Erythromycin-resistant Enterococci Research Articles

Loss of erm(B)-Mediated rRNA Dimethylation and Restoration of Erythromycin Susceptibility in Erythromycin-Resistant Enterococci Following Induced Linezolid Resistance.

Linezolid has been reported to restore erythromycin susceptibility in erythromycin-resistant Staphylococcus aureus. This phenomenon has not been reported in enterococci and the mechanisms involved therein are still unknown. The purpose of this study was to investigate the mechanisms involved and the effect of combining linezolid with erythromycin on erythromycin-resistant enterococci. Checkerboard techniques were used to determine drug interactions, and 12 of 14 isolates showed a synergistic effect between erythromycin and linezolid (fractional inhibitory concentration <0.5). We observed that the erm(B) gene, which encodes a dimethyltransferase responsible for erythromycin resistance, was expressed from transposon Tn1545 in the tested erythromycin-resistant enterococci. After exposure to linezolid, erm(B)-mediated rRNA dimethylation at A2071 could not be detected, and the erm(B) gene was lost following acquisition of erythromycin susceptibility. Thus, in conclusion, linezolid combined with erythromycin exerts a synergistic effect against erythromycin-resistant enterococci. Linezolid treatment suppressed erm(B)-mediated rRNA dimethylation at A2071, which could lead to loss of the erm(B) gene.

Association between Tulathromycin Treatment for Bovine Respiratory Disease and Antimicrobial Resistance Profiles among Gut Commensals and Foodborne Bacterial Pathogens Isolated from Feces of Beef Steers

This study was conducted to evaluate the association between a therapeutic dose of tulathromycin for bovine respiratory disease in beef steers and the antimicrobial and multidrug resistance profiles of the gastrointestinal tract commensals Escherichia coli and Enterococcus spp. and the foodborne pathogens Salmonella enterica and Campylobacter spp. isolated from fecal samples. Individual fecal samples were collected on days 0, 14, and 28 from 70 beef steers that were housed in a single pen and had been treated or not treated with tulathromycin. Samples were cultured for bacterial isolation, and isolates were tested for antimicrobial susceptibility with the broth microdilution method to determine the MICs of clinically relevant antimicrobials used in both human and veterinary medicine. Generalized linear mixed effects models were fitted to estimate the prevalence of the bacterial species and the prevalence of resistant isolates over time and between treated and nontreated cattle and of multidrug-resistant isolates. Model-adjusted mean prevalences of E. coli, Enterococcus spp., S. enterica, and Campylobacter spp. were 99.5, 85.9, 1.5, and 17.7%, respectively. The prevalence of erythromycin-resistant Enterococcus spp. was significantly higher on day 14 (59.7%) than on day 28 (22.2%). A higher prevalence of erythromycin-resistant Enterococcus spp. was found in samples from treated (59.3%) than in samples from nontreated (27.6%) animals. Multidrug resistance (three or more antimicrobial classes) was observed in 8.4% of E. coli isolates and 62.7% of Enterococcus isolates. The administration of tulathromycin was significantly associated with an increased prevalence of erythromycin-resistant Enterococcus spp. isolates.

A Small Multihost Plasmid Carrying erm(T) Identified in Enterococcus faecalis

The aim of this study was to determine the mobile genetic elements involved in the horizontal transfer of erm(T) in Enterococcus faecalis, and its transmission ability in heterologous hosts. A total of 159 erythromycin-resistant enterococci isolates were screened for the presence of macrolide resistance genes by PCR. Whole genome sequencing for erm(T)-carrying E. faecalis E165 was performed. The transmission ability in heterologous hosts was explored by conjugation, transformation, and fitness cost. The erm(T) gene was detected only in an E. faecalis isolate E165 (1/159), which was located on a 4,244-bp small plasmid, designed pE165. Using E. faecalis OG1RF as the recipient strain, pE165 is transferable. Natural transformation experiments using Streptococcus suis P1/7 and Streptococcus mutans UA159 as the recipients indicated it is transmissible, which was also observed by electrotransformation using Staphylococcus aureus RN4220 as a recipient. The erm(T)-carrying pE165 can replicate in the heterologous host including E. faecalis OG1RF, S. suis P1/7, S. mutans UA159, and S. aureus RN4220 and conferred resistance to erythromycin and clindamycin to all hosts. Although there is no disadvantage of pE165 in the recipient strains in growth curve experiments, all the pE165-carrying recipients had a fitness cost compared to the corresponding original recipients in growth competition experiments. In brief, an erm(T)-carrying plasmid was for the first time described in E. faecalis and as transmissible to heterologous hosts.

Differences in chlorine and peracetic acid disinfection kinetics of Enterococcus faecalis and Escherichia fergusonii and their susceptible strains based on gene expressions and genomics

This study was conducted to investigate mechanisms of cross-resistance to chlorine and peracetic acid (PAA) disinfectants by antibiotic-resistant bacteria. Our study evaluated chlorine and PAA based disinfection kinetics of erythromycin-resistant Enterococcus faecalis, meropenem-resistant Escherichia fergusonii, and susceptible strains of these species. Using the integrated second-order disinfectant decay model and first-order Chick-Watson's Law, it was found that the meropenem-resistant Escherichia fergusonii strain showed significantly less log inactivation compared to the susceptible E. fergusonii strain in response to both chlorine and PAA disinfection (p-value = 0.059, 3.5 × 10−6). On the other hand, the susceptible Enterococcus faecalis strain showed similar log inactivation compared to the erythromycin-resistant strain in response to either treatment (p-value = 0.075, 0.28). Meropenem-resistant E. fergusonii showed an increase in gene expression of New Delhi metallo-β-lactamase (blaNDM-1) gene to chlorine, but there was no increase in expression to PAA. Whole genome sequencing (WGS) was then conducted to elucidate the differences in genes among both resistant and susceptible table E. fergusonii strains. The average nucleotide identity (ANI) analysis of the draft genomes (>97% similarity) suggests that meropenem-resistant E. fergusonii (S1) and meropenem-susceptible E. fergusonii (S2) are the same species but different strains. Both strains have the same genes for oxidative stress pathways, oxidative scavenger genes, and nearly 40 different antibiotic efflux pump genes. The chromosomal and plasmid draft genomes of meropenem-resistant and susceptible E. fergusonii strains each have 65 and 52 antibiotic resistance genes, respectively. Of these, the resistant E. fergusonii strain harbored the extended-spectrum beta-lactamases blaCTX-M-15 and blaTEM-1 genes located on the chromosome, and a blaTEM-1 gene on the plasmid. The overall findings of this study are significant, as they reveal that antibiotic-resistant and susceptible strains of E. fergusonii exhibit different responses towards chlorine and PAA disinfection.

A Comparative Quantitative Assessment of Human Exposure to Various Antimicrobial-Resistant Bacteria among U.S. Ground Beef Consumers

Consumption of animal-derived meat products is suspected as an important exposure route to antimicrobial resistance, as the presence of antimicrobial-resistant bacteria (ARB) along the beef supply chain is well documented. A retail-to-fork quantitative exposure assessment was established to compare consumers' exposure to various ARB due to the consumption of ground beef with and without "raised without antibiotics" claims and to inform potential exposure mitigation strategies related to consumer practices. The microbial agents evaluated included Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant E. coli,Salmonella enterica, TETrS. enterica, third-generation cephalosporin-resistant S. enterica, nalidixic acid-resistant S. enterica, Enterococcus spp., TETrEnterococcus spp., erythromycin-resistant Enterococcus spp., Staphylococcus aureus, and methicillin-resistant S. aureus. The final model outputs were the probability of exposure to at least 0 to 6 log CFU microorganisms per serving of ground beef at the time of consumption. It was estimated that tetracycline resistance was more prevalent in ground beef compared with other types of resistance, among which the predicted average probability of ingesting TETrEnterococcus was highest (6.2% of ingesting at least 0 log CFU per serving), followed by TETrE. coli (3.1%) and TETrSalmonella (0.0001%), given common product purchase preferences and preparation behaviors among beef consumers in the United States. The effectiveness of consumer-related interventions was estimated by simulating the differences in exposure as a result of changes in consumer practices in purchasing, handling, and preparing ground beef. The results indicated that proper use of recommended safe cooking and food preparation practices mitigates ARB exposure more effectively than choosing raised without antibiotics compared with conventional beef.

Antimicrobial Resistance in U.S. Retail Ground Beef with and without Label Claims Regarding Antibiotic Use

Antibiotics used during food animal production account for approximately 77% of U.S. antimicrobial consumption by mass. Ground beef products labeled as raised without antibiotics (RWA) are perceived to harbor lower levels of antimicrobial-resistant bacteria than conventional (CONV) products with no label claims regarding antimicrobial use. Retail ground beef samples were obtained from six U.S. cities. Samples with an RWA or U.S. Department of Agriculture Organic claim (n = 299) were assigned to the RWA production system. Samples lacking these claims (n = 300) were assigned to the CONV production system. Each sample was cultured for the detection of five antimicrobial-resistant bacteria. Genomic DNA was isolated from each sample, and a quantitative PCR assay was used to determine the abundance of 10 antimicrobial resistance (AMR) genes. Prevalence of tetracycline-resistant Escherichia coli (CONV, 46.3%; RWA, 34.4%; P < 0.01) and erythromycin-resistant Enterococcus (CONV, 48.0%; RWA, 37.5%; P = 0.01) was higher in CONV ground beef. Salmonella was detected in 1.2% of samples. The AMR gene blaCTX-M (CONV, 4.1 log-normalized abundance; RWA, 3.8 log-normalized abundance; P < 0.01) was more abundant in CONV ground beef. The AMR genes mecA (CONV, 4.4 log-normalized abundance; RWA, 4.9 log-normalized abundance; P = 0.05), tet(A) (CONV, 3.9 log-normalized abundance; RWA, 4.5 log-normalized abundance; P < 0.01), tet(B) (CONV, 3.9 log-normalized abundance; RWA, 4.5 log-normalized abundance; P < 0.01), and tet(M) (CONV, 5.4 log-normalized abundance; RWA, 5.8 log-normalized abundance; P < 0.01) were more abundant in RWA ground beef. Although these results suggest that antimicrobial use during U.S. cattle production does not increase human exposure to antimicrobial-resistant bacteria via ground beef, quantitative microbiological risk assessments are required for authoritative determination of the human health impacts of the use of antimicrobial agents during beef production.

Multidrug Resistance in Enterococci Isolated From Wild Pampas Foxes (Lycalopex gymnocercus) and Geoffroy's Cats (Leopardus geoffroyi) in the Brazilian Pampa Biome.

Enterococci are ubiquitous microorganisms present in various environments and within the gastrointestinal tracts of humans and other animals. Notably, fecal enterococci are suitable indicators for monitoring antimicrobial resistance dissemination. Resistant bacterial strains recovered from the fecal samples of wild animals can highlight important aspects of environmental disturbances. In this report, we investigated antimicrobial susceptibility as well as resistance and virulence genes in fecal enterococci isolated from wild Pampas foxes (Lycalopex gymnocercus) (n = 5) and Geoffroy's cats (Leopardus geoffroyi) (n = 4) in the Brazilian Pampa biome. Enterococci were isolated from eight out of nine fecal samples and Enterococcus faecalis was identified in both animals. However, E. faecium and E. durans were only detected in Pampas foxes, while E. hirae was only detected in Geoffroy's cats. Antimicrobial susceptibility analysis showed resistance to rifampicin (94%), erythromycin (72.6%), ciprofloxacin/norfloxacin (40%), streptomycin (38%), and tetracycline (26%). The high frequency of multidrug-resistant enterococci (66%) isolated in this study is a matter of concern since these are wild animals with no history of therapeutic antibiotic exposure. The tetM/tetL and msrC/ermB genes were detected in most tetracycline- and erythromycin-resistant enterococci, respectively. The gelE, ace, agg, esp, and clyA virulence genes were also detected in enterococci. In conclusion, our data suggest that habitat fragmentation and anthropogenic activities in the Pampa biome may contribute to high frequencies of multidrug-resistant enterococci in the gut communities of wild Pampas foxes and Geoffroy's cats. To the best of the authors' knowledge, this is the first report of antimicrobial-resistant enterococci in the Pampa biome.

82 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses &amp; reduction of macrolide-resistant bacteria dissemination

Abstract Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384±13.5kg) consuming a finishing diet (90% concentrate) for 188 days (Final BW= 660±42 kg) were used in a completely randomized design where treatments included: TYL (Tylosin phosphate fed at 90 mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n = 32).Feed intake was recorded daily and BW was recorded every 28 days until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data was analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P = 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P &amp;lt; 0.02) and consequently greater numerical yield grade (P &amp;lt; 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL= 63% vs. IGY= 52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P &amp;lt; 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in the diets of feedlot cattle.

81 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses & reduction of macrolide-resistant bacteria dissemination

Abstract Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum andTrueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384±13.5kg) consuming a finishing diet (90% concentrate) for 188 d (Final BW= 660±42 kg) were used in a completely randomized design where treatments included: TYL (Tylosin phosphate fed at 90 mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n = 32). Feed intake was recorded daily and BW was recorded every 28 d until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data was analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P = 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P &amp;lt; 0.02) and consequently greater numerical yield grade (P &amp;lt; 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL= 63% vs. IGY= 52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P &amp;lt; 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in the diets of feedlot cattle.

83 Evaluation of feeding immunoglobulin-Y to Holstein feeder cattle for the control of liver abscesses &amp; reduction of macrolide-resistant bacteria dissemination

Abstract Despite the regular use of macrolide-antibiotics in the diets of feedlot cattle, liver abscesses persist representing a financial burden to both pre- and post-mortem sectors of the beef industry. Alternative strategies, which simultaneously address liver infections and reduce the dissemination of antibiotic-resistant bacteria, are needed. An immunoglobulin-Y (IGY) product, developed to target Fusobacterium necrophorum and Trueperella pyogenes, was evaluated for the control of liver abscesses and its effect on macrolide-resistant Enterococcus growth from feces. Sixty-four Holstein steers (Initial BW= 384± 13.5kg) consuming a finishing diet (90% concentrate) for 188 days (Final BW= 660± 42 kg) were used in a completely randomized design where steer was experimental unit and treatments included: TYL (Tylosin phosphate fed at 90mg/hd/day; n = 32) or IGY (fed at 2.5g/hd/day; n= 32). Feed intake was recorded daily and BW was recorded every 28 days until slaughter. After 150 d on treatment fecal samples were randomly obtained from each treatment group four times (n = 40) and cultured on bile-esculine agar enriched with 8 μg/mL of erythromycin. Continuous variables of feedlot growth performance, carcass data and bacterial growth were analyzed using the MIXED procedure of SAS with the fixed effect of treatment and random effects of steer or sampling day, respectively. Ordinal carcass data were analyzed using the FREQ procedure of SAS observing the Cochran-Mantel-Haenszel value. Treatment did not affect DMI (P= 0.21), ADG (P = 0.50) or G:F (P = 0.36). Treatment neither affected hot carcass weight (P = 0.37) nor longissimus muscle area (P = 0.25). However, TYL resulted in greater back fat thickness than IGY (0.32 vs. 0.23 in.; P &amp;lt; 0.02) and consequently greater numerical yield grade (P &amp;lt; 0.02). Quality grade was not affected by treatment (P = 0.65). No treatment difference was observed for liver abscesses prevalence (TYL=63% vs. IGY=52%; P = 0.40) or severity (P = 0.23). There was a greater proportion of erythromycin-resistant Enterococcus growth for TYL than IGY (42.6 vs. 21.9%; P &amp;lt; 0.04). Specifically formulated IGY feed additives may be viable alternatives to antibiotics in feedlot cattle diets.

The effect of tylosin on antimicrobial resistance in beef cattle enteric bacteria: A systematic review and meta-analysis

BackgroundTylosin is a commonly used in-feed antimicrobial and is approved in several countries to reduce the incidence of liver abscesses in beef cattle. Macrolides are critically important antimicrobials in human health and used to treat some foodborne bacterial diseases, such as Campylobacter jejuni and Salmonella. Feeding tylosin could select for resistant enteric bacteria in cattle, which could contaminate beef products at slaughter and potentially cause foodborne illness. We conducted a systematic review and meta-analysis to evaluate the impact of feeding tylosin to cattle on phenotypic and genotypic resistance in several potential zoonotic enteric bacteria: Enterococcus species, Escherichia coli, Salmonella enterica subspecies enterica, and Campylobacter species. This review was registered with PROSPERO (#CRD42018085949). ResultsEleven databases were searched for primary research studies that fed tylosin at approved doses to feedlot cattle and tested bacteria of interest for phenotypic or genotypic resistance. We screened 1,626 citations and identified 13 studies that met the inclusion criteria. Enterococcus species were tested in seven studies, Escherichia coli was isolated in five studies, three studies reported on Salmonella, and two studies reported on Campylobacter species. Most studies relied on phenotypic antimicrobial susceptibility testing and seven also reported resistance gene testing. A random-effects meta-analyses of erythromycin-resistant enterococci from four studies had significant residual heterogeneity. Only two studies were available for a meta-analysis of tylosin-resistant enterococci. A semi-quantitative analysis demonstrated an increase in macrolide-resistant enterococci after long durations of tylosin administration (>100 days). Semi-quantitative analyses of other bacteria-antimicrobial combinations revealed mixed results, but many comparisons found no effect of tylosin administration. However, about half of these no-effect comparisons did not record the cumulative days of tylosin administration or the time since the last dose. ConclusionsWhen fed at approved dosages for typical durations, tylosin increases the proportion of macrolide-resistant enterococci in the cattle gastrointestinal tract, which could pose a zoonotic risk to human beef consumers. Feeding tylosin for short durations may mitigate the impact on macrolide-resistant enterococci and further studies are encouraged to determine the effect of minimizing or eliminating tylosin use in beef cattle. There may also be an impact on other bacteria and other antimicrobial resistances but additional details or data are needed to strengthen these comparisons. We encourage authors of antimicrobial-resistance studies to follow reporting guidelines and publish details of all comparisons to strengthen future meta-analyses.

In-Feed Tylosin Phosphate Administration to Feedlot Cattle Minimally Affects Antimicrobial Resistance

The macrolide class antimicrobial tylosin (trade name Tylan) is approved by the U.S. Food and Drug Administration for continuous inclusion in feed for liver abscess prevention. To address concerns that this antimicrobial application may threaten human health, a population of feedlot steers was split into a control treatment (n = 42) and a tylosin treatment (n = 42). Feed rations were identical except for the inclusion of tylosin at 60 to 90 mg per head per day. Fecal swab (n = 335), pen surface material (n = 256), feed (n = 56), and water trough (n = 32) samples were obtained over four sample occasions: November (1 day before the start of tylosin inclusion in feed), January (80 days of tylosin in feed), April (167 days), and June (253 days). These samples were cultured for Escherichia coli, tetracycline-resistant E. coli, third-generation cephalosporin-resistant E. coli, Enterococcus, tetracycline-resistant Enterococcus, and erythromycin-resistant Enterococcus. Metagenomic DNA was isolated from each June fecal swab and pen surface material sample. Metagenomic DNA samples were pooled by pen for 14 fecal and 14 pen surface material samples. Quantitative PCR was employed to assess the abundances of the following 10 antimicrobial resistance genes: aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M). Nasal swab samples (n = 335) were obtained from each steer during each sample period and cultured for the presence of Staphylococcus aureus and methicillin-resistant S. aureus. Of these measurements, only January and June mean fecal swab erythromycin-resistant Enterococcus colony counts for tylosin-treated cattle were significantly higher (P ≤ 0.05) than the range of mean values for control treatments. These results suggest that in-feed tylosin through the end of finishing has a narrow and minimal antimicrobial resistance impact.

Antimicrobial Resistance in Retail Ground Beef with and Without a “Raised Without Antibiotics” Claim

ObjectivesThe occurrences of human bacterial infections complicated by antimicrobial resistance (AMR) have increased in recent decades. Concerns have been raised that food-animal production practices that incorporate antimicrobials contribute significantly to human AMR exposures since food-animal production accounts for approximately 81% of U.S. antimicrobial consumption by mass. Although empirical studies comparing AMR levels in meat products, including ground beef, are scant ground beef products with Raised without Antibiotics (RWA) label claims are perceived to harbor less AMR than “conventional” (CONV) products with no label claims regarding antimicrobial use. The objective of this research was to determine AMR levels in retail ground beef with and without an RWA label claims.Materials and MethodsRetail ground beef samples were obtained from 6 U.S. cities. Samples were obtained on the following dates: 9/18/2017, 10/30/2017, 11/27/2017. 1/29/2018. 3/5/2018, and 6/11/2018. A total of 599 samples were obtained. Samples with a “Raised without Antibiotics” or USDA Organic claim (N = 299) were assigned to the RWA production system. Samples lacking a “Raised without Antibiotics” claim (N = 300) were assigned to the CONV production system. Each sample was cultured for the detection of five antimicrobial resistant bacteria (ARB). Genomic DNA was isolated from each sample and qPCR was used to determine the abundance of ten antimicrobial resistance genes (ARGs). The impacts of production system and city on ARB detection were assessed by the Likelihood-ratio chi-squared test. The impacts of production system and city on ARG abundance was assessed by two-way ANOVA.ResultsTetracycline-resistant Escherichia coli (CONV = 46.3%; RWA = 34.4%) and erythromycin-resistant Enterococcus (CONV = 48.0%; RWA = 37.5%) were more frequently (P &lt; 0.01) detected in CONV. Detection of third generation cephalosporin-resistant E. coli (CONV = 5.7%; RWA = 1.0%), vancomycin-resistant Enterococcus (CONV = 0.0%; RWA = 0.0%) and methicillin-resistant Staphylococcus aureus (CONV = 1.3%; RWA = 0.7%) did not differ (P = 1.00). The blaCTX-M ARG was more abundant in CONV (2.4 vs. 2.1 log copies/gram, P = 0.01) but the tet(A) (2.4 vs. 2.5 log copies/gram, P = 0.02) and tet(M) (3.6 vs. 3.9 log copies/gram, P &lt; 0.01) ARGs were more abundant in RWA. aadA1, blaCMY-2, mecA, erm(B), and tet(B) abundances did not differ significantly (Fig. 5) (P &gt; 0.05). Abundances of aac (6’)-Ie-aph (2”)-Ia and blaKPC-2 were not analyzed since they were quantified in less than 5% of the samples.ConclusionU.S. retail CONV and RWA ground beef harbor generally similar levels of AMR since only 5 of 15 AMR measurements were statistically different between production systems. Three AMR measurements were higher in CONV, while 2 AMR measurements were higher in RWA. These results are in general agreement with a recently published study authored by our group that examined antimicrobial resistance in CONV and RWA ground beef obtained from U.S. foodservice suppliers (Vikram et al., J. Food Prot. 81:2007–2018. 2018.). Together these studies suggest that antimicrobial use during U.S. cattle production has minimal to no impact on human exposure to AMR via ground beef.Figure 5.

Food Service Pork Chops from Three U.S. Regions Harbor Similar Levels of Antimicrobial Resistance Regardless of Antibiotic Use Claims

Pork products from animals "raised without antibiotics" (RWA) are assumed to harbor lower levels of antimicrobial resistance (AMR) than conventional (CONV) pork products with no claims regarding use of antimicrobial agents during production. A total of 372 pork chop samples from CONV (n = 190) and RWA (n = 182) production systems were collected over 13 months from three food service suppliers. The following bacteria were cultured: Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant (3GCr) E. coli, Salmonella enterica, TETr Salmonella, 3GCr Salmonella, nalidixic acid-resistant Salmonella, Enterococcus spp., TETr Enterococcus, erythromycin-resistant Enterococcus, Staphylococcus aureus, and methicillin-resistant S. aureus. Production system did not significantly impact the detection of cultured bacteria (P > 0.05). Metagenomic DNA was isolated from each sample, and equal amounts of metagenomic DNA were pooled by supplier, month, and production system for 75 pooled samples (38 CONV, 37 RWA). Quantitative PCR was used to assess the abundances of the following 10 AMR genes: aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M). For all 10 AMR genes, abundances did not differ significantly (P > 0.05) between production systems. These results suggest that use of antimicrobial agents during swine production minimally impacts the AMR of bacteria in pork chops.

Antimicrobial Resistance in Retail Ground Beef with and Without a “Raised Without Antibiotics” Claim

ObjectivesThe occurrences of human bacterial infections complicated by antimicrobial resistance (AMR) have increased in recent decades. Concerns have been raised that food-animal production practices that incorporate antimicrobials contribute significantly to human AMR exposures since food-animal production accounts for approximately 81% of U.S. antimicrobial consumption by mass. Although empirical studies comparing AMR levels in meat products, including ground beef, are scant ground beef products with Raised without Antibiotics (RWA) label claims are perceived to harbor less AMR than “conventional” (CONV) products with no label claims regarding antimicrobial use. The objective of this research was to determine AMR levels in retail ground beef with and without an RWA label claims.Materials and MethodsRetail ground beef samples were obtained from 6 U.S. cities. Samples were obtained on the following dates: 9/18/2017, 10/30/2017, 11/27/2017. 1/29/2018. 3/5/2018, and 6/11/2018. A total of 599 samples were obtained. Samples with a “Raised without Antibiotics” or USDA Organic claim (N = 299) were assigned to the RWA production system. Samples lacking a “Raised without Antibiotics” claim (N = 300) were assigned to the CONV production system. Each sample was cultured for the detection of five antimicrobial resistant bacteria (ARB). Genomic DNA was isolated from each sample and qPCR was used to determine the abundance of ten antimicrobial resistance genes (ARGs). The impacts of production system and city on ARB detection were assessed by the Likelihood-ratio chi-squared test. The impacts of production system and city on ARG abundance was assessed by two-way ANOVA.ResultsTetracycline-resistant Escherichia coli (CONV = 46.3%; RWA = 34.4%) and erythromycin-resistant Enterococcus (CONV = 48.0%; RWA = 37.5%) were more frequently (P < 0.01) detected in CONV. Detection of third generation cephalosporin-resistant E. coli (CONV = 5.7%; RWA = 1.0%), vancomycin-resistant Enterococcus (CONV = 0.0%; RWA = 0.0%) and methicillin-resistant Staphylococcus aureus (CONV = 1.3%; RWA = 0.7%) did not differ (P = 1.00). The blaCTX-M ARG was more abundant in CONV (2.4 vs. 2.1 log copies/gram, P = 0.01) but the tet(A) (2.4 vs. 2.5 log copies/gram, P = 0.02) and tet(M) (3.6 vs. 3.9 log copies/gram, P < 0.01) ARGs were more abundant in RWA. aadA1, blaCMY-2, mecA, erm(B), and tet(B) abundances did not differ significantly (Fig. 5) (P > 0.05). Abundances of aac (6’)-Ie-aph (2”)-Ia and blaKPC-2 were not analyzed since they were quantified in less than 5% of the samples.ConclusionU.S. retail CONV and RWA ground beef harbor generally similar levels of AMR since only 5 of 15 AMR measurements were statistically different between production systems. Three AMR measurements were higher in CONV, while 2 AMR measurements were higher in RWA. These results are in general agreement with a recently published study authored by our group that examined antimicrobial resistance in CONV and RWA ground beef obtained from U.S. foodservice suppliers (Vikram et al., J. Food Prot. 81:2007–2018. 2018.). Together these studies suggest that antimicrobial use during U.S. cattle production has minimal to no impact on human exposure to AMR via ground beef.Figure 5.

Similar Levels of Antimicrobial Resistance in U.S. Food Service Ground Beef Products with and without a “Raised without Antibiotics” Claim

U.S. ground beef with "raised without antibiotics" (RWA) label claims are perceived as harboring fewer bacteria with antimicrobial resistance (AMR) than are found in conventional (CONV) ground beef with no such label claim. A total of 370 ground beef samples from CONV ( n = 191) and RWA ( n = 179) production systems were collected over 13 months from three food service suppliers. The following bacteria were cultured: Escherichia coli, tetracycline-resistant (TETr) E. coli, third-generation cephalosporin-resistant (3GCr) E. coli, Salmonella enterica, TETr S. enterica, 3GCr S. enterica, nalidixic acid-resistant S. enterica, Enterococcus spp., erythromycin-resistant Enterococcus spp., TETr Enterococcus spp., Staphylococcus aureus, and methicillin-resistant S. aureus. TETr E. coli was more frequently detected in CONV ground beef (CONV, 54.2%; RWA, 35.2%; P < 0.01), but supplier ( P < 0.01) and production system × suppler interaction ( P < 0.01) effects were also significant. Metagenomic DNA was isolated from each sample, and equal amounts of metagenomic DNA were pooled by supplier, month, and production system for 75 pooled samples (38 CONV, 37 RWA). The abundance of aac(6')-Ie-aph(2″)-Ia, aadA1, blaCMY-2, blaCTX-M, blaKPC-2, erm(B), mecA, tet(A), tet(B), and tet(M) genes was assessed by quantitative PCR. The tet(A) (2.9-log2-fold change, P = 0.04) and tet(B) (5.6-log2-fold change) ( P = 0.03) genes were significantly more abundant in RWA ground beef. Phylogenetic analyses revealed that ground beef microbiomes differed more by supplier than by production system. These results were consistent with prior research suggesting antimicrobial use in U.S. beef cattle has minimal impact on the AMR of bacteria found in these products. These results should spur a reevaluation of assumptions regarding the impact of antimicrobial use during U.S. beef production on the AMR of bacteria in ground beef.

Species distribution, antibiotic resistance and virulence traits in canine and feline enterococci in Tunisia.

In order to investigate the possible role of dogs and cats in the carriage and potential dissemination of resistant enterococci, seventy faecal samples from dogs and cats were tested for enterococci. Fifty-eight enterococci were recovered. Isolates were identified as Enterococcus faecium (n = 31) and E. faecalis (n = 14) E. durans (n = 6), E. casseliflavus (n = 2), E. hirae and E. gallinarum (2 isolates each). Enterococcal isolates showed resistance to ciprofloxacin (n = 35), erythromycin (n = 31), tetracycline (n = 25), kanamycin (n = 15), streptomycin (n = 13), pristinamycin (n = 11), gentamicin (n = 10), chloramphenicol (n = 8), and linezolid (n = 6). The gene erm(B) was detected in 22 out of 31 erythromycin-resistant enterococci. All tetracycline-resistant enterococci carried tet(M) and/or tet(L) genes. The gene aac(6')-Ie-aph(2″)-Ia was identified in five of high-level gentamicin-resistant isolates, the genes aph(3')-IIIa and/or aac(6')-Ie-aph(2″)-Ia in eleven high-level kanamycin-resistant isolates and the gene ant(6)-Ia in eleven high-level streptomycin-resistant isolates. Only one strain harboured cat(A) gene, and five strains contained vat(E) or vat(D) genes. Virulence genes gel(E) (21 strains), esp (11 strains) and cylA/cylB (5 strains) were detected. High genetic diversity was demonstrated among E. faecium isolates by pulsed-field gel electrophoresis (PFGE). Dogs and cats can be carriers of antibiotic-resistant enterococci in their faeces that could shed into the household environment.

Antimicrobial-Resistant Fecal Bacteria from Ceftiofur-Treated and Nonantimicrobial-Treated Comingled Beef Cows at a Cow-Calf Operation.

We compared the occurrences of 3rd-generation cephalosporin-resistant (3GCr), tetracycline-resistant (TETr), and trimethoprim-sulfamethoxazole-resistant (COTr) Escherichia coli, 3GCr and nalidixic acid-resistant (NALr) Salmonella enterica, and erythromycin-resistant (ERYr) enterococci from the fecal samples of ceftiofur-treated (n = 162) and nonantimicrobial-treated (n = 207) comingled beef cows ≥8 years old, for which complete antimicrobial treatment records were available. The prevalence of 3GCr (17%; n = 369), TETr (88%), COTr E. coli (22%), and ERYr enterococci (69%) was not significantly (p > 0.05) associated with ceftiofur treatment, prior history of other antimicrobial treatments, or duration of time between last antimicrobial treatment and sampling. 3GCr and NALr S. enterica were not detected. The prevalence of tetB was significantly (p < 0.05) higher compared with tetA among TETr E. coli. However, the prevalence of tetA was significantly (p < 0.05) higher than tetB among 3GCr and COTr E. coli. There was a significant (p < 0.05) association between tetM and ermB among ERYr enterococci. In conclusion, occurrences of 3GCr, TETr, and COTr E. coli and ERYr enterococci in comingled antimicrobial-treated and nonantimicrobial-treated beef cows were not associated with ceftiofur or other antimicrobial use, indicating that other factors influenced the observed levels of antimicrobial-resistant bacteria in feces of beef cows.

Antibiotic resistance and virulence of enterococci isolates from healthy humans in Tunisia

The aim of this work was to determine the occurrence of different enterococcal species and the prevalence of antimicrobial resistance and virulence genes in enterococci isolates recovered from faecal samples of 98 healthy human volunteers in Tunisia. Isolates were tested for antibiotic resistance phenotypes, genotypes and virulence genes. In addition, high-level aminoglycoside resistant (HLAR) Enterococcus faecalis and Enterococcus faecium isolates were tested for clonal diversity by pulsed field gel electrophoresis (PFGE). The following species were detected among the 98 enterococci obtained: 51 Enterococcus faecalis, 40 Enterococcus faecium, 4 Enterococcus mundtii and 3 Enterococcus gallinarum. Antibiotic resistance was detected as follows (% in E. faecalis/E. faecium): erythromycin (51 %/55 %), tetracycline (51 %/15 %), pristinamycin (51 %/27.5 %), gentamicin (19.6 %/10 %), kanamycin (25.5 %/12.5 %), streptomycin (29.4 %/7.5 %), chloramphenicol (7.8 %/2.5 %). The tet(M)+/−tet(L) genes were found in 92 % and 83 % of tetracycline-resistant E. faecalis and E. faecium isolates, respectively. High-level resistance for kanamycin and gentamicin was mediated by aph(3′)-IIIa and aac(6′)-aph(2″) genes. Of erythromycin-resistant enterococci, 85 % harboured the erm(B) gene. The erm(C) gene was found in one erythromycin-resistant E. mundtii isolate. Most high-level gentamicin and/or streptomycin E. faecalis and E. faecium isolates showed unrelated patterns by PFGE. Fifty-two percent of enterococci showed a multidrug-resistant phenotype. The following virulence genes were detected among the 98 enterococci: gelE (13.2 %), esp (20.4 %), cyl (9 %, showing haemolytic activity), ace (48.9 %), and hyl (0 %). In conclusion, E. faecalis and E. faecium are the predominant enterococcal species in the faecal environment of healthy humans, and they present high rates of resistance for antibiotics of clinical relevance, such as aminoglycosides.

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

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