Phenotypic and Genotypic Study of Gelatinase and β‐Haemolysis Activities in Faecal Enterococci of Poultry in Portugal

The detection of gelatinase and β‐haemolysis activities was carried out in 83 faecal enterococci (43 Enterococcus faecalis, 33 E. faecium, five E. durans and two E. hirae) of poultry origin. In addition, the presence of genes of the gelE–fsrABC locus and of the cyl operon (cylL_L, cylL_S, cylA, cylB and cylM) were studied by polymerase chain reaction and correlated with gelatinase and β‐haemolysis production, respectively. Most of our E. faecalis isolates were gelatinase‐positive (88%), being this activity not frequent in the other enterococcal species (2.5%). Only one of the 33 E. faecium isolates showed a positive gelatinase reaction. All enterococci that showed gelatinase activity harboured the gelE and fsrABC genes, although these genes were also detected in four E. faecalis and one E. durans gelatinase‐negative isolates. Most of our non‐E. faecalis gelatinase‐negative isolates did not harbour gelE–fsrABC genes. A high proportion of faecal enterococci of poultry origin harboured genes of the cyl operon (71%), although only 7% contained the five cyl tested genes (all of them E. faecalis). Only one isolate of our series could express β‐haemolysis, harbouring the whole cyl operon. The cylL_S genotype was the most prevalent in our enterococci (39%) and also the most prevalent among our E. faecalis isolates (60%). Other genotypes detected were the following ones (% of enterococci): cylA + cylB + cylM (13%), cylL_L + cylA (4%), cylL_L (4%), cylL_L + cylA + cylB + cylM (2%), cylL_L + cylA + cylM (1%) and cylA + cylM (1%). Both phenotypic and genotypic assays are important to evaluate the virulence potential of enterococci.     

Antimicrobial resistance and virulence profile of enterococci isolated from poultry and cattle sources in Nigeria

This study investigated the occurrence, antimicrobial resistance and virulence of Enterococcus from poultry and cattle farms. Three hundred and ninety samples: cloacal/rectal swabs (n?=?260) and manure (n?=?130] were processed for recovery of Enterococcus species. Standard bacteriological methods were used to isolate, identify and characterize Enterococcus species for antimicrobial susceptibility and expression of virulence traits. Detection of antibiotic resistance and virulence genes was carried out by polymerase chain reaction. Enterococcus was recovered from 167 (42.8%) of the 390 samples tested with a predominance of Enterococcus faecium (27.7%). Other species detected were Enterococcus gallinarum, Enterococcus faecalis, Enterococcus hirae, Enterococcus raffinosus, Enterococcus avium, Enterococcus casseliflavus, Enterococcus mundtii and Enterococcus durans. All the isolates tested were susceptible to vancomycin, but resistance to tetracycline, erythromycin, ampicillin and gentamicin was also observed among 61.0, 61.0, 45.1 and 32.7% of the isolates, respectively. Sixty (53.1%) of the isolates were multidrug resistant presenting as 24 different resistance patterns with resistance to gentamicin-erythromycin-streptomycin-tetracycline (CN-ERY-STR-TET) being the most common (n?=?11) pattern. In addition to expression of virulence traits (haemolysin, gelatinase, biofilm production), antibiotic resistance (tetK, tetL, tetM, tetO and ermB) and virulence (asa1, gelE, cylA) genes were detected among the isolates. Also, in vitro transfer of resistance determinants was observed among 75% of the isolates tested. Our data revealed poultry, cattle and manure in this area are hosts to varying Enterococcus species harbouring virulence and resistance determinants that can be transferred to other organisms and also are important for causing nosocomial infection.     

Prevalence and antimicrobial resistance among Escherichia coli and Salmonella in Ontario smallholder chicken flocks

Surveillance is an important component of an overall strategy to address antimicrobial resistant bacteria in food animals and the food chain. The poultry market has many points of entry into the Canadian food chain, and some production practices are underrepresented in terms of surveillance. For example, pathogen carriage and antimicrobial resistance surveillance data are limited in smallholder chicken flocks raised for slaughter at provincially inspected abattoirs. In Canada, antimicrobial resistance in Escherichia coli and Salmonella isolated from commercial broiler chicken flocks, slaughtered at federally inspected abattoirs, is monitored by the Canadian Integrated Program for Antimicrobial Resistance Surveillance (CIPARS ). The objective of this study was to establish baseline information of antimicrobial resistance presence in E. coli and Salmonella isolated from smallholder flocks in Ontario, utilizing CIPARS collection and isolation methodologies, and to compare findings with CIPARS federally inspected abattoir data from Ontario, Canada. Five chickens per flock were sampled from 205 smallholder flocks. Of 1,025 samples, the E. coli prevalence was 99% (1,022/1,025), and 47% (483/1,022) of positive E. coli isolates were resistant to one or more of the 14 antimicrobials. Furthermore, as compared to results reported for the CIPARS commercial flocks, E. coli isolates from smallholder flocks had significantly lower resistance prevalence to six of 14 individual antimicrobials. Recovery of E. coli did not differ between federally inspected and provincially inspected flocks. Salmonella prevalence at the bird level in smallholder flocks was 0.3% (3/1,025), significantly lower (p  ? 0.0001, 95% CI 0.080%–0.86%) than federally inspected commercial flocks. The overall differences found between the commercial and smallholder flocks may be explained by differences in poultry husbandry practices and hatchery sources.     

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,Virulence Genes and Antibiotic Resistance of Enteropathogenic Escherichia coli (EPEC) from Twelve Bovine Farms in the North‐East of Ireland

Cattle faecal samples (n = 480) were collected from a cluster of 12 farms, and PCR screened for the presence of the intimin gene (eae). Positive samples were cultured, and colonies were examined for the presence of eae and verocytotoxin (vtx) genes. Colonies which were positive for the intimin gene and negative for the verocytotoxin genes were further screened using PCR for a range of virulence factors including bfpA, espA, espB, tir ehxA, toxB, etpD, katP, saa, iha, lpfA_{O157/OI‐141} and lpfA_{O157/OI‐154.} Of the 480 faecal samples, 5.8% (28/480) were PCR positive, and one isolate was obtained from each. All 28 isolates obtained were bfpA negative and therefore atypical EPEC (aEPEC). The serotypes detected included O2:H27, O8:H36, O15:H2, O49:H+, O84:H28, O105:H7 and O132:H34 but half of the isolates could not be serogrouped using currently available antisera. Twenty‐two (79%) of the isolates carried the tir gene but only 25% were espB positive, and all other virulence genes tested for were scarce or absent. Several isolates showed intermediate resistance to ciprofloxacin, kanamycin, nalidixic acid, minocycline and tetracycline; full resistance to nalidixic acid or tetracycline with one isolate (O?:H8) displaying resistance to aminoglycosides (kanamycin and streptomycin), quinolones (nalidixic acid) and sulphonamides. This study provides further evidence that cattle are a potential source of aEPEC and add to the very limited data currently available on virulence genes and antibiotic resistance in this pathogenic E. coli group in animals.     

Drug use and antimicrobial resistance among Escherichia coli and Enterococcus spp. isolates from chicken and turkey flocks slaughtered in Quebec,Canada

An observational study was conducted of chicken and turkey flocks slaughtered at federal processing plants in the province of Quebec, Canada. The objectives were to estimate prevalence of drug use at hatchery and on farm and to identify antimicrobial resistance (AMR) in cecal Escherichia coli and Enterococcus spp. isolates and factors associated with AMR. Eighty-two chicken flocks and 59 turkey flocks were sampled. At the hatchery, the most used antimicrobial was ceftiofur in chickens (76% of flocks) and spectinomycin in turkeys (42% of flocks). Virginiamycin was the antimicrobial most frequently added to the feed in both chicken and turkey flocks. At least 1 E. coli isolate resistant to third-generation cephalosporins was present in all chicken flocks and in a third of turkey flocks. Resistance to tetracycline, streptomycin, and sulfisoxazole was detected in > 90% of flocks for E. coli isolates. Antimicrobial resistance (AMR) was observed to bacitracin, erythromycin, lincomycin, quinupristin-dalfopristin, and tetracycline in both chicken and turkey flocks for Enterococcus spp. isolates. No resistance to vancomycin was observed. The use of ceftiofur at hatchery was significantly associated with the proportion of ceftiofur-resistant E. coli isolates in chicken flocks. In turkey flocks, ceftiofur resistance was more frequent when turkeys were placed on litter previously used by chickens. Associations between drug use and resistance were observed with tetracycline (turkey) in E. coli isolates and with bacitracin (chicken and turkey), gentamicin (turkey), and tylosin (chicken) in Enterococcus spp. isolates. Further studies are needed to provide producers and veterinarians with alternative management practices and tools in order to reduce the use of antimicrobial feed additives in poultry.     

Classification and Antimicrobial Susceptibilities of Enterococcus Species Isolated from Apparently Healthy Food‐Producing Animals in Japan

Summary A total of 1024 enterococci were recovered from faeces of healthy animals from 178 cattle, 178 pig and 156 broiler farms. Enterococcus faecium was the predominant species recovered (35.8%), followed by E. faecalis (31.3%) and E. hirae (25.6%). Oxytetracycline resistance was most frequently found among E. faecalis (85.9%), E. faecium (58.8%) and E. hirae (48.1%). Resistance rates to almost all antimicrobials were higher in E. faecalis than E. faecium and E. hirae. Isolates from cattle were more susceptible to the antimicrobials studied than those from pigs and broilers. VanA‐ or VanB‐type vancomycin‐resistant enterococci have not been found since the ban of avoparcin use 5 years ago.     

Antimicrobial resistance and virulence profiles of Enterococcus spp. isolated from horses in korea

Antimicrobial-resistant (AR) enterococci have emerged as leading nosocomial pathogens. Transmission of AR Enterococci from animals to humans has been demonstrated. However, there is limited information on the transmission of enterococci from horses to humans. To address this issue, we characterized 260 enterococci isolated from horse-associated samples in Korea in 2013 based on their AR profiles and virulence traits. AR profiling revealed an average ratio of AR enterococci of 23.8%. Seven isolates (2.7%) were multidrug-resistant Enterococcus faecalis. Most tetracycline-resistant enterococci harbored either tetM or tetL or both genes; genes conferring resistance to other antimicrobials were detected at low rates. Biofilm formation and gelatinase activity were observed in 51.1% and 47.7% of isolates, respectively; most were E. faecalis harboring the gelE gene. Evidence of transmission of AR enterococci between horses and their environments was provided by pulsed-field gel electrophoresis, and highlights the risk of AR enterococcus transmission to horse riders and handlers through close contact.     

Phylogenetic background and virulence genes of Escherichia coli isolates from colisepticemic and healthy broiler chickens in Iran

The purposes of this study were to determine the phylogenetic background and the virulence gene profiles of Escherichia coli isolates from colisepticemic and feces of healthy (AFEC) broiler chickens. In this study, 253 E. coli isolates including 141 avian pathogenic E. coli (APEC) and 112 AFEC isolates were examined by PCR. In general, 253 E. coli isolates distributed among group A (51.8%), B1 (15.8%), B2 (8.7%), and D (23.7%). Ten (8.9%) AFEC isolates segregated in to B1 phylo-group and 102 (91.1%) isolates fell into six different phylogenetic subgroups. Distribution of colisepticemic and fecal isolates differed significantly in their assignments to A and B1 phylo-groups. The three most prevalent virulence genes were crl, fimH, and aer in isolates between both groups. The four genetic markers aer, papC, afa, and sfa were detected significantly more often among colisepticemic isolates than in fecal isolates from healthy broilers. The presence of stx ₂ gene in fecal isolates were significantly differs among the colisepticemic isolates. F17 fimbrial family encoding gene and eae gene were detected in APEC and AFEC isolates, respectively. The colisepticemic and fecal isolates possessed the virulence genes were detected in all of the four phylogenetic groups. Several combination patterns of the virulence genes were detected in APEC and AFEC isolates. In colisepticemic isolates the combination of aer, crl, and fimH genes was the most prevalent pattern. None of the examined isolates harbored the cdt, cnf1, ipaH, and stx ₁ virulence gene sequences.     

Enterococcus faecalis of human and poultry origin share virulence genes supporting the zoonotic potential of E. faecalis

Enterococcus faecalis is a major cause of nosocomial infections in humans and has been linked to severe extra‐intestinal infections in poultry. A zoonotic potential has been suggested and the aim of the present study was to investigate similarities in virulence gene profiles of E. faecalis originating from infections in humans and poultry respectively. A total of 106 isolates of E. faecalis [26 human clinical isolates, 60 poultry clinical isolates (including two small‐colony variants (SCVs) and 20 poultry cloacal isolates] were investigated for presence of seven virulence‐associated genes: ace, asa1, cylA, efaA, EF0591, esp and gelE. For each gene, the PCR‐amplification product was sequenced from one isolate in each group to explore intragenic variations between genes of human and poultry origin. Haemolytic and protease activities were assessed and isolates were assigned a sequence type (ST). Three of the seven genes investigated (ace, efaA and gelE) were present in all isolates. The asa1 was detected in 63/80 and 13/26 isolates of poultry and human origin respectively. For cylA, the numbers were 46/80 and 14/26 respectively. Among poultry isolates, esp and EF0591 were the least frequently observed genes (1/80 and 20/80 respectively); the prevalences among human isolates were 1/26 and 18/26 respectively. A high degree of similarity between genes in human and poultry isolates were confirmed by sequencing of amplification products. None of the cylA‐positive isolates demonstrated haemolytic activity, while the phenotypic expression of gelatinase varied. The ST16 was the only ST shared by human and poultry isolates. The SCV isolates did not show a unique virulence profile or phylogeny. In conclusion, regardless of the distinct phylogenetic background of most E. faecalis isolates of human and poultry origin, we found major similarities in virulence gene profile and gene sequences in isolates from the two sources, supporting the zoonotic risk associated with this organism.     

Determination of antibiotic resistance pattern and virulence genes in Escherichia coli isolated from bovine with subclinical mastitis in southwest of Iran

The aims of the present study were to investigate the prevalence of some virulence genes and also determine the antimicrobial resistance pattern of E. coli isolated from bovine with subclinical mastitis. The milk of 502 cows was collected from 8 dairy herds in the southwest of Iran. Conventional biochemical tests were used for identification of E. coli at the species level. Antimicrobial susceptibility patterns of E. coli isolates were determined by disc agar diffusion method and polymerase chain reaction (PCR) was used for detection of seven virulence genes including f17A, afaE-8, afaD-8, eaeA, cnf1, cnf2, and iucD. Seventy (13.94%) isolates of E. coli were identified in 502 milk samples. The highest rate of resistance was observed against tetracycline (18.6%), while none of the isolates were resistant to streptomycin. Eight (11.5%) out of 70 E. coli isolates carried at least one of the virulence genes. The afaD-8 was the most prevalent gene detected in 5 (7.1%) isolates. The afaE-8, iucD, and eaeA were detected in 3, 3, and 2 isolates respectively. Low prevalence of virulence factors may be indicating that most of the E. coli isolates originated from the commensal flora of cows and enter to the udders via environment contamination with feces.

     

Phenotypic and genotypic properties of Escherichia coli isolated from colisepticemic cases of Japanese quail

This study was conducted to characterize the Escherichia coli isolates from colisepticemic Japanese quails. One hundred and nine E. coli were isolated in pure culture from heart blood of dead Japanese quails. The sampled birds were originated from four different farms. Antibiotic resistance pattern of E. coli isolates were determined against nine antibacterial agents. Phylotype and virulence genes of the isolates were detected by polymerase chain reaction. By disc diffusion method, all of the isolates showed resistance to three or more antibiotics, and 19 different patterns of multiple drug resistance were observed. Phylotyping of the most prevalent multiple drug-resistant isolates revealed that they mostly belonged to phylogroups A (A₁ subgroup). The E. coli isolates belong to four phylogenetic groups: A (55.0%), B1 (18.3%), B2 (17.4%), and D (9.2%). Eighty-nine (81.7%) isolates were distributed in five phylogenetic subgroups including 22 (20.2%) in A₀, 38 (34.9%) in A₁, 19 (17.4%) in B2₃, 7 (6.4%) in D₁, and 3 (2.8%) in D₂. The examined E. coli isolates exhibit at least one of the virulence genes tested, whereas three most prevalent genes were crl (94.5%), fimH (89.0%), and iutA (51.4%), respectively. The genetic marker for Afa (afaI B-C), S (sfa/focD-E), and P (papE-F) fimbriae were found in one, four, and ten isolates, respectively. Thirteen different combinations of virulence gene were observed, where combination of crl and fimH genes was the most prevalent pattern. None of the isolates contained the ipaH, stx1, stx2, and eaeA genetic markers. In conclusion, E. coli strains could be considered as a causative agent of mortality in quail farms. In conclusion, E. coli isolates from colisepticemic quails are distributed in different phylogroups, are resistant to combinations of antibiotic agents, and contain several virulence genes.     

Comparative Analysis on Antibiotic Resistance Characteristics of Listeria spp. and Enterococcus spp. Isolated From Laying Hens and Eggs in Conventional and Organic Keeping Systems in Bavaria,Germany

By investigating the prevalence and antimicrobial resistance characteristics of Gram‐positive bacteria from organic and conventional keeping systems of laying hens, it was to be determined to what extent these properties are influenced by the different systems. For this purpose, a total of 799 cloacal swabs and 800 egg samples were examined. Prevalences for all selected bacteria from cloacal swabs were much the same for both organic and caged birds: Listeria spp. 1.3%_[org] versus 1.6%_[con]; Enterococcus spp. 95.5%_[org] versus 97.5%_[con]. Egg contents and eggshells were generally contaminated to a lesser extent, primarily with Enterococcus spp. Listeria isolates were susceptible to almost all tested antibiotics, only three Listeria innocua from conventional keepings were resistant to clindamycin; one isolate additionally to imipenem. High percentages of Enterococcus faecalis were resistant to doxycycline and macrolides. Enterococcus faecium proved to have high resistance rates to clindamycin, fosfomycin and erythromycin; 9.1% were even resistant to the reserve antibiotic synercid. Further, Enterococcus spp. showed higher resistance rates to doxycycline, erythromycin, fosfomycin and rifampicin. No glycopeptide resistant enterococci were detected. A correlation between keeping system and resistance/susceptibility rates could be demonstrated. In detail, E. faecalis from organic laying hen husbandries showed significant lower resistance prevalences to tylosin, streptomycin and doxycycline; susceptibility rates were higher for enrofloxacin and ciprofloxacin. Rifampicin and imipenem were more effective in isolates from conventional keepings (P < 0.05). The amounts of resistant isolates of the Enterococcus raffinosus from organic farms were significantly lower, the amounts of sensitive isolates were significantly higher than from conventional farms concerning eight antibiotics (P < 0.05). When comparing the susceptibility/resistance rates, as well as the mean minimum inhibitory concentrations values, the consistent tendency is that bacteria from organic layer flocks are more susceptible to antimicrobials. These results show that organic livestock farming plays a part in contributing to reduced antibiotic resistance.     

Antimicrobial susceptibility and distribution of antimicrobial-resistance genes among Enterococcus and coagulase-negative Staphylococcus isolates recovered from poultry litter

Data on the prevalence of antimicrobial resistant enterococci and staphylococci from the poultry production environment are sparse in the United States. This information is needed for science-based risk assessments of antimicrobial use in animal husbandry and potential public-health consequences. In this study, we assessed the susceptibility of staphylococci and enterococci isolated from poultry litter, recovered from 24 farms across Georgia, to several antimicrobials of veterinary and human health importance. Among the 90 Enterococcus isolates recovered, E. hirae (46%) was the most frequently encountered species, followed by E. faecium (27%), E. gallinarum (12%), and E. faecalis (10%). Antimicrobial resistance was most often observed to tetracycline (96%), followed by clindamycin (90%), quinupristin-dalfopristin (62%), penicillin (53%), erythromycin (50%), nitrofurantoin (49%), and clarithromycin (48%). Among the 110 staphylococci isolates recovered, only coagulase-negative staphylococci (CNS) were identified with the predominant Staphylococcus species being S. sciuri (38%), S. lentus (21%), S. xylosus (14%) and S. simulans (12%). Resistance was less-frequently observed among the Staphylococcus isolates for the majority of antimicrobials tested, as compared with Enterococcus isolates, and was primarily limited to clarithromycin (71%), erythromycin (71%), clindamycin (48%), and tetracycline (38%). Multidrug resistance (MDR) phenotypes were prevalent in both Enterococcus and Staphylococcus; however, Enterococcus exhibited a statistically significant difference in the median number of antimicrobials to which resistance was observed (median = 5.0) compared with Staphylococcus species (median = 3.0). Because resistance to several of these antimicrobials in gram-positive bacteria may be attributed to the shuttling of common drug-resistance genes, we also determined which common antimicrobial-resistance genes were present in both enterococci and staphylococci. The antimicrobial resistance genes vat(D) and erm(B) were present in enterococci, vgaB in staphylococci, and mobile genetic elements Tn916 and pheromone-inducible plasmids were only identified in enterococci. These data suggest that the disparity in antimicrobial-resistance phenotypes and genotypes between enterococci and staphylococci isolated from the same environment is, in part, because of barriers preventing exchange of mobile DNA elements.     

Genomic Description of Antibiotic Resistance in Escherichia coli and Enterococci Isolates from Healthy Lusitano Horses

Antibiotic resistance is a global problem, and it is known that commensal bacteria can act as reservoir of antibiotic resistance genes of clinical importance. The aim of the present study was to determine the antibiotic resistance phenotype and mechanisms implicated in resistance of Escherichia coli and Enterococcus spp. isolates collected from fecal samples of 90 Lusitano horses from Portugal. Sixteen of the 71 E. coli isolates (22.5%) recovered showed resistance to at least one of the antibiotics tested. The number of E. coli isolates resistant to streptomycin, tetracycline, chloramphenicol, ampicillin, trimethoprim-sulfamethoxazole, and gentamicin was 9, 7, 6, 3, 2, and 1, respectively. The bla_TEM-1 and bla_OXA-1 genes were detected in ampicillin-resistant isolates and the sul2 and dfrA1 genes in trimethoprim-sulfamethoxazole-resistant, while the aac(3)-I, floR and tet(A) were found in the gentamicin, chloramphenicol and tetracycline-resistant isolates, respectively. Twenty-two of the 71 (31%) recovered enterococci showed antibiotic resistance for at least one of the tested antibiotics, and resistant isolates were identified as Enterococcus faecium (n = 14), E. faecalis (n = 3), E. hirae (n = 2), and Enterococcus spp. (n = 3). The erm(B) and erm(C) genes were identified in erythromycin-resistant enterococci and the tet(M) and/or tet(L) genes in tetracycline-resistant isolates. The slight prevalence of antibiotic resistance among commensal bacteria of healthy Lusitano horses can improve the treatment of upcoming infections in these horses because these microorganisms can be considered as antimicrobial indicator bacteria.     

Characterization of antibiotic resistance genes and virulence factors in faecal enterococci of wild animals in Portugal

Antibiotic susceptibility was tested in 140 non-selected enterococci (73 Enterococcus faecalis, 45 E. faecium and 22 of other species) recovered from faecal samples of 77 wild animals in Portugal. Susceptibility testing for 11 antibiotics (vancomycin, teicoplanin, ampicillin, streptomycin, gentamicin, kanamycin, chloramphenicol, tetracycline, erythromycin, quinupristin-dalfopristin and ciprofloxacin) was determined by disk diffusion and agar dilution methods. Forty-four isolates (31.4%) showed susceptibility to all the antibiotics tested (5.5% of E. faecalis; 62.2% of E. faecium; and 78.6% of E. hirae). Neither ampicillin-resistance nor acquired-vancomycin-resistance was detected and 1.4% of the isolates showed high-level-resistance for gentamicin or streptomycin. Tetracycline and erythromycin resistances were shown in 28.6% and 20.1% of the isolates, respectively. Antibiotic resistance genes were studied by polymerase chain reaction (PCR) and sequencing and tet(M) + tet(L), erm(B) or aac(6')-aph(2') genes were detected in most of tetracycline-, erythromycin- or gentamicin-resistant enterococci respectively. Genes encoding virulence factors were studied by PCR and a wide variety of virulence genes were detected in most of E. faecalis isolates but were rarely found in E. faecium and not detected in the other species. The prevalence of genes encoding virulence factors in E. faecalis was as follows: cpd (98.6%), gelE (75.3%), agg (30.1%), fsr (17.8%), ace (9.6%) and esp (4.1%). Low percentages of antibiotic resistance was found in the faecal enterococci of wild animals but a wide variety of virulence genes were detected among E. faecalis isolates although were rare in the other species.     

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.     

Prevalence and antibiotic resistance profiles of Enterococcus species in chicken at slaughter level; absence of vanA and vanB genes in E. faecalis and E. faecium

The prevalence of enterococci in neck skin samples of poultry from Ankara region in Turkey was investigated and their antibiotic resistance patterns were determined. In the study, 83 of 106 analyzed neck skin samples were positive for Enterococcus, with E. faecium as the most prevalent species (48%) followed by E. durans (23%) and E. faecalis (19%). Lower numbers were detected for E. gallinarum, E. hirae, E. mundtii and E. casseliflavus. Using the disc diffusion method, it was established that over 90% of E. faecium and E. faecalis isolates were high-level resistant against erythromycin and tetracycline. Four E. faecium isolates were additionally resistant to chloramphenicol, gentamicin and streptomycin, though they were susceptible to penicillin G. The most frequently observed multiple resistance in E. faecium (25%) was against erythromycin, tetracycline, chloramphenicol and streptomycin. Of the E. faecalis isolates, 44% were multiple resistant to erythromycin, tetracycline and streptomycin. Vancomycin resistance could not be demonstrated phenotypically and vanA or vanB genes were not detected by multiplex PCR in any of the isolates. Nevertheless, the observed resistance patterns are of concern for public health.     

Antimicrobial Resistance Profiles of Campylobacter spp. Isolated from Broiler Chicken Meat of Estonian,Latvian and Lithuanian Origin at Estonian Retail Level and from Patients with Severe Enteric Infections in Estonia

The resistance patterns of Campylobacter spp. isolated from retail broiler chicken meat originating either from Estonia, Lithuania or Latvia collected in Estonia were determined. Additionally, in collaboration with the laboratories of several Estonian hospitals, antimicrobial susceptibility patterns were determined for Campylobacter isolates from patients with severe Campylobacter enteric infections. The isolates were identified at the species level by the PCR method. Respectively, 88.8% of the isolates were C. jejuni, and 11.2% were C. coli. In total, 126 Campylobacter isolates of broiler chicken meat and human origin were tested for minimal inhibitory concentrations (MICs) with the broth microdilution VetMIC^TH method (National Veterinary Institute; Uppsala, Sweden) for a total of six antimicrobials. Resistance to one or more antimicrobials was detected in 62 (63.3%) of Campylobacter broiler chicken meat isolates and in 20 (71.4%) of human‐origin isolates. Large proportions of the broiler chicken meat isolates were resistant to ciprofloxacin (60.2%). Multidrug resistance (i.e. to three or more unrelated antimicrobials) was detected in five (5.1%) C. jejuni isolates. Among the human isolates, 20 (71.4%) were resistant to fluoroquinolones, and two (7.1%) C. jejuni isolates exhibited multidrug resistance. The chicken meat isolates of Estonian origin were the most susceptible. However, a high proportion of fluoroquinolone‐resistant C. jejuni isolates were found in Latvian and Lithuanian products. The results of this study indicate that the problems caused by the inappropriate use of antimicrobials extend beyond the country in which a food originates; therefore, both domestic and international interventions and agreements are required to implement common policies on antimicrobial usage and to minimize the emergence of Campylobacter drug resistance.     

Study of Vancomycin Resistance in Faecal Enterococci from Healthy Humans and Dogs in Spain a Decade after the Avoparcin Ban in Europe

One hundred and 26 faecal samples from healthy dogs (2009) and 157 faecal samples from healthy humans (2007) from La Rioja region (Spain) were tested to know the carriage of vancomycin‐resistant enterococci (VRE). VRE with intrinsic resistance (vanC) were found in 12% of healthy dogs and humans (29 Enterococcus gallinarum and four Enterococcus casseliflavus). Nevertheless, VRE with acquired mechanism of resistance were not detected among these samples. Four Enterococcus faecalis isolates with vancomycin MIC of 8‐16 mg L^?1 were recovered in human samples, but no single organism with known mechanism of acquired resistance could be identified. These 37 VRE isolates (33 E. gallinarum/E. casseliflavus and four E. faecalis) of dog and human origin were further characterized (PCR detection of antibiotic resistance, virulence and bacteriocin genes). High prevalence of tetracycline resistance was identified (70%), especially among dog isolates harbouring tet(M) ± tet(L) genes; erythromycin resistance was also higher among isolates from dogs and they harboured the erm(B) gene, associated with erm(A) gene in one case. Virulence genes were only identified among E. faecalis isolates of human origin (agg, cpd and/or gelE) and never among E. gallinarum/E. casseliflavus of human or dog origin. Five E. gallinarum isolates of dog and three E. faecalis of human origin expressed bacteriocin activity; among them, only one E. faecalis presented activity against Listeria monocytogenes. The bacteriocin structural gene ef1097 was identified in 3 bacteriocin‐producing E. faecalis isolates, associated with ent1071 in one of them.