Daniel A. Tadesse

Antimicrobial Drug Resistance in Escherichia coli from Humans and Food Animals, United States, 1950-2002

Determining drug resistance trends will optimize treatment and public health responses.

WGS accurately predicts antimicrobial resistance in Escherichia coli

OBJECTIVES The objective of this study was to determine the effectiveness of WGS in identifying resistance genotypes of MDR Escherichia coli and whether these correlate with observed phenotypes. METHODS Seventy-six E. coli strains were isolated from farm cattle and measured for phenotypic resistance to 15 antimicrobials with the Sensititre(®) system. Isolates with resistance to at least four antimicrobials in three classes were selected for WGS using an Illumina MiSeq. Genotypic analysis was conducted with in-house Perl scripts using BLAST analysis to identify known genes and mutations associated with clinical resistance. RESULTS Over 30 resistance genes and a number of resistance mutations were identified among the E. coli isolates. Resistance genotypes correlated with 97.8% specificity and 99.6% sensitivity to the identified phenotypes. The majority of discordant results were attributable to the aminoglycoside streptomycin, whereas there was a perfect genotype-phenotype correlation for most antibiotic classes such as tetracyclines, quinolones and phenicols. WGS also revealed information about rare resistance mechanisms, such as structural mutations in chromosomal copies of ampC conferring third-generation cephalosporin resistance. CONCLUSIONS WGS can provide comprehensive resistance genotypes and is capable of accurately predicting resistance phenotypes, making it a valuable tool for surveillance. Moreover, the data presented here showing the ability to accurately predict resistance suggest that WGS may be used as a screening tool in selecting anti-infective therapy, especially as costs drop and methods improve.

Genetic relatedness and molecular characterization of multidrug resistant Acinetobacter baumannii isolated in central Ohio, USA

BackgroundOver the last decade, nosocomial infections due to Acinetobacter baumannii have been described with an increasing trend towards multidrug resistance, mostly in intensive care units. The aim of the present study was to determine the clonal relatedness of clinical isolates and to elucidate the genetic basis of imipenem resistance.MethodsA. baumannii isolates (n = 83) originated from two hospital settings in central Ohio were used in this study. Pulsed-field gel electrophoresis genotyping and antimicrobial susceptibility testing for clinically relevant antimicrobials were performed. Resistance determinants were characterized by using different phenotypic (accumulation assay for efflux) and genotypic (PCR, DNA sequencing, plasmid analysis and electroporation) approaches.ResultsThe isolates were predominantly multidrug resistant (>79.5%) and comprised of thirteen unique pulsotypes, with genotype VII circulating in both hospitals. The presence of blaOXA-23 in 13% (11/83) and ISAba1linked blaOXA-66 in 79.5% (66/83) of clinical isolates was associated with high level imipenem resistance. In this set of OXA producing isolates, multidrug resistance was bestowed by blaADC-25, class 1 integron-borne aminoglycoside modifying enzymes, presence of sense mutations in gyrA/parC and involvement of active efflux (with evidence for the presence of adeB efflux gene).ConclusionThis study underscores the major role of carbapenem-hydrolyzing class D β-lactamases, and in particular the acquired OXA-23, in the dissemination of imipenem-resistant A. baumannii. The co-occurrence of additional resistance determinant could also be a significant threat.

Longitudinal Study of Salmonella Dispersion and the Role of Environmental Contamination in Commercial Swine Production Systems

ABSTRACT This study investigated the roles of various environmental sources, such as truck-washing systems, waste-processing lagoons, and other sources, as potential contributors to the exposure and dissemination of Salmonella in commercial swine production systems. Four cohorts of nursery age swine herds which originated from distinct farm flows were selected. In addition, cross-sectional sampling of four truck wash stations selected based on the types of disinfectants and sources of water used for sanitizing trucks were tested. Salmonella isolates were recovered from pigs (feces, cecal contents, and mesenteric lymph nodes) and environmental sources (barn floor, lagoon, barn flush, trucks, and holding pens). Antimicrobial susceptibility testing and genotyping were conducted using Kirby-Bauer disk diffusion and amplified fragment length polymorphism, respectively. Salmonella prevalence significantly increased with age from late nursery to slaughter for all of the cohorts (P = 0.007). In two of three instances, all three pig holding pens (lairage) sampled at processing were Salmonella positive. The predominant antibiotypes for all sources included ACSSuT (51.8%), SSuT (16.8%), T (6%), and pansusceptible (7.4%). For the isolates obtained at the farms, the ACSSuT phenotype was 5.6 times more likely to be found in the animals than in the environment (95% confidence interval, 4.4 to 7.2 times). Serogroup B was the most common serogroup (79%), followed by serogroup E (10.4%). Despite the fact that the four production flows were independent, 1 of the 11 genotypic clusters (cluster A1) was commonly detected in any type of sample regardless of its origin. Five of the genotypic clusters (clusters A3, A4, A5, A6, and A7) contained isolates that originated from trucks and lairage swabs and also from cecal contents and/or mesenteric lymph nodes. More interestingly, genotypic clusters A3, A4, and A6 (but not clusters A5 and A7) were not detected on the farms. They originated from the trucks and lairage swabs and then were identified from the cecal contents and/or mesenteric lymph nodes. These findings underscore the significance of various environmental factors, including inadequate truck-washing systems, and emphasize the role of lairage contamination by Salmonella that has food safety significance.

Characterization of Multidrug-Resistant Salmonella enterica Serovar Heidelberg Isolated from Humans and Animals

Salmonella enterica serovar Heidelberg has been recognized as one of the most common serovar associated with foodborne infections in the United States. It is also frequently isolated from nonhuman sources and has increasingly shown resistance to various antimicrobial agents. The present study was undertaken to identify the predominant antimicrobial resistance phenotypes and genotypes of Salmonella Heidelberg (n = 95) isolates of human, swine, and turkey origin. Antimicrobial susceptibility was done using Kirby-Bauer disk diffusion method with a panel of 12 antimicrobials. Pulsed-field gel electrophoresis genotyping was used to determine the diversity of the isolates. The antimicrobial resistance genes and carriage of Class 1 and 2 integrons were determined by polymerase chain reaction. All Salmonella Heidelberg isolates from swine were resistant to one or more of the antimicrobials tested and the majority (73.3%) showed multidrug resistance to streptomycin, tetracycline, and kanamycin (R-type: StTeKm). About 80% of the Salmonella Heidelberg isolates of human origin were pan-susceptible, however, one isolate showed multidrug resistance to 10 of 12 antimicrobials tested. Among the multidrug-resistant (MDR) Salmonella Heidelberg isolates, Class 1 integrons with variable sizes of 1.2 to 1.5 kb were detected in six isolates (three each) from humans and swine. DNA sequencing revealed that Class 1 integrons of both human and swine origin carried a gene encoding aminoglycoside adenyltransferase (aadA). Resistance genes identified in other loci include aphA1-Iab, strA, bla(TEM), and tetA (B). Both human and swine MDR strains of Salmonella Heidelberg carried the resistance phenotypes on self-transferable plasmids. Dendrogram analysis of pulsotypes indicated possible clonality of Salmonella Heidelberg between isolates of human and swine origin. The findings in this study indicate the increasing significance of swine as reservoirs of emerging MDR serovars, such as MDR Salmonella Heidelberg, is of public health significance.

Occurrence of spvA Virulence Gene and Clinical Significance for Multidrug-Resistant Salmonella Strains

Nontyphoidal Salmonella strains are important reservoirs of antimicrobial resistance. An important issue that has not been investigated is whether the multiresistant Salmonella strains are more virulent than their susceptible counterparts. Salmonella isolates collected from clinical human (n = 888) and porcine (n = 2,120) cases at the same time period and geographic location were investigated. Antimicrobial susceptibility, PCR analysis for the spvA virulence gene, and pulsed-field gel electrophoresis (PFGE) genotyping were done. Carriage of spvA was associated with multidrug-resistant (MDR) type ACSSuT strains (odds ratio, 7.1; P < 0.05), a type often implicated in bacteremic human cases. PFGE revealed that clinical isolates from pigs were more clonally related to those of human origin than the nonclinical porcine isolates. The findings suggest that MDR strains that also carry specific virulence factors are more likely to be of clinical significance.

Prevalence and Antimicrobial Resistance Profile of Campylobacter Spp. Isolated from Conventional and Antimicrobial-Free Swine Production Systems from Different U.S. Regions

We conducted a study to compare the prevalence and antimicrobial resistance profile of Campylobacter isolated from 34 farm-slaughter pair cohorts of pigs raised in conventional and antimicrobial-free (ABF) production systems. Isolates originated from four different states of two geographic regions (region 1--Ohio and Michigan; region 2--Wisconsin and Iowa). A total of 838 fecal and 1173 carcass samples were examined. Campylobacter isolates were speciated using multiplex polymerase chain reaction targeting ceuE and hipO genes. The minimum inhibitory concentration was determined using agar dilution to a panel of six antimicrobials: chloramphenicol, erythromycin, gentamicin, ciprofloxacin, nalidixic acid, and tetracycline. Campylobacter spp. was isolated from 472 of 838 pigs (56.3%). Campylobacter prevalence did not vary significantly based on production system (conventional [58.9%] and ABF [53.7%], odds ratio [OR] 1.4, 95% confidence interval [CI] 0.8-2.6, p = 0.24) or geographic region (region 1 [54.1%] and region 2 [58.2%], OR 1.02, 95% CI 0.6-1.9, p = 0.92). At slaughter plant, Campylobacter prevalence varied based on processing stages (19.4% at pre-evisceration, 25.3% at postevisceration, and 3.2% at postchill). Resistance was common to tetracycline (64.5%), erythromycin (47.9%), and nalidixic acid (23.5%). Campylobacter isolates from conventional production systems were more likely to be erythromycin resistant than from ABF (OR 3.2, 95% CI 1.4-7.2, p = 0.01). The proportion of ciprofloxacin-resistant Campylobacter coli isolates were 3.7% and 1.2% from ABF and conventional production systems, respectively. Thirty-seven out of 1257 C. coli (2.9%) were resistant to both erythromycin and ciprofloxacin, drugs of choice for treatment of invasive human campylobacteriosis. The finding of ciprofloxacin resistance, particularly from ABF herds, has significant implications on the potential role of risk factors other than mere antimicrobial use for production purposes.

Yersinia enterocolitica of Porcine Origin: Carriage of Virulence Genes and Genotypic Diversity

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against β-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.

Environmental Salmonella surveillance in the Ohio State University Veterinary Teaching Hospital.

Sampling was conducted at The Ohio State Universitys Veterinary Teaching Hospital (OSU-VTH) to evaluate the extent of environmental contamination with Salmonella enterica, at 1-week intervals beginning March 19, 2007, through May 21, 2007. Environmental samples were collected from various surface and floor locations in the equine and food animal areas using sterile moistened gauze sponges. All samples were processed using standard bacteriologic culture to identify the presence of Salmonella spp. Genetic relatedness of isolates was assessed using amplified fragment length polymorphism (AFLP) procedures, and minimum inhibitory concentrations to a panel of antimicrobial drugs were determined using microbroth dilution. A total of 16 Salmonella isolates were recovered from 270 (5.9%) cultured environmental and animal samples, with prevalence ranging from 0% to 32% on individual sampling dates. A total of 9% of the samples from the food animal section and 2.5% of the samples from the equine section tested positive for Salmonella (p = 0.03). The 16 isolates represented seven different clonal strains and four different serotypes (Paratyphi B var. L-tartate n = 3, Kentucky n = 5, Cerro n = 7, Montevideo n = 1), most of which were pansusceptible to a panel of antimicrobial drugs. Our results indicate that animal treatment areas with a high population of animals or cases within the veterinary teaching hospital can become contaminated with Salmonella, especially in high traffic areas that may facilitate horizontal dissemination. The most common occurrence appears to be infected agricultural animals that contaminate the hospital environment, but normal cleaning and disinfection appears to effectively prevent long-term contamination.

Prevalence of Yersinia enterocolitica in Antimicrobial-Free and Conventional Antimicrobial Use Swine Production

Swine are the primary reservoir for foodborne illness associated with Yersinia enterocolitica. The use of antimicrobials in animal agriculture has been hypothesized as having a potential role in the increase in prevalence of zoonotic pathogens. The objective of this study was to compare the frequency of Y. enterocolitica fecal shedding in swine reared on farms with conventional antimicrobial use policies to farms that were antimicrobial free (ABF). Swine farms were selected from three regions in the United States. In each region, farms were categorized based on antimicrobial use policy. Fecal samples were collected from pigs on-farm within 48 h of harvest. The overall proportion of Y. enterocolitica and ail-harboring Y. enterocolitica-positive pigs was 10.9% and 4.0%, respectively. There were increased odds (odds ratio [OR] 6.8, 95% confidence interval [CI] 3.46-13.28) for a pig to be Y. enterocolitica positive if it was reared on an ABF farm as compared to a conventional farm. There was no significant association between farm antimicrobial use policy and isolation of an ail-harboring Y. enterocolitica from an individual pig (OR 1.8, 95% CI 0.90-3.61). The association of antimicrobial use policy with Y. enterocolitica shedding in feces should be interpreted cautiously, as antimicrobial use cannot be separated from other management factors (e.g., confinement or outdoor housing), which may be associated with risk of Y. enterocolitica in swine.