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Antimicrobial resistance (AMR) is a significant public health threat. With the rise of affordable whole genome sequencing, in silico approaches to assessing AMR gene content can be used to detect known resistance mechanisms and potentially identify novel mechanisms. To enable accurate assessment of AMR gene content, as part of a multi-agency collaboration, NCBI developed a comprehensive AMR gene database, the Bacterial Antimicrobial Resistance Reference Gene Database and the AMR gene detection tool AMRFinder. Here, we describe the expansion of the Reference Gene Database, now called the Reference Gene Catalog, to include putative acid, biocide, metal, stress resistance genes, in addition to virulence genes and species-specific point mutations. Genes and point mutations are classified by broad functions, as well as more detailed functions. As we have expanded both the functional repertoire of identified genes and functionality, NCBI released a new version of AMRFinder, known as AMRFinderPlus. This new tool allows users the option to utilize only the core set of AMR elements, or include stress response and virulence genes, too. AMRFinderPlus can detect acquired genes and point mutations in both protein and nucleotide sequence. In addition, the evidence used to identify the gene has been expanded to include whether nucleotide or protein sequence was used, its location in the contig, and presence of an internal stop codon. These database improvements and functional expansions will enable increased precision in identifying AMR genes, linking AMR genotypes and phenotypes, and determining possible relationships between AMR, virulence, and stress response.

We report isolation of a New Delhi metallo-β-lactamase-5-producing carbapenem-resistant Escherichia coli sequence type 167 from companion animals in the United States. Reports of carbapenem-resistant Enterobacteriaceae in companion animals are rare. We describe a unique cluster of bla -producing E. coli in a veterinary hospital.

Background Klebsiella spp. is an important human and animal pathogen, and it is commonly found with resistance to clinically important antimicrobials worldwide. The main goals of this study were to determine the prevalence of antimicrobial resistance genes in our study population and to assess the relatedness between Klebsiella spp. isolated from humans and animals. Isolates were collected in 2019 and 2020 from various animal hosts that presented to veterinary hospitals in the U.S. that participate in the FDA’s Center for Veterinary Medicine Veterinary Laboratory Investigation and Response Network’s antimicrobial resistance monitoring program. Results We sequenced a total of 204 Klebsiella spp. isolates. A majority of isolates were identified as K. pneumoniae (149/204, 73.0%), followed by K. quasipneumoniae (30/204, 14.7%), K. variicola (15/204, 7.4%), K. aerogenes (5/204, 2.5%), K. oxytoca (4/204, 2.0%), and K. grimontii (1/204, 0.5%). Out of 204 isolates, 138 were recovered from dogs, 25 from horses, 17 from cats, 6 from avian species, 5 from cows and 3 from pigs. The remaining 10 isolates were recovered from a few other mammal species. Klebsiella spp. isolates were very diverse. In silico multilocus sequence typing (MLST), using WGS data, identified a total of 88 known sequence types across all isolates. Seventeen isolates were not assigned an MLST sequence type due to combinations of alleles not previously found in the PubMLST database. 45 of the 204 isolates were assigned to 20 different single nucleotide polymorphism (SNP) clusters in the National Center for Biotechnology Information (NCBI) Pathogen Detection browser, and out of those, four isolates were assigned SNP clusters that also contained human isolates, all from dogs. The closest human isolate was 29 SNPs from a dog isolate. A total of 36 resistance genes were identified. The three most common resistance genes were oqxAB , fosA , and bla SHV . None of the isolates had carbapenem resistance genes, although one isolate from a goat had mcr-8.1 , a colistin resistance gene. Conclusions To our knowledge, this is the largest collection of sequenced Klebsiella from sick animals ever assembled, and the results found limited relatedness between these isolates and those from humans, despite the diversity of sequenced isolates.

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.

From the first cases in 2019, COVID-19 infections caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) have resulted in over 6 million human deaths in a worldwide pandemic. SARS-CoV-2 is commonly spread from human to human through close contact and is capable of infecting both humans and animals. Worldwide, there have been over 675 animal outbreaks reported that resulted in over 2000 animal infections including domestic and wild animals. As the role of animal infections in the transmission, pathogenesis, and evolution of SARS-CoV-2 is still unfolding, accurate and reliable animal diagnostic tests are critical to aid in managing both human and animal health. This review highlights key animal samples and the three main diagnostic approaches used for animal testing: PCR, serology, and Next Generation Sequencing. Diagnostic results help inform (often difficult) clinical decision-making, but also possible ways to mitigate spread among pets, food supplies, or wildlife. A One Health approach has been key to monitoring the SARS-CoV-2 pandemic, as consistent human-animal interactions can lead to novel variants. Having multiple animal diagnostic tests for SARS-CoV-2 available is critical to ensure human, animal, and environmental health.

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. Lociq is a computational method that generates typing schema for plasmid characterization to aid investigations into the origin, evolution and epidemiology of multidrug resistant plasmids.

This cross-sectional study determined the serovars, antimicrobial resistance genes, and virulence factors of Salmonella isolated from hatcheries, broiler farms, processing plants, and retail outlets in Trinidad and Tobago. Salmonella in silico serotyping detected 23 different serovars where Kentucky 20.5% (30/146), Javiana 19.2% (28/146), Infantis 13.7% (20/146), and Albany 8.9% (13/146) were the predominant serovars. There was a 76.0% (111/146) agreement between serotyping results using traditional conventional methods and whole-genome sequencing (WGS) in in silico analysis. In silico identification of antimicrobial resistance genes conferring resistance to aminoglycosides, cephalosporins, peptides, sulfonamides, and antiseptics were detected. Multidrug resistance (MDR) was detected in 6.8% (10/146) of the isolates of which 100% originated from broiler farms. Overall, virulence factors associated with secretion systems and fimbrial adherence determinants accounted for 69.3% (3091/4463), and 29.2% (1302/4463) counts, respectively. Ten of 20 isolates of serovar Infantis (50.0%) showed MDR and contained the blaCTX-M-65 gene. This is the first molecular characterization of Salmonella isolates detected along the entire broiler production continuum in the Caribbean region using WGS. The availability of these genomes will help future source tracking during epidemiological investigations associated with Salmonella foodborne outbreaks in the region and worldwide.

Disease causing bacteria often manipulate host cells in a way that facilitates the infectious process. Many pathogenic gram-negative bacteria accomplish this by using type III secretion systems. In these complex secretion pathways, bacterial chaperones direct effector proteins to a needle-like secretion apparatus, which then delivers the effector protein into the host cell cytosol. The effector protein ExoU and its chaperone SpcU are components of the Pseudomonas aeruginosa type III secretion system. Secretion of ExoU has been associated with more severe infections in both humans and animal models. Here we describe the 1.92 Å X-ray structure of the ExoU-SpcU complex, a full-length type III effector in complex with its full-length cognate chaperone. Our crystallographic data allow a better understanding of the mechanism by which ExoU kills host cells and provides a foundation for future studies aimed at designing inhibitors of this potent toxin.

Extraintestinal pathogenic Escherichia coli (ExPEC) cause urinary tract and potentially life-threatening invasive infections. Unfortunately, the origins of ExPEC are not always clear. We used genomic data of E. coli isolates from five U.S. government organizations to evaluate potential sources of ExPEC infections. Virulence gene analysis of 38,032 isolates from human, food animal, retail meat, and companion animals classified the subset of 8142 non-diarrheagenic isolates into 40 virulence groups. Groups were identified as low, medium, and high relative risk of containing ExPEC strains, based on the proportion of isolates recovered from humans. Medium and high relative risk groups showed a greater representation of sequence types associated with human disease, including ST-131. Over 90% of food source isolates belonged to low relative risk groups, while >60% of companion animal isolates belonged to medium or high relative risk groups. Additionally, 18 of the 26 most prevalent antimicrobial resistance determinants were more common in high relative risk groups. The associations between antimicrobial resistance and virulence potentially limit treatment options for human ExPEC infections. This study demonstrates the power of large-scale genomics to assess potential sources of ExPEC strains and highlights the importance of a One Health approach to identify and manage these human pathogens.

Whole-genome sequencing (WGS) has transformed our understanding of antimicrobial resistance, yielding new insights into the genetics underlying resistance. To date, most studies using WGS to study antimicrobial resistance have focused on gram-negative bacteria in the family Enterobacteriaceae, such as Salmonella spp. and Escherichia coli, which have well-defined resistance mechanisms. In contrast, relatively few studies have been performed on gram-positive organisms. We sequenced 197 strains of Enterococcus from various animal and food sources, including 100 Enterococcus faecium and 97 E. faecalis. From analyzing acquired resistance genes and known resistance-associated mutations, we found that resistance genotypes correlated with resistance phenotypes in 96.5% of cases for the 11 drugs investigated. Some resistances, such as those to tigecycline and daptomycin, could not be investigated due to a lack of knowledge of mechanisms underlying these phenotypes. This study showed the utility of WGS for predicting antimicrobial resistance based on genotype alone.