Characterization of Salmonella enterica serovar Heidelberg from Turkey-Associated Sources

Salmonella enterica serovar Heidelberg strains are frequently associated with food-borne illness, with recent isolates showing higher rates of resistance to multiple antimicrobial agents. One hundred eighty S. enterica serovar Heidelberg isolates, collected from turkey-associated production and processing sources, were tested for antimicrobial susceptibility and compared by pulsed-field gel electrophoresis (PFGE) and plasmid profile analysis. The potential for the transfer of resistance between strains was studied by conjugation experiments. PFGE analysis using XbaI digestion identified eight clusters (based on 90% similarity), with the largest containing 71% of the isolates. Forty-two percent of the isolates were resistant to at least 1 of the 15 antimicrobial agents tested, and 4% of the isolates were resistant to 8 or more antimicrobial agents. Resistances to streptomycin (32%), tetracycline (30%), and kanamycin (24%) were most commonly detected. Interestingly, the XbaI PFGE profiles of selective multidrug-resistant strains (n = 22) of S. enterica serovar Heidelberg from turkey-associated sources were indistinguishable from the predominant profile (JF6X01.0022) detected in isolates associated with human infections. These isolates were further differentiated into seven distinct profiles following digestion with the BlnI enzyme, with the largest cluster comprising 15 isolates from veterinary diagnostic and turkey processing environments. Conjugation experiments indicated that resistance to multiple antimicrobial agents was transferable among strains with diverse PFGE profiles.     

Role of Calf-Adapted Escherichia coli in Maintenance of Antimicrobial Drug Resistance in Dairy Calves

The prevalence of antimicrobial drug-resistant bacteria is typically highest in younger animals, and prevalence is not necessarily related to recent use of antimicrobial drugs. In dairy cattle, we hypothesize that antimicrobial drug-resistant, neonate-adapted bacteria are responsible for the observed high frequencies of resistant Escherichia coli in calves. To explore this issue, we examined the age distribution of antimicrobial drug-resistant E. coli from Holstein cattle at a local dairy and conducted an experiment to determine if low doses of oxytetracycline affected the prevalence of antimicrobial drug-resistant E. coli. Isolates resistant to tetracycline (>4 μg/ml) were more prevalent in <3-month-old calves (79%) compared with lactating cows (14%). In an experimental trial where calves received diets supplemented with or without oxytetracycline, the prevalence of tetracycline-resistant E. coli was slightly higher for the latter group (P = 0.039), indicating that drug use was not required to maintain a high prevalence of resistant E. coli. The most common resistance pattern among calf E. coli isolates included resistance to streptomycin (>12 μg/ml), sulfadiazine (>512 μg/ml), and tetracycline (>4 μg/ml) (SSuT), and this resistance pattern was most prevalent during the period when calves were on milk diets. To determine if prevalence was a function of differential fitness, we orally inoculated animals with nalidixic acid-resistant strains of SSuT E. coli and susceptible E. coli. Shedding of SSuT E. coli was significantly greater than that of susceptible strains in neonatal calves (P < 0.001), whereas there was no difference in older animals (P = 0.5). These data support the hypothesis that active selection for traits linked to the SSuT phenotype are responsible for maintaining drug-resistant E. coli in this population of dairy calves.     

Longitudinal Characterization of Resistant Escherichia coli in Fecal Deposits from Cattle Fed Subtherapeutic Levels of Antimicrobials

Model fecal deposits from cattle fed or not fed antimicrobial growth promoters were examined over 175 days in the field for growth and persistence of total Escherichia coli and numbers and proportions of ampicillin-resistant (Amp^r) and tetracycline-resistant (Tet^r) E. coli. In addition, genotypic diversity and the frequency of genetic determinants encoded by Amp^r and Tet^r E. coli were investigated. Cattle were fed diets containing chlortetracycline (44 ppm; A44 treatment group), chlortetracycline plus sulfamethazine (both at 44 ppm; AS700 treatment group), or no antibiotics (control). Fecal deposits were sampled 12 times over 175 days. Numbers of Tet^r E. coli in A44 and AS700 deposits were higher (P < 0.001) than those of controls and represented up to 35.6% and 20.2% of total E. coli, respectively. A time-by-treatment interaction (P < 0.001) was observed for the numbers of Tet^r and Amp^r E. coli. Except for Amp^r E. coli in control deposits, all E. coli numbers increased (P < 0.001) in deposits up to day 56. Even after 175 days, high Tet^r E. coli numbers were detected in A44 and AS700 deposits [5.9 log₁₀ CFU (g dry matter)⁻¹ and 5.4 log₁₀ CFU (g dry matter)⁻¹, respectively]. E. coli genotypes, as determined by pulsed-field gel electrophoresis, were diverse and were influenced by the antimicrobial growth promoter and the sampling time. Of the determinants screened, bla_TEM1, tetA, tetB, tetC, sul1, and sul2 were frequently detected. Occurrence of determinants was influenced by the feeding of antimicrobials. Fecal deposits remain a source of resistant E. coli even after a considerable period of environmental exposure.In North America antibiotics are widely used in beef cattle production as prophylactics or antimicrobial growth promoters (AGP). Used in this manner, antibiotics are generally administered in the diet either at times of high disease risk or on a continuous basis to improve feed efficiency. Employment of AGP in this manner may increase the prevalence of commensal antimicrobial-resistant (AR) bacteria (1, 41).There is evidence that resistant bacteria can be transferred from livestock to humans (5, 39, 55), and consequently the use of AGP has raised public health concerns. While the modes of transmission of AR bacteria and gene determinants are complex (33), survivability in agriculture-related matrices may be a critical factor in their dissemination (54). Most research on the persistence of AR bacteria in livestock waste has focused on large-scale management systems, such as land-applied manure (20, 44) or storage lagoons and pits (28, 49). In some instances, these systems have been shown to decrease the survival of select bacteria and the presence of AR genes (35, 51). Nevertheless, there is limited knowledge of the fate of AR bacteria or resistance determinants in feces shed from individuals, such as the fecal deposits that are formed in feedlot pens on intensively managed farms. Previous work has shown that fecal deposits provide an environment that is conducive to the growth and survival of pathogenic and commensal Escherichia coli (4, 48, 57). Because cattle fed AGP excrete antimicrobial residues in their feces (3), the residues may exert a selection pressure on bacteria after deposition of feces and potentially confer a growth advantage to resistant bacteria.The objective of the current study was to investigate both the longitudinal phenotypic and genotypic ecologies of AR E. coli in fecal deposits arising from groups of cattle administered either no AGP or one of two tetracycline-based AGP that are commonly used in the North American industry. We focused on ampicillin-resistant (Amp^r) and tetracycline-resistant (Tet^r) E. coli because both AGP treatments contained tetracycline, and we have previously found a link between the use of tetracycline-based AGP and Amp^r E. coli (1). We hypothesized that resistant E. coli would be more numerous in fecal deposits from animals previously fed AGP.     

Antibiotic Resistance of Enterococci in American Bison (Bison bison) from a Nature Preserve Compared to That of Enterococci in Pastured Cattle

Enterococci isolated from a bison population on a native tall-grass prairie preserve in Kansas were characterized and compared to enterococci isolated from pastured cattle. The species diversity was dominated by Enterococcus casseliflavus in bison (62.4%), while Enterococcus hirae was the most common isolate from cattle (39.7%). Enterococcus faecalis was the second most common species isolated from bison (16%). In cattle, E. faecalis and Enterococcus faecium were isolated at lower percentages (3.2% and 1.6%, respectively). No resistance to ampicillin, chloramphenicol, gentamicin, or high levels of vancomycin was detected from either source. Tetracycline and erythromycin resistance phenotypes, encoded by tetO and ermB, respectively, were common in cattle isolates (42.9% and 12.7%, respectively). A significant percentage of bison isolates (8% and 4%, respectively) were also resistant to these two antibiotics. The tetracycline resistance genes from both bison and cattle isolates resided on mobile genetic elements and showed a transfer frequency of 10⁻⁶ per donor, whereas erythromycin resistance was not transferable. Resistance to ciprofloxacin was found to be higher in enterococci from bison (14.4%) than in enterococci isolated from cattle (9.5%). The bison population can serve as a sentinel population for studying the spread and origin of antibiotic resistance.     

Antimicrobial Resistance in Escherichia coli Isolates from Swine and Wild Small Mammals in the Proximity of Swine Farms and in Natural Environments in Ontario, Canada

Wild animals not normally exposed to antimicrobial agents can acquire antimicrobial agent-resistant bacteria through contact with humans and domestic animals and through the environment. In this study we assessed the frequency of antimicrobial resistance in generic Escherichia coli isolates from wild small mammals (mice, voles, and shrews) and the effect of their habitat (farm or natural area) on antimicrobial resistance. Additionally, we compared the types and frequency of antimicrobial resistance in E. coli isolates from swine on the same farms from which wild small mammals were collected. Animals residing in the vicinity of farms were five times more likely to carry E. coli isolates with tetracycline resistance determinants than animals living in natural areas; resistance to tetracycline was also the most frequently observed resistance in isolates recovered from swine (83%). Our results suggest that E. coli isolates from wild small mammals living on farms have higher rates of resistance and are more frequently multiresistant than E. coli isolates from environments, such as natural areas, that are less impacted by human and agricultural activities. No Salmonella isolates were recovered from any of the wild small mammal feces. This study suggests that close proximity to food animal agriculture increases the likelihood that E. coli isolates from wild animals are resistant to some antimicrobials, possibly due to exposure to resistant E. coli isolates from livestock, to the resistance genes of these isolates, or to antimicrobials through contact with animal feed.     

Antimicrobial Resistance in Salmonella enterica Serovar Heidelberg Isolates from Retail Meats, Including Poultry, from 2002 to 2006

Salmonella enterica serovar Heidelberg frequently causes food-borne illness in humans. There are few data on the prevalence, antimicrobial susceptibility, and genetic diversity of Salmonella serovar Heidelberg isolates in retail meats. We compared the prevalences of Salmonella serovar Heidelberg in a sampling of 20,295 meats, including chicken breast (n = 5,075), ground turkey (n = 5,044), ground beef (n = 5,100), and pork chops (n = 5,076), collected during 2002 to 2006. Isolates were analyzed for antimicrobial susceptibility and compared genetically using pulsed-field gel electrophoresis (PFGE) and PCR for the bla_CMY gene. A total of 298 Salmonella serovar Heidelberg isolates were recovered, representing 21.6% of all Salmonella serovars from retail meats. One hundred seventy-eight (59.7%) were from ground turkey, 110 (36.9%) were from chicken breast, and 10 (3.4%) were from pork chops; none was found in ground beef. One hundred ninety-eight isolates (66.4%) were resistant to at least one compound, and 49 (16.4%) were resistant to at least five compounds. Six isolates (2.0%), all from ground turkey, were resistant to at least nine antimicrobials. The highest resistance in poultry isolates was to tetracycline (39.9%), followed by streptomycin (37.8%), sulfamethoxazole (27.7%), gentamicin (25.7%), kanamycin (21.5%), ampicillin (19.8%), amoxicillin-clavulanic acid (10.4%), and ceftiofur (9.0%). All isolates were susceptible to ceftriaxone and ciprofloxacin. All ceftiofur-resistant strains carried bla_CMY. PFGE using XbaI and BlnI showed that certain clones were widely dispersed in different types of meats and meat brands from different store chains in all five sampling years. These data indicate that Salmonella serovar Heidelberg is a common serovar in retail poultry meats and includes widespread clones of multidrug-resistant strains.     

Influence of Antibiotic Selection on Genetic Composition of Escherichia coli Populations from Conventional and Organic Dairy Farms

The widespread agricultural use of antimicrobials has long been considered a crucial influence on the prevalence of resistant genes and bacterial strains. It has been suggested that antibiotic applications in agricultural settings are a driving force for the development of antimicrobial resistance, and epidemiologic evidence supports the view that there is a direct link between resistant human pathogens, retail produce, farm animals, and farm environments. Despite such concerns, little is understood about the population processes underlying the emergence and spread of antibiotic resistance and the reversibility of resistance when antibiotic selective pressure is removed. In this study, hierarchical log-linear modeling was used to assess the association between farm type (conventional versus organic), age of cattle (calf versus cow), bacterial phenotype (resistant versus susceptible), and the genetic composition of Escherichia coli populations (E. coli Reference Collection [ECOR] phylogroup A, B1, B2, or D) among 678 susceptible and resistant strains from a previously published study of 60 matched dairy farms (30 conventional and 30 organic) in Wisconsin. The analysis provides evidence for clonal resistance (ampicillin resistance) and genetic hitchhiking (tetracycline resistance [Tet^r]), estimated the rate of compositional change from conventional farming to organic farming (mean, 8 years; range, 3 to 15 years), and discovered a significant association between low multidrug resistance, organic farms, and strains of the numerically dominant phylogroup B1. These data suggest that organic farming practices not only change the frequency of resistant strains but also impact the overall population genetic composition of the resident E. coli flora. In addition, the results support the hypothesis that the current prevalence of Tet^r loci on dairy farms has little to do with the use of this antibiotic.     

Measurement of Antimicrobial-Resistant Escherichia coli in Pig Feces with a Hydrophobic Grid Membrane Filter Interpreter System

Hydrophobic grid membrane filter technology was used to measure resistance among Escherichia coli in pig fecal samples to ampicillin, sulfisoxazole, and tetracycline. The method accurately measured resistance, with sensitivities ranging from 96.5 to 99.5% and specificities ranging from 87.0 to 98.3%, and it identified E. coli with 96% confidence.     

Presence and Sources of Fecal Coliform Bacteria in Epilithic Periphyton Communities of Lake Superior

Epilithic periphyton communities were sampled at three sites on the Minnesota shoreline of Lake Superior from June 2004 to August 2005 to determine if fecal coliforms and Escherichia coli were present throughout the ice-free season. Fecal coliform densities increased up to 4 orders of magnitude in early summer, reached peaks of up to 1.4 × 10⁵ CFU cm⁻² by late July, and decreased during autumn. Horizontal, fluorophore-enhanced repetitive-PCR DNA fingerprint analyses indicated that the source for 2% to 44% of the E. coli bacteria isolated from these periphyton communities could be identified when compared with a library of E. coli fingerprints from animal hosts and sewage. Waterfowl were the major source (68 to 99%) of periphyton E. coli strains that could be identified. Several periphyton E. coli isolates were genotypically identical (≥92% similarity), repeatedly isolated over time, and unidentified when compared to the source library, suggesting that these strains were naturalized members of periphyton communities. If the unidentified E. coli strains from periphyton were added to the known source library, then 57% to 81% of E. coli strains from overlying waters could be identified, with waterfowl (15 to 67%), periphyton (6 to 28%), and sewage effluent (8 to 28%) being the major potential sources. Inoculated E. coli rapidly colonized natural periphyton in laboratory microcosms and persisted for several weeks, and some cells were released to the overlying water. Our results indicate that E. coli from periphyton released into waterways confounds the use of this bacterium as a reliable indicator of recent fecal pollution.     

Salmonella enterica Burden in Harvest-Ready Cattle Populations from the Southern High Plains of the United States

Our objectives were to quantify the Salmonella enterica burdens in harvest-ready cattle and to identify specific at-risk populations of cattle most likely to harbor multiply resistant S. enterica. Hide swabs were collected in abattoirs from three cohorts of cattle (feedlot origin cattle that had achieved desirable harvest characteristics and dairy- and beef-type cows harvested because of poor productivity). Feces were collected from two cohorts housed in feedlots (cattle that had achieved desirable harvest characteristics and animals identified for salvage recovery because of poor productivity). Facilities were visited on four occasions over a 12-month period. Salmonella enterica isolates were recovered, and organisms were quantified using standard microbiological methodologies. Susceptibility to antimicrobial drugs and serotype were determined for one S. enterica isolate per sample. Salmonella enterica was recovered from 55.6% of 1,681 samples. The prevalences on hides and in feces were 69.6% and 30.3%, respectively. The concentrations of S. enterica organisms averaged (as determined by the most probable number technique) 1.82 log₁₀/100 cm² of hides and 0.75 log₁₀/g of feces. None of the isolates recovered from cattle that had achieved desirable harvest characteristics were resistant to four or more drugs. For isolates recovered from animals with poor productivity characteristics, 6.5% were resistant to four or more drugs. Twenty-two serovars were identified, with the most common being Salmonella enterica serovar Anatum (25.5%), Salmonella enterica serovar Montevideo (22.2%), and Salmonella enterica serovar Cerro (12.5%). High-level resistance, i.e., resistance to four or more drugs, was clustered within a few relatively uncommon serovars. These results demonstrate that even though S. enterica isolates are readily recoverable from harvest-ready cattle, multiply resistant variants are rare and are associated with specific serovars in cattle harvested because of poor productivity characteristics.     

Influx of Enterococci and Associated Antibiotic Resistance and Virulence Genes from Ready-To-Eat Food to the Human Digestive Tract

The influx of enterococcal antibiotic resistance (AR) and virulence genes from ready-to-eat food (RTEF) to the human digestive tract was assessed. Three RTEFs (chicken salad, chicken burger, and carrot cake) were sampled from five fast-food restaurants five times in summer (SU) and winter (WI). The prevalence of enterococci was significantly higher in SU (92.0% of salad samples and 64.0% of burger samples) than in WI (64.0% of salad samples and 24.0% of burger samples). The overall concentrations of enterococci during the two seasons were similar (~10³ CFU/g); the most prevalent were Enterococcus casseliflavus (41.5% of isolates) and Enterococcus hirae (41.5%) in WI and Enterococcus faecium (36.8%), E. casseliflavus (27.6%), and Enterococcus faecalis (22.4%) in SU. Resistance in WI was detected primarily to tetracycline (50.8%), ciprofloxacin (13.8%), and erythromycin (4.6%). SU isolates were resistant mainly to tetracycline (22.8%), erythromycin (22.1%), and kanamycin (13.0%). The most common tet gene was tet(M) (35.4% of WI isolates and 11.9% of SU isolates). The prevalence of virulence genes (gelE, asa1, cylA, and esp) and marker genes for clinical isolates (EF_0573, EF_0592, EF_0605, EF_1420, EF_2144, and pathogenicity island EF_0050) was low (≤12.3%). Genotyping of E. faecalis and E. faecium using pulsed-field gel electrophoresis revealed that the food contamination likely originated from various sources and that it was not clonal. Our conservative estimate (single AR gene copy per cell) for the influx of tet genes alone to the human digestive tract is 3.8 × 10⁵ per meal (chicken salad). This AR gene influx is frequent because RTEFs are commonly consumed and that may play a role in the acquisition of AR determinants in the human digestive tract.     

Antimicrobial Resistance-Conferring Plasmids with Similarity to Virulence Plasmids from Avian Pathogenic Escherichia coli Strains in Salmonella enterica Serovar Kentucky Isolates from Poultry

Salmonella enterica, a leading cause of food-borne gastroenteritis worldwide, may be found in any raw food of animal, vegetable, or fruit origin. Salmonella serovars differ in distribution, virulence, and host specificity. Salmonella enterica serovar Kentucky, though often found in the food supply, is less commonly isolated from ill humans. The multidrug-resistant isolate S. Kentucky {"type":"entrez-protein","attrs":{"text":"CVM29188","term_id":"985925135","term_text":"CVM29188"}}CVM29188, isolated from a chicken breast sample in 2003, contains three plasmids (146,811 bp, 101,461 bp, and 46,121 bp), two of which carry resistance determinants (pCVM29188_146 [strAB and tetRA] and pCVM29188_101 [bla_CMY-2 and sugE]). Both resistance plasmids were transferable by conjugation, alone or in combination, to S. Kentucky, Salmonella enterica serovar Newport, and Escherichia coli recipients. pCVM29188_146 shares a highly conserved plasmid backbone of 106 kb (>90% nucleotide identity) with two virulence plasmids from avian pathogenic Escherichia coli strains (pAPEC-O1-ColBM and pAPEC-O2-ColV). Shared avian pathogenic E. coli (APEC) virulence factors include iutA iucABCD, sitABCD, etsABC, iss, and iroBCDEN. PCR analyses of recent (1997 to 2005) S. Kentucky isolates from food animal, retail meat, and human sources revealed that 172 (60%) contained similar APEC-like plasmid backbones. Notably, though rare in human- and cattle-derived isolates, this plasmid backbone was found at a high frequency (50 to 100%) among S. Kentucky isolates from chickens within the same time span. Ninety-four percent of the APEC-positive isolates showed resistance to tetracycline and streptomycin. Together, our findings of a resistance-conferring APEC virulence plasmid in a poultry-derived S. Kentucky isolate and of similar resistance/virulence plasmids in most recent S. Kentucky isolates from chickens and, to lesser degree, from humans and cattle highlight the need for additional research in order to examine the prevalence and spread of combined virulence and resistance plasmids in bacteria in agricultural, environmental, and clinical settings.Nontyphoidal Salmonella enterica infections are one of the leading causes of bacterial food-borne gastroenteritis worldwide and an important public health problem in the United States, causing an estimated 1.4 million cases of infection, 15,000 hospitalizations, and more than 400 deaths annually in the United States alone (41). Although Salmonella infection usually presents with self-limiting diarrhea, in some patient populations, such as the immunocompromised, it can lead to life-threatening systemic infections that require effective and immediate antimicrobial therapy (21). The global emergence of multidrug resistance in S. enterica isolates from agricultural and clinical settings has therefore raised concerns and resulted in the establishment of several national resistance surveillance programs, such as the European Antimicrobial Resistance Surveillance System and the National Antimicrobial Monitoring System (NARMS) in the United States.In the United States, antimicrobial compounds are widely used in agriculture, not only to treat and prevent disease in plants, fruits, vegetables, and animals but also to promote growth in poultry and other livestock (25, 38). As a consequence, multidrug resistance is commonly detected in enterobacteria isolated from veterinary sources, including nontyphoidal Salmonella and other food-borne pathogens (40). Several studies have indicated the possibility that resistance reservoirs in animals can promote the transmission of resistance determinants from agricultural to clinical settings via food contaminants (1, 2, 45). Whether antimicrobial use in agriculture enhances the distribution and spectrum of antimicrobial resistance phenotypes in clinical settings has been the focus of vigorous debate within the public health and research communities (15, 19, 24, 27, 43). The extent of multidrug resistance in food-borne pathogens (10, 40), however, remains a concern. In 2006, only 17.7%, 25.0%, 38.8%, and 73.7% of the nontyphoidal Salmonella isolates from ground turkey, pork chop, chicken breast, and ground beef samples, respectively, showed susceptibility to all 16 antimicrobial compounds tested as part of the NARMS program (10). On the other hand, the overall prevalence of antimicrobial resistance phenotypes in nontyphoidal Salmonella isolates from human sources has slightly decreased, from 33.8% of all 876 isolates tested in 1996 to 19.4% of all 1,654 isolates tested in 2005 displaying a detectable resistance phenotype to at least one out of five antimicrobial subclasses as defined by the Clinical and Laboratory Standards Institute. However, in the same interval, increases from 0.4% to 2.4% and 0.2% to 2.9% in resistance to the clinically important subclasses of quinolones (nalidixic acid [Nal]) and cephalosporins (ceftiofur), respectively, were observed for the same set of human Salmonella isolates (6). Altogether, these reports demonstrate the need for further investigations on the influence of antimicrobial selection on the evolution, distribution, and transmission of resistance and virulence phenotypes among enteric bacteria derived from agricultural and clinical settings in order to prevent or at least limit the future spread of resistant zoonotic pathogens between these environments.Salmonella enterica subsp. enterica serovar Kentucky is widespread in poultry meats but is relatively uncommon in human cases of salmonellosis (7). S. Kentucky did not rank among the 20 most frequent Salmonella serotypes isolated from human sources in 2006 (7). In food-related sources, however, it is often found in animal samples and has been the most common serotype isolated from chickens (48.8%) (40) and chicken meat (38.8%) (10). At lower proportions, it is also present in turkey and cattle (2.6% and 3.6% of all nontyphoidal Salmonella isolates, respectively) (40). In the past decade, the fraction of S. Kentucky isolates from chickens, compared with other serotypes, has been increasing steadily, from 25% in 1997 to almost 50% in 2006 (40). Interestingly, while on average only 63 (0.174%) S. Kentucky isolates were reported between 1996 and 2004 among all nontyphoidal salmonellae from human samples, this number increased in 2005 (81 isolates [0.224%]) and 2006 (123 isolates [0.302%]) (7). Antimicrobial resistance phenotypes in S. Kentucky isolates from chicken meat are overrepresented compared to those in other serovars, with resistance to tetracycline (72.9%) and streptomycin (69.5%) being most commonly found (10). Although a causal connection between the increase of S. Kentucky in chickens and the number of human infections caused by the same serovar has not been demonstrated, further investigation is warranted, particularly in light of the high prevalence of antimicrobial resistance phenotypes in S. Kentucky isolates from chickens and the increasing resistance to beta-lactam compounds.Here, we describe the plasmid component of the genome of S. Kentucky {"type":"entrez-protein","attrs":{"text":"CVM29188","term_id":"985925135","term_text":"CVM29188"}}CVM29188, a multidrug-resistant strain that was isolated in 2003 from a chicken meat sample with resistance to streptomycin, tetracycline, ampicillin, and ceftiofur. Using a combination of in silico and in vivo approaches, including comparative plasmid sequence analysis, conjugative plasmid transfer, and PCR-based plasmid screenings, we present new insights into the genetic basis for multidrug resistance phenotypes of this isolate that provide new clues about virulence evolution and host adaptation in this Salmonella serovar.     

Molecular Subtyping and Genetic Analysis of the Enterohemolysin Gene (ehxA) from Shiga Toxin-Producing Escherichia coli and Atypical Enteropathogenic E. coli

Analyses of the distribution of virulence factors among different Escherichia coli pathotypes, including Shiga toxin-producing E. coli (STEC), may provide some insight into the mechanisms by which different E. coli strains cause disease and the evolution of distinct E. coli types. The aim of this study was to examine the DNA sequence of the gene for enterohemolysin, a plasmid-encoded toxin that readily causes the hemolysis of washed sheep erythrocytes, and to assess the distribution of enterohemolysin subtypes among E. coli isolates from various human and animal sources. The 2,997-bp ehxA gene was amplified from 227 (63.8%) of 356 stx- and/or eae-positive E. coli strains isolated from cattle and sheep and from 24 (96.0%) of 25 STEC strains isolated from humans with diarrheal disease. By using PCR and restriction fragment length polymorphism (RFLP) analysis of ehxA, six distinct PCR-RFLP types (A to F) were observed, with strains of subtypes A and C constituting 91.6% of all the ehxA-positive strains. Subtype A was associated mainly with ovine strains with stx only (P < 0.001), and subtype C was associated with bovine eae-positive strains (P < 0.001). Eleven ehxA alleles were fully sequenced, and the phylogenetic analysis indicated the presence of three closely related (>95.0%) ehxA sequence groups, one including eae-positive strains (subtypes B, C, E, and F) and the other two including mainly eae-negative STEC strains (subtypes A and D). In addition to being widespread among STEC strains, stx-negative, eae-positive strains (atypical enteropathogenic E. coli strains) isolated from cattle and sheep have similar ehxA subtypes and hemolytic activities.     

Genotype Analysis of Escherichia coli Strains Isolated from Children and Chickens Living in Close Contact

Escherichia coli isolates from rectal swabs from 62 chickens and stools from 42 children living in close contact with chickens on the same farms in Kiambu district, Kenya, were compared for their genetic relatedness. Antibiotic susceptibility profiles broadly categorized isolates from the children and from the chickens into two separate clusters: the majority (144; 85.5%) of the E. coli isolates from children were multidrug resistant, while the majority (216; 87.1%) of the E. coli isolates from chickens were either fully susceptible or resistant only to tetracycline. Sixty- and 100- to 110-MDA plasmids were found to encode the transferable resistance to co-trimoxazole and tetracycline. HindIII restriction endonuclease digestion of the 60- and 100- to 110-MDA plasmids produced four distinct patterns for isolates from children and three distinct patterns for isolates from chickens. XbaI digestion of genomic DNA followed by pulsed-field gel electrophoresis (PFGE) analysis produced 14 distinct clusters. There were six distinct PFGE clusters among the isolates from children, while among the isolates from chickens there were seven distinct clusters. Only one PFGE cluster contained isolates from both children and chickens, with the isolates displaying an approximately 60% coefficient of similarity. This study showed that although several different genotypes of E. coli were isolated from children and chickens from the same farms, the E. coli strains from these two sources were distinct.     

Determinants of Antimicrobial Resistance in Escherichia coli Strains Isolated from Faeces and Urine of Women with Recurrent Urinary Tract Infections

For women with recurrent urinary tract infections (rUTI), the contribution of antibiotic use versus patient-related factors in determining the presence of antimicrobial resistance in faecal and urinary Escherichia coli, obtained from the same patient population, has not been assessed yet. Within the context of the ‘Non-antibiotic prophylaxis for recurrent urinary tract infections’ (NAPRUTI) study, the present study assessed determinants of antimicrobial resistance in E. coli isolated from urinary and faecal samples of women with rUTIs collected at baseline. Potential determinants of resistance were retrieved from self-administered questionnaires. From 434 asymptomatic women, 433 urinary and 424 faecal samples were obtained. E. coli was isolated from 146 (34%) urinary samples and from 336 (79%) faecal samples, and subsequently tested for antimicrobial susceptibility. Multivariable analysis showed trimethoprim/sulfamethoxazole (SXT) use three months prior to inclusion to be associated with urine E. coli resistance to amoxicillin (OR 3.6, 95% confidence interval: 1.3–9.9), amoxicillin-clavulanic acid (OR 4.4, 1.5–13.3), trimethoprim (OR 3.9, 1.4–10.5) and SXT (OR 3.2, 1.2–8.5), and with faecal E. coli resistance to trimethoprim (OR 2.0, 1.0–3.7). The number of UTIs in the preceding year was correlated with urine E. coli resistance to amoxicillin-clavulanic acid (OR 1.11, 1.01–1.22), trimethoprim (OR 1.13, 1.03–1.23) and SXT (OR 1.10, 1.01–1.19). Age was predictive for faecal E. coli resistance to amoxicillin (OR 1.02, 1.00–1.03), norfloxacin and ciprofloxacin (both OR 1.03, 1.01–1.06). In conclusion, in women with rUTI different determinants were found for urinary and faecal E. coli resistance. Previous antibiotic use and UTI history were associated with urine E. coli resistance and age was a predictor of faecal E. coli resistance. These associations could best be explained by cumulative antibiotic use.     

Prevalence and Virulence Factors of Escherichia coli Serogroups O26, O103, O111, and O145 Shed by Cattle in Scotland

A national survey was conducted to determine the prevalence of Escherichia coli O26, O103, O111, and O145 in feces of Scottish cattle. In total, 6,086 fecal pats from 338 farms were tested. The weighted mean percentages of farms on which shedding was detected were 23% for E. coli O26, 22% for E. coli O103, and 10% for E. coli O145. The weighted mean prevalences in fecal pats were 4.6% for E. coli O26, 2.7% for E. coli O103, and 0.7% for E. coli O145. No E. coli O111 was detected. Farms with cattle shedding E. coli serogroup O26, O103, or O145 were widely dispersed across Scotland and were identified most often in summer and autumn. However, on individual farms, fecal shedding of E. coli O26, O103, or O145 was frequently undetectable or the numbers of pats testing positive were small. For serogroup O26 or O103 there was clustering of positive pats within management groups, and the presence of an animal shedding one of these serogroups was a positive predictor for shedding by others, suggesting local transmission of infection. Carriage of vtx was rare in E. coli O103 and O145 isolates, but 49.0% of E. coli O26 isolates possessed vtx, invariably vtx₁ alone or vtx₁ and vtx₂ together. The carriage of eae and ehxA genes was highly associated in all three serogroups. Among E. coli serogroup O26 isolates, 28.9% carried vtx, eae, and ehxA—a profile consistent with E. coli O26 strains known to cause human disease.     

Effects of Dried Distillers’ Grain on Fecal Prevalence and Growth of Escherichia coli O157 in Batch Culture Fermentations from Cattle

Distillers’ grains (DG), a by-product of ethanol production, are fed to cattle. Associations between Escherichia coli O157 prevalence and feeding of DG were investigated in feedlot cattle (n = 379) given one of three diets: steam-flaked corn (SFC) and 15% corn silage with 0 or 25% dried distillers’ grains (DDG) or SFC with 5% corn silage and 25% DDG. Ten fecal samples were collected from each pen weekly for 12 weeks to isolate E. coli O157. Cattle fed 25% DDG with 5 or 15% silage had a higher (P = 0.01) prevalence of E. coli O157 than cattle fed a diet without DDG. Batch culture ruminal or fecal microbial fermentations were conducted to evaluate the effect of DDG on E. coli O157 growth. The first study utilized microbial inocula from steers fed SFC or dry-rolled corn with 0 or 25% DDG and included their diet as the substrate. Ruminal microbial fermentations from steers fed DDG had higher E. coli O157 contents than ruminal microbial fermentations from steers fed no DDG (P < 0.05) when no substrate was included. Fecal fermentations showed no DDG effect on E. coli O157 growth. In the second study with DDG as a substrate, ruminal fermentations with 0.5 g DDG had higher (P < 0.01) E. coli O157 concentrations at 24 h than ruminal fermentations with 0, 1, or 2 g DDG. In fecal fermentations, 2 g DDG resulted in a higher concentration (P < 0.05) at 24 h than 0, 0.5, or 1 g DDG. The results indicate that there is a positive association between DDG and E. coli O157 in cattle, and the findings should have important ramifications for food safety.     

Antimicrobial resistance,virulence genes and PFGE-profiling of Escherichia coli isolates from South Korean cattle farms

To estimate the prevalence of Escherichia coli with potential pathogenicity in cattle farm in South Korea, a total of 290 E. coli isolates were isolated from cattle farms over a period of 2 years in South Korea. These were examined for phenotypic and genotypic characteristics including antimicrobial susceptibility, serotype, and gene profiles of virulence and antimicrobial resistance. The most dominant virulence gene was f17 (26.2%), followed by stx2 (15.9%), ehxA (11.0%), stx1 (8.3%), eae (5.2%), and sta (4.1%). Some shiga-toxin producing E. coli isolates possessed eae (15.9%). All isolates except for one showed resistance to one or more antimicrobials, with 152 isolates exhibiting multidrug-resistance. The most prevalent resistance phenotype detected was streptomycin (63.1%), followed by tetracycline (54.5%), neomycin (40.3%), cephalothin (32.8%), amoxicillin (30.0%), ampicillin (29.7%), and sulphamethoxazole/trimethoprim (16.6%). The associated resistance determinants detected were strA-strB (39.0%), tet(E) (80.0%), tet(A) (27.6%), aac(3)-IV (33.1%), aphA1 (21.4%), bla _TEM (23.8%), and sul2 (22.1%). When investigated by O serotyping and PFGE molecular subtyping, the high degree of diversity was exhibited in E. coli isolates. These results suggest that E. coli isolates from South Korean cattle farms are significantly diverse in terms of virulence and antimicrobial resistance. In conclusion, the gastroinstestinal flora of cattle could be a significant reservoir of diverse virulence and antimicrobial resistance determinants, which is potentially hazardous to public health.     

Effects of Subtherapeutic Administration of Antimicrobial Agents to Beef Cattle on the Prevalence of Antimicrobial Resistance in Campylobacter jejuni and Campylobacter hyointestinalis

The influence of antimicrobial agents on the development of antimicrobial resistance (AMR) in Campylobacter isolates recovered from 300 beef cattle maintained in an experimental feedlot was monitored over a 315-day period (11 sample times). Groups of calves were assigned to one of the following antimicrobial treatments: chlortetracycline and sulfamethazine (CS), chlortetracycline alone (Ct), virginiamycin, monensin, tylosin phosphate, and no antimicrobial agent (i.e., control treatment). In total, 3,283 fecal samples were processed for campylobacters over the course of the experiment. Of the 2,052 bacterial isolates recovered, 92% were Campylobacter (1,518 were Campylobacter hyointestinalis and 380 were C. jejuni). None of the antimicrobial treatments decreased the isolation frequency of C. jejuni relative to the control treatment. In contrast, C. hyointestinalis was isolated less frequently from animals treated with CS and to a lesser extent from animals treated with Ct. The majority (≥94%) of C. jejuni isolates were sensitive to ampicillin, erythromycin, and ciprofloxacin, but more isolates with resistance to tetracycline were recovered from animals fed Ct. All of the 1,500 isolates of C. hyointestinalis examined were sensitive to ciprofloxacin. In contrast, 11%, 10%, and 1% of these isolates were resistant to tetracycline, erythromycin, and ampicillin, respectively. The number of animals from which C. hyointestinalis isolates with resistance to erythromycin and tetracycline were recovered differed among the antimicrobial treatments. Only Ct administration increased the carriage rates of erythromycin-resistant isolates of C. hyointestinalis, and the inclusion of CS in the diet increased the number of animals from which tetracycline-resistant isolates were recovered. The majority of C. hyointestinalis isolates with resistance to tetracycline were obtained from cohorts within a single pen, and most of these isolates were recovered from cattle during feeding of a forage-based diet as opposed to a grain-based diet. The findings of this study show that the subtherapeutic administration of tetracycline, alone and in combination with sulfamethazine, to feedlot cattle can select for the carriage of resistant strains of Campylobacter species. Considering the widespread use of in-feed antimicrobial agents and the high frequency of beef cattle that shed campylobacters, the development of AMR should be monitored as part of an on-going surveillance program.