Gene markers of generic Escherichia coli associated with colonization and persistence of Escherichia coli O157 in cattle

Enterohemorrhagic Escherichia coli (EHEC) O157 are important foodborne pathogens whose major reservoir are asymptomatic cattle. There is evidence suggesting that nonpathogenic E. coli and bacteriophages in the gastro-intestinal tract can influence the pathogenicity of EHEC O157. The factors contributing to the onset and persistence of shedding EHEC O157 in cattle are not completely elucidated. This study used Bayesian network analysis to identify genetic markers of generic E. coli associated with shedding of EHEC O157 in cattle from data generated during an oral experimental challenge study in 4 groups of 6 steers inoculated with three different EHEC O157 strains. The quantification of these associations was accomplished using mixed effects logistic regression. The results showed that the concurrent presence of generic E. coli carrying the prophage marker R4-N and the virulence marker stx2 increased the odds of the onset of EHEC O157 shedding. The presence of prophage markers z2322 and X011C increased, while C1.N decreased the odds of shedding EHEC O157 two days later. A significant antagonist interaction effect between the presence of the virulence marker stx2 on the day of shedding EHEC O157 and two days before shedding was also found. In terms of the persistence of EHEC O157 shedding, the presence of prophage marker R4-N (OR = 16, and 95% confidence interval (CI): 1.1, 252) was found to increase the odds of stopping EHEC O157 shedding, whereas prophage marker C1.N (OR = 0.16, CI: 0.03, 0.7) and the enterohemolysin gene hly (OR = 0.03, CI: 0.001, 0.8) were found to significantly decrease the odds of stopping EHEC O157 shedding. In conclusion, the study found that the presence of certain genetic markers in the generic E. coli genome can influence the pathogenicity of EHEC O157.     

Prevalence of antimicrobial resistance in enteric Escherichia coli from domestic pets and assessment of associated risk markers using a generalized linear mixed model

Antimicrobial resistance (AMR) is a growing global public health problem, which is caused by the use of antimicrobials in both human and animal medical practice. The objectives of the present cross-sectional study were as follows: (1) to determine the prevalence of resistance in Escherichia coli isolated from the feces of pets from the Porto region of Portugal against 19 antimicrobial agents and (2) to assess the individual, clinical and environmental characteristics associated with each pet as risk markers for the AMR of the E. coli isolates.     

Resistance mechanisms and farm-level distribution of fecal Escherichia coli isolates resistant to extended-spectrum cephalosporins in pigs in Spain

Introduction

Fecal Escherichia coli isolates showing a phenotype of reduced susceptibility or resistance to extended-spectrum cephalosporins are common among pigs in Spain. The aim of this study was to describe the main beta-lactam resistance mechanisms carried by these strains and their distribution at farm-level.

Materials and methods

Twenty-nine E. coli isolates showing reduced susceptibility or resistance to extended-spectrum cephalosporins were collected from a sampling frame of 80 pig farms distributed over 13 Spanish provinces. The survey was carried out at the slaughterhouse level in 2004.

Results

Of the 29 isolates, 21 (72%) met the criteria for a positive phenotypic confirmatory test for extended-spectrum beta-lactamases (ESBL). The following ESBLs were detected: SHV-12 (12 isolates, 41%), CTX-M-1 (three isolates, 10%), CTX-M-9 (three isolates, 10%), and CTX-M-14 (three isolates, 10%). The remaining eight isolates (28%) were phenotypically non-ESBL, with seven of them (24%) showing mutations on the chromosomal ampC gene promoter at positions −42 (C → T), −18 (G → A), −1 (C → T), and +58 (C → T). A multiplex PCR for detection of plasmidic class C beta-lactamases was negative for all isolates.

Conclusion

Different ESBLs and other mechanisms linked to extended-spectrum cephalosporin resistance are widely distributed among fecal E. coli from slaughter pigs in Spain.     

Phenotypic and genotypic characteristics of antimicrobial resistant Escherichia coli isolated from symbovine flies, cattle and sympatric insectivorous house martins from a farm in the Czech Republic (2006-2007)

The prevalence of antimicrobial resistant Escherichia coli was tested in symbovine flies and sympatric house martins (Delichon urbica) at a dairy farm. Antimicrobial resistant E. coli was detected in 89% (n = 147) of isolates from flies within a calf barn. Isolates with the same antimicrobial resistance phenotypes, genes, and pulsotypes were found between both fly and calf E. coli isolates, suggesting that the calves were the initial source of the antimicrobial resistant strains in fly isolates. Symbovine flies were considered as important reservoirs of antimicrobial resistant E. coli strains at a dairy farm, due to their intensive contact with cattle feces and manure. House martin fecal samples from the same farm contained 4.5% (n = 393) of antimicrobial resistant E. coli. House martin isolates displayed different macrorestriction profiles than fly isolates and the significance of house martins as a reservoir and vector of antimicrobial resistant E. coli appears low.     

牛肉源大肠杆菌的耐药性检测及相关耐药基因分布

为了解牛肉源大肠杆菌的耐药性并检测其相关耐药基因分布,本研究选取了117株牛肉源大肠杆菌,经药敏纸片法对11种抗菌药物进行了药敏检测,并根据耐药表型利用普通和(或)多重PCR技术对耐四环素菌中tet(A)、tet(B)和tet(C)基因,耐氨苄西林菌中blaTEM1、blaPSE1和blaOXA1基因,耐链霉素菌中strA-strB、aadA1基因,耐磺胺类药菌中sul1、sul2和sul3基因进行了调查分析。结果显示,117株大肠杆菌对四环素、氨苄西林、链霉素和磺胺异恶唑的耐药率较高,分别为89%、42%、38%和22%。tet(A)基因是所有四环素耐药基因中最为流行的一种基因(55%);在检测的3个β-内酰胺类药物耐药基因中,最流行的为blaTEM1基因(73%);链霉素的耐药性主要由strA-strB基因(38%)编码;sul2基因在耐磺胺异恶唑菌中的检出率最高(77%)。结果表明本次分离的牛肉源大肠杆菌耐药非常严重,进一步肯定了肉牛业生产中抗菌药的使用对大肠杆菌耐药性的产生和发展所发挥的重要作用,提示食品动物养殖应严格控制饲料中抗菌药的滥用。     

Phylogenetic distribution of virulence genes in Escherichia coli isolated from bovine mastitis in Iran

One hundred and twenty seven Escherichia coli isolates from bovine mastitis were examined to detect the phylogenetic group/subgroups and a selection of virulence associated genes. Forty nine (38.58%) isolates belonged to group B1 the remaining isolates fell into four phylogenetic subgroups: A₀ (18.11%), A₁ (26.77%), D₁ (6.29%) and D₂ (10.23%). None of the isolates belonged to B2 group. Forty seven (37.00%) isolates were positive for at least one virulence gene, among them f17A was the most common gene, found in 20.47% of the isolates. Among the E. coli isolates, 11.81% had iucD, 9.44% f17c-A, 9.44% cnf2, 7.87% f17b-A, 6.29% afaD-8 and afaE-8, 3.14% f17d-A, 0.78% cnf1 and 0.78% clpG genes. All of the detected virulence genes were present alone or in combination with each other except clpG and f17d-A genes that were only found alone. None of the isolates contained the genes for F17a-A, intimin, P or S fimbriae.     

Staphylococcus aureus and Escherichia coli elicit different innate immune responses from bovine mammary epithelial cells

Escherichia coli and Staphylococcus aureus are the most important pathogenic bacteria causing bovine clinical mastitis and subclinical mastitis, respectively. However, little is known about the molecular mechanisms underlying the different host response patterns caused by these bacteria. The aim of this study was to characterize the different innate immune responses of bovine mammary epithelium cells (MECs) to heat-inactivated E. coli and S. aureus. Gene expression of Toll-like receptor 2 (TLR2) and TLR4 was compared. The activation of nuclear factor kappa B (NF-κB) and the kinetics and levels of cytokine production were analyzed. The results show that the mRNA for TLR2 and TLR4 was up-regulated when the bovine MECs were stimulated with heat-inactivated E. coli, while only TLR2 mRNA was up-regulated when the bovine MECs were stimulated with heat-inactivated S. aureus. The expression of tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6 and IL-8 increased more rapidly and higher when the bovine MECs were stimulated with heat-inactivated E. coli than when they were stimulated with heat-inactivated S. aureus. E. coli strongly activated NF-κB in the bovine MECs, while S. aureus failed to activate NF-κB. Heat-inactivated S. aureus could induce NF-κB activation when bovine MECs cultured in medium without fetal calf serum. These results were confirmed using TLR2- and TLR4/MD2-transfected HEK293 cells and suggested that differential TLR recognition and the lack of NF-κB activation account for the impaired immune response elicited by heat-inactivated S. aureus.     

Detection of genes encoding for virulence and adherence factors in Escherichia coli isolated in slaughtered Sarda breed sheep

In order to investigate the pathogenic profile of Escherichia coli hosted in “Sarda” sheep, autochthonous race present in Sardinia, thirty-seven E. coli strains collected from different sources (fleeces, carcass swabs and gut mucosa) of pre-chill slaughtered sheep (ewes and lambs) were serotyped using pheno- and genotypic methods. Furthermore, the presence of genes encoding for virulence factors and mediating for localized mucosal adherence factors was investigated, and pulsed-field gel electrophoresis (PFGE) characterization was performed.     

A longitudinal field trial assesing the impact of feeding waste milk containing antibiotic residues on the prevalence of ESBL-producing Escherichia coli in calves

A longitudinal field trial was carried out on a farm known to harbour cefotaximase (CTX-M)-positive Escherichia coli, in order to assess the impact of feeding waste milk containing antibiotic residues (WM + AR) on the prevalence of these bacteria in the faeces of calves. Fifty calves were alternately assigned to one of two groups at birth and fed either milk replacer (control group) or WM + AR (treatment group). Faecal samples were collected from all calves daily for the first week after enrolment, twice weekly until weaning, then weekly for a further six weeks. Environmental samples from the calf housing were collected weekly. WM + AR and powdered milk samples were examined for antibiotic residues and CTX-M-positive E. coli. Total E. coli and CTX-M-positive E. coli in faecal samples were enumerated using selective media. Regression analyses were performed on the bacterial count data using a population-averaged approach based on generalised estimating equations (GEE) to account for repeated measurements on individual calves over time.     

Avian colibacillosis caused by an intestinal pathogenic Escherichia coli isolate from calf diarrhea

An intestinal pathogenic Escherichia coli isolate from calf diarrhea, containing the iutA, f17A, afa-8D, and cnf2 genes, was able to cause avian colibacillosis after experimental infection in chickens. Intra-tracheal inoculation and spray of this strain caused 10% of mortality and gross lesions, including airsacculitis, pericarditis, and perihepatitis. These results suggest that some bovine pathogenic E. coli can cause extra-intestinal infections in other animal species.     

Diversity of Campylobacter jejuni and Campylobacter coli Genotypes from Human and Animal Sources from Rio de Janeiro, Brazil

To compare the genotypes of Campylobacter jejuni and Campylobacter coli isolates of human and animal origin collected in Rio de Janeiro City, 30 C. jejuni and 35 C. coli isolates from animal sources (n = 45) and human patients with gastroenteritis (n = 20) were genotyped by PCR-based techniques, namely random amplified polymorphic DNA (RAPD-PCR) and enterobacterial repetitive intergenic consensus sequence (ERIC-PCR). RAPD-PCR identified 50 types and ERIC-PCR identified 22 genotypes, among the 65 Campylobacter isolates. Both PCR methods discriminated the C. jejuni and C. coli groups of isolates. Combining the results of both methods, no single genotype was shared between isolates from human and animal sources. Two groups of two C. coli isolates each with identical genotypes were found among poultry and pig isolates. A high level of genetic diversity observed among the Campylobacter isolates suggests lack of overlap between isolates from different sources.     

First Report of Methicillin-Resistant Staphylococcus aureus ST5 and ST398 from Purebred Lusitano Horses

Methicillin-resistant Staphylococcus aureus (MRSA) was first described in horses in 1996. The frequency of MRSA colonization in horses varies among European countries, but it is unknown in Portugal. The aim of this study was to screen for MRSA nasal carriage in a sample of horses entering the Equine Unit, Large Animal Veterinary Teaching Hospital of the Faculty of Veterinary Medicine, Lisbon, Portugal. Seventy-one horses were swabbed, and MRSA was identified by selective isolation on a chromogenic medium. Two S aureus isolates showed resistance to oxacillin (minimum inhibitory concentration >4 μg/mL) and contained the mecA gene. Both strains were isolated from purebred Lusitano horses that lived in farms with more than 20 equines. These MRSA strains represented two different clones: isolate FMVA3/10 was an MRSA sequence type ST5 with a staphylococcal cassette chromosome mec VI, coresistant to erythromycin and clindamycin; and isolate FMVA16/10 was sequence type ST398, with a staphylococcal cassette chromosome mec IV, coresistant to tetracycline, gentamicin, and trimethoprim. Isolate FMVA3/10 represents a human epidemic clone not previously reported among horses in Europe, which once again reinforces the fact that transmission of MRSA clones between horses and humans occurs. Isolate FMVA16/10 represents the first report of the detection of MRSA ST398 among horses in Portugal. Lusitano horses can carry animal and human MRSA in the nostrils, acting as reservoirs, which can potentially be transmitted to humans.     

Effect of susceptibility to enterotoxigenic Escherichia coli F4 and of dietary tryptophan on gut microbiota diversity observed in healthy young pigs

Healthy weaned pigs susceptible to enterotoxigenic Escherichia coli F4 (ETEC) require more tryptophan (Trp) to maximize their performance. This may be related to an effect on intestinal microbiota. We studied the intestinal bacterial diversity of healthy pigs with different susceptibility to ETEC and fed different Trp levels. Thirty-six littermate weaned pigs were selected to obtain a set potentially formed of 50% ETEC-susceptible and 50% non-susceptible pigs, based on a Mucin 4 gene polymorphism. Pigs were fed a diet with 0.17 (TrpL) or 0.22 (TrpH) standardized ileal digestible Trp:Lys ratio for 21 days. Slaughtered pigs were classified into non-susceptible, mildly susceptible, and susceptible, by testing ETEC adhesion to intestinal villi. Bacterial diversity in jejunum content was assessed by the 16S rRNA gene-targeted denaturing gradient gel electrophoresis (DGGE) fingerprinting analysis and expressed by the Shannon index. Susceptible pigs had a reduced bacterial diversity, particularly with TrpL diet (p = 0.003). The ETEC adhesion class affected the quantification of enterobacteria DNA (p = 0.027). One DGGE band, which referred to Clostridium bartlettii, was not evidenced in all the susceptible pigs; less DNA from this microbe was quantified by RT-PCR in the jejunum from TrpH susceptible pigs (p = 0.025) compared to TrpL. The gene expression for β-galactoside α-2,3-sialyltransferase 1 was higher in jejunal tissue of ETEC-susceptible pigs (p = 0.019). In studies on pig gut microbiota, the presence of intestinal receptors for ETEC should be considered because of their contribution to a reduced bacterial diversity. This effect could be partially reversed by dietary Trp addition.     

Interaction between attaching-effacing Escherichia coli O26:K60 and O157:H7 in young lambs

Recent surveys have shown that Escherichia coli O26 is prevalent in ruminants compared with E. coli O157. These serogroups share common colonisation factors and we hypothesised that prior colonisation by E. coli O26 may show reduced colonisation by E. coli O157. To test this hypothesis, strains of E. coli O26:K60 and O157:H7 were tested in competitive in vitro and in vivo studies. Using an established 6-week-old lamb model, an experimental group of lambs was dosed orally with E. coli O26:K60 and then E. coli O157:H7 four days later. The faecal shedding of O26:K60 and O157:H7 organisms from this experimental group was compared with that from animals dosed with either O26:K60 alone or O157:H7 alone. Shedding data indicated that counts for O157:H7 were unaffected by the competition from O26:K60, whereas the O26:K60 counts were lower when competing with O157:H7.     

Field trial evaluating changes in prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers medicated with enrofloxacin, apramycin and amoxicillin

The present study investigates, under field conditions, the influence of antimicrobial administration on prevalence and patterns of antimicrobial resistance among Escherichia coli and Enterococcus spp. isolated from growing broilers. For this purpose, a group of 16,000 commercial broiler chickens was treated with enrofloxacin from day 1 to day 3, gentamicin from day 19 to day 21, and ampicillin from day 26 to day 28. A control group of 16,000 broilers was placed in the same controlled environment poultry house. Fecal (from both groups) and feed samples were collected at regular intervals. Few E. coli isolates were obtained from either farm environment or poultry feed samples, while enterococci were found to be ubiquitous among these samples. The frequency of resistance against most antimicrobials tested was significantly higher (P < 0.05) in E. coli isolated from broilers receiving intermittent antimicrobial pressure than that from non-medicated broilers, whereas in enterococci these differences were only observed among structurally related antimicrobial drugs and over a short period of time. By the time the broilers reached market age (33 days), several multi-resistant E. coli and enterococci were detected in the feces of the medicated group. Results suggest that antimicrobial resistance in E. coli was mainly medication-dependent, whereas among enterococci, changes observed over time were apparently influenced by factors apart from antimicrobial exposure, namely the resistance organisms previously present in farm environment and those present in feedstuffs.     

Occurrence of weak mutators among avian pathogenic Escherichia coli (APEC) isolates causing salpingitis and peritonitis in broiler breeders

A collection of 46 avian pathogenic Escherichia coli (APEC) isolates was examined for the presence of mutators by determining the rate of mutation to rifampicin resistance. The collection included 34 E. coli isolates obtained in pure culture from chronic lesions of salpingitis and peritonitis in 34 broiler breeders, of which 12 were associated with the development of secondary septicemia. Twelve additional isolates were obtained from a clonal outbreak (ST95) of E. coli peritonitis syndrome (EPS), the lesions of which changed gradually over time into a subacute/chronic form. The hypothesis of the present study was that mutation rates would be higher for chronic infection isolates than for isolates from acute infections/exacerbations. The distribution of mutation rates followed a pattern similar to that found for other clinical isolates of E. coli, with a modal/median value of 1.47 × 10⁻⁸. Of the 46 isolates, 24% (n = 11) were weakly hypermutable (2.00 × 10⁻⁸ ≤ μ < 2.00 × 10⁻⁷), however, no strong mutators were detected (μ ≥ 2.00 × 10⁻⁷). Chronic salpingitis isolates had the highest proportion (45%, P = 0.001) of weak mutators and also, significantly higher mutation rates (P = 0.003) compared to isolates that caused septicemia (4%). In addition, mutation rates were significantly lower among ST95 isolates (P < 0.0005), and among isolates from the same clonal group as ST95 (P = 0.027), when compared to isolates from other groups. Although a clear association with the time phase of infection (as lesions of EPS became more chronic) could not be observed (ρ = 0.523, P = 0.081), a higher frequency of weak mutators among chronic infection isolates suggests that increased mutation rates play a role in adaptation of APEC to long-term persistence in an infected host environment.     

食品动物源大肠杆菌多重耐药性监测

为掌握畜禽源大肠杆菌的耐药情况,了解其对各种抗菌药物的敏感程度,自广东地区临床分离鉴定出294株食品动物源大肠杆菌,采用琼脂稀释法测定294株大肠杆菌对14种抗菌药物的敏感性。结果显示,食品动物源大肠杆菌对各抗菌药的敏感性不同,且多重耐药现象较严重;受试大肠杆菌对四环素和复方新诺明的耐药率较高,均达到80%以上;对第3代头孢菌素类耐药率相对较低。     

Prevalence and characterization of Staphylococcus aureus and Staphylococcus pseudintermedius isolated from companion animals and environment in the veterinary teaching hospital in Zambia,Africa

The Republic of Zambia consists of only one veterinary teaching school at the University of Zambia (UNZA) where students and veterinarians are exposed to many bacterial pathogens including Staphylococcus aureus (SA) and Staphylococcus pseudintermedius (SP). The aim of this study was the characterization and antimicrobial susceptibility profile of eleven SA and 48 SP isolates from the veterinary hospitals’ in- and outpatients and the environment. No isolate was resistant to cefoxitin by disk diffusion test and the corresponding resistance gene mecA was not found. In contrast, the resistance rates of SA to penicillin (63.6%) and trimethoprim-sulfamethoxazole (36.4%) and SP to penicillin (52.1%) and tetracycline (25.0%) were the highest. A variety of sequence types (STs) without a predominant type including numerous novel types were determined, especially for SP (39.6%). The spa typing provided a clonal assignment for all SAs (100%) and 24 SPs (50%) with three and two novel types, respectively. This study has provided an overview of SA and SP in the veterinary teaching hospital at UNZA. However, for a better understanding of these species regarding pathogenesis and transmission, further studies on the prevalence and characterization of SA and SP from veterinary staff, pet owners, and farm animals in Zambia is needed.     

Effect of bacitracin on tetracycline resistance in Escherichia coli and Salmonella

Tetracyclines and bacitracin are used extensively as a growth promotant and a therapeutic, respectively in livestock. In this study, we test whether there is an interaction between bacitracin and tetracycline that can select for an increase in microbial tetracycline sensitivity, using Escherichia coli and Salmonella as a model. We found via in vitro studies that bacitracin at sublethal concentrations preferentially selects for outgrowth of tetracycline-sensitive bacteria from among a population of tetracycline-resistant and tetracycline-sensitive E. coli and Salmonella strains. Most of the bacterial strains employed in our study were shown by PCR to possess the tetracycline resistance gene tet(A) or tet(C), either of whose activity is associated with tetracycline efflux. Bacitracin could be substituted for the lipophylic chelator, fusaric acid, used as a positive selective agent for tetracycline sensitivity. Together, these observations suggest that bacitracin-mediated selection for tetracycline sensitivity alters the function of tetracycline efflux proteins. Using bacitracin and tetracycline simultaneously may improve the effectiveness of the antibiotics, while decreasing the risk of selecting for a population of tetracycline-resistant microorganisms.