The spread of pESI-mediated extended-spectrum cephalosporin resistance in Salmonella serovars-Infantis, Senftenberg, and Alachua isolated from food animal sources in the United States.

The goal of this study is to investigate the origin, prevalence, and evolution of the pESI megaplasmid in Salmonella isolated from animals, foods, and humans. We queried 510,097 Salmonella genomes under the National Center for Biotechnology Information (NCBI) Pathogen Detection (PD) database for the presence of potential sequences containing the pESI plasmid in animal, food, and environmental sources. The presence of the pESI megaplasmid was confirmed by using seven plasmid-specific markers (rdA, pilL, SogS, TrbA, ipf, ipr2 and IncFIB(pN55391)). The plasmid and chromosome phylogeny of these isolates was inferred from single nucleotide polymorphisms (SNPs). Our search resolved six Salmonella clusters carrying the pESI plasmid. Four were emergent Salmonella Infantis clusters, and one each belonged to serovar Senftenberg and Alachua. The Infantis cluster with a pESI plasmid carrying blaCTX-M-65 gene was the biggest of the four emergent Infantis clusters, with over 10,000 isolates. This cluster was first detected in South America and has since spread widely in United States. Over time the composition of pESI in United States has changed with the average number of resistance genes showing a decrease from 9 in 2014 to 5 in 2022, resulting from changes in gene content in two integrons present in the plasmid. A recent and emerging cluster of Senftenberg, which carries the blaCTX-M-65 gene and is primarily associated with turkey sources, was the second largest in the United States. SNP analysis showed that this cluster likely originated in North Carolina with the recent acquisition of the pESI plasmid. A single Alachua isolate from turkey was also found to carry the pESI plasmid containing blaCTX-M-65 gene. The study of the pESI plasmid, its evolution and mechanism of spread can help us in developing appropriate strategies for the prevention and further spread of this multi-drug resistant plasmid in Salmonella in poultry and humans.

Metagenomic survey of antimicrobial resistance (AMR) in Maryland surface waters differentiated by high and low human impact.

Here, we examine surface waters as a modality to better understand baseline antimicrobial resistance (AMR) across the environment to supplement existing AMR monitoring in pathogens associated with humans, foods, and animals. Data from metagenomic and quasimetagenomic (shotgun sequenced enrichments) are used to describe AMR in Maryland surface waters from high and low human impact classifications.

Genomic analysis of azithromycin-resistant Salmonella from food animals at slaughter and processing, and retail meats, 2011-2021, United States.

IMPORTANCE: Macrolides of different ring sizes are critically important antimicrobials for human medicine and veterinary medicine, though the widely used 15-membered ring azithromycin in humans is not approved for use in veterinary medicine. We document here the emergence of azithromycin-resistant Salmonella among the NARMS culture collections between 2011 and 2021 in food animals and retail meats, some with co-resistance to ceftriaxone or decreased susceptibility to ciprofloxacin. We also provide insights into the underlying genetic mechanisms and genomic contexts, including the first report of a novel combination of azithromycin resistance determinants and the characterization of multidrug-resistant plasmids. Further, we highlight the emergence of a multidrug-resistant Salmonella Newport clone in food animals (mainly cattle) with both azithromycin resistance and decreased susceptibility to ciprofloxacin. These findings contribute to a better understating of azithromycin resistance mechanisms in Salmonella and warrant further investigations on the drivers behind the emergence of resistant clones.

A comparison of methods to detect low levels of Salmonella enterica in surface waters to support antimicrobial resistance surveillance efforts performed in multiple laboratories.

Developing effective and sensitive detection methods for antimicrobial resistant Salmonella enterica from surface water is a goal of the National Antimicrobial Resistance Monitoring System (NARMS). There are no specified methods for recovery of S. enterica in surface waters in the U.S. A multi-laboratory evaluation of four methods - bulk water enrichment (BW), vertical Modified Moore Swab (VMMS), modified Standard Method 9260.B2 (SM), and dead-end ultrafiltration (DEUF) - was undertaken to recover S. enterica from surface water. In Phase 1, one-liter volumes of water were collected from the same site on five different dates. Water was shipped and analyzed at four different laboratory locations (A, B, C, and D) for recovery of 1) inoculated fluorescent S. Typhimurium strain (ca. 30 CFU/L) and 2) Salmonella present in the water sampled. At each location, BW, VMMS, or SM recovery was performed on five separate 1 L water samples. Twenty 1 L water samples were subjected to each recovery method, and overall, sixty 1 L samples were assayed for Salmonella. Inoculated, fluorescent Salmonella Typhimurium and environmental Salmonella spp. were recovered from 65 % (39/60) and 45 % (27/60) of water samples, respectively. BW, VMMS, and SM recovered fluorescent S. Typhimurium from 60 %, 60 %, and 75 % of inoculated samples, respectively. Analysis by Chi-squared test determined laboratory location had a significant (p < 0.05) effect on fluorescent S. Typhimurium recovery compared to method or date of water collection. In Phase 2, recovery of inoculated fluorescent S. Typhimurium from 1 L samples by SM and DEUF was compared at laboratory locations B and D. SM and DEUF recovered fluorescent S. Typhimurium from 100 % (20/20) and 95 % (19/20) of inoculated water samples, respectively; laboratory location (p > 0.05) did not affect Salmonella recovery. Uniform laboratory methodology and training should be prioritized in conducting Salmonella recovery from surface water in laboratories.

Lociq provides a loci-seeking approach for enhanced plasmid subtyping and structural characterization.

Antimicrobial resistance (AMR) monitoring for public health is relying more on whole genome sequencing to characterize and compare resistant strains. This requires new approaches to describe and track AMR that take full advantage of the detailed data provided by genomic technologies. The plasmid-mediated transfer of AMR genes is a primary concern for AMR monitoring because plasmid rearrangement events can integrate new AMR genes into the plasmid backbone or promote hybridization of multiple plasmids. To better monitor plasmid evolution and dissemination, we developed the Lociq subtyping method to classify plasmids by variations in the sequence and arrangement of core plasmid genetic elements. Subtyping with Lociq provides an alpha-numeric nomenclature that can be used to denominate plasmid population diversity and characterize the relevant features of individual plasmids. Here we demonstrate how Lociq generates typing schema to track and characterize the origin, evolution and epidemiology of multidrug resistant plasmids.

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.

Long-Read Sequencing Reveals Evolution and Acquisition of Antimicrobial Resistance and Virulence Genes in Salmonella enterica.

Salmonella enterica is a significant and phylogenetically diverse zoonotic pathogen. To understand its genomic heterogeneity and antimicrobial resistance, we performed long-read sequencing on Salmonella isolated from retail meats and food animals. A collection of 134 multidrug-resistant isolates belonging to 33 serotypes were subjected to PacBio sequencing. One major locus of diversity among these isolates was the presence and orientation of Salmonella pathogenic islands (SPI), which varied across different serotypes but were largely conserved within individual serotypes. We also identified insertion of an IncQ resistance plasmid into the chromosome of fourteen strains of serotype I 4,[5],12:i:- and the Salmonella genomic island 1 (SGI-1) in five serotypes. The presence of various SPIs, SGI-1 and integrated plasmids contributed significantly to the genomic variability and resulted in chromosomal resistance in 55.2% (74/134) of the study isolates. A total of 93.3% (125/134) of isolates carried at least one plasmid, with isolates carrying up to seven plasmids. We closed 233 plasmid sequences of thirteen replicon types, along with twelve hybrid plasmids. Some associations between Salmonella isolate source, serotype, and plasmid type were seen. For instance, IncX plasmids were more common in serotype Kentucky from retail chicken. Plasmids IncC and IncHI had on average more than five antimicrobial resistance genes, whereas in IncX, it was less than one per plasmid. Overall, 60% of multidrug resistance (MDR) strains that carried >3 AMR genes also carried >3 heavy metal resistance genes, raising the possibility of co-selection of antimicrobial resistance in the presence of heavy metals. We also found nine isolates representing four serotypes that carried virulence plasmids with the spv operon. Together, these data demonstrate the power of long-read sequencing to reveal genomic arrangements and integrated plasmids with a high level of resolution for tracking and comparing resistant strains from different sources. Additionally, the findings from this study will help expand the reference set of closed Salmonella genomes that can be used to improve genome assembly from short-read data commonly used in One Health antimicrobial resistance surveillance.

A National Antimicrobial Resistance Monitoring System Survey of Antimicrobial-Resistant Foodborne Bacteria Isolated from Retail Veal in the United States.

ABSTRACT: Little is known about the prevalence of antimicrobial-resistant (AMR) bacteria in veal meat in the United States. We estimated the prevalence of bacterial contamination and AMR in various veal meats collected during the 2018 U.S. National Antimicrobial Resistance Monitoring System (NARMS) survey of retail outlets in nine states and compared the prevalence with the frequency of AMR bacteria from other cattle sources sampled for NARMS. In addition, we identified genes associated with resistance to medically important antimicrobials and gleaned other genetic details about the resistant organisms. The prevalence of Campylobacter, Salmonella, Escherichia coli, and Enterococcus in veal meats collected from grocery stores in nine states was 0% (0 of 358), 0.6% (2 of 358), 21.1% (49 of 232), and 53.5% (121 of 226), respectively, with ground veal posing the highest risk for contamination. Both Salmonella isolates were resistant to at least one antimicrobial agent as were 65.3% (32 of 49) of E. coli and 73.6% (89 of 121) of Enterococcus isolates. Individual drug and multiple drug resistance levels were significantly higher (P < 0.05) in E. coli and Enterococcus from retail veal than in dairy cattle ceca and retail ground beef samples from 2018 NARMS data. Whole genome sequencing was conducted on select E. coli and Salmonella from veal. Cephalosporin resistance (blaCMY and blaCTX-M), macrolide resistance (mph), and plasmid-mediated quinolone resistance (qnr) genes and gyrA mutations were found. We also identified heavy metal resistance genes ter, ars, mer, fieF, and gol and disinfectant resistance genes qac and emrE. An stx1a-containing E. coli was also found. Sequence types were highly varied among the nine E. coli isolates that were sequenced. Several plasmid types were identified in E. coli and Salmonella, with the majority (9 of 11) of isolates containing IncF. This study illustrates that veal meat is a carrier of AMR bacteria.

Antimicrobial resistance in non-Typhoidal Salmonella from retail poultry meat by antibiotic usage-related production claims - United States, 2008-2017.

Antimicrobial resistance (AMR) in non-typhoidal Salmonella from poultry is a public health concern. Injudicious use of antibiotics in humans and agriculture fuels the emergence of resistance. The objective of this study was to characterize the prevalence, antibiotic susceptibility profiles and genetic resistance mechanisms of Salmonella isolated from US retail poultry meat samples with and without antibiotic-related claims. We reviewed data from 46,937 poultry meat samples collected from 2008 to 2017 through the FDA NARMS retail meat program. Antibiotic usage claims on the poultry packaging were used to categorize the sample as 'conventionally raised' or 'reduced or no antibiotic use'. The results show that the prevalence of Salmonella in conventional poultry samples (8.6%) was higher than reduced or no antibiotic use poultry samples (5.1%). The odds of resistance to three or more antimicrobial classes (multi-drug resistant) were 2.61 times higher for Salmonella isolates from conventional samples, compared to isolates from reduced antibiotic use samples. The frequency of the aminoglycoside resistance gene, strB, and the beta-lactam resistant gene, blaCMY-2, were higher in isolates from conventional meat. This study suggests that conventionally raised poultry meat was more likely to be contaminated with multi-drug resistant Salmonella, and those Salmonella are more likely to carry genes for antibiotics resistance.

Predicting antimicrobial susceptibility from the bacterial genome: A new paradigm for one health resistance monitoring.

The laboratory identification of antibacterial resistance is a cornerstone of infectious disease medicine. In vitro antimicrobial susceptibility testing has long been based on the growth response of organisms in pure culture to a defined concentration of antimicrobial agents. By comparing individual isolates to wild-type susceptibility patterns, strains with acquired resistance can be identified. Acquired resistance can also be detected genetically. After many decades of research, the inventory of genes underlying antimicrobial resistance is well known for several pathogenic genera including zoonotic enteric organisms such as Salmonella and Campylobacter and continues to grow substantially for others. With the decline in costs for large scale DNA sequencing, it is now practicable to characterize bacteria using whole genome sequencing, including the carriage of resistance genes in individual microorganisms and those present in complex biological samples. With genomics, we can generate comprehensive, detailed information on the bacterium, the mechanisms of antibiotic resistance, clues to its source, and the nature of mobile DNA elements by which resistance spreads. These developments point to a new paradigm for antimicrobial resistance detection and tracking for both clinical and public health purposes.

Comparative Genomic Analysis of 450 Strains of Salmonellaenterica Isolated from Diseased Animals.

Salmonella is a leading cause of bacterial infections in animals and humans. We sequenced a collection of 450 Salmonella strains from diseased animals to better understand the genetic makeup of their virulence and resistance features. The presence of Salmonella pathogenicity islands (SPIs) varied by serotype. S. Enteritidis carried the most SPIs (n = 15), while S. Mbandaka, S. Cerro, S. Meleagridis, and S. Havana carried the least (n = 10). S. Typhimurium, S. Choleraesuis, S. I 4,5,12:i:-, and S. Enteritidis each contained the spv operon on IncFII or IncFII-IncFIB hybrid plasmids. Two S. IIIa carried a spv operon with spvD deletion on the chromosome. Twelve plasmid types including 24 hybrid plasmids were identified. IncA/C was frequently associated with S. Newport (83%) and S. Agona (100%) from bovine, whereas IncFII (100%), IncFIB (100%), and IncQ1 (94%) were seen in S. Choleraesuis from swine. IncX (100%) was detected in all S. Kentucky from chicken. A total of 60 antimicrobial resistance genes (ARGs), four disinfectant resistances genes (DRGs) and 33 heavy metal resistance genes (HMRGs) were identified. The Salmonella strains from sick animals contained various SPIs, resistance genes and plasmid types based on the serotype and source of the isolates. Such complicated genomic structures shed light on the strain characteristics contributing to the severity of disease and treatment failures in Salmonella infections, including those causing illnesses in animals.

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.

The mcr-9 Gene of Salmonella and Escherichia coli Is Not Associated with Colistin Resistance in the United States.

Reports of transmissible colistin resistance show the importance of comprehensive colistin resistance surveillance. Recently, a new allele of the mobile colistin resistance (mcr) gene family designated mcr-9, which shows variation in genetic context and colistin susceptibility, was reported. We tested over 100 Salmonella enterica and Escherichia coli isolates with mcr-9 from the National Antimicrobial Resistance Monitoring System (NARMS) in the United States for their susceptibility to colistin and found that every isolate was susceptible, with an MIC of ≤1 µg/ml. Long-read sequencing of 12 isolates revealed mcr-9 on IncHI plasmids that were either independent or integrated into the chromosome. Overall, these results demonstrate that caution is necessary when determining the clinical relevance of new resistance genes.

Diverse Fluoroquinolone Resistance Plasmids From Retail Meat E. coli in the United States.

Fluoroquinolones are used to treat serious bacterial infections, including those caused by Escherichia coli and Salmonella enterica. The emergence of plasmid-mediated quinolone resistance (PMQR) represent a new challenge to the successful treatment of Gram-negative infections. As part of a long-term strategy to generate a reference database of closed plasmids from antimicrobial resistant foodborne bacteria, we performed long-read sequencing of 11 E. coli isolates from retail meats that were non-susceptible to ciprofloxacin. Each of the isolates had PMQR genes, including qnrA1, qnrS1, and qnrB19. The four qnrB19 genes were carried on two distinct ColE-type plasmids among isolates from pork chop and ground turkey and were identical to plasmids previously identified in Salmonella. Seven other plasmids differed from any other sequences in GenBank and comprised IncF and IncR plasmids that ranged in size from 48 to 180 kb. These plasmids also contained different combinations of resistance genes, including those conferring resistance to beta-lactams, macrolides, sulfonamides, tetracycline, and heavy metals. Although relatively few isolates have PMQR genes, the identification of diverse plasmids in multiple retail meat sources suggests the potential for further spread of fluoroquinolone resistance, including through co-selection. These results highlight the value of long-read sequencing in characterizing antimicrobial resistance genes of public health concern.

Using Genomics to Track Global Antimicrobial Resistance.

The recent advancements in rapid and affordable DNA sequencing technologies have revolutionized diagnostic microbiology and microbial surveillance. The availability of bioinformatics tools and online accessible databases has been a prerequisite for this. We conducted a scientific literature review and here we present a description of examples of available tools and databases for antimicrobial resistance (AMR) detection and provide future perspectives and recommendations. At least 47 freely accessible bioinformatics resources for detection of AMR determinants in DNA or amino acid sequence data have been developed to date. These include, among others but not limited to, ARG-ANNOT, CARD, SRST2, MEGARes, Genefinder, ARIBA, KmerResistance, AMRFinder, and ResFinder. Bioinformatics resources differ for several parameters including type of accepted input data, presence/absence of software for search within a database of AMR determinants that can be specific to a tool or cloned from other resources, and for the search approach employed, which can be based on mapping or on alignment. As a consequence, each tool has strengths and limitations in sensitivity and specificity of detection of AMR determinants and in application, which for some of the tools have been highlighted in benchmarking exercises and scientific articles. The identified tools are either available at public genome data centers, from GitHub or can be run locally. NCBI and European Nucleotide Archive (ENA) provide possibilities for online submission of both sequencing and accompanying phenotypic antimicrobial susceptibility data, allowing for other researchers to further analyze data, and develop and test new tools. The advancement in whole genome sequencing and the application of online tools for real-time detection of AMR determinants are essential to identify control and prevention strategies to combat the increasing threat of AMR. Accessible tools and DNA sequence data are expanding, which will allow establishing global pathogen surveillance and AMR tracking based on genomics. There is however, a need for standardization of pipelines and databases as well as phenotypic predictions based on the data.

Validating the AMRFinder Tool and Resistance Gene Database by Using Antimicrobial Resistance Genotype-Phenotype Correlations in a Collection of Isolates.

Antimicrobial resistance (AMR) is a major public health problem that requires publicly available tools for rapid analysis. To identify AMR genes in whole-genome sequences, the National Center for Biotechnology Information (NCBI) has produced AMRFinder, a tool that identifies AMR genes using a high-quality curated AMR gene reference database. The Bacterial Antimicrobial Resistance Reference Gene Database consists of up-to-date gene nomenclature, a set of hidden Markov models (HMMs), and a curated protein family hierarchy. Currently, it contains 4,579 antimicrobial resistance proteins and more than 560 HMMs. Here, we describe AMRFinder and its associated database. To assess the predictive ability of AMRFinder, we measured the consistency between predicted AMR genotypes from AMRFinder and resistance phenotypes of 6,242 isolates from the National Antimicrobial Resistance Monitoring System (NARMS). This included 5,425 Salmonella enterica, 770 Campylobacter spp., and 47 Escherichia coli isolates phenotypically tested against various antimicrobial agents. Of 87,679 susceptibility tests performed, 98.4% were consistent with predictions. To assess the accuracy of AMRFinder, we compared its gene symbol output with that of a 2017 version of ResFinder, another publicly available resistance gene detection system. Most gene calls were identical, but there were 1,229 gene symbol differences (8.8%) between them, with differences due to both algorithmic differences and database composition. AMRFinder missed 16 loci that ResFinder found, while ResFinder missed 216 loci that AMRFinder identified. Based on these results, AMRFinder appears to be a highly accurate AMR gene detection system.

Using Machine Learning To Predict Antimicrobial MICs and Associated Genomic Features for Nontyphoidal Salmonella.

Nontyphoidal Salmonella species are the leading bacterial cause of foodborne disease in the United States. Whole-genome sequences and paired antimicrobial susceptibility data are available for Salmonella strains because of surveillance efforts from public health agencies. In this study, a collection of 5,278 nontyphoidal Salmonella genomes, collected over 15 years in the United States, was used to generate extreme gradient boosting (XGBoost)-based machine learning models for predicting MICs for 15 antibiotics. The MIC prediction models had an overall average accuracy of 95% within ±1 2-fold dilution step (confidence interval, 95% to 95%), an average very major error rate of 2.7% (confidence interval, 2.4% to 3.0%), and an average major error rate of 0.1% (confidence interval, 0.1% to 0.2%). The model predicted MICs with no a priori information about the underlying gene content or resistance phenotypes of the strains. By selecting diverse genomes for the training sets, we show that highly accurate MIC prediction models can be generated with less than 500 genomes. We also show that our approach for predicting MICs is stable over time, despite annual fluctuations in antimicrobial resistance gene content in the sampled genomes. Finally, using feature selection, we explore the important genomic regions identified by the models for predicting MICs. To date, this is one of the largest MIC modeling studies to be published. Our strategy for developing whole-genome sequence-based models for surveillance and clinical diagnostics can be readily applied to other important human pathogens.

Novel linezolid resistance plasmids in Enterococcus from food animals in the USA.

Objectives: To sequence the genomes and determine the genetic mechanisms for linezolid resistance identified in three strains of Enterococcus isolated from cattle and swine caecal contents as part of the US National Antimicrobial Resistance Monitoring System (NARMS) surveillance programme. Methods: Broth microdilution was used for in vitro antimicrobial susceptibility testing to assess linezolid resistance. Resistance mechanisms and plasmid types were identified from data generated by WGS on Illumina® and PacBio® platforms. Conjugation experiments were performed to determine whether identified mechanisms were transmissible. Results: Linezolid resistance plasmids containing optrA were identified in two Enterococcus faecalis isolates and one Enterococcus faecium. The E. faecium isolate also carried the linezolid resistance gene cfr on the same plasmid as optrA. The linezolid resistance plasmids had various combinations of additional resistance genes conferring resistance to phenicols (fexA), aminoglycosides [spc and aph(3')-III] and macrolides [erm(A) and erm(B)]. One of the plasmids was confirmed to be transmissible by conjugation, resulting in linezolid resistance in the transconjugant. Conclusions: To the best of our knowledge, this is the first identification of linezolid resistance in the USA in bacteria isolated from food animals. The oxazolidinone class of antibiotics is not used in food animals in the USA, but the genes responsible for resistance were identified on plasmids with other resistance markers, indicating that there may be co-selection for these plasmids due to the use of different antimicrobials. The transmissibility of one of the plasmids demonstrated the potential for linezolid resistance to spread horizontally. Additional surveillance is necessary to determine whether similar plasmids are present in human strains of Enterococcus.