One Health Threat of Treated Wastewater Discharge in Urban Ohio Rivers: Implications for Surface Water and Fish Gut Microbiome and Resistome.

Wastewater treatment plants (WWTPs) are thought to be a major disseminating source of antibiotic resistance (AR) to the environment, establishing a crucial connection between human and environmental resistome. The objectives of this study were to determine how wastewater effluents impact microbiome and resistome of freshwater and fish, and identify potential AR-carrying clinically relevant pathogens in these matrices. We analyzed wastewater influent and effluent from four WWTPs in three metropolitan areas of Ohio, USA via shotgun metagenomic sequencing. We also sequenced river water and fish guts from three reaches (upstream, at the WWTP outfall, and downstream). Notably, we observed a decline in microbiome diversity and AR gene abundance from wastewater to the receiving river. We also found significant differences by reach and trophic level (diet) in beta-diversity of the fish gut microbiomes. SourceTracker revealed that 0.443 and 0.248 more of the of the fish gut microbiome was sourced from wastewater effluent in fish from the outfall and downstream locations, respectively, compared to upstream fish. Additionally, AR bacteria of public health concern were annotated in effluent and river water samples, indicating potential concern for human exposure. In summary, our findings show the continued role of wastewater as a significant AR reservoir and underscores the considerable impact of wastewater discharge on aquatic wildlife, which highlights the One Health nature of this issue.

Transcontinental Dissemination of Enterobacterales Harboring blaNDM-1 in Retail Frozen Shrimp.

The global food trade provides a means of disseminating antimicrobial resistant (AMR) bacteria and genes. Using selective media, carbapenem-resistant species of Enterobacterales (Providencia sp. and Citrobacter sp.), were detected in a single package of imported frozen shrimp purchased from a grocery store in Ohio, USA. Polymerase chain reaction confirmed that both isolates harbored blaNDM-1 genes. Following PacBio long read sequencing, the sequences were annotated using the NCBI Prokaryotic Genome Annotation Pipeline. The blaNDM-1 genes were found in IncC plasmids, each with different antimicrobial resistance island configuration. We found that the blaNDM-1 AMR islands had close relationships with previously reported environmental, food, and clinical isolates detected in Asia and the United States, highlighting the importance of the food chain in the global dissemination of antimicrobial resistance.

A complex cyclical One Health pathway drives the emergence and dissemination of antimicrobial resistance.

Since their commercialization, scientists have known that antimicrobial use kills or inhibits susceptible bacteria while allowing resistant bacteria to survive and expand. Today there is widespread antimicrobial resistance (AMR), even to antimicrobials of last resort such as the carbapenems, which are reserved for use in life-threatening infections. It is often convenient to assign responsibility for this global health crisis to the users and prescribers of antimicrobials. However, we know that animals never treated with antimicrobials carry clinically relevant AMR bacteria and genes. The causal pathway from bacterial susceptibility to resistance is not simple, and dissemination is cyclical rather than linear. Amplification of AMR occurs in healthcare environments and on farms where frequent exposure to antimicrobials selects for resistant bacterial populations. The recipients of antimicrobial therapy release antimicrobial residues, resistant bacteria, and resistance genes in waste products. These are reduced but not removed during wastewater and manure treatment and enter surface waters, soils, recreational parks, wildlife, and fields where animals graze and crops are grown for human and animal consumption. The cycle is complete when a patient carrying AMR bacteria is treated with antimicrobials that amplify the resistant bacterial populations. Reducing the development and spread of AMR requires a One Health approach with the combined commitment of governments, medical and veterinary professionals, agricultural industries, food and feed processors, and environmental scientists. In this review and in the companion Currents in One Health by Ballash et al, JAVMA, April 2024, we highlight just a few of the steps of the complex cyclical causal pathway that leads to the amplification, dissemination, and maintenance of AMR.

The One Health dissemination of antimicrobial resistance occurs in both natural and clinical environments.

Once considered to be a simple cause-and-effect relationship with localized impact, the concept of how antimicrobial use drives antimicrobial resistance is now recognized as a complex, transdisciplinary problem on a global scale. While the issue of antimicrobial resistance is often studied and addressed at the antimicrobial-human or antimicrobial-animal treatment interface, the role of the environment in the One Health dynamics of antimicrobial resistance is not as well understood. Antimicrobial-resistant bacteria, including those resistant to carbapenem drugs, are emerging in veterinary clinical environments, on farms, and in natural habitats. These multidrug-resistant bacteria can colonize our livestock and companion animals and are later disseminated into the environment, where they contaminate surface waters and colonize wildlife. From here, the One Health transmission cycle of antimicrobial-resistant bacteria is completed as environmental reservoirs can serve as sources of antimicrobial resistance transmission into human or animal healthcare settings. In this review, we utilize a One Health perspective to evaluate how environments become contaminated and, in turn, become reservoirs that can colonize and infect our veterinary species, and how the veterinary field is combating environmental contamination with antimicrobial stewardship regulations and program implementation. The companion Currents in One Health by Parker et al, AJVR, April 2024, addresses the intensive research that justifies this One Health cycle of antimicrobial resistance transmission and emerging techniques that are dissecting the complex interactions at the One Health interface.

Longitudinal examination of urine pH, specific gravity, protein, culture, and antimicrobial resistance profiles in healthy dogs.

BACKGROUND: Urine is routinely evaluated in dogs to assess health. Reference ranges for many urine properties are well established, but the scope of variation in these properties over time within healthy dogs is not well characterized. OBJECTIVES: Longitudinally characterize urine properties in healthy dogs over 3 months. ANIMALS: Fourteen healthy client-owned dogs. METHODS: In this prospective study, dogs were evaluated for health; then, mid-stream free-catch urine was collected from each dog at 12 timepoints over 3 months. Urine pH, urine specific gravity (USG), protein, cultures, and antimicrobial resistance profiles were assessed at each timepoint. RESULTS: Urine pH varied within and between dogs over time (Friedman's test: within P = .03; between P < .005). However, USG, protein, and bacterial diversity of urine were consistent within dogs over time, and only varied between dogs (Kruskal-Wallis: between all P < .005). Antimicrobial resistant isolates were identified in 12 out of 14 dogs with 34 of 48 of the isolates demonstrating resistance to amoxicillin. CONCLUSIONS AND CLINICAL IMPORTANCE: Urine pH should be assessed at multiple timepoints via pH meter before making clinical decisions. Mid-stream free-catch urine with high concentrations of bacteria (>105 CFU/mL) should not be considered the only indicator of urinary tract infection. Bacterial isolates from dogs in this study had widespread resistance to amoxicillin/oxacillin underscoring the need for antimicrobial stewardship.

Previous Antibiotic Exposure Reshapes the Population Structure of Infecting Uropathogenic Escherichia coli Strains by Selecting for Antibiotic Resistance over Urovirulence.

Antibiotic therapy is the standard of care for urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC). However, previous antibiotic therapy may impart a selective pressure that influences the population structure and pathogenic potential of infecting UPEC strains. Here, we conducted a 3-year study using whole-genome-sequencing analysis and retrospective medical record review to characterize how antibiotic exposure influenced the phenotypic antibiotic resistance, acquired resistome, virulome, and population structure of 88 UTI-causing E. coli strains from dogs. A majority of UTI-associated E. coli strains were from phylogroup B2 and clustered within sequence type 372. Previous antibiotic exposure was associated with a population shift toward UPEC from phylogroups other than the typical urovirulent phylogroup B2. The specific virulence profiles within the accessory virulome that were associated with antibiotic use were elicited by the effect of antibiotics on UPEC phylogenetic structure. Among phylogroup B2, antibiotic exposure increased the quantity of genes within the resistome and the odds of developing reduced susceptibility to at least one antibiotic. Non-B2 UPEC strains harbored a more diverse and greater resistome that conferred reduced susceptibility to multiple antibiotic classes following antibiotic exposure. Collectively, these data suggest that previous antibiotic exposure establishes an environment that provides a selective edge to non-B2 UPEC strains through their diverse and abundant antibiotic resistance genes, despite their lack of urovirulence genes. Our findings highlight the necessity for judicious use of antibiotics as we uncover another mechanism by which antibiotic exposure and resistance can influence the dynamics of bacterial infectious disease. IMPORTANCE Urinary tract infections (UTIs) are one of the most common infections of dogs and humans. While antibiotic therapy is the standard of care for UTIs and other infections, antibiotic exposure may influence the pathogenic profile of subsequent infections. We used whole-genome sequencing and retrospective medical record review to characterize the effect of systemic antibiotic therapy on the resistance, virulence, and population structure of 88 UTI-causing UPEC strains isolated from dogs. Our results indicate that antibiotic exposure alters the population structure of infecting UPEC strains, providing a selective edge for non-B2 phylogroups that harbor diverse and abundant resistance gene catalogues but fewer urovirulence genes. These findings highlight how antibiotic resistance can influence pathogen infection dynamics and have clinical implications for the judicious use of antibiotics for bacterial infections.

Prevalence Estimation, Antimicrobial Susceptibility, and Serotyping of Salmonella enterica Recovered from New World Non-Human Primates (Platyrrhini), Feed, and Environmental Surfaces from Wildlife Centers in Costa Rica.

Concern about zoonoses and wildlife has increased. Few studies described the role of wild mammals and environments in the epidemiology of Salmonella. Antimicrobial resistance is a growing problem associated with Salmonella that threatens global health, food security, the economy, and development in the 21st century. The aim of this study is to estimate the prevalence and identify antibiotic susceptibility profiles and serotypes of non-typhoidal Salmonella enterica recovered from non-human primate feces, feed offered, and surfaces in wildlife centers in Costa Rica. A total of 180 fecal samples, 133 environmental, and 43 feed samples from 10 wildlife centers were evaluated. We recovered Salmonella from 13.9% of feces samples, 11.3% of environmental, and 2.3% of feed samples. Non-susceptibility profiles included six isolates from feces (14.6%): four non-susceptible isolates (9.8%) to ciprofloxacin, one (2.4%) to nitrofurantoin, and one to both ciprofloxacin and nitrofurantoin (2.4%). Regarding the environmental samples, one profile was non-susceptible to ciprofloxacin (2.4%) and two to nitrofurantoin (4.8%). The serotypes identified included Typhimurium/I4,[5],12:i:-, S. Braenderup/Ohio, S. Newport, S. Anatum/Saintpaul, and S. Westhampton. The epidemiological surveillance of Salmonella and antimicrobial resistance can serve in the creation of strategies for the prevention of the disease and its dissemination throughout the One Health approach.

Antimicrobial-Resistant Enterobacterales Recovered from the Environment of Two Zoological Institutions Include Enterobacter cloacae Complex ST171 Producing KPC-4 Carbapenemase.

Environmental surfaces can serve as reservoirs for pathogens and antimicrobial-resistant (AMR) bacteria in healthcare settings. Although active surveillance programs are used in veterinary and human healthcare, unconventional settings like zoological facilities are often overlooked, even though antimicrobials are used to maintain the health of their animal collections. Here, we used electrostatic cloths to conduct active environmental surveillance over a 2-year period at two zoological institutions to determine contamination prevalence of human-only and mixed animal-human touch environments with AMR bacteria. We recovered Enterobacterales isolates that expressed quinolone resistance, an AmpC-like phenotype, and an extended-spectrum ß-lactamase phenotype from 144 (39%), 141 (38.2%), and 72 (19.5%) of the environmental samples, respectively. The zoological institutions, areas and exhibits within the zoological facility, and sampling surface type affected the odds of recovering AMR bacteria from the environment. Three carbapenemase-producing Enterobacter cloacae complex ST171 isolates recovered from one zoological facility harbored an IncH12 plasmid with a Tn4401b-KPC-4 transposon conferring multidrug resistance. One isolate maintained three tandem repeats of a Tn4401b-KPC-4 element on an IncHI2 plasmid, although this isolate was susceptible to the four carbapenem drugs tested. These three isolates and their IncH12 plasmids shared significant genomic similarity with two E. cloacae complex isolates recovered from canine patients at a regional veterinary hospital during year 2 of this study. Our results indicated that surface environments at zoological institutions can serve as reservoirs for AMR bacteria and their genes and have implications for animal and public health. IMPORTANCE Environmental surfaces can be a source of antimicrobial-resistant (AMR) bacteria that pose a risk to human and animal health. Zoological institutions are unique environments where exotic animals, staff, and visitors intermingle and antimicrobials are used to maintain animal health. However, zoological environments are often overlooked as reservoirs of AMR bacteria. Here, we show that zoological environments can serve as reservoirs of fluoroquinolone-resistant and extended-spectrum cephalosporin-resistant bacteria. In addition, we isolated three carbapenemase-producing Enterobacter cloacae complex strains carrying blaKPC-4, including one with a unique, tandem triplicate of the Tn4401b-KPC-4 element. Comparative whole genomics of these isolates with two E. cloacae complex isolates from patients at a regional veterinary hospital highlighted the possibility of local KPC-4 spread between animal environments. Our results suggest that environments at zoological institutions serve as reservoirs for AMR bacteria and pose a hypothetical One Health risk to the public, staff, and the wild animal populations in captivity.

Frequency and diversity of carbapenemase-producing Enterobacterales recovered from untreated wastewater impacted by selective media containing cefotaxime and meropenem in Ohio, USA.

As safe agents of last resort, carbapenems are reserved for the treatment of infections caused by multidrug-resistant organisms. The impact of ß-lactam antibiotics, cefotaxime, and meropenem on the frequency and diversity of carbapenemase-producing organisms recovered from environmental samples has not been fully established. Therefore, this methodological study aimed at determining ß-lactam drugs used in selective enrichment and their impact on the recovery of carbapenemase-producing Enterobacterales (CPE) from untreated wastewater. We used a longitudinal study design where 1L wastewater samples were collected weekly from wastewater treatment plant (WWTP) influent and quarterly from contributing sanitary sewers in Columbus, Ohio USA with 52 total samples collected. Aliquots of 500 mL were passed through membrane filters of decreasing pore sizes to enable all the water to pass through and capture bacteria. For each sample, the resulting filters were placed into two modified MacConkey (MAC) broths, one supplemented with 0.5 µg/mL of meropenem and 70 µg/mL of ZnSO4 and the other supplemented with 2 µg/mL cefotaxime. The inoculated broth was then incubated at 37° C overnight, after which they were streaked onto two types of correspondingly-modified MAC agar plates supplemented with 0.5 µg/mL and 1.0 µg/mL of meropenem and 70 µg/mL of ZnSO4 and incubated at 37°C overnight. The isolates were identified based on morphological and biochemical characteristics. Then, up to four distinct colonies of each isolate's pure culture per sample were tested for carbapenemase production using the Carba-NP test. Matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS) MALDI-TOF MS was used to identify carbapenemase-producing organisms. In total 391 Carba-NP positive isolates were recovered from the 52 wastewater samples: 305 (78%) isolates had blaKPC, 73 (19%) carried blaNDM, and 14 (4%) harbored both blaKPC and blaNDM resistance genes. CPE genes of both blaKPC and blaNDM were recovered in both types of modified MAC broths, with 84 (21%) having a blaKPC gene, 22 (6%) carrying blaNDM and 9 (2%) harbored both a blaKPC and blaNDM of isolates recovered from MAC medium incorporated with 0.5ug/mL meropenem and 70ug/mL ZnSO4. The most prevalent isolates were Klebsiella pneumoniae, Escherichia coli, and Citrobacter spp.

Risk of anesthesia-related complications in draft horses: a retrospective, single-center analysis.

OBJECTIVE: To report anesthetic-related complications and determine risks associated with anesthesia in draft horses. STUDY DESIGN: Retrospective study. ANIMALS: A total of 401 anesthetic records for draft horse breeds that underwent general anesthesia from January 2010 through December 2020 were reviewed; horses euthanized during general anesthesia were excluded. METHODS: Demographics, perioperative drugs used, procedure type and duration, time to extubation, number of attempts to stand, use of sling in recovery and perioperative morbidity and mortality were investigated. Morbidity and mortality statistical evaluation included univariable logistic regression analysis and ordinal regression analysis. RESULTS: American Society of Anesthesiologists (ASA) status I-II, ASA III-V and total mortality rate for all cases was 0.69% (2/288), 6.19% (7/113) and 2.24% (9/401), respectively, with Belgian horses being overrepresented (6/9). Cardiac arrest occurred in six out of nine horses that died without euthanasia, and five out of six of these horses underwent colic surgery. Factors associated with increased mortality risk included ASA status of III-V, increased body weight, emergency status and horses presenting for colic. Hypotension, hypercarbia and hypoxemia occurred in 56% (224/401), 46% (186/401) and 14% (58/401) of horses, respectively. During recovery from anesthesia, lighter horses and horses undergoing shorter anesthetic procedures were more likely to be successful on the first or second attempt to stand and were less likely to require a sling in recovery. CONCLUSIONS AND CLINICAL RELEVANCE: Draft horses undergoing general anesthesia had a higher mortality rate than previously reported for all types and breeds of horses.

Fish as sentinels of antimicrobial resistant bacteria, epidemic carbapenemase genes, and antibiotics in surface water.

Surface waters, especially those receiving wastewater flows, can disseminate antimicrobial resistant bacteria (ARB), antimicrobial resistance genes (ARG), and antibiotics. In the Scioto River of central Ohio, United States, we evaluated fishes as potential sentinels of ARB and antimicrobial contamination and investigated the influence of antimicrobial exposure on the fish intestinal resistome. Seventy-seven fish were collected from river reaches receiving inputs from two wastewater treatment plants that serve the greater Columbus Metropolitan Area. Fish were screened for the presence of cephalosporin-resistant (CeRO) and carbapenem-resistant (CRO) organisms, epidemic carbapenemase genes, and antibiotic drugs and metabolites using culture methods, droplet digital PCR, and ultra-high performance liquid chromatography tandem mass spectroscopy (UHPLC-MS/MS). Nearly 21% of fish harbored a CeRO in their resistome, with 19.4% exhibiting bacteria expressing an AmpC genotype encoded by blaCMY, and 7.7% with bacteria expressing an extended-spectrum ß-lactamase phenotype encoded by blaCTX-M. blaKPC and blaNDM were present in 87.7% (57/65) and 80.4% (37/46) of the intestinal samples at an average abundance of 104 copies. Three antibiotics-lincomycin (19.5%), azithromycin (31.2%) and sulfamethoxazole (3.9%)-were found in hepatic samples at average concentrations between 25-31 ng/g. Fish harboring blaCTX-M and those exposed to azithromycin were at greater odds of being downstream of a wastewater treatment plant. Fish that bioconcentrated antibiotics in their liver were not at greater odds of harboring CeRO, CRO, or epidemic carbapenemase gene copies in their resistome. Our findings confirm that fishes can be effective bioindicators of surface waters contaminated with ARB, ARG, and antibiotics. Moreover, our findings highlight the varying importance of different mechanisms that facilitate establishment of ARB in aquatic ecosystems.

Pathogenomics and clinical recurrence influence biofilm capacity of Escherichia coli isolated from canine urinary tract infections.

Biofilm formation enhances bacteria's ability to colonize unique niches while protecting themselves from environmental stressors. Escherichia coli that colonize the urinary tract can protect themselves from the harsh bladder environment by forming biofilms. These biofilms promote persistence that can lead to chronic and recurrent urinary tract infections (UTI). While biofilm formation is frequently studied among urinary E. coli, its association with other pathogenic mechanisms and adaptations in certain host populations remains poorly understood. Here we utilized whole genome sequencing and retrospective medical record analysis to investigate associations between the population structure, phenotypic resistance, resistome, virulome, and patient demographic and clinical findings of 104 unique urinary E. coli and their capacity to form biofilms. We show that population structure including multilocus sequence typing and Clermont phylogrouping had no association with biofilm capacity. Among clinical factors, exposure to multiple antibiotics within that past 30 days and a clinical history of recurrent UTIs were positively associated with biofilm formation. In contrast, phenotypic antimicrobial reduced susceptibility and corresponding acquired resistance genes were negatively associated with biofilm formation. While biofilm formation was associated with increased virulence genes within the cumulative virulome, individual virulence genes did not influence biofilm capacity. We identified unique virulotypes among different strata of biofilm formation and associated the presence of the tosA/R-ibeA gene combination with moderate to strong biofilm formation. Our findings suggest that E. coli causing UTI in dogs utilize a heterogenous mixture of virulence genes to reach a biofilm phenotype, some of which may promote robust biofilm capacity. Antimicrobial use may select for two populations, non-biofilm formers that maintain an arsenal of antimicrobial resistance genes to nullify treatment and a second that forms durable biofilms to avoid therapeutic insults.

Comparison of environmental microbiomes in an antibiotic resistance-polluted urban river highlights periphyton and fish gut communities as reservoirs of concern.

Natural waterways near urban areas are heavily impacted by anthropogenic activities, including their microbial communities. A contaminant of growing public health concern in rivers is antibiotic resistant genes (ARGs), which can spread between neighboring bacteria and increase the potential for transmission of AR bacteria to animals and humans. To identify the matrices of most concern for AR, we compared ARG burdens and microbial community structures between sample types from the Scioto River Watershed, Ohio, the United States, from 2017 to 2018. Five environmental matrices (water, sediment, periphyton, detritus, and fish gut) were collected from 26 river sites. Due to our focus on clinically relevant ARGs, three carbapenem resistance genes (blaKPC, blaNDM, and blaOXA-48) were quantified via DropletDigital™ PCR. At a subset of nine urbanized sites, we conducted16S rRNA gene sequencing and functional gene predictions. Carbapenem resistance genes were quantified from all matrices, with blaKPC being the most detected (88 % of samples), followed by blaNDM (64 %) and blaOXA-48 (23 %). Fish gut samples showed higher concentrations of blaKPC and blaNDM than any other matrix, indicating potential ARG bioaccumulation, and risk of broader dissemination through aquatic and nearshore food webs. Periphyton had higher concentrations of blaNDM than water, sediment, or detritus. Microbial community analysis identified differences by sample type in community diversity and structure. Sediment samples had the most diverse microbial communities, and detritus, the least. Spearman correlations did not reveal significant relationships between the concentrations of the monitored ARGs and microbial community diversity. However, several differentially abundant taxa and microbial functions were identified by sample type that is definitive of these matrices' roles in the river ecosystem and habitat type. In summary, the fish gut and periphyton are a concern as AR reservoirs due to their relatively high concentration of carbapenem resistance genes, diverse microbial communities, and natural functions that promote AR.

Colonization of White-Tailed Deer (Odocoileus virginianus) from Urban and Suburban Environments with Cephalosporinase- and Carbapenemase-Producing Enterobacterales.

Wildlife play a role in the acquisition, maintenance, and dissemination of antimicrobial resistance (AMR). This is especially true at the human-domestic animal-wildlife interface, like urbanized areas, where interactions occur that can promote the cross-over of AMR bacteria and genes. We conducted a 2-year fecal surveillance (n = 783) of a white-tailed deer (WTD) herd from an urban park system in Ohio to identify and characterize cephalosporin-resistant and carbapenemase-producing bacteria using selective enrichment. Using generalized linear mixed models we found that older (OR = 2.3, P < 0.001), male (OR = 1.8, P = 0.001) deer from urbanized habitats (OR = 1.4, P = 0.001) were more likely to harbor extended-spectrum cephalosporin-resistant Enterobacterales. In addition, we isolated two carbapenemase-producing Enterobacterales (CPE), a Klebsiella quasipneumoniae harboring blaKPC-2 and an Escherichia coli harboring blaNDM-5, from two deer from urban habitats. The genetic landscape of the plasmid carrying blaKPC-2 was unique, not clustering with other reported plasmids encoding KPC-2, and only sharing 78% of its sequence with its nearest match. While the plasmid carrying blaNDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the blaNDM-5, has been reported infrequently. Both carbapenemase genes were successfully conjugated to a J53 recipient conferring a carbapenem-resistant phenotype. Our findings highlight that urban environments play a significant role on the transmission of AMR bacteria and genes to wildlife and suggest WTD may play a role in the dissemination of clinically and epidemiologically relevant antimicrobial resistant bacteria. IMPORTANCE The role of wildlife in the spread of antimicrobial resistance is not fully characterized. Some wildlife, including white-tailed deer (WTD), can thrive in suburban and urban environments. This may result in the exchange of antimicrobial resistant bacteria and resistance genes between humans and wildlife, and lead to their spread in the environment. We found that WTD living in an urban park system carried antimicrobial resistant bacteria that were important to human health and resistant to antibiotics used to treat serious bacterial infections. This included two deer that carried bacteria resistant to carbapenem antibiotics which are critically important for treatment of life-threatening infections. These two bacteria had the ability to transfer their AMR resistance genes to other bacteria, making them a threat to public health. Our results suggest that WTD may contribute to the spread of antimicrobial resistant bacteria in the environment.

Detection of Zoonotic Bacteria and Paragonimus kellicotti in Red Swamp Crayfish (Procambarus clarkii) and the Assessment of Traditional Crayfish Boils.

ABSTRACT: Studies of red swamp crayfish (Procambarus clarkii) outside of the United States confirm the presence of a variety of zoonotic pathogens, but it is unknown whether these same pathogens occur in P. clarkii in the United States. The U.S. commercial crayfish industry generates $200 million yearly, underscoring the need to evaluate this consumer commodity. The study objectives were to evaluate specific zoonotic pathogens present on P. clarkii from Alabama and Louisiana, states in the southeastern United States, and to determine the effectiveness of traditional food preparation methods to reduce pathogens. Experiment A evaluated the presence of Escherichia coli, Salmonella, Staphylococcus aureus, and Vibrio spp. in crayfish and environmental samples over a 2-month collection period (May to June 2021). Crayfish sampling consisted of swabbing the cephalothorax region; 15 samples were tested for E. coli, Salmonella, and S. aureus, and an additional 15 samples for Vibrio spp. Additionally, crayfish shipping materials were sampled. In experiment B, 92 crayfish were evaluated for Paragonimus kellicotti. Experiment C compared live and boiled crayfish for the presence of Vibrio spp. In experiments A and B, all 60 (100%) crayfish samples and 13 (81.25%) of 16 environmental samples showed growth characteristic of Vibrio spp. Three (5%) of 60 samples showed E. coli growth, with no statistical difference (P = 0.5536) between farms. P. kellicotti, Salmonella, and S. aureus were not recovered from any samples. In experiment C, all 10 (100%) of the live preboiled crayfish samples showed characteristic growth, whereas 1 (10%) of 10 samples of crayfish boiled in unseasoned water showed Vibrio growth (P < 0.0001). These results confirm that Vibrio spp. and E. coli may be present on U.S. commercial crayfish and that care should be taken when handling any materials that come into contact with live crayfish because they can potentially be contaminated.

Temporal Trends in Antimicrobial Resistance of Fecal Escherichia coli from Deer.

The changing epidemiologic role of wildlife as reservoirs of antimicrobial-resistant bacteria (ARB) is poorly understood. In this study, we characterize the phenotypic resistance of commensal Escherichia coli from fecal samples of 879 individual white-tailed (Odocoileus virginianus; WTD) over a ten-year period and analyze resistance patterns. Our results show commensal E. coli from WTD had significant linear increases in reduced susceptibility to 5 of 12 antimicrobials, including broad-spectrum cephalosporins and fluoroquinolones, from 2006 to 2016. In addition, the relative frequency distribution of minimal inhibitory concentrations of two additional antimicrobials shifted towards higher values from across the study period. The prevalence of multidrug-resistant commensal E. coli increased over the study period with a prevalence of 0%, 2.2%, and 3.7% in 2006, 2012, and 2016, respectively. WTD may be persistently and increasingly exposed to antibiotics or their residues, ARB, and/or antimicrobial resistance genes via contaminated environments like surface water receiving treated wastewater effluent.

Antimicrobial resistant bacteria recovered from retail ground meat products in the US include a Raoultella ornithinolytica co-harboring blaKPC-2 and blaNDM-5.

Retail beef and pork, including processed products, can serve as vehicles for the zoonotic foodborne transmission of pathogens and antimicrobial resistant bacteria. However, processed and seasoned products like sausages, are not often included in research and surveillance programs. The objective of this study was to investigate retail ground beef and pork, including processed products, for the presence of common foodborne pathogens and antimicrobial resistant bacteria. We purchased 763 packages of fresh and fully cooked retail meat products during 29 visits to 17 grocery stores representing seven major grocery chains located in west and central Ohio. Each package of meat was evaluated for contamination with methicillin-resistant Staphylococcus aureus (MRSA), Salmonella spp., Enterobacteriaceae expressing extended-spectrum cephalosporin resistance, and carbapenemase-producing organisms (CPO). Only 3 of the 144 (2.1%) packages of fully cooked meat products contained any of these organisms, 1 with an extended-spectrum ß-lactamase-producing (ESBL) Enterobacteriaceae and 2 with CPO. Among the 619 fresh meat products, we found that 85 (13.7%) packages were contaminated with MRSA, 19 (3.1%) with Salmonella, 136 (22.0%) with Enterobacteriaceae expressing an AmpC (blaCMY) resistance genotype, 25 (4.0%) with Enterobacteriaceae expressing an ESBL (blaCTX-M) resistance genotype, and 31 (5.0%) with CPO, primarily environmental organisms expressing intrinsic carbapenem resistance. However, one CPO, a Raoultella ornithinolytica, isolated from pork sausage co-harbored both blaKPC-2 and blaNDM-5 on IncN and IncX3 plasmids, respectively. Our findings suggest that fresh retail meat, including processed products can be important vehicles for the transmission of foodborne pathogens and antimicrobial resistant bacteria, including those with epidemic carbapenemase-producing genotypes.

Suspect screening of pharmaceuticals in fish livers based on QuEChERS extraction coupled with high resolution mass spectrometry.

The presence of pharmaceuticals and personal care products (PPCPs) in aquatic environments is of increasing concern due to the presence of residues in fish and aquatic organisms, and emerging antibiotic resistance. Wastewater release is an important contributor to the presence of these compounds in aquatic ecosystems, where they may accumulate in food webs. The traditional environmental surveillance approach relies on the targeted analysis of specific compounds, but more suspect screening methods have been developed recently to allow for the identification of a variety of contaminants. In this study, a method based on QuEChERS extraction - using acetonitrile/water mixture as solvent and PSA/C18 for sample clean-up - was applied to identify pharmaceuticals and their metabolites in fish livers. Both target and suspect screening workflows were used and fish were sampled upstream and downstream of wastewater treatment plants from the Scioto River, Ohio (USA). The method performed well in terms of extraction of some target PPCPs, with recoveries generally above 90%, good repeatability (<20%), and linearity. Based on target analysis, lincomycin and sulfamethoxazole were two antibiotics identified in fish livers at average concentrations of 30.3 and 25.6 ng g-1 fresh weight, respectively. Using suspect screening, another antibiotic, azithromycin and an antidepressant metabolite, erythrohydrobupropion were identified (average concentrations: 27.8 and 13.8 ng g-1, respectively). The latter, reported, to the best of our knowledge, for the first time in fish livers, was also found at higher concentrations in fish livers sampled downstream vs. upstream. The higher frequency of detection for azithromycin in benthic feeding fish species (63%) as well as clusters identified between different foraging groups suggest that foraging behavior may be an important mechanism in the bioaccumulation of PPCPs. This study shows how suspect screening is effective in identifying new contaminants in fish livers, notably using differential analysis among different spatially distributed samples.

Prevalence of extended-spectrum cephalosporin-, carbapenem-, and fluoroquinolone-resistant members of the family Enterobacteriaceae isolated from the feces of horses and hospital surfaces at two equine specialty hospitals.

OBJECTIVE: To estimate the prevalence of extended-spectrum cephalosporin-, carbapenem-, and fluoroquinolone-resistant bacteria of the family Enterobacteriaceae in the feces of hospitalized horses and on hospital surfaces. SAMPLE: Fecal and environmental samples were collected from The Ohio State University Galbreath Equine Center (OSUGEC) and a private referral equine hospital in Kentucky (KYEH). Feces were sampled within 24 hours after hospital admission and after 48 hours and 3 to 7 days of hospitalization. PROCEDURES: Fecal and environmental samples were enriched, and then selective media were inoculated to support growth of Enterobacteriaceae bacteria that expressed resistance phenotypes to extended-spectrum cephalosporins, carbapenems, and fluoroquinolones. RESULTS: 358 fecal samples were obtained from 143 horses. More samples yielded growth of Enterobacteriaceae bacteria that expressed resistance phenotypes (AmpC ß-lactamase, OR = 4.2; extended-spectrum beta-lactamase, OR = 3.2; and fluoroquinolone resistance, OR = 4.0) after 48 hours of hospitalization, versus within 24 hours of hospital admission. Horses hospitalized at KYEH were at greater odds of having fluoroquinolone-resistant bacteria (OR = 2.2). At OSUGEC, 82%, 64%, 0%, and 55% of 164 surfaces had Enterobacteriaceae bacteria with AmpC ß-lactamase phenotype, extended-spectrum beta-lactamase phenotype, resistance to carbapenem, and resistance to fluoroquinolones, respectively; prevalences at KYEH were similarly distributed (52%, 32%, 1%, and 35% of 315 surfaces). CONCLUSIONS AND CLINICAL RELEVANCE: Results indicated that antimicrobial-resistant Enterobacteriaceae may be isolated from the feces of hospitalized horses and from the hospital environment. Hospitalization may lead to increased fecal carriage of clinically important antimicrobial-resistance genes.

Prevalence and Antimicrobial Susceptibility of Salmonella Serovars Isolated from U.S. Retail Ground Pork.

One objective of this study was to determine overall prevalence of Salmonella in ground pork from U.S. retail stores over three seasons including both case-ready and store-ground packages. Package types collected included: overwrap, chub, modified atmosphere packaging, and other (plastic or wax paper wrapped). Because package type represents different production systems and are subject to varied microbiological government regulation and testing methodologies, both USDA-FSIS and FDA Salmonella isolation protocols were performed. Another objective of the study was to determine serotypes and antimicrobial susceptibility profiles of the isolates obtained from the ground pork samples. Ground pork aliquots were subjected to real-time PCR. Recovered isolates were serotyped and minimum inhibitory concentration analysis to 15 antimicrobials was determined using microbroth dilution. Overall prevalence of Salmonella in ground pork from the 865 samples collected was 1.39%. Prevalence was not affected by package type (p = 0.29) nor grind location (case-ready vs. store-ground; p = 0.17). Season affected Salmonella prevalence (p = 0.05) with most isolates found during fall, and there was a tendency for geographic region to affect prevalence (p = 0.07). The USDA Salmonella isolation method was more effective at recovering isolates (p = 0.01) compared with the FDA methodology and yielded a kappa statistic of 0.26 as a measure of agreement. The serotypes isolated included: Infantis, 4,5,12:i:-, Brandenburg, Typhimurium var 5-, Seftenberg, and Johannesburg with only two packages containing multiple serotypes. No isolates were resistant to antibiotics commonly used to treat human Salmonella infections including extended spectrum cephalosporins or fluoroquinolones. Although the recovery of Salmonella from retail ground pork samples was rare, Salmonella Typhimurium (and its monophasic variant 4,5,12:i:-), which are among the most common serovars recovered from human infections, were recovered. Therefore, more effective strategies to further reduce or eliminate these pathogens from retail pork products are warranted.