Temporal shedding patterns and virulence factors of Escherichia coli serogroups O26, O103, O111, O145, and O157 in a cohort of beef calves and their dams

This study investigated the shedding of Escherichia coli O26, O103, O111, O145, and O157 in a cohort of beef calves from birth over a 5-month period and assessed the relationship between shedding in calves and shedding in their dams, the relationship between shedding and scouring in calves, and the effect of housing on shedding in calves. Fecal samples were tested by immunomagnetic separation and by PCR and DNA hybridization assays. E. coli O26 was shed by 94% of calves. Over 90% of E. coli O26 isolates carried the vtx(1), eae, and ehl genes, 6.5% carried vtx(1) and vtx(2), and one isolate carried vtx(2) only. Serogroup O26 isolates comprised seven pulsed-field gel electrophoresis (PFGE) patterns but were dominated by one pattern which represented 85.7% of isolates. E. coli O103 was shed by 51% of calves. Forty-eight percent of E. coli O103 isolates carried eae and ehl, 2% carried vtx(2), and none carried vtx(1). Serogroup O103 isolates comprised 10 PFGE patterns and were dominated by two patterns representing 62.5% of isolates. Shedding of E. coli O145 and O157 was rare. All serogroup O145 isolates carried eae, but none carried vtx(1) or vtx(2). All but one serogroup O157 isolate carried vtx(2), eae, and ehl. E. coli O111 was not detected. In most calves, the temporal pattern of E. coli O26 and O103 shedding was random. E. coli O26 was detected in three times as many samples as E. coli O103, and the rate at which calves began shedding E. coli O26 for the first time was five times greater than that for E. coli O103. For E. coli O26, O103, and O157, there was no association between shedding by calves and shedding by dams within 1 week of birth. For E. coli O26 and O103, there was no association between shedding and scouring, and there was no significant change in shedding following housing.     

Molecular epidemiology of antimicrobial-resistant commensal Escherichia coli strains in a cohort of newborn calves

Pulsed-field gel electrophoresis (PFGE) was used to investigate the dissemination and diversity of ampicillin-resistant (Amp(r)) and nalidixic acid-resistant (Nal(r)) commensal Escherichia coli strains in a cohort of 48 newborn calves. Calves were sampled weekly from birth for up to 21 weeks and a single resistant isolate selected from positive samples for genotyping and further phenotypic characterization. The Amp(r) population showed the greatest diversity, with a total of 56 different genotype patterns identified, of which 5 predominated, while the Nal(r) population appeared to be largely clonal, with over 97% of isolates belonging to just two different PFGE patterns. Distinct temporal trends were identified in the distribution of several Amp(r) genotypes across the cohort, with certain patterns predominating at different points in the study. Cumulative recognition of new Amp(r) genotypes within the cohort was biphasic, with a turning point coinciding with the housing of the cohort midway through the study, suggesting that colonizing strains were from an environmental source on the farm. Multiply resistant isolates dominated the collection, with >95% of isolates showing resistance to at least two additional antimicrobials. Carriage of resistance to streptomycin, sulfamethoxazole, and tetracycline was the most common combination, found across several different genotypes, suggesting the possible spread of a common resistance element across multiple strains. The proportion of Amp(r) isolates carrying sulfamethoxazole resistance increased significantly over the study period (P < 0.05), coinciding with a decline in the most common genotype pattern. These data indicate that calves were colonized by a succession of multiply resistant strains, with a probable environmental source, that disseminated through the cohort over time.     

Comparison of diversities of Escherichia coli O157 shed from a cohort of spring-born beef calves at pasture and in housing

A cohort of spring-born beef calves demonstrated limited genetic and phenotypic diversity of Escherichia coli O157 when kept in a state of isolation. Despite this, there was a difference in the pulsed-field gel electrophoresis and phage types of isolates shed by cattle at pasture compared with those shed by the same cattle when weaned and housed.     

Natural transmission of Cryptosporidium parvum between dams and calves on a dairy farm

The transmission of Cryptosporidium parvum between dams and their respective calves was studied. For this purpose, fecal specimens taken from the rectum of preparturient, parturient, and postparturient dams were analyzed for C. parvum oocysts. Fecal specimens were taken from the newborn calf 4 hr after birth. Because the environment can be a source of contamination to the animals, specimens taken from inside and outside the barn were analyzed. The sucrose concentration method together with the Zielh-Nielsen acid-fast staining method were employed to increase the chances of oocyst detection. We are reporting that at parturition, the dams shed a higher number of oocysts by comparison to the preparturient and postparturient periods. Neonates acquire the infection at birth mainly because of the high number of oocysts shed by the dams at parturition. The management practice of moving calves 4 hr after birth away from the dams and the barn reduces the number of clinical cases because they are no longer in contact with an environment that is highly contaminated. We hypothesize that the increase in the number of oocysts sheds by dams at parturition might be due to a depression of the T helper 1-type of immune response during that period.     

High-level genotypic variation and antibiotic sensitivity among Escherichia coli O157 strains isolated from two Scottish beef cattle farms

Escherichia coli O157:H7 is a human pathogen that is carried and transmitted by cattle. Scotland is known to have one of the highest rates of E. coli O157 human infections in the world. Two hundred ninety-three isolates were obtained from naturally infected cattle and the environment on two farms in the Scottish Highlands. The isolates were typed by pulsed-field gel electrophoresis (PFGE) with XbaI restriction endonuclease enzyme, and 19 different variations in patterns were found. There was considerable genomic diversity within the E. coli O157 population on the two farms. The PFGE pattern of one of the observed subtypes matched exactly with that of a strain obtained from a Scottish patient with hemolytic-uremic syndrome. To examine the stability of an individual E. coli O157 strain, continuous subculturing of a strain was performed 110 times. No variation from the original PFGE pattern was observed. We found three indistinguishable subtypes of E. coli O157 on both study farms, suggesting common sources of infection. We also examined the antibiotic resistance of the isolated strains. Phenotypic studies demonstrated resistance of the strains to sulfamethoxazole (100%), chloramphenicol (3.07%), and at a lower rate, other antibiotics, indicating the preservation of antibiotic sensitivity in a rapidly changing population of E. coli O157.     

Applicability of phylogenetic methods for characterizing the public health significance of verocytotoxin-producing Escherichia coli strains

Two phylogenetic methods (multilocus sequence typing [MLST] and a multiplex PCR) were investigated to determine whether phylogenetic classification of verocytotoxin-producing Escherichia coli serotypes correlates with their classification into groups (seropathotypes A to E) based on their relative incidence in human disease and on their association with outbreaks and serious complications. MLST was able to separate 96% of seropathotype D and E serotypes from those that cause serious disease (seropathotypes A to C), whereas the multiplex PCR lacked this level of seropathotype discrimination.     

The effect of the management of calves on the prevalence of antibiotic-resistant strains of Escherichia coli in their faeces

Escherichia coli isolated from six calves, in two groups of three which were managed differently and which received no antibiotics, were differentiated into strains on the basis of O serotype, biotype and resistance pattern. Keeping the calves in the maternal environment for 10, rather than 4 d, was associated with a delay in the time taken for strains of E. coli resistant to antibacterial agents to outnumber, in the faeces, those that were sensitive. These findings indicate that the prevalence of resistant E. coli strains in the intestinal microbiota reflect, in part, their prevalence in the environment and that their level may be influenced by the management of the animal.     

Virulence-related DNA sequences and adherence patterns in strains of enteropathogenic Escherichia coli

The presence of the genes for Escherichia coli adherence factor (EAF), attaching and effacing lesion (eae) and bundle-forming pili (bfp) in 72 strains identified as enteropathogenic E. coli (EPEC) by slide agglutination was evaluated using hybridization and PCR. The adherence property of these strains was assayed using 3h HeLa cells adherence assay. The results obtained indicated that virulence-associated genes were present in 65% of the strains but only ten (13.9%) isolates were positive for all the three markers (typical EPEC), 37 (51.4%) isolates carried either one or two of these determinants (atypical EPEC) and the remaining 25 (34.7%) were negative for all these genes. In vitro adherence assay showed that 44 (61.1%) strains adhered to HeLa cells with a defined pattern, 13 (18.1%) isolates adhered loosely with no definite pattern and the remaining 15 (20.8%) were non-adherent. Analysis of the results showed a statistically significant association between the presence of the virulence-related genes with adherence of the strains with a defined pattern (P相似文献   

Binding of milk oligosaccharides by several enterotoxigenic Escherichia coli strains isolated from calves

Milk oligosaccharides have been proposed to play an important role in newborn defense, blocking bacterial adhesion to the intestinal mucosa and preventing infections. Some studies have been performed on human milk oligosaccharides. Here we checked whether bovine milk oligosaccharides would achieve the same protective action against the most common calf enteric pathogens. Seven enterotoxigenic Escherichia coli strains, isolated from diarrheic calves, were selected. All strains managed to agglutinate horse erythrocytes, and we therefore used the inhibition of hemagglutination in the presence of oligosaccharides as an indicator of the union between oligosaccharide and bacterial adhesins. Oligosaccharides from different stages of bovine lactation and standard oligosaccharides were assayed. Midlactation milk, in particular that corresponding to the transition period, proved to be the most efficient at inhibiting hemagglutination. The standard oligosaccharides used pointed to the preference of several strains (K99-, F41-, and F17-fimbriated) for 2,6-linked sialic acid. By contrast, B23 fimbriae exhibited higher affinity for 2,3-sialylated isomers and B64 seemed to require N-acetylglucosamine for binding.Our results suggest a general trend for milk oligosaccharides. Probably they participate in the protection of newborn mammals from pathogens.     

Isolation and characterisation of dog uropathogenic Escherichia coli strains and their fimbriae

A number of Escherichia coli strains have been isolated from dogs with urinary tract infections. These strains have been characterised with respect to their O, K, H, and fimbrial antigens, colicin production, antibiotic resistance, plasmid content and their ability to haemagglutinate erythrocytes from various species. Crossed immunoelectrophoresis of fimbrial extracts, as well as the reaction of partly purified fimbriae of a number of these strains with monoclonal antibodies revealed homology or a strong crossereaction with an F12 fimbrial subunit protein of human uropathogenic E. coli strains. Unlike human F12 fimbriae producing strains, the dog isolates did agglutinate dog erythrocytes in the presence of D-mannose but not human erythrocytes, indicating that the adhesin carried by these strains is different from the adhesin on fimbriae of human uropathogenic E. coli. Similar indications were obtained from experiments with latex beads coated with the receptor for P-fimbriae. These beads were agglutinated by Escherichia coli strains from human urinary tract infections, but not by the dog isolates described here. Preliminary adhesion experiments of human and dog Escherichia coli to human bladder epithelial and canine kidney epithelial cells also showed differences in adhesion depending on the origin of the strain tested.     

Shedding of Escherichia coli O157:H7 in calves is reduced by prior colonization with the homologous strain

Enterohemorrhagic Escherichia coli O157:H7 has a natural reservoir in the intestinal tracts of cattle. Colonization is asymptomatic and transient, but it is not clear if protective immunity is induced. This study demonstrates that prior colonization induces humoral immune responses to bacterial antigens and reduces bacterial shedding after experimental challenge with the homologous strain.     

Comparison of antibiotic resistance patterns in collections of Escherichia coli and Proteus mirabilis uropathogenic strains

Escherichia coli and Proteus mirabilis are important urinary tract pathogens. The constant increase in the antibiotic resistance of clinical bacterial strains has become an important clinical problem. The aim of this study was to compare the antibiotic resistance of 141 clinical (Sweden and Poland) and 42 laboratory (Czech Republic) P. mirabilis strains and 129 clinical (Poland) uropathogenic E. coli strains. The proportion of unique versus diverse patterns in Swedish clinical and laboratory P. mirabilis strain collections was comparable. Notably, a similar proportion of unique versus diverse patterns was observed in Polish clinical P. mirabilis and E. coli strain collections. Mathematical models of the antibiotic resistance of E. coli and P. mirabilis strains based on Kohonen networks and association analysis are presented. In contrast to the three clinical strain collections, which revealed complex associations with the antibiotics tested, laboratory P. mirabilis strains provided simple antibiotic association diagrams. The monitoring of antibiotic resistance patterns of clinical E. coli and P. mirabilis strains plays an important role in the treatment procedures for urinary tract infections and is important in the context of the spreading drug resistance in uropathogenic strain populations. The adaptability and flexibility of the genomes of E. coli and P. mirabilis strains are discussed.     

Estimation of Escherichia coli in raw ground beef

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.     

DNA-based subtyping of verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2 strains from human and raw meat sources

AIMS: To investigate subtyping methods for verocytotoxin-producing Escherichia coli (VTEC) O128ab:H2. METHODS AND RESULTS: Eleven human and food strains isolated over a 15-year period were examined. All were intimin (eae)-negative, but all possessed enterohaemolysin and VT1-encoding sequences which in nine strains were vtx1c variant. Ten strains had VT2 genes which were all vtx2d. Plasmid profiles and randomly amplified polymorphic DNA-PCR were not discriminatory. Long-PCR restriction fragment length polymorphism of amplicons bound by the p gene and the VT2A subunit had screening potential. Pulsed field gel electrophoresis (PFGE) using XbaI gave fine discrimination although VT2 sequences were located on a 220 kbp fragment conserved in nine strains and on a 200 kbp fragment in the 10th. CONCLUSIONS: As a result of apparent clonality, PFGE proved essential for differentiation. Long-PCR has promise for screening but requires further evaluation of inter-strain variable sequences. SIGNIFICANCE AND IMPACT OF THE STUDY: A combined phenotypic and genotypic screen, and PFGE for selected strains was effective.     

The ecology of Escherichia coli in market calves fed a milk-substitute diet

Dynamic changes in the Escherichia coli population in the calf gut were studied over 21 days in a group of 18 intensively-reared market calves. Isolates were identified by O-serogrouping, biotyping and resistogram patterns. Seventy O-serogroups were identified among nearly 3000 E. coli isolates examined and these were subdivided into 416 strains by means of their biotype and resistogram. Seventy-five per cent of these strains were detected only once or twice, which points to the continual replacement of the E. coli flora with strains that showed low persistence in the gut. The rise in the frequency of antibiotic resistance observed during the study was not due to a change in the proportion of resistant to sensitive strains in the gut flora. It was a consequence of the displacement of the original flora by multiply-resistant strains, which presumably originated from the calves' environment.     

The ecology of Escherichia coli in market calves fed a milk-substitute diet

Dynamic changes in the Escherichia coli population in the calf gut were studied over 21 days in a group of 18 intensively-reared market calves. Isolates were identified by O-serogrouping, biotyping and resistogram patterns. Seventy O-serogroups were identified among nearly 3000 E. coli isolates examined and these were subdivided into 416 strains by means of their biotype and resistogram. Seventy-five per cent of these strains were detected only once or twice, which points to the continual replacement of the E. coli flora with strains that showed low persistence in the gut. The rise in the frequency of antibiotic resistance observed during the study was not due to a change in the proportion of resistant to sensitive strains in the gut flora. It was a consequence of the displacement of the original flora by multiply-resistant strains, which presumably originated from the calves' environment.     

Estimation of Escherichia coli in raw ground beef.

This study was undertaken to establish and evaluate more rapid methods of estimating Escherichia coli in ground beef than the standard most probable number (MPN) technique. Direct inoculation of and modifications to EC medium gave unreliable estimates of the presumptive E. coli count. Solid media incubated at an elevated temperature were compared to the MPN technique. Anderson and Baird-Parker's tryptone bile agar (TBA) method and prepoured plates of Endo, Levine eosin methylene blue (EMB), and violet red bile (VRBA) agars incubated at 44 degree C gave equivalent counts to the standard MPN method. Anderson and Baird-Parker TBA was the most selective solid medium for E. coli estimation, but all selective media incubated at elevated temperature reduced apparent E. coli counts by as much as 50%. Indole-producing and lactose-fermenting Enterobacteriaceae, capable of growth at elevated temperature, were tested for their growth on TBA, EMB, and VRBA at elevated temperature. TBA was selective for E. coli biotype I compared to other Enterobacteriaceae that predominate in meats. VRBA and EMB incubated at elevated temperature were not as selective as TBA, but differences in colonies could be observed between typical E. coli colonies and other Enterobacteriaceae on these media. Therefore, VRBA incubated at elevated temperature is proposed as a quality assurance screening test for presumptive E. coli in ground meat. Resuscitation techniques and prepoured plates with VRBA increased recovery levels of presumptive E. coli, but, under the conditions of this study, not to levels that represented a significant practical difference.