Antimicrobial-Resistant Escherichia coli, Enterobacter cloacae, Enterococcus faecium, and Salmonella Kentucky Harboring Aminoglycoside and Beta-Lactam Resistance Genes in Raw Meat-Based Dog Diets, USA.

The practice of feeding raw meat-based diets to dogs has grown in popularity worldwide in recent years. However, there are public health risks in handling and feeding raw meat-based dog diets (RMDDs) to dogs since there are no pathogen reduction steps to reduce the microbial load, which may include antimicrobial-resistant pathogenic bacteria. A total of 100 RMDDs from 63 suppliers were sampled, and selective media were used to isolate bacteria from the diets. Bacterial identification, antimicrobial susceptibility testing, and whole-genome sequencing (WGS) were conducted to identify antimicrobial resistance (AMR). The primary meat sources for RMDDs included in this study were poultry (37%) and beef (24%). Frozen-dry was the main method of product production (68%). In total, 52 true and opportunistic pathogens, including Enterobacterales (mainly Escherichia coli, Enterobacter cloacae) and Enterococcus faecium, were obtained from 30 RMDDs. Resistance was identified to 19 of 28 antimicrobials tested, including amoxicillin/clavulanic acid (23/52, 44%), ampicillin (19/52, 37%), cephalexin (16/52, 31%), tetracycline (7/52, 13%), marbofloxacin (7/52, 13%), and cefazolin (6/52, 12%). All 19 bacterial isolates submitted for WGS harbored at least one type of AMR gene. The identified AMR genes were found to mediate resistance to aminoglycoside (gentamicin, streptomycin, amikacin/kanamycin, gentamicin/kanamycin/tobramycin), macrolide, beta-lactam (carbapenem, cephalosporin), tetracycline, fosfomycin, quinolone, phenicol/quinolone, and sulfonamide. In conclusion, the results of this study suggest that feeding and handling RMDDs may pose a significant public health risk due to the presence of antimicrobial-resistant pathogens, and further research and intervention may be necessary to minimize these risks.

GalaxyTrakr: a distributed analysis tool for public health whole genome sequence data accessible to non-bioinformaticians.

BACKGROUND: Processing and analyzing whole genome sequencing (WGS) is computationally intense: a single Illumina MiSeq WGS run produces ~ 1 million 250-base-pair reads for each of 24 samples. This poses significant obstacles for smaller laboratories, or laboratories not affiliated with larger projects, which may not have dedicated bioinformatics staff or computing power to effectively use genomic data to protect public health. Building on the success of the cloud-based Galaxy bioinformatics platform ( http://galaxyproject.org ), already known for its user-friendliness and powerful WGS analytical tools, the Center for Food Safety and Applied Nutrition (CFSAN) at the U.S. Food and Drug Administration (FDA) created a customized 'instance' of the Galaxy environment, called GalaxyTrakr ( https://www.galaxytrakr.org ), for use by laboratory scientists performing food-safety regulatory research. The goal was to enable laboratories outside of the FDA internal network to (1) perform quality assessments of sequence data, (2) identify links between clinical isolates and positive food/environmental samples, including those at the National Center for Biotechnology Information sequence read archive ( https://www.ncbi.nlm.nih.gov/sra/ ), and (3) explore new methodologies such as metagenomics. GalaxyTrakr hosts a variety of free and adaptable tools and provides the data storage and computing power to run the tools. These tools support coordinated analytic methods and consistent interpretation of results across laboratories. Users can create and share tools for their specific needs and use sequence data generated locally and elsewhere. RESULTS: In its first full year (2018), GalaxyTrakr processed over 85,000 jobs and went from 25 to 250 users, representing 53 different public and state health laboratories, academic institutions, international health laboratories, and federal organizations. By mid-2020, it has grown to 600 registered users and processed over 450,000 analytical jobs. To illustrate how laboratories are making use of this resource, we describe how six institutions use GalaxyTrakr to quickly analyze and review their data. Instructions for participating in GalaxyTrakr are provided. CONCLUSIONS: GalaxyTrakr advances food safety by providing reliable and harmonized WGS analyses for public health laboratories and promoting collaboration across laboratories with differing resources. Anticipated enhancements to this resource will include workflows for additional foodborne pathogens, viruses, and parasites, as well as new tools and services.

Enhancing the one health initiative by using whole genome sequencing to monitor antimicrobial resistance of animal pathogens: Vet-LIRN collaborative project with veterinary diagnostic laboratories in United States and Canada.

BACKGROUND: Antimicrobial resistance (AMR) of bacterial pathogens is an emerging public health threat. This threat extends to pets as it also compromises our ability to treat their infections. Surveillance programs in the United States have traditionally focused on collecting data from food animals, foods, and people. The Veterinary Laboratory Investigation and Response Network (Vet-LIRN), a national network of 45 veterinary diagnostic laboratories, tested the antimicrobial susceptibility of clinically relevant bacterial isolates from animals, with companion animal species represented for the first time in a monitoring program. During 2017, we systematically collected and tested 1968 isolates. To identify genetic determinants associated with AMR and the potential genetic relatedness of animal and human strains, whole genome sequencing (WGS) was performed on 192 isolates: 69 Salmonella enterica (all animal sources), 63 Escherichia coli (dogs), and 60 Staphylococcus pseudintermedius (dogs). RESULTS: We found that most Salmonella isolates (46/69, 67%) had no known resistance genes. Several isolates from both food and companion animals, however, showed genetic relatedness to isolates from humans. For pathogenic E. coli, no resistance genes were identified in 60% (38/63) of the isolates. Diverse resistance patterns were observed, and one of the isolates had predicted resistance to fluoroquinolones and cephalosporins, important antibiotics in human and veterinary medicine. For S. pseudintermedius, we observed a bimodal distribution of resistance genes, with some isolates having a diverse array of resistance mechanisms, including the mecA gene (19/60, 32%). CONCLUSION: The findings from this study highlight the critical importance of veterinary diagnostic laboratory data as part of any national antimicrobial resistance surveillance program. The finding of some highly resistant bacteria from companion animals, and the observation of isolates related to those isolated from humans demonstrates the public health significance of incorporating companion animal data into surveillance systems. Vet-LIRN will continue to build the infrastructure to collect the data necessary to perform surveillance of resistant bacteria as part of fulfilling its mission to advance human and animal health. A One Health approach to AMR surveillance programs is crucial and must include data from humans, animals, and environmental sources to be effective.

Multidrug-Resistant Campylobacter jejuni Outbreak Linked to Puppy Exposure - United States, 2016-2018.

Campylobacter causes an estimated 1.3 million diarrheal illnesses in the United States annually (1). In August 2017, the Florida Department of Health notified CDC of six Campylobacter jejuni infections linked to company A, a national pet store chain based in Ohio. CDC examined whole-genome sequencing (WGS) data and identified six isolates from company A puppies in Florida that were highly related to an isolate from a company A customer in Ohio. This information prompted a multistate investigation by local and state health and agriculture departments and CDC to identify the outbreak source and prevent additional illness. Health officials from six states visited pet stores to collect puppy fecal samples, antibiotic records, and traceback information. Nationally, 118 persons, including 29 pet store employees, in 18 states were identified with illness onset during January 5, 2016-February 4, 2018. In total, six pet store companies were linked to the outbreak. Outbreak isolates were resistant by antibiotic susceptibility testing to all antibiotics commonly used to treat Campylobacter infections, including macrolides and quinolones. Store record reviews revealed that among 149 investigated puppies, 142 (95%) received one or more courses of antibiotics, raising concern that antibiotic use might have led to development of resistance. Public health authorities issued infection prevention recommendations to affected pet stores and recommendations for testing puppies to veterinarians. This outbreak demonstrates that puppies can be a source of multidrug-resistant Campylobacter infections in humans, warranting a closer look at antimicrobial use in the commercial dog industry.

New variants of porcine epidemic diarrhea virus with large deletions in the spike protein, identified in the United States, 2016-2017.

Four types of porcine epidemic diarrhea virus (PEDV) variants with a large deletion in the spike protein were detected, together with the original US PEDV, from pig fecal and oral fluid samples collected during 2016-2017 in the US. Two of the variants are similar to those identified in Japan: one contains a 194-aa deletion, the same as PEDV variant TTR-2/JPN/2014, while the other contains a 204-aa deletion, the same as PEDV variant JKa-292/CS1de204. Two new S1 NTD-del PEDV variants were found: one contains a 201-aa deletion located at residues 30-230 and the other contains a 202-aa deletion located at residues 24-225 of the S protein. This is the first report on coinfection of S1 NTD-del PEDV variants and the original US PEDV strain in US pigs, indicating that PEDV continues to evolve in pigs and might be responsible for disease pattern changes.

Monitoring emerging pathogens using negative nucleic acid test results from endemic pathogens in pig populations: Application to porcine enteric coronaviruses.

This study evaluated the use of endemic enteric coronaviruses polymerase chain reaction (PCR)-negative testing results as an alternative approach to detect the emergence of animal health threats with similar clinical diseases presentation. This retrospective study, conducted in the United States, used PCR-negative testing results from porcine samples tested at six veterinary diagnostic laboratories. As a proof of concept, the database was first searched for transmissible gastroenteritis virus (TGEV) negative submissions between January 1st, 2010, through April 29th, 2013, when the first porcine epidemic diarrhea virus (PEDV) case was diagnosed. Secondly, TGEV- and PEDV-negative submissions were used to detect the porcine delta coronavirus (PDCoV) emergence in 2014. Lastly, encountered best detection algorithms were implemented to prospectively monitor the 2023 enteric coronavirus-negative submissions. Time series (weekly TGEV-negative counts) and Seasonal Autoregressive-Integrated Moving-Average (SARIMA) were used to control for outliers, trends, and seasonality. The SARIMA's fitted and residuals were then subjected to anomaly detection algorithms (EARS, EWMA, CUSUM, Farrington) to identify alarms, defined as weeks of higher TGEV-negativity than what was predicted by models preceding the PEDV emergence. The best-performing detection algorithms had the lowest false alarms (number of alarms detected during the baseline) and highest time to detect (number of weeks between the first alarm and PEDV emergence). The best-performing detection algorithms were CUSUM, EWMA, and Farrington flexible using SARIMA fitted values, having a lower false alarm rate and identified alarms 4 to 17 weeks before PEDV and PDCoV emergences. No alarms were identified in the 2023 enteric negative testing results. The negative-based monitoring system functioned in the case of PEDV propagating epidemic and in the presence of a concurrent propagating epidemic with the PDCoV emergence. It demonstrated its applicability as an additional tool for diagnostic data monitoring of emergent pathogens having similar clinical disease as the monitored endemic pathogens.

Genomic Study on Blood Culture Isolates From Patients With Staphylococcus Infection-associated Glomerulonephritis.

Introduction: Staphylococcus infection-associated glomerulonephritis (SAGN), is an autoimmune sequela of infection affecting a subset of infected patients without specific predictive factors, frequently presenting with acute nephritic syndrome and propensity for chronic kidney disease. We performed a comparative genotypic and phenotypic analysis of S. aureus isolates from patients that did and those that did not develop SAGN. Methods: We had 22 culture-proven cases of SAGN from Ohio State University Wexner Medical Center (OSUWMC) from 2004 to 2016, 9 of 22 being blood cultures, with archived isolates. These, along with blood culture isolates from 12 patients with no clinical evidence of SAGN (between ages 40 to 80 years) over the same period were used for genotyping. For host demographic comparison, we used all available SAGN cases (n = 85, including those with positive cultures other than blood; and patients with kidney biopsies received from referring hospitals) and all OSUWMC patients with positive Staphylococcus cultures without glomerulonephritis (GN) (n = 23,496). Results: Multiple sequence types (STs) suggesting strain diversity was seen in the GN isolates with mainly clonal complexes (CC) 5 and 59. Mutations in the agr operon were identified in significantly higher number of the GN isolates (83%) than non-GN isolates (16%). Significant differences in ß-hemolysis and biofilm formation was also observed between the groups. Conclusion: The functionality of these agr mutants remains to be seen, but the presently known effects of reduced agr function, namely increased surface adhesins, biofilm formation, and persistent bacteremia could be important microbial factors predisposing to SAGN and testing for them early during infection could help to predict its development.

Multi-laboratory evaluation of the Illumina iSeq platform for whole genome sequencing of Salmonella, Escherichia coli and Listeria.

There is a growing need for public health and veterinary laboratories to perform whole genome sequencing (WGS) for monitoring antimicrobial resistance (AMR) and protecting the safety of people and animals. With the availability of smaller and more affordable sequencing platforms coupled with well-defined bioinformatic protocols, the technological capability to incorporate this technique for real-time surveillance and genomic epidemiology has greatly expanded. There is a need, however, to ensure that data are of high quality. The goal of this study was to assess the utility of a small benchtop sequencing platform using a multi-laboratory verification approach. Thirteen laboratories were provided the same equipment, reagents, protocols and bacterial reference strains. The Illumina DNA Prep and Nextera XT library preparation kits were compared, and 2×150 bp iSeq i100 chemistry was used for sequencing. Analyses comparing the sequences produced from this study with closed genomes from the provided strains were performed using open-source programs. A detailed, step-by-step protocol is publicly available via protocols.io (https://www.protocols.io/view/iseq-bacterial-wgs-protocol-bij8kcrw). The throughput for this method is approximately 4-6 bacterial isolates per sequencing run (20-26 Mb total load). The Illumina DNA Prep library preparation kit produced high-quality assemblies and nearly complete AMR gene annotations. The Prep method produced more consistent coverage compared to XT, and when coverage benchmarks were met, nearly all AMR, virulence and subtyping gene targets were correctly identified. Because it reduces the technical and financial barriers to generating WGS data, the iSeq platform is a viable option for small laboratories interested in genomic surveillance of microbial pathogens.

Next-generation sequencing capacity and capabilities within the National Animal Health Laboratory Network.

With the cost of next-generation sequencing (NGS) decreasing, this technology is rapidly being integrated into the workflows of veterinary clinical and diagnostic laboratories nationwide. The mission of the U.S. Department of Agriculture-National Animal Health Laboratory Network (NAHLN) is in part to evaluate new technologies and develop standardized processes for deploying these technologies to network laboratories for improving detection and response to emerging and foreign animal diseases. Thus, in 2018, the NAHLN identified the integration of NGS into the network as a top priority. In order to assess the current state of preparedness across NAHLN laboratories and to identify which have the capability for performing NGS, a questionnaire was developed by the NAHLN Methods Technical Working Group and submitted to all NAHLN laboratories in December 2018. Thirty of 59 laboratories completed the questionnaire, of which 18 (60%) reported having some sequencing capability. Multiple sequencing platforms and reagents were identified, and limited standardized quality control parameters were reported. Our results confirm that NGS capacity is available within the NAHLN, but several gaps remain. Gaps include not having sufficient personnel trained in bioinformatics and data interpretation, lack of standardized methods and equipment, and maintenance of sufficient computing capacity to meet the growing demand for this technology.

Metagenomic Sequencing for Identification of Xylella fastidiosa from Leaf Samples.

Xylella fastidiosa (Xf) is a globally distributed plant-pathogenic bacterium. The primary control strategy for Xf diseases is eradicating infected plants; therefore, timely and accurate detection is necessary to prevent crop losses and further pathogen dispersal. Conventional Xf diagnostics primarily relies on quantitative PCR (qPCR) assays. However, these methods do not consider new or emerging variants due to pathogen genetic recombination and sensitivity limitations. We developed and tested a metagenomics pipeline using in-house short-read sequencing as a complementary approach for affordable, fast, and highly accurate Xf detection. We used metagenomics to identify Xf to the strain level in single- and mixed-infected plant samples at concentrations as low as 1 pg of bacterial DNA per gram of tissue. We also tested naturally infected samples from various plant species originating from Europe and the United States. We identified Xf subspecies in samples previously considered inconclusive with real-time PCR (quantification cycle [Cq], >35). Overall, we showed the versatility of the pipeline by using different plant hosts and DNA extraction methods. Our pipeline provides taxonomic and functional information for Xf diagnostics without extensive knowledge of the disease. This pipeline demonstrates that metagenomics can be used for early detection of Xf and incorporated as a tool to inform disease management strategies. IMPORTANCE Destructive Xylella fastidiosa (Xf) outbreaks in Europe highlight this pathogen's capacity to expand its host range and geographical distribution. The current disease diagnostic approaches are limited by a multiple-step process, biases to known sequences, and detection limits. We developed a low-cost, user-friendly metagenomic sequencing tool for Xf detection. In less than 3 days, we were able to identify Xf subspecies and strains in field-collected samples. Overall, our pipeline is a diagnostics tool that could be easily extended to other plant-pathogen interactions and implemented for emerging plant threat surveillance.

Patterns of gene expression in swine macrophages infected with classical swine fever virus detected by microarray.

Infection of domestic swine with highly virulent, classical swine fever virus (CSFV) strain Brescia, causes lethal disease in all infected animals. However, the molecular mechanisms involved in modulating the host cellular processes and evasion of the immune response have not been clearly established. To gain insight into, the early host response to CSFV, we analyzed the pattern of gene expression in infected swine macrophages, using custom designed swine microarrays. Macrophages, the target cell for CSFV infection, were isolated from primary cultures of peripheral blood mononuclear cells, allowing us to utilize identical uninfected macrophages at the same time points as CSFV-infected macrophages, allowing only genes induced by CSFV to be identified. First, microarray probes were optimized by screening 244,000 probes for hybridization with RNA from infected and uninfected macrophages. Probes that hybridized and passed quality control standards were used to design a 44,000 probe microarray for this study. Changes in expression levels of 79 genes (48 up- and 31 down-regulated) during the first 48h post-infection were observed. As expected many of the genes with an altered pattern of expression are involved in the development of an innate immune response. Several of these genes had differential expression in an attenuated strain NS4B.VGIv, suggesting that some of these differences are responsible for virulence. The observed gene expression profile might help to explain the immunological and pathological changes associated with infection of pigs with CSFV Brescia.