate of misassignments when nonlibrary isolates (i.e. isolates from animals not used in building the identification library) are present. METHODS AND RESULTS: E. coli and Enterococcus isolates from 784 human, cow, deer, dog, chicken, and gull faecal samples were fingerprinted using BOX-PCR. Jack-knife analysis of the fingerprints showed that the overall rate of correct assignment (ORCA) of 867 E. coli isolates was 67% compared with 82% for 1020 Enterococcus isolates. In a separate blind test using similarity value and quality factor thresholds, the ORCA of 130 E. coli and 131 Enterococcus isolates were 70% and 98%, respectively. The use of these thresholds reduced misassignment of 262 nonlibrary enterococcal isolates from horses, goats, pigs, bats, squirrels, ducks, geese, and migratory song birds. Misassignment was reduced from 100% when thresholds were not used, to 47% using similarity threshold alone, and to 12% when both thresholds were used. CONCLUSIONS: The use of enterococci provides higher rates of correct source assignment compared with E. coli. The use of similarity thresholds to decide whether to accept source assignments made by computer programmes reduces the rate of misassignment of nonlibrary isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: Although both E. coli and Enterococcus spp. are still used in microbial source tracking, the use of enterococci should be preferred over the use of E. coli in DNA fingerprint-based efforts. In addition, because environmental isolates are not limited to those from animals used to build source tracking libraries, similarity thresholds should be used during source assignments to reduce the rate of misassignments.     

The use of ribotyping and antibiotic resistance patterns for identification of host sources of Escherichia coli strains

AIMS: To compare antibiotic resistance and ribotyping patterns ability to identify triplicate isolates sent from a group of 40 Escherichia coli taken from seven host sources. METHODS AND RESULTS: Of the 120 isolates, 22 isolates were resistant to ampicillin, streptomycin, tetracycline and trimethoprim and 98 isolates were susceptible. Antibiotic patterns identified 33 of the triplicates and three of the six groups had isolates from multiple hosts. Ribotyping divided the isolates into 27 ribotype groups with all triplicates grouped into the same ribotype group with one host per group. CONCLUSIONS: Antibiotic susceptibility pattern placed 98 of the isolates in a single group with 50% of the antibiotic susceptibility pattern groups containing multiple host species. Ribotyping groups were host specific with each host having one to seven ribotype groups. SIGNIFICANCE AND IMPACT OF THE STUDY: Antibiotic susceptibility pattern groups have been used for environmental source identification and faecal pollution tracking, however these groups do not always distinguish between host species. Stability of the markers is a potential concern and this system can only be used if antibiotic resistance levels are high in the isolates studied. All isolates have a ribotype group which was stable and like other molecular methods has advantages over antibiotic susceptibility pattern groups which uses a phenotypic method.     

Genetic diversity of Escherichia coli recovered from the oral cavity of beef cattle and their relatedness to faecal E. coli

AIMS: To determine the genetic diversity of generic Escherichia coli recovered from the oral cavities of beef cattle and their relatedness to E. coli isolated from the faeces of cattle during pasture grazing and feedlot finishing. METHODS AND RESULTS: A total of 484 E. coli (248 oral and 236 faecal isolates) were obtained from eight beef cattle after 1 and 5 months of grazing on pasture and after 1 and 5 months in a feedlot. The random amplification of polymorphic DNA (RAPD) method was used to genetically characterize these isolates. The RAPD patterns showed that ca 60% of E. coli recovered from the oral cavities and faeces during pasture and feedlot shared a close genetic relatedness. A number of E. coli with unique RAPD types were also found either in the oral cavities or faeces. Most of the E. coli RAPD types recovered from the oral cavities were shared among animals, but there were also RAPD types which were unique to individual animals. The E. coli populations of the oral cavities were genetically diverse and changed over time. CONCLUSIONS: This study indicates that there are large numbers of E. coli carried in the oral cavities of beef cattle and those E. coli are closely related to strains found in the faeces. The oral cavities of cattle harbour a genetically diverse E. coli population. SIGNIFICANCE AND IMPACT OF THE STUDY: The oral cavity may be an important reservoir of enteric pathogens which may transfer to meat during carcass dressing. A better understanding of the molecular ecology of E. coli in cattle would assist the design of approaches to control pathogenic strains during beef production and processing.     

Prevalence, antibiotic susceptibility, and diversity of Escherichia coli O157:H7 isolates from a longitudinal study of beef cattle feedlots

Prevalence, antibiotic susceptibility, and genetic diversity were determined for Escherichia coli O157:H7 isolated over 11 months from four beef cattle feedlots in southwest Kansas. From the fecal pat (17,050) and environmental (7,134) samples collected, 57 isolates of E. coli O157:H7 were identified by use of bacterial culture and latex agglutination (C/LA). PCR showed that 26 isolates were eaeA gene positive. Escherichia coli O157:H7 was identified in at least one of the four feedlots in 14 of the 16 collections by C/LA and in 9 of 16 collections by PCR, but consecutive positive collections at a single feedlot were rare. Overall prevalence in fecal pat samples was low (0.26% by C/LA, and 0.08% by PCR). No detectable differences in prevalence or antibiotic resistance were found between isolates collected from home pens and those from hospital pens, where antibiotic use is high. Resistant isolates were found for six of the eight antibiotics that could be used to treat E. coli infections in food animals, but few isolates were multidrug resistant. The high diversity of isolates as measured by random amplification of polymorphic DNA and other characteristics indicates that the majority of isolates were unique and did not persist at a feedlot, but probably originated from incoming cattle. The most surprising finding was the low frequency of virulence markers among E. coli isolates identified initially by C/LA as E. coli O157:H7. These results demonstrate that better ways of screening and confirming E. coli O157:H7 isolates are required for accurate determination of prevalence.