Prevalence and molecular characterization of Escherichia coli O157:H7 on Irish lamb carcasses, fleece and in faeces samples

AIMS: To determine the prevalence, seasonal variation and virulence characteristics of Escherichia coli O157:H7 in lambs presented for slaughter in Ireland. METHODS AND RESULTS: Over a 13-month period, pre- and postchill carcass swabs, faeces and fleece samples from 1600 lambs were examined for the presence of E. coli O157:H7. Escherichia coli O157:H7 was isolated from 5.75% (23/400) of fleece samples, 1.5% (6/400) of pre- and 1% (4/400) of postchill carcass swabs but was not isolated in faeces (0/400). The present study detected no evidence of seasonal variation. Polymerase chain reaction analysis showed that both the vt1 and vt2 genes associated with clinical illness were carried by five of the E. coli O157:H7 isolates, while 24 of the remaining isolates carried the vt2 gene only. Phage typing detected four different subtypes: PT 32 (48.48%), PT 8 (12.12%), PT 31 (12.12%) and PT 21/28 (12.12%). CONCLUSIONS: Escherichia coli O157:H7 is present in lambs at slaughter in Irish abattoirs and the virulence profiles of these isolates reveals that they are potentially harmful to humans. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides crucial information indicating that sheep may be a significant contributing source to human E. coli O157:H7 infection.     

In vitro inhibition activity of different bacteriocin-producing Escherichia coli against Salmonella strains isolated from clinical cases

Aims:  To compare in vitro the inhibitory activity of four bacteriocin-producing Escherichia coli to a well-characterized panel of Salmonella strains, recently isolated from clinical cases in Switzerland.
Methods and Results:  A panel of 68 nontyphoidal Salmonella strains was characterized by pulsed-field gel electrophoresis analysis and susceptibility to antibiotics. The majority of tested strains were genetically different, with 40% resistant to at least one antibiotic. E. coli Mcc24 showed highest in vitro activity against Salmonella (100%, microcin 24), followed by E. coli L1000 (94%, microcin B17), E. coli 53 (49%, colicin H) and E. coli 52 (21%, colicin G) as revealed using a cross-streak activity assay.
Conclusions:  Escherichia coli Mcc24, a genetically modified organism producing microcin 24, and E. coli L1000, a natural strain isolated from human faeces carrying the mcb -operon for microcin B17-production, were the most effective strains in inhibiting in vitro both antibiotic resistant and sensitive Salmonella isolates.
Significance and Impact of the Study:  Due to an increasing prevalence of antibiotic resistant Salmonella strains, alternative strategies to fight these foodborne pathogens are needed. E. coli L1000 appears to be a promising candidate in view of developing biotechnological alternatives to antibiotics against Salmonella infections.     

The effect of chlortetracycline treatment and its subsequent withdrawal on multi-resistant Salmonella enterica serovar Typhimurium DT104 and commensal Escherichia coli in the pig

AIMS: To investigate the effect of a therapeutic and sub-therapeutic chlortetracycline treatment on tetracycline-resistant Salmonella enterica serovar Typhimurium DT104 and on the commensal Escherichia coli in pig. METHODS AND RESULTS: Salmonella Typhimurium DT104 was orally administered in all pigs prior to antibiotic treatment, and monitored with the native E. coli. Higher numbers of S. Typhimurium DT104 were shed from treated pigs than untreated pigs. This lasted up to 6 weeks post-treatment in the high-dose group. In this group, there was a 30% increase in E. coli with a chlortetracycline minimal inhibitory concentration (MIC) > 16 mg l-1 and a 10% increase in E. coli with an MIC > 50 mg l-1 during and 2 weeks post-treatment. This effect was less-pronounced in the low-dose group. PCR identified the predominant tetracycline resistance genes in the E. coli as tetA, tetB and tetC. The concentration of chlortetracycline in the pig faeces was measured by HPLC and levels reached 80 microg g-1 faeces during treatment. CONCLUSION: Chlortetracycline treatment increases the proportion of resistant enteric bacteria beyond the current withdrawal time. SIGNIFICANCE AND IMPACT OF THE STUDY: Treated pigs are more likely to enter abattoirs with higher levels of resistant bacteria than untreated pigs promoting the risk of these moving up the food chain and infecting man.     

Effect of the growth promoter avilamycin on emergence and persistence of antimicrobial resistance in enteric bacteria in the pig

AIM: To assess the effect of the growth promoter avilamycin on emergence and persistence of resistance in enteric bacteria in the pig. METHODS AND RESULTS: Pigs (treated with avilamycin for 3 months and controls) were challenged with multi-resistant Salmonella Typhimurium DT104 and faecal counts were performed for enterococci, Escherichia coli, S. Typhimurium and Campylobacter (before, during and 5 weeks post-treatment). Representative isolates were tested for antibiotic resistance and for the presence of resistance genes. Avilamycin-resistant Enterococci faecalis (speciated by PCR) were isolated from the treated pigs and continued to be detected for the first week after treatment had ceased. The avilamycin-resistance gene was characterized by PCR as the emtA gene and speciation by PCR. MIC profiling confirmed that more than one strain of Ent. faecalis carried this gene. There was no evidence of increased antimicrobial resistance in the E. coli, Salmonella and Campylobacter populations, although there was a higher incidence of tetB positive E. coli in the treated pigs than the controls. CONCLUSION: Although avilamycin selects for resistance in the native enterococci population of the pig, no resistant isolates were detected beyond 1 week post-treatment. This suggests that resistant isolates were unable to persist once selective pressure was removed and were out-competed by the sensitive microflora. SIGNIFICANCE AND IMPACT OF THE STUDY: Our data suggest the risk of resistant isolates becoming carcass contaminants and infecting humans could be minimized by introducing a withdrawal period after using avilamycin and prior to slaughter.     

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.     

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.     

The prevalence and spread of Escherichia coli O157:H7 at a commercial beef abattoir

AIMS: To investigate the prevalence and virulence characteristics of Escherichia coli O157:H7 after a number of beef process operations at a commercial Irish abattoir. METHODS AND RESULTS: Two 12-month studies were carried out. The first study (study 1) examined the prevalence of E. coli O157:H7 at up to six sites on carcasses at eight stages of the dressing, washing, chilling and boning process. The second study (study 2) examined the prevalence of E. coli O157:H7 in bovine faeces and rumen contents post-slaughter and on dressed, washed carcasses. Isolates from both studies were phage-typed and the presence of genes encoding verocytotoxin, enterohaemolysin and intimin production was determined. E. coli O157:H7 was isolated from four of 36 carcasses in study 1. E. coli O157:H7 was detected during hide removal and was detected at multiple carcass sites and multiple process stages, including boning. On two carcasses, contamination was first detected at the bung following its freeing and tying. All isolates from study 1 were phage type (PT) 2, eaeAO157 and ehlyA positive, but were verocytotoxin 1 (VT1) and verocytotoxin 2 (VT2) negative. In study 2, E. coli O157:H7 was isolated from 2.4% of faecal, 0.8% of rumen and 3.2% of carcass samples. In some cases, isolates recovered from the faeces of a particular animal, the resulting carcass and adjacent carcasses on the line had the same phage typing and virulence characteristic profile patterns. All isolates from study 2 were eaeAO157 and ehlyA positive and only one isolate was VT1 and VT2 negative. Most isolates were PT 32. A higher frequency of positive isolations was noted from samples taken during spring and late summer. CONCLUSION: These studies show that in a typical Irish beef abattoir, carcass contamination with E. coli O157:H7 can occur during hide removal and bung tying and this contamination can remain on the carcass during subsequent processing. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides data that is necessary for the understanding of how E. coli O157:H7 contamination of beef occurs.     

Trimethoprim resistance plasmids in Escherichia coli isolated from cases of diarrhoea in cattle, pigs and sheep

A total of 1572 isolates of Escherichia coli obtained from the faeces of young farm animals with diarrhoea over the period 1980–1983 were screened for resistance to trimethoprim (Tp). Resistance to Tp was detected in263/954 (28%) of bovine isolates,59/441 (13%) of porcine isolates and15/177 (9%) of ovine isolates. Seventy-five resistant isolates from separate outbreaks of infection on farms within a 25 mile radius of Nottingham were examined in detail. Sixty-eight (91%) of the 75 isolates were resistant to > 1024 mg Tp/1 and 34 (50%) of these 'highly resistant' isolates (45% of total resistant isolates) transferred their Tp resistance to E. coli K12. A further 13 (17%) isolates were demonstrated to carry non-self-transferable plasmids which were capable of being mobilized to E. coli K12 by the broad host range plasmid RP4. Thirty-one self-transferable Tp R plasmids were divided between the following incompatibility groups: IncB (14 plasmids), IncF_***H (4 plasmids), IncH₂ (1 plasmid), IncIaP (10 plasmids), IncIdT (1 plasmid) and IncP (1 plasmid). In terms of antibiotic resistance patterns and incompatibility properties, many of these plasmids closely resembled those isolated from human patients in the same area, suggesting that there may be a common pool of Tp R plasmids.     

Distribution of insertion sequence IS1 in multiple-antibiotic resistant clinical Enterobacteriaceae strains

The presence of insertion sequence IS1 in 70 multiple-antibiotic resistant clinical strains was determined. This 70-strain collection comprised 46 Escherichia coli, 18 Salmonella and 6 Shigella strains. The presence of IS1 was detected in the chromosome and plasmids of 73% and 63% of the strains, respectively, and 51% of the strains carried IS1 in both. The frequency of IS1 was higher in Salmonella than in E. coli and Shigella strains. A total of 31 strains carried large plasmids with IS1; 10 of these strains (32.3%) were able to transfer all or some of the antibiotic resistance markers to E. coli K12 or S. typhimurium recipient strains. Resistance markers of all clinical strains were maintained stably after several generations of growth. The presence of IS1 in a relatively high percentage of plasmids of multiple-antibiotic resistant clinical isolates, suggests a role for this sequence in the dissemination of genes which code for antibiotic resistance.     

The prevalence of Salmonella spp. in bovine faecal,rumen and carcass samples at a commercial abattoir

AIMS: To determine the prevalence, serotype and antibiotic resistance profile of Salmonella isolates in cattle and on carcasses at a commercial Irish abattoir. METHODS AND RESULTS: Faecal, rumen and carcass samples were collected from a beef abattoir over a 12-month period and examined for the presence of Salmonella spp. Isolates were serotyped, phage typed (when serotype was found to be S. Typhimurium) and tested for susceptibility to a panel of antibiotics. Salmonella was isolated from 2% of faecal, 2% of rumen and 7.6% of carcass samples. Salmonella was most frequently isolated from samples taken during the period August to October. S. Dublin was isolated from 72% of positive samples. S. Agona and S. Typhimurium definitive type (DT)104 were each isolated from 14% of positive samples. All S. Typhimurium DT104 isolates were resistant to ampicillin, chloramphenicol, streptomycin, sulphafurazole and tetracycline (ACSSuT). On occasion, from a single animal, the same serotype was isolated from more than one sample (i.e. faeces and rumen; faeces and carcass; rumen and carcass; faeces, rumen and carcass). CONCLUSIONS: Salmonella is present in cattle at slaughter and on beef carcasses at an Irish abattoir, with a higher frequency of occurrence during the period August to October. Most isolates from the study are not commonly associated with human clinical infection, with the exception of S. Typhimurium DT104 (R-type ACSSuT). SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides epidemiological data that is necessary for the understanding of beef as a source of human Salmonella infection.     

Antimicrobial susceptibility and factors affecting the shedding of E. coli O157:H7 and Salmonella in dairy cattle

AIMS: To examine factors affecting faecal shedding of the foodborne pathogens Escherichia coli O157:H7 and Salmonella in dairy cattle and evaluate antimicrobial susceptibility of these isolates. METHODS: Faecal samples were obtained in replicate from lactating (LAC; n = 60) and non-lactating (NLAC; n = 60) Holstein cattle to determine influence of heat stress, parity, lactation status (LAC vs NLAC) and stage of lactation [60 days in milk (DIM)] and cultured for E. coli O157:H7 and Salmonella. A portion of the recovered isolates were examined for antimicrobial susceptibility using the broth microdilution technique. RESULTS: No effects of heat stress were observed. Lactating cows shed more (P < 0.01) E. coli O157:H7 than NLAC cows (43% vs 32%, respectively). Multiparous LAC cows tended to shed more (P = 0.06) Salmonella than primiparous LAC cows (39% vs 27%, respectively). Parity did not influence (P > 0.10) bacterial shedding in NLAC cows. Cows 60 DIM. Seventeen Salmonella serotypes were identified with the most prevalent being Senftenberg (18%), Newport (17%) and Anatum (15%). Seventy-nine of the Salmonella isolates were resistant to at least one of the seven antibiotics. Escherichia coli O157:H7 isolates were resistant to 11 different antibiotics with multiple resistance to nine or more antibiotics observed in five isolates. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated differences in the shedding patterns of foodborne pathogens due to the stage of the milk production cycle and may help identify times when on-farm pathogen control would be the most effective.     

High prevalence of antimicrobial-resistant genes and integrons in Escherichia coli isolates from Black-headed Gulls in the Czech Republic

AIMS: To carry out an assessment of the occurrence of resistance to antimicrobials in Escherichia coli that has been isolated from young Black-headed Gulls in three nesting colonies. METHODS AND RESULTS: A total of 257 isolates were tested for sensitivity to eight antibacterial substances by disk diffusion method. The polymerase chain reaction was used for detecting specific genes of antibacterial resistance and class 1 integrons in resistant E. coli isolates. A total 75 (29.9%) of 257 isolates were resistant to one or more antimicrobial agents. The dominant type of resistance was to tetracycline, detected in 49 (19.1%) isolates. Resistance to ampicillin was detected in 30 (11.7%), cephalothin in 11 (4.3%), streptomycin in 24 (9.3%), sulphonamides in 20 (7.8%) and chloramphenicol in 5 (1.9%) isolates. Nine isolates carrying integrons were detected. CONCLUSIONS: The study suggests that young Black-headed Gulls are an important host reservoir of resistant E. coli strains, probably reflecting the presence of such strains in their sources of food and/or water. SIGNIFICANCE AND IMPACT OF THE STUDY: Although Black-headed Gulls do not naturally come into contact with antibiotics, these birds can be infected with resistant E. coli and potentially serve as their reservoirs, vectors and bioindicators in the environment.     

Prevalence of bacterial faecal pathogens in separated and unseparated stored pig slurry

AIMS: To examine the prevalence and diversity of bacterial faecal pathogens in unseparated slurry, separated solids and liquid fractions from a commercial pig farm. METHODS: A total of 43 stored slurry specimens originating from a fattening house over the period February-April 2002 were analysed, consisting of unseparated (n = 14) slurry, separated solids (n = 16) and separated liquid (n = 13). Specimens were examined for the presence of five bacterial pathogens including Salmonella spp., Shigella spp., Campylobacter spp., Escherichia coli O157 and Yersinia enterocolitica. Selective enrichment and plating methods were employed for detection of Salmonella spp. and Campylobacter spp. and conventional selective plating techniques for the remaining genera. Antibiogram profiles to 12 antibiotic agents were obtained for all Salmonella isolates obtained. RESULTS: Salmonella spp. were identified in all components of the slurry specimens, whereas Campylobacter spp. was only recovered from the unseparated and separated liquid fractions. In both cases, the separated liquid fraction had the highest prevalence of pathogens and the separated solid fraction had the lowest prevalence. None of the slurry specimens examined were positive for E. coli O157:H7, Shigella spp. or Y. enterocolitica. Twenty-nine isolates of Salmonella were recovered from the slurry specimens, comprising seven serovars, of which Salmonella manhattan was the most prevalent, accounting for over half [15 of 29 (51.7%)] of all Salmonella isolates. Salmonella anatum, Salm. derby, Salm. give, Salm. heidelberg, Salm. simi and Salm. stanley serovars were also recovered. All Salmonella isolates were sensitive to ampicillin, augmentin (amoxicillin/clavulanic acid), chloramphenicol, ciprofloxacin, gentamicin, kanamycin and trimethoprim, but has variable resistance to tetracycline (100%), sulphonamides (84.6%), furazolidone (38.5%), nalidixic acid (15.4%) and streptomycin (15.4%). The majority (57.7%) of isolates displayed antibiotic resistance to at least two antibiotic agents, followed by 34.6% of isolates being resistant to three agents and the remainder (7.7%) being resistant to four antibiotics. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated a marked reduction in the prevalence of Campylobacter and Salmonella in the solids component of separated pig slurry. The adoption of control processes such as aeration of slurry prior to its spread onto agricultural land and newer approaches to pathogen reduction should be investigated, to reduce the transmission of pathogens from pig slurry to the environment.     

Enumeration and antibiotic resistance patterns of fecal indicator organisms isolated from migratory Canada geese (Branta canadensis)

Thermotolerant fecal indicator organisms carried by migratory waterfowl may serve as reservoirs of antibiotic resistance. To determine the extent to which such antibiotic resistance markers were present in migratory Canada geese (Branta canadensis) on the Maryland Eastern Shore, we isolated Enterococcus spp. and Escherichia coli from fresh feces and examined the antibiotic resistance profiles of these bacteria. Samples were obtained in October 2002, January 2003, and March 2003. Thermotolerant E. coli counts ranged from 0 to 1.0x10(7) colony forming units (CFU)/0.1g (g-1) wet weight of feces, whereas Enterococcus spp. counts ranged from 1.0x10(2)-1.0x10(7) CFU g-1 wet weight of feces. Primary isolates of each indicator organism were tested against a panel of 10 antibiotics. Greater than 95% of E. coli isolates were resistant to penicillin G, ampicillin, cephalothin, and sulfathiazole; no E. coli were resistant to ciprofloxacin. Enterococcal isolates showed highest resistance to cephalothin, streptomycin, and sulfathiazole; no enterococci were resistant to chloramphenicol. The tetracyclines, streptomycin, and gentamycin provided the greatest discrimination among E. coli isolates; chlortetracycline, cephalothin, and gentamycin resistance patterns provided the greatest discrimination between enterococcal strains. Multiple antibiotic resistance (MAR) profiles were calculated: fall (E. coli=0.499; enterococci=0.234), winter (E. coli=0.487; enterococci=0.389), and spring (E. coli=0.489; enterococci=0.348). E. faecalis and E. faecium, which are recognized human nosocomial pathogens, were cultured from winter (44 and 56%, respectively) and spring (13 and 31%, respectively) fecal samples.     

Occurrence of antibiotic resistance in Escherichia coli from surface waters and fecal pollution sources near Hamilton, Ontario

Antibiotic resistance was examined in 462 Escherichia coli isolates from surface waters and fecal pollution sources around Hamilton, Ontario. Escherichia coli were resistant to the highest concentrations of each of the 14 antibiotics studied, although the prevalence of high resistance was mostly low. Two of 12 E. coli isolates from sewage in a CSO tank had multiple resistance to ampicillin, ciprofloxacin, gentamicin, and tetracycline above their clinical breakpoints. Antibiotic resistance was less prevalent in E. coli from bird feces than from municipal wastewater sources. A discriminant function calculated from antibiotic resistance data provided an average rate of correct classification of 68% for discriminating E. coli from bird and wastewater fecal pollution sources. The preliminary microbial source tracking results suggest that, at times, bird feces might be a more prominent contributor of E. coli to Bayfront Park beach waters than municipal wastewater sources.     

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.     

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.     

Antimicrobial resistance in generic Escherichia coli isolates from wild small mammals living in swine farm, residential, landfill, and natural environments in southern Ontario, Canada

To assess the impacts of different types of human activity on the development of resistant bacteria in the feces of wild small mammals, we compared the prevalences and patterns of antimicrobial resistance and resistance genes in generic Escherichia coli and Salmonella enterica isolates from fecal samples collected from wild small mammals living in four environments: swine farms, residential areas, landfills, and natural habitats. Resistance to antimicrobials was observed in E. coli isolates from animals in all environments: 25/52 (48%) animals trapped at swine farms, 6/69 (9%) animals trapped in residential areas, 3/20 (15%) animals trapped at landfills, and 1/22 (5%) animals trapped in natural habitats. Animals trapped on farms were significantly more likely to carry E. coli isolates with resistance to tetracycline, ampicillin, sulfisoxazole, and streptomycin than animals trapped in residential areas. The resistance genes sul2, aadA, and tet(A) were significantly more likely to be detected in E. coli isolates from animals trapped on farms than from those trapped in residential areas. Three S. enterica serotypes (Give, Typhimurium, and Newport) were recovered from the feces of 4/302 (1%) wild small mammals. All Salmonella isolates were pansusceptible. Our results show that swine farm origin is significantly associated with the presence of resistant bacteria and resistance genes in wild small mammals in southern Ontario, Canada. However, resistant fecal bacteria were found in small mammals living in all environments studied, indicating that environmental exposure to antimicrobials, antimicrobial residues, resistant bacteria, or resistance genes is widespread.     

Geographical variation in antibiotic resistance profiles of Escherichia coli isolated from swine, poultry, beef and dairy cattle farm water retention ponds in Florida

AIMS: The aim of this study was to assess geographical variation in multiple antibiotic resistance (MAR) profiles of livestock Escherichia coli as well as to evaluate the ability of MAR profiles to differentiate sources of faecal pollution. METHODS AND RESULTS: More than 2000 E. coli isolates were collected from water retention ponds and manure of swine, poultry, beef and dairy farms in south, central and north Florida, and analysed for MAR using nine antibiotics. There were significant differences in antibiotic resistance of E. coli by season and livestock type for more than one antibiotic, but regional differences were significant only for ampicillin. Over the three regions, discriminant analysis using MAR profiles correctly classified 27% of swine, 49% of poultry, 56% of beef and 51% of dairy isolates. CONCLUSIONS: Regional variations in MAR combined with moderate discrimination success suggest that MAR profiles of E. coli may only be marginally successful in identifying sources of faecal pollution. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrates the existence of regional and seasonal differences in MAR profiles as well as the limited ability of MAR profiles to discriminate among livestock sources.     

Laboratory heating studies with Salmonella spp. and Escherichia coli in organic matter,with a view to decontamination of poultry houses

AIMS: To determine a temperature-humidity-time treatment that eliminates Salmonella and Escherichia coli in substrates representing organic matter in poorly cleaned poultry houses, i.e. worst case scenario laboratory tests. METHODS AND RESULTS: Organic matter (poultry faeces and feed) in a 2.5-cm layer was inoculated with 2 x 10(5)-3 x 10(6) Salmonella g(-1), left undried or dried at ca. 30% relative humidity (RH) during a 10-day period, and temperature increased at 1 degrees C h(-)1 to the final heating temperature of 50, 55, 60, 65 or 70 degrees C and held at 16-30 or 100% RH. All samples were tested for Salmonella according to predetermined sampling time schedules and faecal samples were also tested for naturally occurring E. coli. Overall, humidity was an important factor in the elimination of Salmonella and E. coli. Results for recovery of Salmonella and E. coli were highly associated. CONCLUSIONS: The application of >/=60 degrees C and 100% RH during a 24-h period eliminated Salmonella and E. coli in all samples. Escherichia coli could be used as an indicator bacterium for the elimination of Salmonella. SIGNIFICANCE AND IMPACT OF THE STUDY: The results from worst case scenario laboratory tests could be applied in steam heating of persistently Salmonella-infected poultry houses. The use of E. coli as an indicator bacterium for the validation of Salmonella results should be considered.