Antimicrobial resistant bacteria in wild mammals and birds: a coincidence or cause for concern?

Background

The emergence and dissemination of antimicrobial resistance (AMR) is a growing concern to public and animal health. The contribution attributable to wildlife remains unclear. In this study two unrelated wildlife species herring gulls (Larus argentatus) and a hybrid deer (Cervus elaphus x Cervus nippon) were investigated for the presence of Escherichia coli expressing an AMR phenotype.

Findings

Bacterial isolates resistant to β-lactam compounds were identified in both animal species and the production of functional β-lactamase was confirmed using nitrocefin. The prevalence of resistant isolates was higher in herring gulls (87%) compared to deer (31%). Resistance to this class of antibiotic was found only in non-pathogenic E. coli in herring gulls and in both pathogenic and non-pathogenic E. coli strains in deer.

Conclusions

The presence of AMR in wildlife has implications for public health, food safety and potable water source protection among others.     

Diversity of Plasmids and Antimicrobial Resistance Genes in Multidrug‐Resistant Escherichia coli Isolated from Healthy Companion Animals

The presence and transfer of antimicrobial resistance genes from commensal bacteria in companion animals to more pathogenic bacteria may contribute to dissemination of antimicrobial resistance. The purpose of this study was to determine antimicrobial resistance gene content and the presence of genetic elements in antimicrobial resistant Escherichia coli from healthy companion animals. In our previous study, from May to August, 2007, healthy companion animals (155 dogs and 121 cats) from three veterinary clinics in the Athens, GA, USA area were sampled and multidrug‐resistant E. coli (n = 36; MDR, resistance to ≥2 antimicrobial classes) were obtained. Of the 25 different plasmid replicon types tested by PCR, at least one plasmid replicon type was detected in 94% (34/36) of the MDR E. coli; four isolates contained as many as five different plasmid replicons. Nine replicon types (FIA, FIB, FII, I2, A/C, U, P, I1 and HI2) were identified with FIB, FII, I2 as the most common pattern. The presence of class I integrons (intI) was detected in 61% (22/36) of the isolates with eight isolates containing aminoglycoside‐ and/or trimethoprim‐resistance genes in the variable cassette region of intI. Microarray analysis of a subset of the MDR E. coli (n = 9) identified the presence of genes conferring resistance to aminoglycosides (aac, aad, aph and strA/B), β‐lactams (ampC, cmy, tem and vim), chloramphenicol (cat), sulfonamides (sulI and sulII), tetracycline [tet(A), tet(B), tet(C), tet(D) and regulator, tetR] and trimethoprim (dfrA). Antimicrobial resistance to eight antimicrobials (ampicillin, cefoxitin, ceftiofur, amoxicillin/clavulanic acid, streptomycin, gentamicin, sulfisoxazole and trimethoprim‐sulfamethoxazole) and five plasmid replicons (FIA, FIB, FII, I1 and I2) were transferred via conjugation. The presence of antimicrobial resistance genes, intI and transferable plasmid replicons indicate that E. coli from companion animals may play an important role in the dissemination of antimicrobial resistance, particularly to human hosts during contact.     

Antimicrobial resistance of Salmonella and generic Escherichia coli isolated from surface water samples used for recreation and a source of drinking water in southwestern Ontario,Canada

Antimicrobial resistance (AMR) in the aquatic environment represents an important means of introduction and dissemination of resistance genes, and presence of resistant pathogens in surface waters may pose a public health concern to recreational and drinking water users. The purpose of this study was to explore antimicrobial resistance patterns in water samples collected from the Grand River watershed (southwestern Ontario, Canada) to describe the composition, trends and potential risks of AMR in the aquatic environment. As part of FoodNet Canada and the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS), stream water samples were collected bi‐weekly from sampling sites within the Grand River watershed in the Waterloo, Ontario sentinel site and tested for the presence and antimicrobial susceptibility of Salmonella spp. (2005–2013) and generic Escherichia coli (2012–2013). Of all samples tested, 16% of Salmonella and 22% of E. coli isolates were resistant to at least one antimicrobial, including three Salmonella isolates and two E. coli isolates that were resistant to Category I antimicrobials, which are classified as very high importance for the treatment of serious bacterial infections in humans. The greatest proportion of resistant E. coli isolates were observed from the river site upstream of the drinking water intake, while the greatest proportion of resistant Salmonella isolates were from sites upstream in the watershed, and at one recreational water site. Salmonella resistance trends remained fairly stable between 2007 and 2013, with the exception of streptomycin and tetracycline which increased in 2010 and 2013. Continued surveillance of antimicrobial resistance patterns and exploration of risk factor data will allow for a better understanding of resistance transmission in the aquatic environment.     

Investigations of the Distribution and Persistence of Salmonella and Ciprofloxacin‐Resistant Escherichia coli in Turkey Hatcheries in the UK

This study aimed at gaining information on the presence of Salmonella in UK turkey hatcheries and possible epidemiological links between breeding farms, hatcheries and finishing farms. The presence of ciprofloxacin‐resistant E. coli in hatchery samples, as well as in faecal samples from farms, and trends in occurrence of resistance were also investigated. Over a 2 year‐period, four British turkey hatcheries were visited and intensively sampled for the presence of Salmonella and ciprofloxacin‐resistant E. coli. In two hatcheries, a link could be demonstrated between the presence of certain Salmonella serovars in the hatcheries and on breeding and finishing farms. Within the hatcheries, serovars linked to breeding farms were found more frequently in the poult processing and dispatch areas, whereas serovars identified as ‘resident hatchery contaminants’ were predominantly found inside the hatcher cabinets. Ciprofloxacin‐resistant isolates of S. Senftenberg were identified in one hatchery, which coincided with enrofloxacin treatment of some of the breeding flocks. Ciprofloxacin‐resistant E. coli was found in two hatcheries, and the majority of these isolates showed multidrug resistance.     

Molecular and Statistical Analysis of Campylobacter spp. and Antimicrobial‐Resistant Campylobacter Carriage in Wildlife and Livestock from Ontario Farms

The objectives of this study were to (i) compare the carriage of Campylobacter and antimicrobial‐resistant Campylobacter among livestock and mammalian wildlife on Ontario farms, and (ii) investigate the potential sharing of Campylobacter subtypes between livestock and wildlife. Using data collected from a cross‐sectional study of 25 farms in 2010, we assessed associations, using mixed logistic regression models, between Campylobacter and antimicrobial‐resistant Campylobacter carriage and the following explanatory variables: animal species (beef, dairy, swine, raccoon, other), farm type (swine, beef, dairy), type of sample (livestock or wildlife) and Campylobacter species (jejuni, coli, other). Models included a random effect to account for clustering by farm where samples were collected. Samples were subtyped using a Campylobacter‐specific 40 gene comparative fingerprinting assay. A total of 92 livestock and 107 wildlife faecal samples were collected, and 72% and 27% tested positive for Campylobacter, respectively. Pooled faecal samples from livestock were significantly more likely to test positive for Campylobacter than wildlife samples. Relative to dairy cattle, pig samples were at significantly increased odds of testing positive for Campylobacter. The odds of isolating Campylobacter jejuni from beef cattle samples were significantly greater compared to dairy cattle and raccoon samples. Fifty unique subtypes of Campylobacter were identified, and only one subtype was found in both wildlife and livestock samples. Livestock Campylobacter isolates were significantly more likely to exhibit antimicrobial resistance (AMR) compared to wildlife Campylobacter isolates. Campylobacter jejuni was more likely to exhibit AMR when compared to C. coli. However, C. jejuni isolates were only resistant to tetracycline, and C.  coli isolates exhibited multidrug resistance patterns. Based on differences in prevalence of Campylobacter spp. and resistant Campylobacter between livestock and wildlife samples, and the lack of similarity in molecular subtypes and AMR patterns, we concluded that the sharing of Campylobacter species between livestock and mammalian wildlife was uncommon.     

Circulation of emerging NDM‐5‐producing Escherichia coli among humans and dogs in Egypt

The emergence of NDM‐producing Escherichia coli has considerably threatened human and animal health worldwide. This study describes for the first time in Egypt, the draft genome sequences of emerging NDM‐5‐producing E. coli from humans and dogs, and investigates genetic relatedness between isolates from both sources. Two E. coli from human urine and seven from environmental clinical samples of dogs exhibited resistance to carbapenems and harbouring bla_NDM were subjected to Illumina Miseq whole‐genome sequencing (WGS). Assembly and analysis of the reads were performed to identify resistance genes, multilocus sequence types (MLST), plasmid replicon types (Inc) and insertion sequences (IS) of the bla_NDM region; core genome MLST (cgMLST) analysis was also performed. Two different NDM alleles were identified; bla_NDM‐5 in E. coli HR119 from the urine of a healthy person and environmental samples of dogs, and bla_NDM‐1 in E. coli HR135 from a human patient's urine. Multiple mobilizable resistance genes to different antimicrobial classes were identified except the colistin resistance gene, mcr. E. coli isolates from humans and dogs were assigned to different sequence types (STs). Using cgMLST, dog isolates clustered together with only 1–2 allellic differences; however, human E. coli showed 1,978 different allelles compared with dog isolates. Plasmidfinder results indicated the presence of an IncX3 replicon in bla_NDM‐5‐producing E. coli; however, bla_NDM‐1 was linked to IncCoIKP3. Notably, the NDM region (3 Kb) in all isolates from humans and dogs was highly similar with variable flanking sequences that represented different IS elements. This study reports the first emergence of NDM‐5‐producing E. coli from dogs in Egypt that shared some genetic features with human isolates and could be considered potential public health threats.     

Low prevalence of zoonotic multidrug‐resistant bacteria in veterinarians in a country with prudent use of antimicrobials in animals

The occurrence of multidrug‐resistant zoonotic bacteria in animals has been increasing worldwide. Working in close contact with livestock increases the risk of carriage of these bacteria. We investigated the occurrence of extended‐spectrum beta‐lactamase (ESBL) and plasmidic AmpC beta‐lactamase producing Enterobacteriaceae (ESBL/pAmpC‐PE) and livestock‐associated methicillin‐resistant Staphylococcus aureus (LA‐MRSA) in Finnish veterinarians (n = 320). In addition to microbiological samples, background information was collected. Bacterial whole genome sequencing was performed to deduce sequence types (STs), spa types and resistance genes of the isolates. In total, 3.0% (9/297) of the veterinarians carried ESBL producing Escherichia coli, with one ESBL producing E. coli isolate producing also AmpC. Seven different STs, sequences of several different plasmid groups as well as several different bla_ESBL/pAmpC genes existed in different combinations. No carbapenemase or colistin resistance genes were detected. MRSA was detected in 0.3% (1/320) of the samples. The strain belonged to LA‐MRSA clonal complex (CC) 398 (ST398, spa type 011, lacking Panton‐Valentine leukocidin genes). In conclusion, this study shows low carriage of multidrug‐resistant zoonotic bacteria in Finnish veterinarians. However, finding LA‐MRSA for the first time in a sample from a veterinarian in a country with prudent use of animal antimicrobials and regarding the recent rise of LA‐MRSA on Finnish pig farms, a strong recommendation to protect people working in close contact with animals carrying LA‐MRSA CC398 is given. Further studies are needed to explain why the prevalence of LA‐MRSA in veterinarians is lower in Finland than in other European countries.     

Low Occurrence of Extended‐Spectrum β‐lactamase‐Producing Escherichia coli in Finnish Food‐Producing Animals

ESBL/AmpC‐producing Escherichia coli is increasingly isolated from humans and animals worldwide. The occurrence of ESBL/AmpC‐producing E. coli was studied in food‐producing animals in Finland, a country with a low and controlled use of antimicrobials in meat production chain. A total of 648 cattle, 531 pig, 495 broiler and 35 turkey faecal samples were collected from four Finnish slaughterhouses to determine the presence of extended‐spectrum β‐lactamase (ESBL/AmpC)‐producing E. coli. In addition, 260 broiler and 15 turkey samples were screened for carbapenemase‐producing E. coli. Susceptibility to different class of cephalosporins and meropenem was determined with disc diffusion tests according to the European Committee on Antimicrobial Susceptibility Testing (EUCAST). Determination of ESBL/AmpC production was performed with a combination disc diffusion test according to the recommendations of the European Food Safety Authority (EFSA). Plasmidic bla_ESBL/AmpC genes were characterized by polymerase chain reaction and sequencing. A collection of isolates producing AmpC enzyme but not carrying plasmidic bla_AmpC was analysed by PCR and sequencing for possible chromosomal ampC promoter area mutations. Altogether ESBL/AmpC‐producing E. coli was recovered from five cattle (0.8%), eight pig (1.5%) and 40 broiler samples (8.1%). No ESBL/AmpC‐producing E. coli was found in turkey samples. Carbapenem resistance was not detected. Altogether ESBL/AmpC‐producing E. coli was found on 4 (2.0%), 3 (4.5%) and 14 (25%) cattle, pig and broiler farms, respectively. From cattle samples 3 (27%) bla_CTX‐M‐1 and from broiler samples 13 (33%) bla_CTX‐M‐1 and 22 (55%) bla_CMY‐2 gene‐carrying isolates were detected. In pigs, no plasmidic bla_ESBL/AmpC gene‐carrying isolates were found. In all analysed isolates, the same mutations in the promoter region of chromosomal ampC were detected. The results showed low occurrence of ESBL/AmpC‐producing E. coli in Finnish food‐producing animals. In pigs, plasmidic bla_ESBL/AmpC‐carrying E. coli was not detected at all.     

Antimicrobial resistance of Escherichia coli isolates from canine urinary tract infections

This study determined the antimicrobial resistance profiles of Escherichia coli isolates from dogs with a presumptive diagnosis of urinary tract infection (UTI). Urine samples from 201 dogs with UTI diagnosed through clinical examination and urinalysis were processed for isolation of Escherichia coli. Colonies from pure cultures were identified by biochemical reactions (n=114) and were tested for susceptibility to 18 antimicrobials. The two most frequent antimicrobials showing resistance in Urinary E. coli isolates were oxytetracycline and ampicillin. Among the resistant isolates, 17 resistance patterns were observed, with 12 patterns involving multidrug resistance (MDR). Of the 69 tetracycline-resistant E. coli isolates, tet(B) was the predominant resistance determinant and was detected in 50.9% of the isolates, whereas the remaining 25.5% isolates carried the tet(A) determinant. Most ampicillin and/or amoxicillin-resistant E. coli isolates carried bla_TEM-1 genes. Class 1 integrons were prevalent (28.9%) and contained previously described gene cassettes that are implicated primarily in resistance to aminoglycosides and trimethoprim (dfrA1, dfrA17-aadA5). Of the 44 quinolone-resistant E. coli isolates, 38 were resistant to nalidixic acid, and 6 were resistant to nalidixic acid, ciprofloxacin and enrofloxacin. Chromosomal point mutations were found in the GyrA (Ser83Leu) and ParC (Ser80Ile) genes. Furthermore, the aminoglycoside resistance gene aacC2, the chloramphenicol resistant gene cmlA and the florfenicol resistant gene floR were also identified. This study revealed an alarming rate of antimicrobial resistance among E. coli isolates from dogs with UTIs.     

Antimicrobial resistance in Escherichia coli isolates from frugivorous (Eidolon helvum) and insectivorous (Nycteris hispida) bats in Southeast Nigeria,with detection of CTX-M-15 producing isolates

Thirty-five Escherichia coli isolates obtained from the liver, spleen and intestines of 180 frugivorous and insectivorous bats were investigated for antimicrobial resistance phenotypes/genotypes, prevalence of Extended-Spectrum beta-lactamase (ESBL) production, virulence gene detection and molecular typing. Eight (22.9 %) of the isolates were multidrug resistant (MDR). Two isolates were cefotaxime-resistant, ESBL-producers and harbored the bla_CTX-M-15 gene; they belonged to ST10184-D and ST2178-B1 lineages. tet(A) gene was detected in all tetracycline-resistant isolates while int1 (n = 8) and bla_TEM (n = 7) genes were also found. Thirty-three of the E. coli isolates were assigned to seven phylogenetic groups, with B1 (45.7 %) being predominant. Three isolates were enteropathogenic E. coli (EPEC) pathovars, containing the eae gene (with the variants gamma and iota), and lacking stx1/stx2 genes. Bats in Nigeria are possible reservoirs of potentially pathogenic MDR E. coli isolates which may be important in the ecology of antimicrobial resistance at the human-livestock-wildlife-environment interfaces. The study reinforces the importance of including wildlife in national antimicrobial resistance monitoring programmes.     

Antimicrobial Resistance Profiles and Clonal Relatedness of Canine and Feline Escherichia coli Pathogens Expressing Multidrug Resistance in the United States

Background: Antimicrobial resistance is increasing among Escherichia coli isolates associated with spontaneous infection in dogs and cats. Objectives: To describe E. coli resistance phenotypes and clonal relatedness and their regional prevalence. Animals: Isolates of E. coli (n = 376) collected from dogs and cats in the United States between May and September 2005. Methods: Isolates submitted from the South, West, Northeast, and Midwest regions of the United States were prospectively studied. Phenotype was based on E‐test susceptibility to 7 antimicrobials. Isolates were classified as no (NDR), single (SDR), or multidrug resistance (MDR). Clonal relatedness was determined by pulsed‐field gel electrophoresis (PFGE). Results: One hundred and ninety‐three (51%) isolates expressed resistance to at least 1 drug, yielding 42 phenotypes. SDR isolates (n = 84; 44%, 8 phenotypes), expressed resistance most commonly to amoxicillin (30%, n = 25) and least commonly to cefpodoxime (1%, n = 1). MDR isolates (n = 109; 56%, 31 phenotypes) were resistant to amoxicillin (96%, n = 105), amoxicillin‐clavulanate (85%, n = 93), and enrofloxacin (64%, n = 70); 18% (n = 20) were resistant to all drugs tested. The frequency of MDR did not differ regionally (P= .066). MDR minimum inhibitory concentrations (MICs) were 6‐fold higher than SDR MICs (P < .0001). Dendrograms of 91 isolates representing 25 phenotypes revealed 62 different PFGE profiles. Conclusions and Clinical Importance: E. coli strains spontaneously infecting dogs and cats are genetically and phenotypically diverse. Given the current prevalence of MDR among clinical isolates of E. coli in United States, implementation of a robust surveillance program is warranted.     

A repeated cross‐sectional study of the epidemiology of Campylobacter and antimicrobial resistant Enterobacteriaceae in free‐living Canada geese in Guelph,Ontario, Canada

From May through October 2016, we conducted a repeated cross‐sectional study examining the effects of temporal, spatial, flock and demographic factors (i.e. juvenile vs. adult) on the prevalence of Campylobacter and antimicrobial resistant Enterobacteriaceae among 344 fresh faecal samples collected from Canada geese (Branta canadensis) from four locations where birds nested in Guelph, Ontario, Canada. The overall prevalence of Campylobacter among all fresh faecal samples was 9.3% and was greatest in the fall when these birds became more mobile following the nesting season. Based on 40 gene comparative genomic fingerprinting (CGF40), the increase in prevalence noted in the fall was matched by an increase in the number of unique CGF40 subtypes identified. Resistance to colistin was detected most commonly, in 6% of Escherichia coli isolates, and was highest in the late summer months. All colistin‐resistant isolates were negative for the mcr‐1 to mcr‐5 genes; a chromosomal resistance mechanism (PmrB) was identified in all of these isolates. The prevalence of samples with E. coli exhibiting multi‐class resistance or extended spectrum beta‐lactamase was low (i.e. <2% of samples). The intra‐class correlation coefficients, estimated from the variance components of multilevel logistic regression models, indicated that the shedding of Campylobacter and antimicrobial resistant E. coli among geese within a flock (i.e. birds collected from the same site on the same day) was moderately correlated. Spatial, temporal, and spatiotemporal clusters identified using the spatial scan statistic, largely supported the findings from our multi‐level models. Salmonella was not isolated from any of the fresh faecal samples collected suggesting that its prevalence in this population of birds was very low.     

A Cross‐Sectional Study Examining the Prevalence and Risk Factors for Anti‐Microbial‐Resistant Generic Escherichia coli in Domestic Dogs that Frequent Dog Parks in Three Cities in South‐Western Ontario,Canada

Anti‐microbial resistance can threaten health by limiting treatment options and increasing the risk of hospitalization and severity of infection. Companion animals can shed anti‐microbial‐resistant bacteria that may result in the exposure of other dogs and humans to anti‐microbial‐resistant genes. The prevalence of anti‐microbial‐resistant generic Escherichia coli in the faeces of dogs that visited dog parks in south‐western Ontario was examined and risk factors for shedding anti‐microbial‐resistant generic E. coli identified. From May to August 2009, canine faecal samples were collected at ten dog parks in three cities in south‐western Ontario, Canada. Owners completed a questionnaire related to pet characteristics and management factors including recent treatment with antibiotics. Faecal samples were collected from 251 dogs, and 189 surveys were completed. Generic E. coli was isolated from 237 of the faecal samples, and up to three isolates per sample were tested for anti‐microbial susceptibility. Eighty‐nine percent of isolates were pan‐susceptible; 82.3% of dogs shed isolates that were pan‐susceptible. Multiclass resistance was detected in 7.2% of the isolates from 10.1% of the dogs. Based on multilevel multivariable logistic regression, a risk factor for the shedding of generic E. coli resistant to ampicillin was attending dog day care. Risk factors for the shedding of E. coli resistant to at least one anti‐microbial included attending dog day care and being a large mixed breed dog, whereas consumption of commercial dry and home cooked diets was protective factor. In a multilevel multivariable model for the shedding of multiclass‐resistant E. coli, exposure to compost and being a large mixed breed dog were risk factors, while consumption of a commercial dry diet was a sparing factor. Pet dogs are a potential reservoir of anti‐microbial‐resistant generic E. coli; some dog characteristics and management factors are associated with the prevalence of anti‐microbial‐resistant generic E. coli in dogs.     

Genetic Characterization of Antibiotic Resistance in Enteropathogenic Escherichia coli Carrying Extended‐Spectrum β‐Lactamases Recovered from Diarrhoeic Rabbits

A total of 52 Escherichia coli strains isolated from diarrhoeic rabbits were investigated for their enteropathogenic E. coli (EPEC) pathotype by PCR amplification of eae and bfp virulence genes. A total of 22 EPEC isolates were identified, serotyped and studied for antibiotic resistance and screened for the detection of extended‐spectrum β‐lactamases (ESBLs). The EPEC isolates belonged to three serogroups (O26, O92 and O103). The most common serogroup (O103:K‐:H2) was observed among 17 EPEC strains, the O92:K‐serogroup in three isolates (the antibiotic sensitive ones) and the remaining O26:K‐serogroup in two isolates (the ESBLs isolates). Resistances to ampicillin and tetracycline were the most frequent and detected followed by resistance to nalidixic acid, streptomycin, trimethoprim–sulphamethoxazole, cefoxitin, gentamicin and ciprofloxacin. All the isolates were sensitive for amikacin, ceftazidime, aztreonam, imipenem, chloramphenicol, tobramycin and amoxicillin + clavulanic acid. Two isolates recovered from two adult animals showed an intermediate susceptibility to cefotaxime, and a positive screening test for ESBL was demonstrated in both. The bla_TEM gene was demonstrated in the majority of ampicillin‐resistant isolates. The aac(3)‐II or aac(3)‐IV genes were detected in the four gentamicin‐resistant isolates. In addition, the aadA gene was detected in 60% of streptomycin‐resistant isolates. The tet(A) or tet(B) genes were identified in all tetracycline‐resistant isolates. A total of nine EPEC isolates showed the phenotype SXT‐resistant, and the sul1 and/or sul2 and/or sul3 genes were detected in all of them. Our findings showed that the molecular detection by the eae and bfp genes by PCR followed by serotyping is useful for monitoring trends in EPEC infections of rabbits allowing the identification of their possible reservoirs. The detection of genes involved in the resistance to antibiotics of different families in a relatively high proportion of faecal E. coli isolates of rabbits is of great interest and could be considered a serious public health problem.     

Prevalence of extended-spectrum beta-lactamase-producing Escherichia coli isolates in faecal samples of broilers

Seventy-six faecal samples were obtained from broilers at slaughterhouse level in Portugal. Samples were inoculated on cefotaxime-supplemented Levine agar plates. Cefotaxime-resistant Escherichia coli isolates were recovered from 32 samples (42.1%), obtaining a total of 34 E. coli isolates (one or two isolates per sample). Susceptibility to 16 antibiotics was studied by disk diffusion method, and 85% of the isolates presented a phenotype of multi-resistance that included antimicrobial agents of at least four different families. Extended-spectrum-beta-lactamases (ESBL) of the TEM and CTX-M groups were detected in 31 ESBL-positive E. coli isolates. Twenty-six isolates harboured the bla_TEM-52 gene and two of them also harboured bla_TEM-1b. The bla_CTX-M-14 gene was identified in three isolates (in association with bla_TEM-1b in one of them), and bla_CTX-M-32 was demonstrated in two additional isolates. Three of the 34 cefotaxime-resistant isolates (9%) did not produce ESBLs, and two of them presented mutations at positions −42 (C → T), −18 (G → A), −1 (C → T), and +58(C → T) of the promoter/attenuator region of ampC gene. tet(A) and/or tet(B) genes were detected in all 34 tetracycline-resistant isolates, aadA in all 26 streptomycin-resistant isolates; cmlA in 3 of 6 chloramphenicol-resistant isolates, and aac(3)-II or aac(3)-I + aac(3)-IV genes in all 4 gentamicin-resistant isolates. Different combinations of sul1, sul2 and sul3 genes were demonstrated among the 22 trimethoprim–sulfamethoxazole-resistant isolates. Amino acid changes in GyrA and ParC proteins were identified in all 18 ciprofloxacin-resistant isolates. The results of this study indicate that the intestinal tract of healthy poultry is a reservoir of ESBL-positive E. coli isolates.     

Detection of multidrug resistant Escherichia coli in the ovaries of healthy broiler breeders with emphasis on extended-spectrum β-lactamases producers

In the last few years, antimicrobial resistant (AMR) Escherichia coli have been detected in newborn chickens suggesting their vertical transmission from breeding birds to their offspring. However, little is known about the presence of AMR E. coli in the reproductive organs of broiler breeders. The aim of this study was to investigate the presence of E. coli in the ovaries of healthy broiler breeders and to study their antimicrobial resistance. Samples from broiler breeders (n = 80) collected from 80 different broiler breeder flocks were included in this study. Antibiotic susceptibility testing was performed using disk diffusion method according to Clinical and Laboratory Standards Institute guidelines. Minimal inhibitory concentrations (MICs) of five antimicrobial agents were determined by Etest. PCR and sequencing were used to detect the bla_ESBL genes. E. coli were detected in the ovaries of thirty seven out of 80 (46.25%) sampled flocks. High levels of resistance to various first-line antimicrobial agents were recorded in E. coli isolates. This study showed that 89.18% of E. coli isolates were multidrug resistant (MDR). Furthermore, MDR extended-spectrum β-lactamases (ESBL)-producing E. coli were detected in the ovaries of four different broiler breeder flocks. Molecular characterization revealed that three isolates harboured bla_CTX-M-1 gene and one isolate expressed bla_SHV-12 gene. In addition, one bla_CTX-M-1 -producing E. coli co-harboured the bla_TEM-1 gene. These findings would contribute to a better epidemiological understanding of MDR E. coli for improve existing preventive strategies in order to reduce the dissemination of antimicrobial resistance in the broiler production system.     

Pathotyping and antimicrobial susceptibility testing of Escherichia coli isolates from neonatal calves

Neonatal calf mortality is a major concern to livestock sector worldwide. Neonatal calf diarrhoea (NCD), an acute severe condition causes morbidity and mortality in calves. Amongst various pathogens involved in NCD, E. coli is considered as one of the major causes. The study was targeted to characterize E. coli isolates from neonatal calves for diarrhoeagenic Escherichia coli (DEC) types (pathotyping), antimicrobial resistance (AMR) profiling and to correlate with epidemiological parameters. From neonates, a total of 113 faecal samples were collected, out of that 308, lactose fermenting colonies were confirmed as E. coli. Pathotypable isolates (12.3%) were represented by STEC (6.1%), EPEC (2.9%), ETEC (1.9%), EAEC (0.9%) and EHEC (0.3%). Occurrence of STEC was more in non-diarrhoeic calves, whereas ETEC was observed more in diarrhoeic calves. EPEC occurrence was observed in both diarrhoeic and non-diarrhoeic calves. Fishers extract test showed no significant association for occurrence of DEC types to type of dairies, health status, species, breed, age and sex of neonatal calves. Two hundred and eighty isolates were tested for antimicrobial susceptibility. The isolates showed maximum resistance towards ampicillin (55.4%) followed by tetracycline (54.3%), while minimum resistance was observed towards meropenem (2.5%). Multidrug resistant E. coli isolates were found to be 139 (49.6%), and Extended-spectrum beta-lactamase (ESBL) producers were 120 (42.9%). DEC pathotypes like STEC, ETEC, EHEC and EAEC that are also multidrug resistant present in neonatal calves have zoonotic potential and hence are of public health significance.

     

Cross-sectional study of antimicrobial-resistant bacteria in horses. Part 1: Prevalence of antimicrobial-resistant Escherichia coli and methicillin-resistant Staphylococcus aureus

Reasons for performing study: The increasing prevalence of antimicrobial‐resistant bacteria such as methicillin‐resistant Staphylococcus aureus (MRSA) and antimicrobial‐resistant Escherichia coli represents a significant problem. However, the carriage of such bacteria by horses in the UK has not been well characterised. Objectives: To estimate the prevalence of nasal carriage of MRSA and faecal carriage of antimicrobial‐resistant E. coli amongst horses in the general equine community of the mainland UK. Methods: A cross‐sectional study of horses recruited by 65 randomly selected equine veterinary practices was conducted, with nasal swabs and faecal samples collected. Faecal samples were cultured for antimicrobial‐resistant E. coli. Nasal swabs were cultured for staphylococcal species; methicillin‐resistant isolates identified as S. aureus were characterised by SCCmec and spa gene typing. Multilevel logistic regression models were used to calculate prevalence estimates with adjustment for clustering at practice and premises levels. Spatial variation in risk of antimicrobial resistance was also examined. Results: In total, 650 faecal samples and 678 nasal swabs were collected from 692 horses located on 525 premises. The prevalence of faecal carriage of E. coli with resistance to any antimicrobial was 69.5% (95% CI 65.9–73.1%) and the prevalence of extended‐spectrum β‐lactamase (ESBL)‐producing E. coli was 6.3% (95% CI 4.1–9.6%). The prevalence of nasal carriage of MRSA was 0.6% (95% CI 0.2–1.5%). Spatial analysis indicated variation across the UK for risk of carriage of resistant and multidrug‐resistant (resistant to more than 3 antimicrobial classes) E. coli. Conclusions and potential relevance: Carriage of MRSA by horses in the community appears rare, but the prevalence of antimicrobial‐resistant E. coli (including ESBL‐producing E. coli) is higher. A high prevalence of antimicrobial‐resistant bacteria could have significant health implications for the horse population of the UK.     

Changes in the prevalence,genotypes and antimicrobial resistance phenotypes of non‐typhoidal Salmonella recovered from mail‐order hatchling poultry sold at US feed stores, 2013–2015

Every year, multiple outbreaks of salmonellosis in humans are linked to contact with mail‐order chicks and ducks. The objective of this study was to describe the temporal changes in the prevalence of serovars, genotypes and antimicrobial resistance (AMR) phenotypes of non‐typhoidal Salmonella (NTS) recovered from shipped boxes of mail‐order hatchling poultry in the United States during 2013 to 2015. In each year, a sample of feed stores belonging to a single national chain participated in the study. The store employees submitted swabs or hatchling pads from hatchling boxes and shipment tracking information of the arriving boxes to the investigators. NTS was cultured from the samples and isolates were sent to the National Veterinary Services Laboratories (Ames, IA) for serotyping, pulsed‐field gel electrophoresis (PFGE) and AMR phenotyping. The PFGE patterns of Salmonella serovars isolated from hatchling boxes were compared with those from human outbreaks of salmonellosis linked to live poultry contact. The box‐level prevalence of NTS was significantly higher in 2015 compared to 2014. Also, the population of Salmonella serovars recovered in 2015 was more diverse and substantially different from those recovered in the previous two years. Of PFGE patterns recovered from hatchling boxes, seven distinct patterns in 2015, three in 2014 and four in 2013 were indistinguishable from the PFGE patterns of human outbreaks‐associated strains in the respective years. Importantly, a significant positive correlation was found between the box‐level prevalence of PFGE patterns and the number of human illnesses associated with the same patterns. Also, the proportion of multidrug‐resistant isolates was higher in 2014 and 2015 compared to that in 2013. The results demonstrate that shipments of mail‐order hatchling poultry are frequently contaminated with Salmonella genotypes indistinguishable from human outbreaks‐associated strains each year, and control efforts at hatchery level are likely to have an important public health impact.     

Antimicrobial Resistance Profiles and Diversity in Salmonella from Humans and Cattle, 2004–2011

Analysis of long‐term anti‐microbial resistance (AMR) data is useful to understand source and transmission dynamics of AMR. We analysed 5124 human clinical isolates from Washington State Department of Health, 391 cattle clinical isolates from the Washington Animal Disease Diagnostic Laboratory and 1864 non‐clinical isolates from foodborne disease research on dairies in the Pacific Northwest. Isolates were assigned profiles based on phenotypic resistance to 11 anti‐microbials belonging to eight classes. Salmonella Typhimurium (ST), Salmonella Newport (SN) and Salmonella Montevideo (SM) were the most common serovars in both humans and cattle. Multinomial logistic regression showed ST and SN from cattle had greater probability of resistance to multiple classes of anti‐microbials than ST and SN from humans (P < 0.0001). While these findings could be consistent with the belief that cattle are a source of resistant ST and SN for people, occurrence of profiles unique to cattle and not observed in temporally related human isolates indicates these profiles are circulating in cattle only. We used various measures to assess AMR diversity, conditional on the weighting of rare versus abundant profiles. AMR profile richness was greater in the common serovars from humans, although both source data sets were dominated by relatively few profiles. The greater profile richness in human Salmonella may be due to greater diversity of sources entering the human population compared to cattle or due to continuous evolution in the human environment. Also, AMR diversity was greater in clinical compared to non‐clinical cattle Salmonella, and this could be due to anti‐microbial selection pressure in diseased cattle that received treatment. The use of bootstrapping techniques showed that although there were shared profiles between humans and cattle, the expected and observed number of profiles was different, suggesting Salmonella and associated resistance from humans and cattle may not be wholly derived from a common population.