Antibiotic Resistance among Escherichia coli O-serotypes from the Gut and Carcasses of Commercially Slaughtered Broiler Chickens: a Potential Public Health Hazard

Presumptive coliform counts and the distribution of Escherichia coli O-serotypes were investigated in chicken rectal contents (175) abdominal cavities (152) and on the carcasses of 44 which had been commercially raised, slaughtered and prepared for sale. Large numbers of E. coli resistant to at least one antibacterial agent were found at each site; comparison of the O-serotypes suggested heavy contamination of the carcass with strains from the gut. The range of O-serotypes was similar to that found in man and some public health implications of cross-infection particularly by handling uncooked birds in the kitchen, are discussed.     

Quantifying Transmission of Campylobacter jejuni in Commercial Broiler Flocks

Since meat from poultry colonized with Campylobacter spp. is a major cause of bacterial gastroenteritis, human exposure should be reduced by, among other things, prevention of colonization of broiler flocks. To obtain more insight into possible sources of introduction of Campylobacter into broiler flocks, it is essential to estimate the moment that the first bird in a flock is colonized. If the rate of transmission within a flock were known, such an estimate could be determined from the change in the prevalence of colonized birds in a flock over time. The aim of this study was to determine the rate of transmission of Campylobacter using field data gathered for 5 years for Australian broiler flocks. We used unique sampling data for 42 Campylobacter jejuni-colonized flocks and estimated the transmission rate, which is defined as the number of secondary infections caused by one colonized bird per day. The estimate was 2.37 ± 0.295 infections per infectious bird per day, which implies that in our study population colonized flocks consisting of 20,000 broilers would have an increase in within-flock prevalence to 95% within 4.4 to 7.2 days after colonization of the first broiler. Using Bayesian analysis, the moment of colonization of the first bird in a flock was estimated to be from 21 days of age onward in all flocks in the study. This study provides an important quantitative estimate of the rate of transmission of Campylobacter in broiler flocks, which could be helpful in future studies on the epidemiology of Campylobacter in the field.     

Genotype Analyses of Campylobacter Isolated from Distinct Segments of the Reproductive Tracts of Broiler Breeder Hens

Campylobacter isolated from feces and from the oviduct of six broiler breeder hens were genotyped by using flaA SVR DNA sequence analyses. A diversity of genotypes was observed among fecal and oviduct isolates. Comparison of isolates from the oviducts of individual hens revealed variable results. In three cases (hen 2, hen 3, and hen 6), analyses indicated that isolates from all regions of the individual hen's reproductive tract were closely related; isolates from hen 1 and hen 4 were diverse. Comparison of the Campylobacter isolates between hens revealed that in two cases, hens 1 and 3 and hens 4 and 6, certain isolates possessed identical flaA SVR sequence types. Comparisons of Campylobacter isolates recovered from a distinct region of the oviduct were found to have increased diversity as sampling progressed down the oviduct. This study further demonstrates that Campylobacter is present within the reproductive tract of breeder hens and that this presence may enable vertical transmission of Campylobacter from the breeder hen to the broiler offspring. Received: 11 January 2002 / Accepted: 13 March 2002     

Correlation of Campylobacter Bacteriophage with Reduced Presence of Hosts in Broiler Chicken Ceca

Campylobacter jejuni and Campylobacter-specific bacteriophage were enumerated from broiler chicken ceca selected from 90 United Kingdom flocks (n = 205). C. jejuni counts in the presence of bacteriophage (mean log₁₀ 5.1 CFU/g) were associated with a significant (P < 0.001) reduction compared to samples with Campylobacter alone (mean log₁₀ 6.9 CFU/g).     

Pathotype and Antibiotic Resistance Gene Distributions of Escherichia coli Isolates from Broiler Chickens Raised on Antimicrobial-Supplemented Diets

The impact of feed supplementation with bambermycin, monensin, narasin, virginiamycin, chlortetracycline, penicillin, salinomycin, and bacitracin on the distribution of Escherichia coli pathotypes in broiler chickens was investigated using an E. coli virulence DNA microarray. Among 256 E. coli isolates examined, 59 (23%) were classified as potentially extraintestinal pathogenic E. coli (ExPEC), while 197 (77%) were considered commensal. Except for chlortetracycline treatment, the pathotype distribution was not significantly different among treatments (P > 0.05). Within the 59 ExPEC isolates, 44 (75%) were determined to be potentially avian pathogenic E. coli (APEC), with the remaining 15 (25%) considered potentially “other” ExPEC isolates. The distribution within phylogenetic groups showed that 52 (88%) of the ExPEC isolates belonged to groups B2 and D, with the majority of APEC isolates classified as group D and most commensal isolates (170, 86%) as group A or B1. Indirect assessment of the presence of the virulence plasmid pAPEC-O2-ColV showed a strong association of the plasmid with APEC isolates. Among the 256 isolates, 224 (88%) possessed at least one antimicrobial resistance gene, with nearly half (107, 42%) showing multiple resistance genes. The majority of resistance genes were distributed among commensal isolates. Considering that the simultaneous detection of antimicrobial resistance tet(A), sulI, and bla_TEM genes and the integron class I indicated a potential presence of the resistance pAPEC-O2-R plasmid, the results revealed that 35 (14%) of the isolates, all commensals, possessed this multigene resistance plasmid. The virulence plasmid was never found in combination with the antimicrobial resistance plasmid. The presence of the ColV plasmid or the combination of iss and tsh genes in the majority of APEC isolates supports the notion that when found together, the plasmid, iss, and tsh serve as good markers for APEC. These data indicate that different resistant E. coli pathotypes can be found in broiler chickens and that the distribution of such pathotypes and certain virulence determinants could be modulated by antimicrobial agent feed supplementation.Several classes of antimicrobial agents, such as glycolipids (bambermycin), cyclic peptides (bacitracin), ionophores (monensin and salinomycin), streptogramins (virginiamycin), and β-lactams (penicillin), are widely used as food additives in modern animal husbandry to prevent infections and promote growth (6). Increasing antimicrobial resistance in animals and its potential threat to human health led to the ban of bacitracin, spiramycin, tylosin, and virginiamycin as feeding additives by the European Union in 1999 (7, 46). Although this precautionary measure is still controversial because of being seen as having a negligible impact on human health, negative consequences for animal health and welfare, including economic losses for farmers, were subsequently observed in Europe (7). In stark contrast, however, the ban has been beneficial in reducing the total quantity of antibiotics administered to food animals (7, 47). Under good production conditions and correct use of antibiotics, poultry production is reported to be competitive (14, 47, 48) and even beneficial in reducing antimicrobial resistance in important food animal reservoirs and thus the potential threat to public health (48).Escherichia coli is generally considered a commensal member of the normal gastrointestinal microflora in humans and animals, yet some strains are known to cause serious morbidity and mortality. The expression of various virulence factors, which affect cellular processes, can result in different clinical diseases, such as cystitis, pyelonephritis, sepsis/meningitis, and gastroenteritis. The possession of different virulence gene subsets can further define the E. coli pathotype (31). The extraintestinal pathogenic E. coli (ExPEC) strains are epidemiologically and phylogenetically distinct from both intestinal pathogenic and commensal strains (43). In North America, annually, several million cases of urinary tract infections, abdominal infections, pelvic infections, pneumonia, meningitis, and sepsis are caused by ExPEC (42). In poultry production, avian pathogenic E. coli (APEC) is responsible for significant economic losses. APEC strains induce extraintestinal diseases such as air sacculitis, colibacillosis, polysorositis, and septicemia in birds (9, 21, 22, 31, 35, 45). Although no specific set of virulence factors has been clearly linked to APEC strains, most identified virulence factors are similar to those frequently associated with ExPEC (36).Bearing in mind that the avian intestinal environment has been considered a reservoir of E. coli having zoonotic potential (15) and the possible contamination of poultry products with such bacteria during slaughter, the impact of antimicrobial feeding additives on the distribution and dissemination of bacterial pathotypes and antibiotic resistance needs to be explored to address human, animal, and environmental health concerns. To this end, an E. coli DNA virulence microarray previously employed to assess the genotypes (virulence and antibiotic resistance genes) of E. coli strains isolated from different environmental ecosystems and from the chicken intestinal tract (1, 10, 19, 20, 33) was used. The aim of the present trial was to investigate the distributions of pathotypes and of virulence and antibiotic resistance genes in E. coli isolates from broilers fed with antimicrobial supplementation diets including bambermycin, penicillin, salinomycin, bacitracin, chlortetracycline, virginiamycin, monensin, and narasin.     

Sources of Campylobacter spp. Colonizing Housed Broiler Flocks during Rearing

The study aimed to identify sources of campylobacter in 10 housed broiler flocks from three United Kingdom poultry companies. Samples from (i) the breeder flocks, which supplied the broilers, (ii) cleaned and disinfected houses prior to chick placement, (iii) the chickens, and (iv) the environments inside and outside the broiler houses during rearing were examined. Samples were collected at frequent intervals and examined for Campylobacter spp. Characterization of the isolates using multilocus sequence typing (MLST), serotyping, phage typing, and flaA restriction fragment length polymorphism typing was performed. Seven flocks became colonized during the growing period. Campylobacter spp. were detected in the environment surrounding the broiler house, prior to as well as during flock colonization, for six of these flocks. On two occasions, isolates detected in a puddle just prior to the birds being placed were indistinguishable from those colonizing the birds. Once flocks were colonized, indistinguishable strains of campylobacter were found in the feed and water and in the air of the broiler house. Campylobacter spp. were also detected in the air up to 30 m downstream of the broiler house, which raises the issue of the role of airborne transmission in the spread of campylobacter. At any time during rearing, broiler flocks were colonized by only one or two types determined by MLST but these changed, with some strains superseding others. In conclusion, the study provided strong evidence for the environment as a source of campylobacters colonizing housed broiler flocks. It also demonstrated colonization by successive campylobacter types determined by MLST during the life of a flock.     

Genomic Diversity of Campylobacter coli and Campylobacter jejuni Isolates Recovered from Free-Range Broiler Farms and Comparison with Isolates of Various Origins

In many industrialized countries, the incidence of campylobacteriosis exceeds that of salmonellosis. Campylobacter bacteria are transmitted to humans mainly in food, especially poultry meat products. Total prevention of Campylobacter colonization in broiler flocks is the best way to reduce (or eliminate) the contamination of poultry products. The aim of this study was to establish the sources and routes of contamination of broilers at the farm level. Molecular typing methods (DNA macrorestriction pulsed-field gel electrophoresis and analysis of gene polymorphism by PCR-restriction fragment length polymorphism) were used to characterize isolates collected from seven broiler farms. The relative genomic diversity of Campylobacter coli and Campylobacter jejuni was determined. Analysis of the similarity among 116 defined genotypes was used to determine clusters within the two species. Furthermore, evidence of recombination suggested that there were genomic rearrangements within the Campylobacter populations. Recovery of related clusters from different broiler farms showed that some Campylobacter strains might be specifically adapted to poultry. Analysis of the Campylobacter cluster distribution on three broiler farms showed that soil in the area around the poultry house was a potential source of Campylobacter contamination. The broilers were infected by Campylobacter spp. between days 15 and 36 during rearing, and the type of contamination changed during the rearing period. A study of the effect of sanitary barriers showed that the chickens stayed Campylobacter spp. free until they had access to the open area. They were then rapidly colonized by the Campylobacter strains isolated from the soil.     

Effect of Bacteriophage Application on Campylobacter jejuni Loads in Commercial Broiler Flocks

Campylobacteriosis is the most frequent food-borne human enteritis. The major source for infection with Campylobacter spp. is broiler meat. Risk assessments consider the reduction of Campylobacter in primary production to be most beneficial for human health. The aim of this study was to test the efficacy of a bacteriophage application under commercial conditions which had proved to be effective in previous noncommercial studies under controlled experimental conditions. A phage cocktail for Campylobacter reduction was tested on three commercial broiler farms each with a control and an experimental group. Colonization of Campylobacter was confirmed prior to phage application in fecal samples. Subsequently, a phage cocktail was applied via drinking water in the experimental group (log₁₀ 5.8 to 7.5 PFU/bird). One day after phage application, Campylobacter counts of one experimental group were reduced under the detection limit (<50 CFU/g, P = 0.0140) in fecal samples. At slaughter, a significant reduction of >log₁₀ 3.2 CFU/g cecal content compared to the control was still detected (P = 0.0011). No significant reduction was observed in the experimental groups of the other trials. However, a significant drop in cecal Campylobacter counts occurred in a phage-contaminated control. These results suggest that maximum reduction of Campylobacter at the slaughterhouse might be achieved by phage application 1 to 4 days prior to slaughter.     

Comparison of Genotypes and Serotypes of Campylobacter jejuni Isolated from Danish Wild Mammals and Birds and from Broiler Flocks and Humans

The incidence of human infection with Campylobacter jejuni is increasing in most developed countries and the reason for this is largely unknown. Although poultry meat is considered to be a major source, it is evident that other reservoirs exist, possibly common to humans and poultry. Environmental sources are believed to be important reservoirs of Campylobacter infection in broiler chicken flocks. We investigated the potential importance of wildlife as a source of infection in commercial poultry flocks and in humans by comparing the serotype distributions, fla types, and macrorestriction profiles (MRPs) of C. jejuni isolates from different sources. The serotype distribution in wildlife was significantly different from the known distributions in broilers and humans. Considerable sero- and genotype diversity was found within the wildlife collection, although two major groups of isolates within serotype O:12 and the O:4 complex were found. Common clonal lines among wildlife, chicken, and/or human isolates were identified within serotype O:2 and the O:4 complex. However, MRPs of O:12 and O:38 strains isolated from wildlife and other sources indicated that some clonal lines propagated in a wide selection of animal species but were not detected in humans or broilers in this study. The applied typing methods successfully identified different clonal groups within a strain collection showing large genomic diversity. However, the relatively low number of wildlife strains with an inferred clonal relationship to human and chicken strains suggests that the importance of wildlife as a reservoir of infection is limited.     

Colonisation of a Phage Susceptible Campylobacter jejuni Population in Two Phage Positive Broiler Flocks

The pathogens Campylobacter jejuni and Campylobacter coli are commensals in the poultry intestine and campylobacteriosis is one of the most frequent foodborne diseases in developed and developing countries. Phages were identified to be effective in reducing intestinal Campylobacter load and this was evaluated, in the first field trials which were recently carried out. The aim of this study was to further investigate Campylobacter population dynamics during phage application on a commercial broiler farm. This study determines the superiority in colonisation of a Campylobacter type found in a field trial that was susceptible to phages in in vitro tests. The colonisation factors, i.e. motility and gamma glutamyl transferase activity, were increased in this type. The clustering in phylogenetic comparisons of MALDI-TOF spectra did not match the ST, biochemical phenotype and phage susceptibility. Occurrence of Campylobacter jejuni strains and phage susceptibility types with different colonisation potential seem to play a very important role in the success of phage therapy in commercial broiler houses. Thus, mechanisms of both, phage susceptibility and Campylobacter colonisation should be further investigated and considered when composing phage cocktails.     

Multiple Antibiotic Resistance Patterns of Escherichia coli Isolates from Swine Farms

Antibiotic resistance of Escherichia coli from sows and pigs was determined to compare patterns between pigs of various ages and degrees of antibiotic use. Resistance patterns differed between farm types and pigs of differing ages, indicating that pig age and degree of antibiotic use affect resistance of fecal E. coli.     

Antimicrobial Resistance Profiles of Campylobacter jejuni Isolates from Wild Birds in Sweden

In order to determine the occurrence and frequency of resistant strains of the bacterium Campylobacter jejuni and to establish baseline MICs in isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137 C. jejuni isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of C. jejuni have the potential to spread to wild bird hosts.     

Enumeration of Salmonella and Campylobacter spp. in Environmental Farm Samples and Processing Plant Carcass Rinses from Commercial Broiler Chicken Flocks

A prospective cohort study was performed to evaluate the prevalences and loads of Salmonella and Campylobacter spp. in farm and processing plant samples collected from 55 commercial broiler chicken flocks. Environmental samples were collected from broiler houses within 48 h before slaughter, and carcass rinses were performed on birds from the same flocks at 4 different stages of processing. Salmonella was detected in farm samples of 50 (90.9%) flocks and in processing samples of 52 (94.5%) flocks. Campylobacter was detected in farm samples of 35 (63.6%) flocks and in processing samples of 48 (87.3%) flocks. There was a significant positive relationship between environmental farm samples and processing plant carcass rinses with respect to both Salmonella and Campylobacter prevalences and loads. Campylobacter loads were significantly higher than Salmonella loads, and the correlations between samples collected from the same flocks were higher for Campylobacter than they were for Salmonella. Boot socks were the most sensitive sample type for detection of Salmonella on the farm, whereas litter samples had the strongest association with Salmonella loads in pre- and postchill carcass rinses. Boot socks, drag swabs, and fecal samples all had similar sensitivities for detecting Campylobacter on the farm, and all were more strongly associated with Campylobacter loads in carcass rinses than were litter samples. Farm samples explained a greater proportion of the variability in carcass rinse prevalences and loads for Campylobacter than they did for Salmonella. Salmonella and Campylobacter prevalences and loads both decreased significantly as birds progressed through the processing plant.     

Genetic Diversity and Quinolone Resistance in Campylobacter jejuni Isolates from Poultry in Senegal

We used the multilocus sequence typing (MLST) method to evaluate the genetic diversity of 46 Campylobacter jejuni isolates from chickens and to determine the link between quinolone resistance and sequence type (ST). There were a total of 16 ST genotypes, and the majority of them belonged to seven clonal complexes previously identified by using isolates from human disease. The ST-353 complex was the most common complex, whereas the ST-21, ST-42, ST-52, and ST-257 complexes were less well represented. The resistance phenotype varied for each ST, and the Thr-86-Ile substitution in the GyrA protein was the predominant mechanism of resistance to quinolone. Nine of the 14 isolates having the Thr-86-Ile substitution belonged to the ST-353 complex. MLST showed that the emergence of quinolone resistance is not related to the diffusion of a unique clone and that there is no link between ST genotype and quinolone resistance. Based on silent mutations, different variants of the gyrA gene were shown to exist for the same ST. These data provide useful information for understanding the epidemiology of C. jejuni in Senegal.     

Effects of Climate on Incidence of Campylobacter spp. in Humans and Prevalence in Broiler Flocks in Denmark

Campylobacter infections are increasing and pose a serious public health problem in Denmark. Infections in humans and broiler flocks show similar seasonality, suggesting that climate may play a role in infection. We examined the effects of temperature, precipitation, relative humidity, and hours of sunlight on Campylobacter incidence in humans and broiler flocks by using lag dependence functions, locally fitted linear models, and cross validation methods. For humans, the best model included average temperature and sunlight 4 weeks prior to infection; the maximum temperature lagged at 4 weeks was the best single predictor. For broilers, the average and maximum temperatures 3 weeks prior to slaughter gave the best estimate; the average temperature lagged at 3 weeks was the best single predictor. The combined effects of temperature and sunlight or the combined effects of temperature and relative humidity predicted the incidence in humans equally well. For broiler flock incidence these factors explained considerably less. Future research should focus on elements within the broiler environment that may be affected by climate, as well as the interaction of microclimatic factors on and around broiler farms. There is a need to quantify the contribution of broilers as a source of campylobacteriosis in humans and to further examine the effect of temperature on human incidence after this contribution is accounted for. Investigations should be conducted into food consumption and preparation practices and poultry sales that may vary by season.     

Survival of Campylobacter Jejuni/Coli in Ground Refrigerated and in Ground Frozen Beef Liver and in Frozen Broiler Carcasses

Survival of 5 strains of Campylobacter jejuni/coli in ground beef liver stored at 4° C and at –20° C was studied. After 6 days of storage at 4° C the beef liver was spoiled, which was indicated by APG log 7.25 and lactobacilli count log 7.0. During this storage Campylobacter counts decreased only slightly. After 12 weeks of storage at –20° C Campylobacter counts decreased by 2–3 logs in frozen ground beef liver. Survival of 4 strains of C. jejuni/coli on frozen broiler carcasses was also studied. Two inoculation levels, 103–104/g and 104–105/g were used. On frozen broiler carcasses Campylobacter counts decreased by 0.5–2.0 logs during 12 weeks at –20° C.     

Antibiotic Resistance Among Cultured Bacterial Isolates from Bioethanol Fermentation Facilities Across the United States

Bacterial contamination of fuel ethanol fermentations by lactic acid bacteria (LAB) can have crippling effects on bioethanol production. Producers have had success controlling bacterial growth through prophylactic addition of antibiotics to fermentors, yet concerns have arisen about antibiotic resistance among the LAB. Here, we report on mechanisms used by 32 LAB isolates from eight different US bioethanol facilities to persist under conditions of antibiotic stress. Minimum inhibitory concentration assays with penicillin, erythromycin, and virginiamycin revealed broad resistance to each of the antibiotics as well as high levels of resistance to individual antibiotics. Phenotypic assays revealed that antibiotic inactivation mechanisms contributed to the high levels of individual resistances among the isolates, especially to erythromycin and virginiamycin, yet none of the isolates appeared to use a β-lactamase. Biofilm formation was noted among the majority of the isolates and may contribute to persistence under low levels of antibiotics. Nearly all of the isolates carried at least one canonical antibiotic resistance gene and many carried more than one. The erythromycin ribosomal methyltransferase (erm) gene class was found in 19 of 32 isolates, yet a number of these isolates exhibit little to no resistance to erythromycin. The erm genes were present in 15 isolates that encoded more than one antibiotic resistance mechanism, suggestive of potential genetic linkages.     

Genetic Determinants of Antibiotic Resistance in Staphylococcus aureus Isolates from Milk of Mastitic Crossbred Cattle

Staphylococcus aureus is one of the major causes of mastitis in dairy animals and its resistance against multiple antimicrobials always remains crucial concern. Present investigation was carried out to detect the distribution of antibiotic-resistant genes of S. aureus isolates. Isolates (128) of S. aureus from mastitic milk were collected, tested for antibiotics with disc-diffusion method, and resistant genes mecA, linA, msrA msrB, vatA, vatB, vatC ermA, ermC tetK, tetM and aacA-D were detected by PCR. The phenotypic antibiotics resistance percent in S. aureus isolates was classified as tetracycline (36.7), gentamycin (30.5), streptomycin (26.6), kanamycin (25.8) and penicillin G (22.7). All the isolates were susceptible to vancomycin. Among isolates, 10.2% were observed as methicillin-resistant. The distribution of antibiotic-resistant genes was linA (51.6) followed by msrB (46.1), tetK + M (34.4), msrA and aacA-D (26.6%). Different antibiotic-resistant genes combinations (mecA/linA-2; mecA/aacA-D/tetK/linA/msrB-3; mecA/linA/msrA/msrB-3; aacA-D/linA/msrA/msrB-4; aacA-D/linA/msrB-7; linA/msrA/msrB-10; tetK/linA/msrA/msrB-11; aacA/tetK/linA/msrB-12 isolates) were observed. All the isolates lacked amplification of vatA, vatB, ermA and ermC genes. Molecular typing resulted genetic variation in protein A (6–12 repeats) and coagulase genes (A–E patterns) were observed. Coagulase A and D genotypes were more prevalent in antibiotic-resistant isolates, while E, B and C in susceptible ones. The significant observation was the prevalence of methicillin-resistant S. aureus, which were resistant to multiple antibiotics. Findings revealed the status of resistant isolates in herd that might be helpful in treatment, controlling of resistant strains and culling of cows for mastitis reduction.     

Arsenite-Induced Multiple Antibiotic Resistance Phenotype in Environmental Isolates of Yersinia enterocolitica

Minimal inhibitory concentrations (MICs) of five antibiotics namely amikacin, gentamicin, tetracycline, ciprofloxacin, and nitrofurantoin for pork isolates of Yersinia enterocolitica increased two- to eightfold after bacteria were grown in the presence of 5 mm arsenite. For Y. enterocolitica isolates obtained from wastewater (sewage effluents), an unequivocal increase in MICs was seen with amikacin and gentamicin. No change was discernible in the outer-membrane proteins after isolates were grown in the presence of arsenite. Received: 14 October 2000 / Accepted: 26 January 2001