Genotypic and Phenotypic Characterization of Antimicrobial and Heavy Metal Tolerance in Salmonella enterica and Escherichia coli Isolates from Swine Feed Mills.

Antimicrobials and heavy metals are commonly used in the animal feed industry. The role of in-feed antimicrobials on the evolution and persistence of resistance in enteric bacteria is not well described. Whole-Genome Sequencing (WGS) is widely used for genetic characterizations of bacterial isolates, including antimicrobial resistance, heavy metal tolerance, virulence factors, and relatedness to other sequenced isolates. The goals of this study were to i) use WGS to characterize Salmonella enterica (n = 33) and Escherichia coli (n = 30) isolated from swine feed and feed mill environments; and ii) investigate their genotypic and phenotypic antimicrobial and heavy metal tolerance. Salmonella isolates belonged to 10 serovars, the most common being Cubana, Senftenberg, and Tennessee. E. coli isolates were grouped into 22 O groups. Phenotypic resistance to at least one antimicrobial was observed in 19 Salmonella (57.6%) and 17 E. coli (56.7%) isolates, whereas multidrug resistance (resistant to ≥3 antimicrobial classes) was observed in four Salmonella (12%) and two E. coli (7%) isolates. Antimicrobial resistance genes were identified in 17 Salmonella (51%) and 29 E. coli (97%), with 11 and 29 isolates possessing genes conferring resistance to multiple antimicrobial classes. Phenotypically, 53% Salmonella and 58% E. coli presented resistance to copper and arsenic. All isolates that possessed the copper resistance operon were resistant to the highest concentration tested (40 mM). Heavy metal tolerance genes to copper and silver were present in 26 Salmonella isolates. Our study showed a strong agreement between predicted and measured resistances when comparing genotypic and phenotypic data for antimicrobial resistance, with an overall concordance of 99% and 98.3% for Salmonella and E. coli, respectively.

Toward the Adoption of Loop-Mediated Isothermal Amplification for Salmonella Screening at the National Antimicrobial Resistance Monitoring System's Retail Meat Sites.

The National Antimicrobial Resistance Monitoring System (NARMS) is a One Health program in the United States that collects data on antimicrobial resistance in enteric bacteria from humans, animals, and the environment. Salmonella is a major pathogen tracked by the NARMS retail meat arm but currently lacks a uniform screening method. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the rapid screening of Salmonella from 69 NARMS retail meat and poultry samples. All samples were processed side by side for culture isolation using two protocols, one from NARMS and the other one described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). Overall, 10 (14.5%) samples screened positive by the Salmonella LAMP assay. Of those, six were culture-confirmed by the NARMS protocol and six by the BAM method with overlap on four samples. No Salmonella isolates were recovered from samples that screened negative with LAMP. These results suggested 100% sensitivity for LAMP in reference to culture. Antimicrobial susceptibility testing and whole-genome sequencing analysis confirmed identities of these isolates. Using the BAM protocol, all Salmonella isolates were recovered from samples undergoing Rappaport-Vassiliadis medium selective enrichment and presumptive colonies (n = 130) were dominated by Hafnia alvei (44.6%), Proteus mirabilis (22.3%), and Morganella morganii (9.9%) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method comparison study clearly demonstrated the benefit of a rapid, robust, and highly sensitive molecular screening method in streamlining the laboratory workflow. Fourteen NARMS retail meat sites further verified the performance of this assay using a portion of their routine samples, reporting an overall specificity of 98.8% and sensitivity of 90%. As of July 2022, the vast majority of NARMS retail meat sites have adopted the Salmonella LAMP assay for rapid screening of Salmonella in all samples.

Prevalence of Antimicrobial Resistance in Select Bacteria From Retail Seafood-United States, 2019.

In 2019, the United States National Antimicrobial Resistance Monitoring System (NARMS) surveyed raw salmon, shrimp, and tilapia from retail grocery outlets in eight states to assess the prevalence of bacterial contamination and antimicrobial resistance (AMR) in the isolates. Prevalence of the targeted bacterial genera ranged among the commodities: Salmonella (0%-0.4%), Aeromonas (19%-26%), Vibrio (7%-43%), Pseudomonas aeruginosa (0.8%-2.3%), Staphylococcus (23%-30%), and Enterococcus (39%-66%). Shrimp had the highest odds (OR: 2.8, CI: 2.0-3.9) of being contaminated with at least one species of these bacteria, as were seafood sourced from Asia vs. North America (OR: 2.7; CI: 1.8-4.7) and Latin America and the Caribbean vs. North America (OR: 1.6; CI: 1.1-2.3) and seafood sold at the counter vs. sold frozen (OR: 2.1; CI: 1.6-2.9). Isolates exhibited pan-susceptibility (Salmonella and P. aeruginosa) or low prevalence of resistance (<10%) to most antimicrobials tested, with few exceptions. Seafood marketed as farm-raised had lower odds of contamination with antimicrobial resistant bacteria compared to wild-caught seafood (OR: 0.4, CI: 0.2-0.7). Antimicrobial resistance genes (ARGs) were detected for various classes of medically important antimicrobials. Clinically relevant ARGs included carbapenemases (bla IMI-2, bla NDM-1) and extended spectrum ß-lactamases (ESBLs; bla CTX-M-55). This population-scale study of AMR in seafood sold in the United States provided the basis for NARMS seafood monitoring, which began in 2020.

Versatility of a Salmonella Loop-Mediated Isothermal Amplification Assay Using Multiple Platforms and Master Mixes in Animal Food Matrices.

BACKGROUND: Improvement in Salmonella detection methods greatly enhances the efficiency of various food testing programs. A Salmonella loop-mediated isothermal amplification (LAMP) assay has been validated in animal food through multi-laboratory validation. OBJECTIVE: The study aimed to demonstrate the versatility of this molecular assay while expanding it to multiple platforms and various reagent choices for use in animal food testing. METHODS: Following the U.S. Food and Drug Administration (FDA)'s Guidelines for the Validation of Analytical Methods for the Detection of Microbial Pathogens in Foods and Feeds, we examined the inclusivity, exclusivity, and LOD of the assay using two platforms (7500 Fast and Genie II) and three LAMP master mixes (GspSSD, GspSSD2.0, and WarmStart) in seven animal food matrixes (dry cat food, dry dog food, cattle feed, dairy feed, horse feed, poultry feed, and swine feed). The FDA's Bacteriological Analytical Manual (BAM) Salmonella culture method was the reference method. RESULTS: Inclusivity and exclusivity data were consistent among all six platform and master mix combinations with a few exceptions. Comparable LODs were observed down to the single-cell level (WarmStart was 10-fold less sensitive). Performance was similar to the BAM method for detecting fractional positive results in seven animal food matrixes. Nonetheless, LAMP time to positive results and annealing/melting temperature differed among master mixes and platforms. CONCLUSION: The Salmonella LAMP assay was successfully validated in two platforms and three master mixes, making it a flexible tool for use by the FDA's field laboratories in regulatory testing of animal food and for adoption by other food testing programs. HIGHLIGHTS: We demonstrated the LAMP assay's versatility on two platforms and three master mixes for the rapid and reliable screening of Salmonella in seven animal food matrixes. GspSSD2.0 was the fastest master mix (time to positive results as early as 3.5 min) while Genie II had several attractive features from a user perspective.

Core Genome MLST for Source Attribution of Campylobacter coli.

Campylobacter species are among the leading foodborne bacterial agents of human diarrheal illness. The majority of campylobacteriosis has been attributed to Campylobacter jejuni (85% or more), followed by Campylobacter coli (5-10%). The distribution of C. jejuni and C. coli varies by host organism, indicating that the contribution to human infection may differ between isolation sources. To address the relative contribution of each source to C. coli infections in humans, core genome multilocus sequence type with a 200-allele difference scheme (cgMLST200) was used to determine cgMLST type for 3,432 C. coli isolated from food animals (n = 2,613), retail poultry meats (n = 389), human clinical settings (n = 285), and environmental sources (n = 145). Source attribution was determined by analyzing the core genome with a minimal multilocus distance methodology (MMD). Using MMD, a higher proportion of the clinical C. coli population was attributed to poultry (49.6%) and environmental (20.9%) sources than from cattle (9.8%) and swine (3.2%). Within the population of C. coli clinical isolates, 70% of the isolates that were attributed to non-cecal retail poultry, dairy cattle, beef cattle and environmental waters came from two cgMLST200 groups from each source. The most common antibiotic resistance genes among all C. coli were tetO (65.6%), bla OXA - 193 (54.2%), aph(3')-IIIa (23.5%), and aadE-Cc (20.1%). Of the antibiotic resistance determinants, only one gene was isolated from a single source: bla OXA - 61 was only isolated from retail poultry. Within cgMLST200 groups, 17/17 cgMLST200-435 and 89/92 cgMLST200-707 isolates encoded for aph(3')-VIIa and 16/16 cgMLST200-319 harbored aph(2')-If genes. Distribution of bla OXA alleles showed 49/50 cgMLST200-5 isolates contained bla OXA - 498 while bla OXA - 460 was present in 37/38 cgMLST200-650 isolates. The cgMLST200-514 group revealed both ant(6)-Ia and sat4 resistance genes in 23/23 and 22/23 isolates, respectively. Also, cgMLST200-266 and cgMLST200-84 had GyrAT86I mutation with 16/16 (100%) and 14/15 (93.3%), respectively. These findings illustrate how cgMLST and MMD methods can be used to evaluate the relative contribution of known sources of C. coli to the human burden of campylobacteriosis and how cgMLST typing can be used as an indicator of antimicrobial resistance in C. coli.

Rapid Screening for Salmonella in Raw Pet Food by Loop-Mediated Isothermal Amplification.

ABSTRACT: Raw pet food, composed of raw meat and vegetables, has increased in popularity in recent years. Multiple surveys and frequent recalls indicate that this commodity has a high risk of contamination with Salmonella and other foodborne pathogens. Improved screening methods are needed to meet the growing demand for testing. This matrix verification study aimed to apply a Salmonella loop-mediated isothermal amplification (LAMP) method, recently completed multilaboratory validation in dry dog food, in several raw pet food matrices, following the U.S. Food and Drug Administration (FDA)'s method validation guidelines. Five types of raw pet food, consisting of freeze-dried beef and chicken treats and frozen beef, pork, and turkey complete foods, were evaluated. For each matrix, two sets of ten 25-g test portions (seven inoculated with ≤30 cells of Salmonella Typhimurium and three uninoculated controls) were examined. One set was preenriched in buffered peptone water and the other one was preenriched in lactose broth, which was followed by LAMP screening using two isothermal master mixes (ISO-001 and ISO-004). All results were confirmed by culture as specified in the FDA Bacteriological Analytical Manual (BAM). The LAMP method accurately detected Salmonella in all inoculated test portions of the five raw pet food samples, regardless of the preenrichment broth used. Positive results could be obtained within 4 min of the LAMP run using the ISO-004 master mix. All uninoculated controls tested negative using LAMP or BAM. In addition, one turkey-based complete pet food sample was found to be already contaminated with three Salmonella serovars harboring multiple antimicrobial resistance genes. The Salmonella LAMP method offers a rapid, reliable, and robust tool for routine screening of Salmonella in raw pet food, which will help better ensure product safety and protect public health.

Loop-Mediated Isothermal Amplification for Screening Salmonella in Animal Food and Confirming Salmonella from Culture Isolation.

Loop-mediated isothermal amplification (LAMP) has emerged as a powerful nucleic acid amplification test for the rapid detection of numerous bacterial, fungal, parasitic, and viral agents. Salmonella is a bacterial pathogen of worldwide food safety concern, including food for animals. Presented here is a multi-laboratory-validated Salmonella LAMP protocol that can be used to rapidly screen animal food for the presence of Salmonella contamination and can also be used to confirm presumptive Salmonella isolates recovered from all food categories. The LAMP assay specifically targets the Salmonella invasion gene (invA) and is rapid, sensitive, and highly specific. Template DNAs are prepared from enrichment broths of animal food or pure cultures of presumptive Salmonella isolates. The LAMP reagent mixture is prepared by combining an isothermal master mix, primers, DNA template, and water. The LAMP assay runs at a constant temperature of 65 °C for 30 min. Positive results are monitored via real-time fluorescence and can be detected as early as 5 min. The LAMP assay exhibits high tolerance to inhibitors in animal food or culture medium, serving as a rapid, reliable, robust, cost-effective, and user-friendly method for screening and confirming Salmonella. The LAMP method has recently been incorporated into the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM) Chapter 5.

Genomic Analysis of Emerging Florfenicol-Resistant Campylobacter coli Isolated from the Cecal Contents of Cattle in the United States.

Genomic analyses were performed on florfenicol-resistant (FFNr) Campylobacter coli isolates recovered from cattle, and the cfr(C) gene-associated multidrug resistance (MDR) plasmid was characterized. Sixteen FFNrC. coli isolates recovered between 2013 and 2018 from beef cattle were sequenced using MiSeq. Genomes and plasmids were found to be closed for three of the isolates using the PacBio system. Single nucleotide polymorphisms (SNPs) across the genome and the structures of MDR plasmids were investigated. Conjugation experiments were performed to determine the transferability of cfr(C)-associated MDR plasmids. The spectrum of resistance encoded by the cfr(C) gene was further investigated by agar dilution antimicrobial susceptibility testing. All 16 FFNr isolates were MDR and exhibited coresistance to ciprofloxacin, nalidixic acid, clindamycin, and tetracycline. All isolates shared the same resistance genotype, carrying aph (3')-III, hph, ΔaadE (truncated), blaOXA-61, cfr(C), and tet(O) genes plus a mutation of GyrA (T86I). The cfr(C), aph (3')-III, hph, ΔaadE, and tet(O) genes were colocated on transferable MDR plasmids ranging in size from 48 to 50 kb. These plasmids showed high sequence homology with the pTet plasmid and carried several Campylobacter virulence genes, including virB2, virB4, virB5, VirB6, virB7, virB8, virb9, virB10, virB11, and virD4 The cfr(C) gene conferred resistance to florfenicol (8 to 32 µg/ml), clindamycin (512 to 1,024 µg/ml), linezolid (128 to 512 µg/ml), and tiamulin (1,024 µg/ml). Phylogenetic analysis showed SNP differences ranging from 11 to 2,248 SNPs among the 16 isolates. The results showed that the cfr(C) gene located in the conjugative pTet MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to several antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. This report highlights the power of genomic antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.IMPORTANCECampylobacter is a leading cause of foodborne diarrheal illness worldwide, with more than one million cases each year in the United States alone. The global emergence of antimicrobial resistance in this pathogen has become a growing public health concern. Florfenicol-resistant (FFNr) Campylobacter has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter coli isolates recovered from cattle in the United States. A gene [cfr(C)] was found to be responsible for resistance not only to florfenicol but also to several other antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. The results showed that cfr(C) is located in a conjugative pTet MDR/virulence plasmid. This report highlights the power of antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.

Disinfectant and Antimicrobial Susceptibility Profiles of Campylobacter coli Isolated in 1998 to 1999 and 2015 from Swine and Commercial Pork Chops.

Susceptibility profiles were determined for 111 Campylobacter coli strains obtained in 1998 to 1999 and 2015 from market age pigs and pork chops against 22 disinfectants and 9 antimicrobials. Resistance to tetracycline (TET) was observed in 44.4% of 1998 to 1999 strains, and the antibiotic resistance profile was TET. But strains obtained in 2015 from swine and retail pork chops had 75% TET resistance and the antibiotic resistance profile was TET, followed by azithromycin-erythromycin-TET-telithromycin-clindamycin. Antimicrobial resistance increased in 2015 strains. All strains were resistant to triclosan, and 84.1% and 95.8% of strains in 1998 to 1999 and 2015, respectively, were chlorhexidine resistant. All strains were susceptible to benzalkonium chloride. There was a shift toward higher susceptibility to chlorhexidine, triclosan, P-128, OdoBan, CPB, and CPC in 2015 swine and pork chop strains compared with 1998 to 1999 strains. The disinfectants Tek-Trol and providone-iodine, tris(hydroxylmethyl)nitromethane (THN) and formaldehyde demonstrated the highest susceptibilities. Didecyldimethylammonium chloride (C10AC) appeared to be about equally effective as benzyldimethyltetradecylammonium chloride (C14BAC) for inhibiting C. coli, and both were more effective than C8AC and C12BAC, but C16BAC was not efficient at inhibiting C. coli. The BACs, C12BAC and C14BAC, were the most effective ingredients in DC&R. Also, C12BAC and C14BAC, or these two in synergy with C10AC were responsible for inhibition of C. coli at high P-128 MICs. No cross-resistance was observed between antibiotics and disinfectants. The continued use of THN and formaldehyde in DC&R should be evaluated since these components are not effective, and their inclusion adds unwanted chemicals in the environment. PRACTICAL APPLICATION: Campylobacter species cause diarrheal disease throughout the world. Disinfectants are often used on the farm, in veterinary medicine, by the food processing industry, in restaurants, and in consumer's homes. Limited information is available in the literature showing how disinfectants or disinfectant components may affect the many different foodborne pathogens, and, specifically, Campylobacter coli studied here. The knowledge generated in this study concerning the interactions of a broad array of disinfectants against C. coli may well affect the types of disinfectants and disinfectant formulations allowable for use by medical personnel, producers, food processors, restaurants, and consumers.

Multi-Laboratory Validation of a Loop-Mediated Isothermal Amplification Method for Screening Salmonella in Animal Food.

Loop-mediated isothermal amplification (LAMP) has gained wide popularity in the detection of Salmonella in foods owing to its simplicity, rapidity, and robustness. This multi-laboratory validation (MLV) study aimed to validate a Salmonella LAMP-based method against the United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method in a representative animal food matrix (dry dog food). Fourteen independent collaborators from seven laboratories in the United States and Canada participated in the study. Each collaborator received two sets of 24 blind-coded dry dog food samples (eight uninoculated; eight inoculated at a low level, 0.65 MPN/25 g; and eight inoculated at a high level, 3.01 MPN/25 g) and initiated the testing on the same day. The MLV study used an unpaired design where different test portions were analyzed by the LAMP and BAM methods using different preenrichment protocols (buffered peptone water for LAMP and lactose broth for BAM). All LAMP samples were confirmed by culture using the BAM method. BAM samples were also tested by LAMP following lactose broth preenrichment (paired samples). Statistical analysis was carried out by the probability of detection (POD) per AOAC guidelines and by a random intercept logistic regression model. Overall, no significant differences in POD between the Salmonella LAMP and BAM methods were observed with either unpaired or paired samples, indicating the methods were comparable. LAMP testing following preenrichment in buffered peptone water or lactose broth also resulted in insignificant POD differences (P > 0.05). The MLV study strongly supports the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of Salmonella in animal food.

Interactions of organic acids with Campylobacter coli from swine.

Campylobacter coli is a bacterial species that is a major cause of diarrheal disease worldwide, and Campylobacter spp. are among the top 5 foodborne pathogens in the United States. During food production organic acids (OAs) are often used to remove bacteria from animal carcasses. The interactions of six OAs with 111 C. coli strains obtained from swine and retail pork chops were studied by determining the molar minimum inhibitory concentrations (MICMs) of the C. coli strains, and the pH at the MICMs. The Henderson-Hasselbalch equation was used to calculate the concentrations of the undissociated and dissociated OAs at the MICMs of the C. coli strains. The results for the 111 different C. coli strains obtained from different locations were treated as a single group for each OA since many of the C. coli strains behaved similarly to each different OA. Inhibition of C. coli was not dependent on pH or on the undissociated OA species, but C. coli inhibition correlated with the dissociated OA species. Therefore, if the concentration of the dissociated OAs decreases from optimum, one may then expect that C. coli bacteria would escape disinfection. The concentration of the dissociated OA should be carefully controlled in a carcass wash. We suggest maintaining a concentration of the dissociated acetic, butyric, citric, formic, lactic and propionic acids at 29, 23, 11, 35, 22 and 25 mM, respectively, when using a carcass wash with these OAs to remove C. coli bacteria. However, due to C. coli utilization of acetate, formate, lactate and propionate, these four OAs may not be the best choice to use for a carcass wash to remove C. coli contamination. Of the six OAs, citric acid was the most efficient at inhibiting C. coli.

Use of whole-genome sequencing for Campylobacter surveillance from NARMS retail poultry in the United States in 2015.

Whole genome sequencing (WGS) has become a rapid and affordable tool for public health surveillance and outbreak detection. In this study, we used the Illuminia MiSeq® to sequence 589 Campylobacter isolates obtained in 2015 from retail poultry meats as part of the National Antimicrobial Resistance Monitoring System (NARMS). WGS data were used to identify the Campylobacter species and to compare the concordance between resistance genotypes and phenotypes. WGS accurately identified 386 C. jejuni and 203 C. coli using gyrA sequence information. Ten resistance genes, including tetO, blaOXA-61, aph(2″)-Ic, aph(2″)-If, aph(2″)-Ig, aph(3')-III, ant(6)-1a, aadE, aph(3")-VIIa, and Inu(C), plus mutations in housekeeping genes (gyrA at position 86, 23S rRNA at position 2074 and 2075), were identified by WGS analysis. Overall, there was a high concordance between phenotypic resistance to a given drug and the presence of known resistance genes. Concordance between both resistance and susceptible phenotypes and genotype was 100% for ciprofloxacin, nalidixic acid, gentamicin, azithromycin, and florfenicol. A few discrepancies were observed for tetracycline, clindamycin, and telithromycin. The concordance between resistance phenotype and genotype ranged from 67.9% to 100%; whereas, the concordance between susceptible phenotype and genotype ranged from 98.0% to 99.6%. Our study demonstrates that WGS can correctly identify Campylobacter species and predict antimicrobial resistance with a high degree of accuracy.

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.

MRSA and multidrug-resistant Staphylococcus aureus in U.S. retail meats, 2010-2011.

Methicillin-resistant Staphylococcus aureus (MRSA) has been detected in retail meats, although large-scale studies are scarce. We conducted a one-year survey in 2010-2011 within the framework of the National Antimicrobial Resistance Monitoring System. Among 3520 retail meats collected from eight U.S. states, 982 (27.9%) contained S. aureus and 66 (1.9%) were positive for MRSA. Approximately 10.4% (107/1032) of S. aureus isolates, including 37.2% (29/78) of MRSA, were multidrug-resistant (MDRSA). Turkey had the highest MRSA prevalence (3.5%), followed by pork (1.9%), beef (1.7%), and chicken (0.3%). Whole-genome sequencing was performed for all 66 non-redundant MRSA. Among five multilocus sequence types identified, ST8 (72.7%) and ST5 (22.7%) were most common and livestock-associated MRSA ST398 was assigned to one pork isolate. Eleven spa types were represented, predominately t008 (43.9%) and t2031 (22.7%). All four types of meats harbored t008, whereas t2031 was recovered from turkey only. The majority of MRSA (84.8%) possessed SCCmec IV and 62.1% harbored Panton-Valentine leukocidin. Pulsed-field gel electrophoresis showed that all ST8 MRSA belonged to the predominant human epidemic clone USA300, and others included USA100 and USA200. We conclude that a diverse MRSA population was present in U.S. retail meats, albeit at low prevalence.

Molecular Subtyping and Source Attribution of Campylobacter Isolated from Food Animals.

Campylobacter spp. commonly cause gastrointestinal illness in humans. Poultry meats have long been considered the predominant source of these infections, but few in-depth Campylobacter source attribution studies have been completed. We analyzed more than 1,300 Campylobacter isolates recovered from a number of animal and food sources, including dairy and beef cattle, pigs, poultry, and retail poultry meat, and compared them with Campylobacter isolates recovered from human clinical samples. Each isolate was subtyped using pulsed-field gel electrophoresis (PFGE) with SmaI and queried against the Centers for Disease Control and Prevention PulseNet database to identify human isolates with indistinguishable patterns. Half (49.5%) of the PFGE patterns from poultry animal and retail meat isolates were indistinguishable from patterns of at least one human isolate. Among the isolates from beef and dairy cows, 56.6 and 65.0%, respectively, of their PFGE patterns were indistinguishable from those of human isolates. Only a small portion of the PFGE patterns of Campylobacter isolated from pigs (9.5%) were found to have PFGE patterns in common with human isolates. These data imply that cattle may be larger contributors to Campylobacter infections than previously recognized and help further our understanding of potential sources of human campylobacteriosis.

Novel gentamicin resistance genes in Campylobacter isolated from humans and retail meats in the USA.

OBJECTIVES: To understand the molecular epidemiology of gentamicin-resistant Campylobacter and investigate aminoglycoside resistance mechanisms. METHODS: One-hundred-and-fifty-one gentamicin-resistant Campylobacter isolates from humans (n = 38 Campylobacter jejuni; n = 41, Campylobacter coli) and retail chickens (n = 72 C. coli), were screened for the presence of gentamicin resistance genes by PCR and subtyped using PFGE. A subset of the isolates (n = 41) was analysed using WGS. RESULTS: Nine variants of gentamicin resistance genes were identified: aph(2″)-Ib, Ic, Ig, If, If1, If3, Ih, aac(6')-Ie/aph(2″)-Ia and aac(6')-Ie/aph(2″)-If2. The aph(2″)-Ib, Ic, If1, If3, Ih and aac(6')-Ie/aph(2″)-If2 variants were identified for the first time in Campylobacter. Human isolates showed more diverse aminoglycoside resistance genes than did retail chicken isolates, in which only aph(2″)-Ic and -Ig were identified. The aph(2″)-Ig gene was only gene shared by C. coli isolates from human (n = 27) and retail chicken (n = 69). These isolates displayed the same resistance profile and similar PFGE patterns, suggesting that contaminated retail chicken was probably the source of human C. coli infections. Human isolates were genetically diverse and generally more resistant than the retail chicken isolates. The most frequent co-resistance was to tetracycline (78/79, 98.7%), followed by ciprofloxacin/nalidixic acid (46/79, 58.2%), erythromycin and azithromycin (36/79, 45.6%), telithromycin (32/79, 40.5%) and clindamycin (18/79, 22.8%). All human and retail meat isolates were susceptible to florfenicol. CONCLUSIONS: This study demonstrated that several new aminoglycoside resistance genes underlie the recent emergence of gentamicin-resistant Campylobacter, and that, in addition to contaminated retail chicken, other sources have also contributed to gentamicin-resistant Campylobacter infections in humans.

Whole-genome sequencing of gentamicin-resistant Campylobacter coli isolated from U.S. retail meats reveals novel plasmid-mediated aminoglycoside resistance genes.

Aminoglycoside resistance in Campylobacter has been routinely monitored in the United States in clinical isolates since 1996 and in retail meats since 2002. Gentamicin resistance first appeared in a single human isolate of Campylobacter coli in 2000 and in a single chicken meat isolate in 2007, after which it increased rapidly to account for 11.3% of human isolates and 12.5% of retail isolates in 2010. Pulsed-field gel electrophoresis analysis indicated that gentamicin-resistant C. coli isolates from retail meat were clonal. We sequenced the genomes of two strains of this clone using a next-generation sequencing technique in order to investigate the genetic basis for the resistance. The gaps of one strain were closed using optical mapping and Sanger sequencing, and this is the first completed genome of C. coli. The two genomes are highly similar to each other. A self-transmissible plasmid carrying multiple antibiotic resistance genes was revealed within both genomes, carrying genes encoding resistance to gentamicin, kanamycin, streptomycin, streptothricin, and tetracycline. Bioinformatics analysis and experimental results showed that gentamicin resistance was due to a phosphotransferase gene, aph(2")-Ig, not described previously. The phylogenetic relationship of this newly emerged clone to other Campylobacter spp. was determined by whole-genome single nucleotide polymorphisms (SNPs), which showed that it clustered with the other poultry isolates and was separated from isolates from livestock.