A one health approach for monitoring antimicrobial resistance: developing a national freshwater pilot effort.

Antimicrobial resistance (AMR) is a world-wide public health threat that is projected to lead to 10 million annual deaths globally by 2050. The AMR public health issue has led to the development of action plans to combat AMR, including improved antimicrobial stewardship, development of new antimicrobials, and advanced monitoring. The National Antimicrobial Resistance Monitoring System (NARMS) led by the United States (U.S) Food and Drug Administration along with the U.S. Centers for Disease Control and U.S. Department of Agriculture has monitored antimicrobial resistant bacteria in retail meats, humans, and food animals since the mid 1990's. NARMS is currently exploring an integrated One Health monitoring model recognizing that human, animal, plant, and environmental systems are linked to public health. Since 2020, the U.S. Environmental Protection Agency has led an interagency NARMS environmental working group (EWG) to implement a surface water AMR monitoring program (SWAM) at watershed and national scales. The NARMS EWG divided the development of the environmental monitoring effort into five areas: (i) defining objectives and questions, (ii) designing study/sampling design, (iii) selecting AMR indicators, (iv) establishing analytical methods, and (v) developing data management/analytics/metadata plans. For each of these areas, the consensus among the scientific community and literature was reviewed and carefully considered prior to the development of this environmental monitoring program. The data produced from the SWAM effort will help develop robust surface water monitoring programs with the goal of assessing public health risks associated with AMR pathogens in surface water (e.g., recreational water exposures), provide a comprehensive picture of how resistant strains are related spatially and temporally within a watershed, and help assess how anthropogenic drivers and intervention strategies impact the transmission of AMR within human, animal, and environmental systems.

Genomic characterization of a WHO critical priority isolate Enterobacter kobei ST2070 harboring OXA-10, KPC-2, and CTX-M-12 recovered from a water irrigation channel in Ecuador.

The discharge of untreated or partially treated wastewater can have detrimental impacts on the quality of water bodies, posing a significant threat to public health and the environment. In Ecuador, previous research indicates a high prevalence of antimicrobial resistant (AMR) bacteria in surface waters affected by human activities, including irrigation channels. In this study, we analyzed sediment samples collected from an irrigation channel utilized for agricultural purposes in northern Ecuador, using microbiological techniques and whole-genome sequencing (WGS). Our investigation revealed the first documented occurrence of E. kobei in Ecuador and the initial report of environmental E. kobei ST2070. Furthermore, we identified the coexistence of OXA-10-type class D ß-lactamase and KPC-2-type class A ß-lactamase in the E. kobei isolate (UTA41), representing the first report of such a phenomenon in this species. Additionally, we detected various antibiotic resistance genes in the E. kobei UTA41 isolate, including blaCTX-M-12, fosA, aac(6')-lb, sul2, msr(E), and mph(A), as well as virulence genes such as bacterial efflux pump and siderophore biosynthesis genes. We also identified two intact prophage regions (Entero_186 and Klebsi_phiKO2) in the isolate. Our study presents the first evidence of E. kobei isolate containing two carbapenemase-encoding genes in environmental samples from Latin America. This finding indicates the potential spread of critical-priority bacteria in water samples originating from anthropogenic sources, such as urban wastewater discharges and livestock facilities.

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.

Rumen Microbial Predictors for Short-Chain Fatty Acid Levels and the Grass-Fed Regimen in Angus Cattle.

The health benefits of grass-fed beef are well documented. However, the rumen microbiome features in beef steers raised in a grass-fed regimen have yet to be identified. This study examined the rumen microbiome profile in the feeding regimes. Our findings show that the rumen microbiome of the grass-fed cattle demonstrated greater species diversity and harbored significantly higher microbial alpha diversity, including multiple species richness and evenness indices, than the grain-fed cattle. Global network analysis unveiled that grass-fed cattle's rumen microbial interaction networks had higher modularity, suggesting a more resilient and stable microbial community under this feeding regimen. Using the analysis of compositions of microbiomes with a bias correction (ANCOM-BC) algorithm, the abundance of multiple unclassified genera, such as those belonging to Planctomycetes, LD1-PB3, SR1, Lachnospira, and Sutterella, were significantly enriched in the rumen of grass-fed steers. Sutterella was also the critical genus able to distinguish the two feeding regimens by Random Forest. A rumen microbial predictor consisting of an unclassified genus in the candidate division SR1 (numerator) and an unclassified genus in the order Bacteroidales (denominator) accurately distinguished the two feeding schemes. Multiple microbial signatures or balances strongly correlated with various levels of SCFA in the rumen. For example, a balance represented by the log abundance ratio of Sutterella to Desulfovibrio was strongly associated with acetate-to-propionate proportions in the rumen (R2 = 0.87), which could be developed as a valuable biomarker for optimizing milk fat yield and cattle growth. Therefore, our findings provided novel insights into microbial interactions in the rumen under different feed schemes and their ecophysiological implications. These findings will help to develop rumen manipulation strategies to improve feed conversion ratios and average daily weight gains for grass- or pasture-fed cattle production.

Author Correction: Template plasmid integration in germline genome-edited cattle.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

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.

Whole-Genome Sequence Analysis of CTX-M Containing Escherichia coli Isolates from Retail Meats and Cattle in the United States.

In recent years, there have been increased reports on the detection of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli and Salmonella strains from food-producing animals and animal products in the United States. We characterized 18 ESBL E. coli isolates from cattle (n = 5), chicken breast (n = 5), ground turkey (n = 6), ground beef (n = 1), and pork chops (n = 1) that were collected by the National Antimicrobial Resistance Monitoring System (NARMS) between 2011 and 2015. In vitro antimicrobial susceptibility testing was done against a panel of 14 antimicrobials followed by a secondary panel of 9 ß-lactam agents. Whole-genome sequencing was used to characterize the resistome, plasmids, and the genetic structures of the ESBL genes. All ESBL-producing E. coli isolates were resistant to at least three antimicrobial classes and carried various blaCTX-M genes. Most of the cattle and ground turkey isolates carried blaCTX-M-27. In chicken breast isolates, blaCTX-M-1 was present as part of an ISEcp1 transposition unit carried on a plasmid that shares sequence similarity with the backbone structure of the IncI plasmid. Isolates carrying the blaCTX-M-14 and blaCTX-M-15 genes, widely distributed in human clinical isolates, were also isolated. To our knowledge, this is the first report of the widely distributed blaCTX-M-14 and blaCTX-M-15 in E. coli isolates from retail meat samples in the United States. Different insertional sequences were identified upstream of these blaCTX-Ms, including ISEcp1, IS26, and IS903-D. CTX-M in E. coli from food animals and retail chicken breast were often present on plasmids with other resistance genes. Other resistance genes identified included aadA, strA, strB, aac(3)-IId, aac(3)-VIa, aph(3')-Ic, blaTEM, blaHERA-3, floR, sul1, sul2, catA1, tetA, tetB, dfrA, and qacE. These data describe the emergence of CTX-M-carrying E. coli isolates in food animals and animal products monitored by NARMS program.

Draft Whole-Genome Sequences of Chryseobacterium piscicola and Chryseobacterium shigense.

We report the draft whole-genome sequences for Chryseobacterium piscicola and Chryseobacterium shigense type strains, bacteria that have been associated with fish gill disease.

Complete Genome Sequences of 14 Salmonella enterica Serovar Enteritidis Strains Recovered from Human Clinical Cases between 1949 and 1995 in the United States.

Salmonella enterica serovar Enteritidis is one of the most commonly isolated foodborne pathogens and is transmitted primarily to humans through consumption of contaminated poultry and poultry products. We are reporting completely closed genome and plasmid sequences of historical S Enteritidis isolates recovered from humans between 1949 and 1995 in the United States.

Draft Whole-Genome Sequences of 18 Flavobacterium spp.

We report here the draft whole-genome sequences for 18 Flavobacterium species type strains that have historically been associated with fish gill disease.

Antimicrobial Resistance in Salmonella in the United States from 1948 to 1995.

We conducted a retrospective study of 2,149 clinicalSalmonellastrains to help document the historical emergence of antimicrobial resistance. There were significant increases in resistance to older drugs, including ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline, which were most common inSalmonella entericaserotype Typhimurium. An increase in multidrug resistance was observed for each decade since the 1950s. These data help show howSalmonellaevolved over the past 6 decades, after the introduction of new antimicrobial agents.

WGS accurately predicts antimicrobial resistance in Escherichia coli.

OBJECTIVES: The objective of this study was to determine the effectiveness of WGS in identifying resistance genotypes of MDR Escherichia coli and whether these correlate with observed phenotypes. METHODS: Seventy-six E. coli strains were isolated from farm cattle and measured for phenotypic resistance to 15 antimicrobials with the Sensititre(®) system. Isolates with resistance to at least four antimicrobials in three classes were selected for WGS using an Illumina MiSeq. Genotypic analysis was conducted with in-house Perl scripts using BLAST analysis to identify known genes and mutations associated with clinical resistance. RESULTS: Over 30 resistance genes and a number of resistance mutations were identified among the E. coli isolates. Resistance genotypes correlated with 97.8% specificity and 99.6% sensitivity to the identified phenotypes. The majority of discordant results were attributable to the aminoglycoside streptomycin, whereas there was a perfect genotype-phenotype correlation for most antibiotic classes such as tetracyclines, quinolones and phenicols. WGS also revealed information about rare resistance mechanisms, such as structural mutations in chromosomal copies of ampC conferring third-generation cephalosporin resistance. CONCLUSIONS: WGS can provide comprehensive resistance genotypes and is capable of accurately predicting resistance phenotypes, making it a valuable tool for surveillance. Moreover, the data presented here showing the ability to accurately predict resistance suggest that WGS may be used as a screening tool in selecting anti-infective therapy, especially as costs drop and methods improve.

Prevalence of Yersinia enterocolitica in antimicrobial-free and conventional antimicrobial use swine production.

Swine are the primary reservoir for foodborne illness associated with Yersinia enterocolitica. The use of antimicrobials in animal agriculture has been hypothesized as having a potential role in the increase in prevalence of zoonotic pathogens. The objective of this study was to compare the frequency of Y. enterocolitica fecal shedding in swine reared on farms with conventional antimicrobial use policies to farms that were antimicrobial free (ABF). Swine farms were selected from three regions in the United States. In each region, farms were categorized based on antimicrobial use policy. Fecal samples were collected from pigs on-farm within 48 h of harvest. The overall proportion of Y. enterocolitica and ail-harboring Y. enterocolitica-positive pigs was 10.9% and 4.0%, respectively. There were increased odds (odds ratio [OR] 6.8, 95% confidence interval [CI] 3.46-13.28) for a pig to be Y. enterocolitica positive if it was reared on an ABF farm as compared to a conventional farm. There was no significant association between farm antimicrobial use policy and isolation of an ail-harboring Y. enterocolitica from an individual pig (OR 1.8, 95% CI 0.90-3.61). The association of antimicrobial use policy with Y. enterocolitica shedding in feces should be interpreted cautiously, as antimicrobial use cannot be separated from other management factors (e.g., confinement or outdoor housing), which may be associated with risk of Y. enterocolitica in swine.

Yersinia enterocolitica of porcine origin: carriage of virulence genes and genotypic diversity.

Yersinia enterocolitica is an important foodborne pathogen, and pigs are recognized as a major reservoir and potential source of pathogenic strains to humans. A total of 172 Y. enterocolitica recovered from conventional and antimicrobial-free pig production systems from different geographic regions (North Carolina, Ohio, Michigan, Wisconsin, and Iowa) were investigated to determine their pathogenic significance to humans. Phenotypic and genotypic diversity of the isolates was assessed using antibiogram, serogrouping, and amplified fragment length polymorphism (AFLP). Carriage of chromosomal and plasmid-borne virulence genes were investigated using polymerase chain reaction. A total of 12 antimicrobial resistance patterns were identified. More than two-thirds (67.4%) of Y. enterocolitica were pan-susceptible, and 27.9% were resistant against ß-lactams. The most predominant serogroup was O:3 (43%), followed by O:5 (25.6%) and O:9 (4.1%). Twenty-two of 172 (12.8%) isolates were found to carry Yersinia adhesion A (yadA), a virulence gene encoded on the Yersinia virulence plasmid. Sixty-nine (40.1%) isolates were found to carry ail gene. The ystA and ystB genes were detected in 77% and 26.2% of the strains, respectively. AFLP genotyping of isolates showed wide genotypic diversity and were grouped into nine clades with an overall genotypic similarity of 66.8-99.3%. AFLP analysis revealed that isolates from the same production system showed clonal relatedness, while more than one genotype of Y. enterocolitica circulates within a farm.

Antimicrobial drug resistance in Escherichia coli from humans and food animals, United States, 1950-2002.

We conducted a retrospective study of Escherichia coli isolates recovered from human and food animal samples during 1950-2002 to assess historical changes in antimicrobial drug resistance. A total of 1,729 E. coli isolates (983 from humans, 323 from cattle, 138 from chickens, and 285 from pigs) were tested for susceptibility to 15 antimicrobial drugs. A significant upward trend in resistance was observed for ampicillin (p<0.001), sulfonamide (p<0.001), and tetracycline (p<0.001). Animal strains showed increased resistance to 11/15 antimicrobial agents, including ampicillin (p<0.001), sulfonamide (p<0.01), and gentamicin (p<0.001). Multidrug resistance (≥3 antimicrobial drug classes) in E. coli increased from 7.2% during the 1950s to 63.6% during the 2000s. The most frequent co-resistant phenotype observed was to tetracycline and streptomycin (29.7%), followed by tetracycline and sulfonamide (29.0%). These data describe the evolution of resistance after introduction of new antimicrobial agents into clinical medicine and help explain the range of resistance in modern E. coli isolates.

Prevalence and antimicrobial resistance profile of Campylobacter spp. isolated from conventional and antimicrobial-free swine production systems from different U.S. regions.

We conducted a study to compare the prevalence and antimicrobial resistance profile of Campylobacter isolated from 34 farm-slaughter pair cohorts of pigs raised in conventional and antimicrobial-free (ABF) production systems. Isolates originated from four different states of two geographic regions (region 1--Ohio and Michigan; region 2--Wisconsin and Iowa). A total of 838 fecal and 1173 carcass samples were examined. Campylobacter isolates were speciated using multiplex polymerase chain reaction targeting ceuE and hipO genes. The minimum inhibitory concentration was determined using agar dilution to a panel of six antimicrobials: chloramphenicol, erythromycin, gentamicin, ciprofloxacin, nalidixic acid, and tetracycline. Campylobacter spp. was isolated from 472 of 838 pigs (56.3%). Campylobacter prevalence did not vary significantly based on production system (conventional [58.9%] and ABF [53.7%], odds ratio [OR] 1.4, 95% confidence interval [CI] 0.8-2.6, p = 0.24) or geographic region (region 1 [54.1%] and region 2 [58.2%], OR 1.02, 95% CI 0.6-1.9, p = 0.92). At slaughter plant, Campylobacter prevalence varied based on processing stages (19.4% at pre-evisceration, 25.3% at postevisceration, and 3.2% at postchill). Resistance was common to tetracycline (64.5%), erythromycin (47.9%), and nalidixic acid (23.5%). Campylobacter isolates from conventional production systems were more likely to be erythromycin resistant than from ABF (OR 3.2, 95% CI 1.4-7.2, p = 0.01). The proportion of ciprofloxacin-resistant Campylobacter coli isolates were 3.7% and 1.2% from ABF and conventional production systems, respectively. Thirty-seven out of 1257 C. coli (2.9%) were resistant to both erythromycin and ciprofloxacin, drugs of choice for treatment of invasive human campylobacteriosis. The finding of ciprofloxacin resistance, particularly from ABF herds, has significant implications on the potential role of risk factors other than mere antimicrobial use for production purposes.

Environmental Salmonella surveillance in the Ohio State University Veterinary Teaching Hospital.

Sampling was conducted at The Ohio State University's Veterinary Teaching Hospital (OSU-VTH) to evaluate the extent of environmental contamination with Salmonella enterica, at 1-week intervals beginning March 19, 2007, through May 21, 2007. Environmental samples were collected from various surface and floor locations in the equine and food animal areas using sterile moistened gauze sponges. All samples were processed using standard bacteriologic culture to identify the presence of Salmonella spp. Genetic relatedness of isolates was assessed using amplified fragment length polymorphism (AFLP) procedures, and minimum inhibitory concentrations to a panel of antimicrobial drugs were determined using microbroth dilution. A total of 16 Salmonella isolates were recovered from 270 (5.9%) cultured environmental and animal samples, with prevalence ranging from 0% to 32% on individual sampling dates. A total of 9% of the samples from the food animal section and 2.5% of the samples from the equine section tested positive for Salmonella (p = 0.03). The 16 isolates represented seven different clonal strains and four different serotypes (Paratyphi B var. L-tartate n = 3, Kentucky n = 5, Cerro n = 7, Montevideo n = 1), most of which were pansusceptible to a panel of antimicrobial drugs. Our results indicate that animal treatment areas with a high population of animals or cases within the veterinary teaching hospital can become contaminated with Salmonella, especially in high traffic areas that may facilitate horizontal dissemination. The most common occurrence appears to be infected agricultural animals that contaminate the hospital environment, but normal cleaning and disinfection appears to effectively prevent long-term contamination.

Occurrence of spvA virulence gene and clinical significance for multidrug-resistant Salmonella strains.

Nontyphoidal Salmonella strains are important reservoirs of antimicrobial resistance. An important issue that has not been investigated is whether the multiresistant Salmonella strains are more virulent than their susceptible counterparts. Salmonella isolates collected from clinical human (n=888) and porcine (n=2,120) cases at the same time period and geographic location were investigated. Antimicrobial susceptibility, PCR analysis for the spvA virulence gene, and pulsed-field gel electrophoresis (PFGE) genotyping were done. Carriage of spvA was associated with multidrug-resistant (MDR) type ACSSuT strains (odds ratio, 7.1; P<0.05), a type often implicated in bacteremic human cases. PFGE revealed that clinical isolates from pigs were more clonally related to those of human origin than the nonclinical porcine isolates. The findings suggest that MDR strains that also carry specific virulence factors are more likely to be of clinical significance.