An assessment of Bacteroides fragilis group organisms as indicators of human faecal pollution

Membrane filtration techniques were used to enumerate Bacteroides fragilis group (BFG) organisms and Escherichia coli in a variety of natural waters, the influents and effluents from three types of sewage treatment plants and faeces of various animals. The results suggest that BFG organisms die off more rapidly than E. coli in water and that animal faeces are not a significant source of BFG. It is suggested that the ratio of BFG to E. coli in water may be used to indicate the proximity of a source of human faecal contamination.     

Development and application of a spiral plating method for the enumeration of Escherichia coli O157 in bovine faeces

AIM: To develop and validate a direct plating method applicable to epidemiological studies for enumerating Escherichia coli O157 in cattle faeces. METHODS AND RESULTS: The spiral plate count method was used to enumerate E. coli O157 in faecal samples. The accuracy and variation of counts was then assessed using faecal samples inoculated with E. coli O157. There was good agreement between inoculated levels of E. coli O157 and those recovered from faeces, particularly when counts were > 10(2) CFU g(-1) of faeces. The method was applied to a small study assessing short-term survival of E. coli O157 in naturally infected cattle faeces. E. coli O157 was found to survive in faeces for over 10 days at concentrations above 10(3) CFU g(-1) of faeces. Populations of E. coli O157 were also found to increase 100-fold in the first few hours after defecation. CONCLUSIONS: The enumeration method is easy to implement and enables a quick throughput of large numbers of samples. The method is accurate and reliable and enables the inherent variation in count data to be explored but needs to be used in combination with a more sensitive method for samples containing < 10(2) CFU g(-1) of faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The method described is appropriate for enumeration of E. coli O157 in cattle faeces in large-scale epidemiological studies.     

Statistical analyses: possible reasons for unreliability of source tracking efforts

Analyses for the presence of indicator organisms provide information on the microbiological quality of water. Indicator organisms recommended by the United States Environmental Protection Agency for monitoring the microbiological quality of water include Escherichia coli, a thermotolerant coliform found in the feces of warm-blooded animals. These bacteria can also be isolated from environmental sources such as the recreational and pristine waters of tropical rain forests in the absence of fecal contamination. In the present study, E. coli isolates were compared to E. coli K12 (ATCC 29425) by restriction fragment length polymorphism using pulsed-field gel electrophoresis. Theoretically, genomic DNA patterns generated by PFGE are highly specific for the different isolates of an organism and can be used to identify variability between environmental and fecal isolates. Our results indicate a different band pattern for almost every one of the E. coli isolates analyzed. Cluster analysis did not show any relations between isolates and their source of origin. Only the discriminant function analysis grouped the samples with the source of origin. The discrepancy observed between the cluster analysis and discriminant function analysis relies on their mathematical basis. Our validation analyses indicate the presence of an artifact (i.e., grouping of environmental versus fecal samples as a product of the statistical analyses used and not as a result of separation in terms of source of origin) in the classification results; therefore, the large genetic heterogeneity observed in these E. coli populations makes the grouping of isolates by source rather difficult, if not impossible.     

A comparison of two pre-enrichment media prior to immunomagnetic separation for the isolation of E. coli O157 from bovine faeces

AIMS: To compare the sensitivity of two pre-enrichment broth media prior to immunomagnetic separation for the isolation of Escherichia coli O157 from cattle faeces. METHODS AND RESULTS: One-gram portions of 721 cattle faeces collected from 43 farms were pre-enriched in buffered peptone water containing vancomycin, cefixime and cefsulodin (BPW-VCC) and buffered peptone water without additives (BPW-WOA), respectively. A total of 137 samples were positive for E. coli O157: 127 pre-enriched with BPW-WOA and 89 pre-enriched in BPW-VCC. Representative isolates were tested for phage type, verotoxin and eae (E. coli attaching and effacing) gene sequences, resulting in the recognition of eight different types. All the E. coli O157 types recognized were isolated by both methods except for three different strains, each of which were isolated only on a single occasion: two by BPW-WOA and another by BPW-VCC. CONCLUSIONS: The results clearly demonstrate, under the conditions of this study, that BPW without antibiotics was the superior pre-enrichment medium for the isolation of E. coli O157 from cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: The use of BPW-WOA in preference to BPW-VCC for the isolation of E. coli O157 from cattle faeces in future research and outbreak studies should lead to a higher number of positive isolates.     

Enumeration of Escherichia coli O157 in cattle faeces using most probable number technique and automated immunomagnetic separation

AIMS: To determine the numbers of Escherichia coli O157 present in the faeces of naturally infected cattle. METHODS AND RESULTS: A combination of the most probable number (MPN) technique and automated immunomagnetic separation (AIMS) was used to enumerate E. coli O157 in cattle faeces from both pasture-fed and grain-fed animals. A total of 22 E. coli O157 positive faecal samples were enumerated for E. coli O157 (10 from pasture-fed and 12 from grain-fed animals). The numbers of E. coli O157 in cattle faeces varied from undetectable (<3 MPN g-1 of faeces) to 2.4 x 104 MPN g-1. There was no significant difference (P = 0.06) between the numbers of E. coli O157 in pasture-fed or grain-fed cattle faeces, although the geometric mean (antilog of the mean of log10 transformed MPN values) was higher in grain-fed (130 MPN g-1) than in pasture-fed (13 MPN g-1). CONCLUSIONS: Although the number of samples tested is small, the results indicate that E. coli O157 make up a small proportion of the total E. coli population present in cattle faeces. SIGNIFICANCE AND IMPACT OF THE STUDY: Information on the numbers of E. coli O157 present in cattle will assist in developing more robust quantitative risk assessments and formulating intervention strategies.     

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.     

Microbial pollution of water by livestock: approaches to risk assessment and mitigation

In this study, we investigate the extent to which the incidence of Escherichia coli O157:H7 can be predicted in human faeces, from human intake and infection via water contaminated by livestock and carrying this zoonotic pathogen in North-East (NE) and South-West (SW) regions of Scotland. In SW Scotland, there is a risk of coastal recreational waters failing EU standards for faecal indicator organisms, and this is assumed to be the main potential waterborne route of infection. In NE Scotland, the main waterborne route is assumed to be the many private drinking water supplies; these are mainly derived from shallow groundwater and surveys show that there is potential for significant levels of microbial contamination from livestock. The risk to human health from these sources has been assessed using a combination of process models, epidemiological risk-assessment methods and survey data. A key assumption in the calculations is the amount of mixing of pathogenic and non-pathogenic E. coli between animal faecal sources and contaminated water intake by humans. Using the probability distributions of the E. coli O157 content of individual faecal pat material (which would imply no mixing between source and human intake), based on three recent surveys of animal faeces in Scotland, led to predicted annual risks of infection slightly higher than observed human infection incidence. Using the geometric mean to represent partial mixing (which theoretically may over- or underestimate incidence with a concave dose-response curve) gave infection rates similar to those observed for two of the three faecal surveys. Using the arithmetic mean led to over-prediction of risk. This is to be expected if the true dose-response curve is (such as the Beta-Poisson curve used here) concave. Other factors that may lead to over-prediction of incidence are discussed, including under-reporting, loss of infectivity as a result of environmental exposure, immunity and the appropriateness of the Beta-Poisson curve. It is concluded that better epidemiological data for calibration of the dose-response curve, better knowledge of the degree of mixing and understanding of immunity are key requirements for progress in process model-based predictions of infection rate. The paper also explores the potential of improved farm and catchment scale management to deliver cost-effective mitigation of pollution of bathing and drinking water by livestock zoonoses.     

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.     

F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin

Faeces of humans, pigs, cattle and chickens were investigated for the presence of somatic coliphages, F-specific bacteriophages and Escherichia coli strains sensitive to infection by F-specific phages. Attention was given to the possible effect of age and use of antibiotics on the prevalence of the FRNA phages and sensitive E. coli strains. Somatic coliphages were often detected in high numbers in all types of faeces. In contrast, FRNA phages were rarely detected in faeces from humans and cattle but more often in faeces from pigs and adult chickens. Samples from young chickens (with or without antibiotics) were consistently positive for FRNA phages (up to 3 x 10(6) pfu/g). F-specific RNA phages were found in substantial numbers (greater than 10(3) pfu/ml) in a variety of wastewaters: domestic, hospital, slaughterhouses and occasionally in 'grey water'. Their origin in wastewater was not clear. Strains from faeces usually belonged to serogroups I and IV. These types were also found in wastewater, as were group II and III strains. Serogroup II phages were abundant in wastewater of human origin but rare in faeces. Escherichia coli strains sensitive to infection by F-specific phages were common in faeces (overall 290/1081: 27%). No strains with fully depressed F-pilus synthesis were detected among the sensitive strains. It is concluded that the occurrence of F-specific RNA bacteriophages in water points to sewage pollution rather than faecal pollution; the mechanism of replication of these organisms in wastewater is not understood.     

Contamination of Pig Carcasses at Two Abattoirs by Escherichia coli with Special Reference to O-serotypes and Antibiotic Resistance

Evidence of the source of carcass contamination of pigs at slaughter was obtained by determining presumptive coliform counts on faeces and on carcass surfaces, and comparing the O-serotypes and antibiotic sensitivity patterns of Escherichia coli from both sites. All of the 16 pig carcasses from the slaughter line of a commercial abattoir were contaminated with presumptive coliform bacilli on most sites examined; the carcasses of six out of eight pigs slaughtered at the Meat Research Institute (MRI) abattoir were also contaminated, but only small numbers of coliforms could be detected on a few of the sites. The proportion of O-serotypes of E. coli present in faeces which were also detected on carcass surfaces, indicating faecal contamination, varied between 0 and 8.6% in MRI slaughtered pigs but reached 66.6% in one group of commercially slaughtered pigs. O-serotypes found on carcass surfaces but not in the faeces of the pigs, were used as an indication of environmental contamination and this was very evident in the commercially slaughtered pigs. A high proportion of E. coli O-serotypes in the gut were resistant to antibiotics and these were also often found on the carcass surface and, since the range of O-serotypes in the pig is similar to that reported in man, the pig must be considered to be a potential reservoir of antibiotic resistant E. coli for man.     

F-specific RNA bacteriophages and sensitive host strains in faeces and wastewater of human and animal origin

Faeces of humans, pigs, cattle and chickens were investigated for the presence of somatic coliphages, F-specific bacteriophages and Escherichia coli strains sensitive to infection by F-specific phages. Attention was given to the possible effect of age and use of antibiotics on the prevalence of the FRNA phages and sensitive E . coli strains. Somatic coliphages were often detected in high numbers in all types of faeces. In contrast, FRNA phages were rarely detected in faeces from humans and cattle but more often in faeces from pigs and adult chickens. Samples from young chickens (with or without antibiotics) were consistently positive for FRNA phages (up to 3 × 10⁶ pfu/g). F-specific RNA phages were found in substantial numbers (> 10³ pfu/ml) in a variety of wastewaters: domestic, hospital, slaughterhouses and occasionally in 'grey water'. Their origin in wastewater was not clear. Strains from faeces usually belonged to serogroups I and IV. These types were also found in wastewater, as were group II and III strains. Serogroup II phages were abundant in wastewater of human origin but rare in faeces. Escherichia coli strains sensitive to infection by F-specific phages were common in faeces (overall 290/1081: 27%). No strains with fully derepressed F-pilus synthesis were detected among the sensitive strains. It is concluded that the occurrence of F-specific RNA bacteriophages in water points to sewage pollution rather than faecal pollution; the mechanism of replication of these organisms in wastewater is not understood.     

An adapted ImmunoMagnetic cell separation method for use in quantification of Escherichia coli O157:H7 from bovine faeces

Detection of Escherichia coli O157:H7 organisms in food, clinical or environmental samples is necessary for diagnosis of infection and epidemiological investigations. However, this pathogen may be present in low numbers and difficult to identify among high numbers of other background bacteria. In order to increase the sensitivity of culture- and PCR detection, pre-enrichment of E. coli O157:H7 in broth culture combined with ImmunoMagnetic cell Separation (IMS) is routinely employed. These methods, although able to detect levels as low as 2 cfu/g (from 10 to 25 g samples), are qualitative detection strategies only. If the actual numbers of E. coli O157:H7 are to be quantified, growth enrichment must be excluded and the organisms isolated directly from the sample of interest. Such quantification is necessary, for example, to determinate contamination levels on beef carcasses and for determination of bacterial numbers in in vivo gene expression studies. In the present study, it was not possible to recover organisms from bovine faecal suspensions using the customary IMS system and so a range of alternative buffers and other paramagnetic beads was tested. Combination of a 6.2-microm diameter bead with a detergent-based buffer gave optimal recovery of E. coli O157:H7 organisms from faecal suspensions. This system was validated for recovery of E. coli O157:H7 by comparing it with that obtained with the standard Dynabeads IMS protocol, using both the traditional broth enrichment method and a quantitative detection approach. We conclude that a 6.2-microm diameter Aureon bead can be used for quantitative isolation of E. coli O157:H7 directly from bovine faeces and, for this purpose, is preferred to the 2.8-microm diameter Dynal bead.     

Waterborne Escherichia coli O157

The waterborne route of Vero cytotoxin-producing E. coli (VTEC) O157 infection was first suggested in two unconnected human cases in 1985. Since then, waterborne VTEC O157 has been identified in sporadic cases and in outbreaks of illness. Recreational waters, private and municipal supplies have been implicated from microbiological, environmental and epidemiological studies of cases. In addition, a research cohort study of farm workers identified exposure to private water supplies as a risk factor for having antibodies to E. coli O157. Sources of contamination are thought to be animal and human faeces or sewage. The presence of low numbers of target organisms in water makes microbiological confirmation difficult, therefore epidemiological evidence has been essential in outbreak investigations. Despite the potential for contamination of water with VTEC O157, waterborne infection is relatively rare largely due to the susceptibility of the organism to water treatment processes. This paper presents the evidence for waterborne VTEC O157 infection, considering current microbiological, environmental and particularly epidemiological information.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli, faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli/faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the microtiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Enumeration by a miniaturized method of Escherichia coli, Streptococcus bovis and enterococci as indicators of the origin of faecal pollution of waters

Counts of Escherichia coli , faecal streptococci and enterococci were made on faecal specimens from human and animal origin and urban raw sewage waters, with microtiter plates containing selective substances. Escherichia coli was more numerous than faecal streptococci and enterococci in 80% of the samples regardless of the origin. Consequently the use of the ratio E. coli /faecal streptococci to distinguish human from animal origin of faecal pollution is questionable. Enterococcus faecalis was predominant in human and poultry faeces, Streptococcus bovis was typical of the bovine faeces and to a lesser extent also of pig faeces whereas Enterococcus durans, Ent. hirae and Ent. faecium did not characterize any faecal source. Streptococcus bovis could be distinguished in the mictrotiter plate by its inability to reduce triphenyl tetrazolium chloride (TTC) in the medium.     

Magnitude of pollution indicator organisms in rural potable water.

A total of 460 water samples were randomly drawn from the potable water supply sources of rural communities in three counties of South Carolina. About 10% of the population, not incorporated in municipalities, was sampled. The samples were tested for total coliforms, Escherichia coli, and fecal streptococci. Significant levels of these pollution indicator organisms were detected in almost all the water supplies. Total coliforms were the most common, and only 7.5% of the water supplies were uncontaminated. E. coli, considered a reliable indicator of recent and dangerous pollution, was observed in 43% of the water supplies. Statistical analyses indicated that the bacterial populations, especially E. coli, were associated with the supply source depth and its distance from the septic tank. Total coliform counts were also weakly correlated to the pH of the water.     

Contamination of milk by enterococci and coliforms from bovine faeces

AIM: To determine the contribution of enterococci and coliforms from bovine faeces and teats to contamination of raw milk. Methods: Putative enterococci (n = 301) and coliforms (n = 365) were isolated from bovine faeces (n = 20), cows' teats (n = 20), the raw milk (n = 1) and the milking environment (n = 4) on one farm. The clonal relationships of each bacterial group were investigated using Pulsed-Field Gel Electrophoresis of genomic macrorestriction fragments. Representatives of the different clusters of enterococci were identified by molecular techniques including rep-PCR, SDS protein profiling, Fluorescent Amplified Fragment Length Polymorphism (FAFLP), phenylalanyl-tRNA synthase (pheS) sequence analysis and/or 16S rDNA gene sequencing. Coliforms were identified by API 20E strips. RESULTS: The majority of the bovine faecal enterococcal isolates were identified as a potential new species of Aerococcus (100 isolates); E. faecium (28 isolates), and Aerococcus viridans (28 isolates) were also found. All coliform isolates from the bovine faeces were identified as Escherichia coli. The coliforms present in the milk were Hafnia alvei, Serratia liquefaciens, Yersinia enterocolitica and Enterobacter amnigenus. No E. coli, Enterococcus or Aerococcus from the bovine faeces were found in the milk. A single clone of H. alvei was found in the water, the milking equipment and the milk, suggesting that the water was the source of the organism in the milk. No vancomycin-resistant aerococci or enterococci were found while most of the isolates tested showed the presence of at least one virulence gene. The milk-sock retained strains that adhered to particulate faecal material. Coliforms were present at approx. 2 orders of magnitude greater than enterococci in the bovine faeces. CONCLUSIONS: The results imply that bovine faeces are not an important source of contamination of raw milk with enterococci or coliforms. SIGNIFICANCE AND IMPACT OF THE STUDY: The results confirm those of two previous studies (Gelsomino et al. 2001, Int J Food Microbiol71, 177-188 and Kagkli et al. 2007, Int J Food Microbiol114, 243-251) on two other farms. The three studies show that contamination of milk by enterococci, lactobacilli and coliforms of bovine faecal origin is extremely low. The results also suggest that where raw milk is implicated in food infection, other factors in addition to faecal contamination must be involved.     

Detection and Incidence of Escherichia coli on Storage Pen Surfaces of Fishing Trawlers

Six methods for the detection and enumeration of Escherichia coli on the storage pen surfaces of commercial fishing trawlers and in harbor wash water were evaluated. E. coli was found consistently present in Boston harbor water used for washing vessel holds and was detected either in small numbers or not at all on storage pen surfaces. Violet Red Bile Agar as a primary enumeration medium was found ineffective for detection of coliforms because of the nonselective development of large numbers of other gram-negative organisms. The use of E. coli broth at 44.5 C for primary most-probable-number determinations, followed by confirmation of E. coli on Levine Eosine Methylene Blue Agar, appears to offer numerous advantages over more conventional methods of detecting E. coli for survey studies of the fishing industry, where coliform-like organisms result in many false-positive presumptives with other methods.     

THE VALUE OF THE ASSOCIATION ESCHERICHIEAE-GROUP D STREPTOCOCCI IN THE DIAGNOSIS OF CONTAMINATION IN FOODS

SUMMARY: The tests for faecal contamination in foods, based on 'indicator species', should be reconsidered for the following reasons. First, in Europe, Escherichia coli is regarded as a specific index of such contamination; but, apparently through the use of antibiotics, the proportion of human faeces containing Klebsiella has risen from 5.2% in 1947 to 48.4% in 1956–7. Moreover, E. coli and other coli-aerogenes organisms may be absent from the gut of certain animals, notably the pig. Second, while the usual methods of counting coli-aerogenes bacteria do not permit the isolation of strains which ferment lactose slowly or not at all (i.e. paracolons), these occur in 35.6% of samples of human faeces. Further, the recommended methods of detecting such strains are not wholly satisfactory. Third, the origin of the coli-aerogenes bacteria is uncertain.
It is thus necessary to consider other organisms which normally inhabit the intestinal tract of humans and other animals. As to Clostridium perfringens , its source is doubtful, for it may come from faeces or from soil, and many foods contain only soil strains. The group D streptococci, on the other hand, are excellent indicators of faecal contamination. They are constant or frequent in the intestines of man and animals, and often more numerous there than coli-aerogenes bacteria. Their specificity as an index of faecal contamination is high and their investigation is easy with the selective media now available. Associated with the coli-aerogenes bacteria, they justify the diagnosis of faecal contamination in a foodstuff.