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.     

A biomarker for the identification of cattle fecal pollution in water using the LTIIa toxin gene from enterotoxigenic Escherichia coli

This research describes a method based on PCR to identify cattle fecal pollution in water using a portion of the heat labile toxin IIA (LTIIa) gene from enterotoxigenic Escherichia coli (ETEC). We describe the development of the primers and target. DNA extracts (221) from different animal fecal and human sewage samples were screened and showed no cross-reactivity. Minimum detection limits using centrifugation and filtration methods to concentrate E. coli seeded into stream, ocean, and secondary effluent waters were found to be at femtogram and attogram levels, respectively. Stability of the biomarker in stream, ocean, and secondary effluent waters was 2-4 weeks for all water types. Finally, 33 farm lagoon and waste samples were collected and 31 tested to validate the method; 93% were positive for the LTIIa trait when >1,000 E. coli were screened and 100% positive when >10(5) E. coli were screened. Prevalence of the toxin gene in the E. coli population affected the outcome of the analyses. The cow biomarker can be used in watershed studies to identify cattle waste with great accuracy if the appropriate numbers of E. coli are screened.     

Diversity and Distribution of Escherichia coli Genotypes and Antibiotic Resistance Phenotypes in Feces of Humans, Cattle, and Horses

Escherichia coli is the most completely characterized prokaryotic model organism and one of the dominant indicator organisms for food and water quality testing, yet comparatively little is known about the structure of E. coli populations in their various hosts. The diversities of E. coli populations isolated from the feces of three host species (human, cow, and horse) were compared by two subtyping methods: ribotyping (using HindIII) and antibiotic resistance analysis (ARA). The sampling effort required to obtain a representative sample differed by host species, as E. coli diversity was consistently greatest in horses, followed by cattle, and was lowest in humans. The diversity of antibiotic resistance patterns isolated from individuals was consistently greater than the diversity of ribotypes. E. coli populations in individuals sampled monthly, over a 7- to 8-month period, were highly variable in terms of both ribotypes and ARA phenotypes. In contrast, E. coli populations in cattle and humans were stable over an 8-h period. Following the cessation of antibiotic therapy, the E. coli population in the feces of one human experienced a rapid and substantial shift, from a multiply antibiotic-resistant phenotype associated with a particular ribotype to a relatively antibiotic-susceptible phenotype associated with a different ribotype. The high genetic diversity of E. coli populations, differences in diversity among hosts, and temporal variability all indicate complex population dynamics that influence the usefulness of E. coli as a water quality indicator and its use in microbial source tracking studies.     

Sensitivity of an immunomagnetic-separation-based test for detecting Escherichia coli O26 in bovine feces

The sensitivity of a test for cattle shedding Escherichia coli serogroup O26 was estimated using several fecal pats artificially inoculated at a range of concentrations with different E. coli O26 strains. The test involves the enrichment of fecal microflora in buffered peptone water, the selective concentration of E. coli O26 using antibody-coated immunomagnetic-separation beads, the identification of E. coli colonies on Chromocult tryptone bile X-glucuronide agar, and confirmation of the serogroup with E. coli serogroup O26-specific antisera using slide agglutination. The effective dose of E. coli O26 for an 80% test sensitivity (ED(80)) was 1.0 x 10(4) CFU g(-1) feces (95% confidence interval, 4.7 x 10(3) to 2.4 x 10(4)). Differences in test sensitivity between different E. coli O26 strains and fecal pats were also observed. Individual estimates of ED(80) for each strain and fecal pat combination ranged from 4.2 x 10(2) to 4.8 x 10(5) CFU g(-1). These results suggest that the test is useful for identifying individuals shedding a large number of E. coli O26 organisms or, if an appropriate number of individuals in a herd are sampled, for identifying affected herds. The study also provides a benchmark estimate of sensitivity that can be used to compare alternative tests for E. coli O26 and a methodological approach that can be applied to tests for other pathogenic members of the Enterobacteriaceae and other sample types.     

Identification of fecal Escherichia coli from humans and animals by ribotyping

Fecal pollution of water resources is an environmental problem of increasing importance. Identification of individual host sources of fecal Escherichia coli, such as humans, pets, production animals, and wild animals, is prerequisite to formulation of remediation plans. Ribotyping has been used to distinguish fecal E. coli of human origin from pooled fecal E. coli isolates of nonhuman origin. We have extended application of this technique to distinguishing fecal E. coli ribotype patterns from human and seven individual nonhuman hosts. Classification accuracy was best when the analysis was limited to three host sources. Application of this technique to identification of host sources of fecal coliforms in water could assist in formulation of pollution reduction plans.     

Practical mechanisms for interrupting the oral-fecal lifecycle of Escherichia coli

Escherichia coli is a common gut inhabitant, but it is usually out numbered by strictly anaerobic bacteria. When fecal material is exposed to oxygen, fermentation acids can be respired, and E. coli numbers increase. E. coli can survive for long periods of time in feces, but subsequent proliferation is dependent on its ability to re-enter the gastrointestinal tract via contaminated water and food. The oral-fecal lifestyle of E. coli is facilitated by its ability to survive the low pH of the human gastric stomach. Most strains of E. coli do not cause human disease, but some strains produce toxins and other virulence factors. Mature cattle carry E. coli O157:H7 without showing signs of infection, and beef can be contaminated with cattle feces at slaughter. Cattle manure is often used as a fertilizer by the vegetable industry, and E. coli from manure can migrate through the soil into water supplies. Sanitation, cooking and chlorination have been used to combat fecal E. coli, but these methods are not always effective. Recent work indicates that cattle diets can be modified overcome the extreme acid resistance of E. coli. When cattle were fed have for only a few days, colonic volatile fatty acid concentrations declined, pH increased, and the E. coli were no longer able to survive a pH shock that mimicked the human gastric stomach. E. coli in stored cattle manure eventually become highly acid resistant even if the cattle were fed hay, but these bacteria could be killed by sodium carbonate (150 mM, pH 8.5). Because the diet manipulations and carbonate treatments affected E. coli in general rather than specific serotypes, there is an increased likelihood of successful field application.     

Origin of contamination and genetic diversity of Escherichia coli in beef cattle

The possible origin of beef contamination and genetic diversity of Escherichia coli populations in beef cattle, on carcasses and ground beef, was examined by using random amplification of polymorphic DNA (RAPD) and PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the fliC gene. E. coli was recovered from the feces of 10 beef cattle during pasture grazing and feedlot finishing and from hides, carcasses, and ground beef after slaughter. The 1,403 E. coli isolates (855 fecal, 320 hide, 153 carcass, and 75 ground beef) were grouped into 121 genetic subtypes by using the RAPD method. Some of the genetic subtypes in cattle feces were also recovered from hides, prechilled carcasses, chilled carcasses, and ground beef. E. coli genetic subtypes were shared among cattle at all sample times, but a number of transient types were unique to individual animals. The genetic diversity of the E. coli population changed over time within individual animals grazing on pasture and in the feedlot. Isolates from one animal (59 fecal, 30 hide, 19 carcass, and 12 ground beef) were characterized by the PCR-RFLP analysis of the fliC gene and were grouped into eight genotypes. There was good agreement between the results obtained with the RAPD and PCR-RFLP techniques. In conclusion, the E. coli contaminating meat can originate from cattle feces, and the E. coli population in beef cattle was highly diverse. Also, genetic subtypes can be shared among animals or can be unique to an animal, and they are constantly changing.     

Occurrence of Rhodococcus coprophilus and associated actinomycetes in feces, sewage, and freshwater.

Freshwater, sewage, and fecal samples from various sources were examined for Rhodococcus coprophilus, associated actinomycetes, Escherichia coli, and fecal streptococci. Rhodococcus coprophilus was isolated consistently from feces of farm animals, poultry reared in proximity to farm animals, freshwater, and wastewater polluted with animal fecal wastes. It was not isolated from samples of human feces. The ratio of R. coprophilus total actinomycetes was higher in feces from cattle, sheep, ducks, and geese than in specimens from pigs, horses, and fowl. In samples from two freshwater streams polluted by fecal material from farm animals, the ratios of R. copropilus to total actinomycetes were similar to those found in fecal specimens from cattle and sheep. Ratios of fecal coliform to fecal streptococci could not distinguish between fresh human and animal fecal samples and, furthermore, were not reflected in the stream waters polluted by animal fecal material. R. coprophilus has potential in water and dairy bacteriology as a specific indicator organism of fecal pollution due to farm animal wastes.     

Genotypic diversity of Escherichia coli in the water and soil of tropical watersheds in Hawaii

High levels of Escherichia coli were frequently detected in tropical soils in Hawaii, which present important environmental sources of E. coli to water bodies. This study systematically examined E. coli isolates from water and soil of several watersheds in Hawaii and observed high overall genotypic diversity (35.5% unique genotypes). In the Manoa watershed, fewer than 9.3% of the observed E. coli genotypes in water and 6.6% in soil were shared between different sampling sites, suggesting the lack of dominant fecal sources in the watershed. High temporal variability of E. coli genotypes in soil was also observed, which suggests a dynamic E. coli population corresponding with the frequently observed high concentrations in tropical soils. When E. coli genotypes detected from the same sampling events were compared, limited sharing between the soil and water samples was observed in the majority of comparisons (73.5%). However, several comparisons reported up to 33.3% overlap of E. coli genotypes between soil and water, illustrating the potential for soil-water interactions under favorable environmental conditions. In addition, genotype accumulation curves for E. coli from water and soil indicated that the sampling efforts in the Manoa watershed could not exhaust the overall genotypic diversity. Comparisons of E. coli genotypes from other watersheds on Oahu, Hawaii, identified no apparent grouping according to sampling locations. The results of the present study demonstrate the complexity of using E. coli as a fecal indicator bacterium in tropical watersheds and highlight the need to differentiate environmental sources of E. coli from fecal sources in water quality monitoring.     

Characterization of Escherichia coli O157:H7 from downer and healthy dairy cattle in the upper Midwest region of the United States

While cattle in general have been identified as a reservoir of Escherichia coli O157:H7, there are limited data regarding the prevalence and clonality of this pathogen in downer dairy cattle and the potential impact to human health that may occur following consumption of meat derived from downer dairy cattle. In the present study, conducted at two slaughter facilities in Wisconsin between May and October of 2001, we established a higher prevalence of E. coli O157:H7 in fecal and/or tissue samples obtained aseptically from intact colons of downer dairy cattle (10 of 203, 4.9%) than in those from healthy dairy cattle (3 of 201, 1.5%). Analyses of 57 isolates, representing these 13 positive samples (one to five isolates per sample), by pulsed-field gel electrophoresis, revealed 13 distinct XbaI restriction endonuclease digestion profiles (REDP). Typically, isolates from different animals displayed distinct REDP and isolates from the same fecal or colon sample displayed indistinguishable REDP. However, in one sample, two different, but highly related, REDP were displayed by the isolates recovered. Antimicrobial susceptibility testing indicated that 10 of the 57 isolates, recovered from 2 (1 downer and 1 healthy animal) of the 13 positive samples, were resistant to at least 1 of 18 antimicrobials tested. However, there was no appreciable difference in the frequency of resistance of isolates recovered from downer and healthy dairy cattle, and not all isolates with the same REDP displayed the same antimicrobial susceptibility profile. Lastly, it was not possible to distinguish between isolates recovered from downer and healthy cattle based on their XbaI REDP or antimicrobial susceptibility. These results indicate that downer cattle had a 3.3-fold-higher prevalence of E. coli O157:H7 than healthy cattle within the time frame and geographic scope of this study.     

Application of ribotyping for differentiating Vibrio cholerae non-O1 isolated from shrimp farms in Thailand.

A collection of 143 Vibrio cholerae non-O1 strains isolated from shrimp farms in Thailand were characterized and grouped by ribotyping. Sixty-four ribotypes were distinguished following digestion of chromosomal DNA with the restriction enzyme BglI, and the reproducibility of the method was 100%. There was no correlation between specific ribotype distributions and the locations of the shrimp farms. Ribotype similarity was examined by cluster analysis, and two main groups with 10 and 54 ribotypes, respectively, were found. Correlation between ribotype and O-antigen expression was shown to exist among those isolates tested. Ribotyping appears to be a suitable method for differentiating environmental V. cholerae non-O1 strains, and comparison of ribotype patterns showed a high degree of genetic divergence within V. cholerae non-O1.     

Tracking the Primary Sources of Fecal Pollution in a Tropical Watershed in a One-Year Study

A study was conducted to determine the primary sources of fecal pollution in a subtropical watershed using host-specific assays developed in temperate regions. Water samples (n = 534) from 10 different sites along the Rio Grande de Arecibo (RGA) watershed were collected mostly on a weekly basis (54 sampling events) during 13 months. DNA extracts from water samples were used in PCR assays to determine the occurrence of fecal bacteria (Bacteroidales, Clostridium coccoides, and enterococci) and human-, cattle-, swine-, and chicken-specific fecal sources. Feces from 12 different animals (n = 340) and wastewater treatment samples (n = 16) were analyzed to determine the specificity and distribution of host-specific assays. The human-specific assay (HF183) was found to be highly specific, as it did not cross-react with nontarget samples. The cattle marker (CF128) cross-reacted to some extent with swine, chicken, and turkeys and was present in 64% of the cattle samples tested. The swine assays showed poor host specificity, while the three chicken assays showed poor host distribution. Differences in the detection of host-specific markers were noted per site. While human and cattle assays showed moderate average detection rates throughout the watershed, areas impacted by wastewater treatment plants and cattle exhibited the highest prevalence of these markers. When conditional probability for positive signals was determined for each of the markers, the results indicated higher confidence levels for the human assay and lower levels for all the other assays. Overall, the results from this study suggest that additional assays are needed, particularly to track cattle, chicken, and swine fecal pollution sources in the RGA watershed. The results also suggest that the geographic stability of genetic markers needs to be determined prior to conducting applied source tracking studies in tropical settings.     

Survival of Candida albicans in tropical marine and fresh waters.

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

Survival of Candida albicans in tropical marine and fresh waters

A survey of Candida albicans indicated that the organism was present at all sites sampled in a rain forest stream and in near-shore coastal waters of Puerto Rico. In the rain forest watershed no relationship existed between densities of fecal coliforms and densities of C. albicans. At two pristine sites in the rain forest watershed both C. albicans and Escherichia coli survived in diffusion chambers for extended periods of time. In near-shore coastal waters C. albicans and E. coli survival times in diffusion chambers were enhanced by effluent from a rum distillery. The rum distillery effluent had a greater effect on E. coli than on C. albicans survival in the diffusion chambers. These studies show that neither E. coli nor C. albicans organisms are good indicators of recent fecal contamination in tropical waters. It further demonstrates that pristine freshwater environments and marine waters receiving organic loading in the tropics can support densities of C. albicans which may be a health hazard.     

Host species-specific metabolic fingerprint database for enterococci and Escherichia coli and its application to identify sources of fecal contamination in surface waters

A metabolic fingerprint database of enterococci and Escherichia coli from 10 host groups of animals was developed to trace the sources of fecal contamination in surface waters. In all, 526 biochemical phenotypes (BPTs) of enterococci and 530 E. coli BPTs were obtained from 4,057 enterococci and 3,728 E. coli isolates tested. Of these, 231 Enterococcus BPTs and 257 E. coli BPTs were found in multiple host groups. The remaining 295 Enterococcus BPTs and 273 E. coli BPTs were unique to individual host groups. The database was used to trace the sources of fecal contamination in a local creek. The mean diversities (Di) of enterococci (Di = 0.76 +/- 0.05) and E. coli (Di = 0.88 +/- 0.04) were high (maximum 1) in water samples, indicating diverse sources of fecal contamination. Overall, 71% of BPTs of enterococci and 67% of E. coli BPTs from water samples were identified as human and animal sources. Altogether, 248 Enterococcus BPTs and 282 E. coli BPTs were found in water samples. Among enterococci, 26 (10%) BPTs were identical to those of humans and 152 BPTs (61%) were identical to those of animals (animal BPTs). Among E. coli isolates, 36 (13%) BPTs were identical to those of humans and 151 (54%) BPTs were identical to those of animals. Of the animal BPTs, 101 (66%) Enterococcus BPTs and 93 (62%) E. coli BPTs were also unique to individual animal groups. On the basis of these unique Enterococcus BPTs, chickens contributed 14% of contamination, followed by humans (10%), dogs (7%), and horses (6%). For E. coli, humans contributed 13% of contamination, followed by ducks (9%), cattle (7%), and chickens (6%). The developed metabolic fingerprint database was able to distinguish between human and animal sources as well as among animal species in the studied catchment.     

Patterns of antimicrobial resistance observed in Escherichia coli isolates obtained from domestic- and wild-animal fecal samples, human septage, and surface water

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286 Escherichia coli strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of E. coli were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general, E. coli isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant E. coli were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.     

Comparison of ribotyping and repetitive extragenic palindromic-PCR for identification of fecal Escherichia coli from humans and animals

This report compares the performances of two popular genotypic methods used for tracking the sources of fecal pollution in water, ribotyping and repetitive extragenic palindromic-PCR (rep-PCR). The rep-PCR was more accurate, reproducible, and efficient in associating DNA fingerprints of fecal Escherichia coli with human and animal hosts of origin.     

Discriminant analysis of ribotype profiles of Escherichia coli for differentiating human and nonhuman sources of fecal pollution.

Estuarine waters receive fecal pollution from a variety of sources, including humans and wildlife. Escherichia coli is a ubiquitous bacterium in the intestines of warm-blooded animals and is used as an indicator of fecal pollution. However, its presence does not specifically differentiate sources of pollution. A total of 238 E. coli isolates from human sources (HS) and nonhuman sources (NHS) were collected from the Apalachicola National Estuarine Research Reserve, from associated sewage treatment plants, and directly from animals and tested for ribotype (RT) profile. HS and NHS isolates showed 41 and 61 RT profiles, respectively. At a similarity index of ca. 50%, HS and NHS isolates demonstrated four clusters, with the majority of HS and NHS isolates located in clusters C and D; isolates obtained directly from human and animal feces also could be grouped within these clusters. Discriminant analysis (DA) of RT profiles showed that 97% of the NHS isolates and 100% of the animal fecal isolates were correctly classified. The average rate of correct classification for HS and NHS isolates was 82%. We conclude that DA of RT profiles may be a useful method for identifying HS and NHS fecal pollution and may potentially facilitate management practices.     

Determining sources of fecal pollution in a rural Virginia watershed with antibiotic resistance patterns in fecal streptococci

Nonpoint sources of pollution that contribute fecal bacteria to surface waters have proven difficult to identify. Knowledge of pollution sources could aid in restoration of the water quality, reduce the amounts of nutrients leaving watersheds, and reduce the danger of infectious disease resulting from exposure to contaminated waters. Patterns of antibiotic resistance in fecal streptococci were analyzed by discriminant and cluster analysis and used to identify sources of fecal pollution in a rural Virginia watershed. A database consisting of patterns from 7,058 fecal streptococcus isolates was first established from known human, livestock, and wildlife sources in Montgomery County, Va. Correct fecal streptococcus source identification averaged 87% for the entire database and ranged from 84% for deer isolates to 93% for human isolates. To field test the method and the database, a watershed improvement project (Page Brook) in Clarke County, Va., was initiated in 1996. Comparison of 892 known-source isolates from that watershed against the database resulted in an average correct classification rate of 88%. Combining all animal isolates increased correct classification rates to > or = 95% for separations between animal and human sources. Stream samples from three collection sites were highly contaminated, and fecal streptococci from these sites were classified as being predominantly from cattle (>78% of isolates), with small proportions from waterfowl, deer, and unidentified sources ( approximately 7% each). Based on these results, cattle access to the stream was restricted by installation of fencing and in-pasture watering stations. Fecal coliforms were reduced at the three sites by an average of 94%, from prefencing average populations of 15,900 per 100 ml to postfencing average populations of 960 per 100 ml. After fencing, <45% of fecal streptococcus isolates were classified as being from cattle. These results demonstrate that antibiotic resistance profiles in fecal streptococci can be used to reliably determine sources of fecal pollution, and water quality improvements can occur when efforts to address the identified sources are made.     

粪便中大肠埃希菌的分离鉴定

2004年4月采集大连金州湾沿岸畜禽养殖场猪、牛、鸡和某中学人粪便样品。经改良的生化鉴定方法鉴定,得大肠埃希菌(Escherichia coli)猪源75株、牛源78株、鸡源69株、人源68株,占粪大肠菌比率分别为85.23%、92.86%、80.23%、80.00%。为验证改良的生化鉴定方法准确性,从中随机选取8株E.coli,扩增16S rRNA序列并与Genebank中E.coli16S序列比对,确定这8株细菌与E.coli同源相似率达99%以上,进而验证改良的生化鉴定方法的可行性。