Prevalence and Antimicrobial Susceptibility of Indicator Organisms Escherichia coli and Enterococcus spp. Isolated from U.S. Animal Food, 2005-2011.

The role animal food plays in the introduction of antimicrobial-resistant bacteria into the human food chain is not well understood. We conducted an analysis of 1025 samples (647 pet food and 378 animal feed) collected across the United States during 2005-2011 for two indicator organisms (Escherichia coli and Enterococcus spp.). The overall prevalence ranged from 12.5% for E. coli to 45.2% for Enterococcus spp., and 11.2% of samples harbored both organisms. Regardless of bacterial genus, animal feed had significantly higher prevalence than pet food (p < 0.001). A general downward trend in prevalence was observed from 2005 to 2009 followed by an upward trend thereafter. Among E. coli isolates (n = 241), resistance was highest to tetracycline (11.2%) and below 5% for fourteen other antimicrobials. Among Enterococcus spp. isolates (n = 1074), Enterococcus faecium (95.1%) was the predominant species. Resistance was most common to tetracycline (30.1%) and ciprofloxacin (10.7%), but below 10% for thirteen other antimicrobials. Multidrug-resistant organisms were observed among both E. coli and Enterococcus spp. isolates at 3.3%. Compared to National Antimicrobial Resistance Monitoring System (NARMS) 2011 retail meat and animal data, the overall resistance for both organisms was much lower in animal food. These findings help establish a historic baseline for the prevalence and antimicrobial resistance among U.S. animal food products and future efforts may be needed to monitor changes over time.

Whole-Genome Sequence Analysis of CTX-M Containing Escherichia coli Isolates from Retail Meats and Cattle in the United States.

In recent years, there have been increased reports on the detection of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Salmonella strains from food-producing animals and animal products in the United States. We characterized 18 ESBL E. coli isolates from cattle (n = 5), chicken breast (n = 5), ground turkey (n = 6), ground beef (n = 1), and pork chops (n = 1) that were collected by the National Antimicrobial Resistance Monitoring System (NARMS) between 2011 and 2015. In vitro antimicrobial susceptibility testing was done against a panel of 14 antimicrobials followed by a secondary panel of 9 ß-lactam agents. Whole-genome sequencing was used to characterize the resistome, plasmids, and the genetic structures of the ESBL genes. All ESBL-producing E. coli isolates were resistant to at least three antimicrobial classes and carried various blaCTX-M genes. Most of the cattle and ground turkey isolates carried blaCTX-M-27. In chicken breast isolates, blaCTX-M-1 was present as part of an ISEcp1 transposition unit carried on a plasmid that shares sequence similarity with the backbone structure of the IncI plasmid. Isolates carrying the blaCTX-M-14 and blaCTX-M-15 genes, widely distributed in human clinical isolates, were also isolated. To our knowledge, this is the first report of the widely distributed blaCTX-M-14 and blaCTX-M-15 in E. coli isolates from retail meat samples in the United States. Different insertional sequences were identified upstream of these blaCTX-Ms, including ISEcp1, IS26, and IS903-D. CTX-M in E. coli from food animals and retail chicken breast were often present on plasmids with other resistance genes. Other resistance genes identified included aadA, strA, strB, aac(3)-IId, aac(3)-VIa, aph(3')-Ic, blaTEM, blaHERA-3, floR, sul1, sul2, catA1, tetA, tetB, dfrA, and qacE. These data describe the emergence of CTX-M-carrying E. coli isolates in food animals and animal products monitored by NARMS program.

Gyrase A Mutations in Campylobacter Associated with Decreased Susceptibility to Different Fluoroquinolones.

Campylobacter is a leading cause of foodborne diarrheal illness worldwide, and the emergence of antimicrobial-resistant strains is a major global public health concern. The goal of this study was to compare the activity of different fluoroquinolone antibiotics against ciprofloxacin-resistant Campylobacter jejuni and Campylobacter coli. Isolates from retail meats collected between 2002 and 2009 were selected based on their in vitro susceptibility testing results against ciprofloxacin. In total, 289 C. jejuni and 165 C. coli were collected and analyzed. All ciprofloxacin-resistant isolates had a single mutation (Thr86Ile) in their gyrase A (gyrA) gene and exhibited decreased susceptibility to all eight fluoroquinolones tested. Gatifloxacin, enrofloxacin, and levofloxacin showed greater activity than the other fluoroquinolone drugs in both ciprofloxacin-sensitive and -resistant strains.

Effects of low concentrations of erythromycin, penicillin, and virginiamycin on bacterial resistance development in vitro.

Distillers grains are co-products of the corn ethanol industry widely used in animal feed. We examined the effects of erythromycin, penicillin, and virginiamycin at low concentrations reflective of those detected in distillers grains on bacterial resistance selection. At 0.1 µg/ml erythromycin, macrolide-resistant mutants were induced in one Campylobacter coli and one Enterococcus faecium strain, while these strains plus three additional C. coli, one additional E. faecium, and one C. jejuni also developed resistance when exposed to 0.25 µg/ml erythromycin. At 0.5 µg/ml erythromycin, a total of eight strains (four Campylobacter and four Enterococcus) obtained macrolide-resistant mutants, including two strains from each genus that were not selected at lower erythromycin concentrations. For penicillin, three of five E. faecium strains but none of five Enterococcus faecalis strains consistently developed resistance at all three selection concentrations. Virginiamycin at two M1:S1 ratios did not induce resistance development in four out of five E. faecium strains; however, increased resistance was observed in the fifth one under 0.25 and 0.5 µg/ml virginiamycin selections. Although not yet tested in vivo, these findings suggest a potential risk of stimulating bacterial resistance development in the animal gut when distillers grains containing certain antibiotic residues are used in animal feed.

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.

Identification and antimicrobial resistance of extraintestinal pathogenic Escherichia coli from retail meats.

Extraintestinal pathogenic Escherichia coli (ExPEC) causes a variety of infections outside the gastrointestinal tract. Retail meats are frequently contaminated with E. coli strains, and they might serve as a vehicle for transmitting ExPEC. A total of 1,275 E. coli isolates recovered from ground beef, ground turkey, chicken breasts, and pork chops obtained in Georgia, Maryland, Oregon, and Tennessee in 2006 were investigated for the presence of ExPEC by using multiplex PCR. Identified ExPEC isolates were assigned to serogroups and phylogenetic groups and then analyzed for antimicrobial susceptibility. Approximately 16% (200 of 1,275) of the E. coli isolates were identified as ExPEC, based on defined genetic criteria. The occurrence of ExPEC was highest in E. coli isolated from ground turkey (23.5%) and chicken breasts (20.2%), and less frequent in isolates from pork chops (8.3%) and ground beef (3.4%). Phylogenetic grouping revealed that most (66.5%) ExPEC isolates fell into the same phylogenetic groups (B2 and D) as did virulent human ExPEC strains. Among the 15 antimicrobial agents tested, resistance to tetracycline (67.0%), sulfisoxazole (59.5%), and streptomycin (46.0%) was most frequent. Most ExPEC isolates (n = 163 [81.5%]) were resistant to at least one antimicrobial agent, and more than half (n = 114 [57%]) exhibited resistance to at least three drugs. This study found that ExPEC strains, including antimicrobial-resistant strains, were frequent among E. coli recovered from retail meats, especially those from chicken and turkey products. These findings indicate a need to better understand the role of certain meat types as potential sources of human ExPEC infection.

Characterization of chloramphenicol resistance in beta-hemolytic Escherichia coli associated with diarrhea in neonatal swine.

Ninety beta-hemolytic Escherichia coli isolates associated with diarrhea in neonatal pigs from multiple farms in Oklahoma were investigated for known associated disease serotypes, virulence factors, ribotypes, and antimicrobial susceptibility phenotypes. Fifteen different serotypes were observed, with 58% of isolates belonging to groups that produce one of three major enterotoxins: O149, O147, and O139. Thirty percent of the swine E. coli isolates possessed a combination of F4 fimbriae and the heat-labile toxin and heat-stable toxin B enterotoxins. Seventy-three percent of the E. coli isolates were resistant to five or more antibiotics. Interestingly, 53% of swine E. coli isolates exhibited resistance to chloramphenicol (CHL), an antibiotic whose use in food animals has been prohibited in the United States since the mid-1980s. The cmlA gene, which encodes a putative CHL efflux pump, was detected by PCR in 47 of the 48 CHL-resistant isolates, and 4 of these also possessed the cat2 gene, which encodes a chloramphenicol acetyltransferase. The one CHL-resistant isolate that did not contain either cmlA or cat-2 possessed the flo gene, which confers resistance to both florfenicol and CHL. To determine whether CHL-resistant swine E. coli isolates represented dissemination of a clonal strain, all 90 isolates were analyzed by ribotyping. Seventeen distinct E. coli ribogroups were identified, with CHL resistance observed among the isolates in all except one of the major ribogroups. The identification of the cmlA gene among diverse hemolytic enterotoxigenic E. coli strains demonstrates its broad dissemination in the swine production environment and its persistence even in the absence of CHL selection pressure.