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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 β-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 Tn 4401b -KPC-4 transposon conferring multidrug resistance. One isolate maintained three tandem repeats of a Tn 4401b -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 bla KPC-4 , including one with a unique, tandem triplicate of the Tn 4401b -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., Competing Interests: The authors declare no conflict of interest.

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 bla KPC-2 and an Escherichia coli harboring bla NDM-5 , from two deer from urban habitats. The genetic landscape of the plasmid carrying bla KPC-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 bla NDM-5 shared sequence similarity with other reported plasmids encoding NDM-5, the intact IS26 mobile genetic elements surrounding multiple drug resistant regions, including the bla NDM-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.

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 β-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 (bla CMY ) resistance genotype, 25 (4.0%) with Enterobacteriaceae expressing an ESBL (bla CTX-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 bla KPC-2 and bla NDM-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.

Wastewater flows from metropolitan areas, especially those with healthcare inputs, can serve as transport reservoirs for the dissemination of clinically-relevant antimicrobial resistant bacteria (ARB) such as carbapenem- (CR) and colistin-resistant (CoR) strains. Pulsed electric field (PEF) is an emerging wastewater management tool for reducing bacterial loads without generating environmentally harmful byproducts, but it's ability to reduce ARB and their genetic determinants is not well reported. We collected 86, 10-L raw wastewater influent samples from a large metropolitan wastewater treatment plant in Columbus, Ohio and subjected them to low (34 kV cm -1 for 67 μsec) and high (36 kV cm -1 for 89 μsec) PEF treatment. We quantified the PEF effectiveness by measuring concentrations of total coliform bacteria, CR and CoR bacteria, and the epidemic carbapenemase gene, bla KPC , before and after PEF treatment. Utilizing marginal linear regression models with generalized estimating equations, we observed that low and high PEF treatment resulted in a 1.94 (95% CI 2.06-1.81; P < 0.001) and 2.32 (95% CI 2.46-2.18; P < 0.001) log reduction of total coliform bacteria concentrations, respectively. Low and high PEF treatment produced similar log reductions between CR E. coli (2.01 (95% CI 2.15-1.86; P < 0.001); 2.14 (95% CI: 5.30-4.61; P < 0.001)) and CR Enterobacteriaceae concentrations (1.55 (95% CI 1.70-1.41; P < 0.001); 1.86 (95% CI 2.05-1.68; P < 0.001)), and resulted in a 1.15 log (95% CI 1.38-0.93, P < 0.001) and 1.28 log (95% CI 1.54-1.03, P < 0.001) reduction of absolute bla KPC concentrations. Log CoR E. coli concentrations were reduced by 2.47 (95% CI 2.78-2.15; P < 0.001) and 2.52 (95% CI 2.91-2.15; P < 0.001) and CoR Enterobacteriaceae by 2.24 (95% CI 2.52-1.95; P < 0.001) and 2.50 (95% CI 2.89-2.11; P < 0.001) following low and high PEF application. PEF can be applied for wastewater management as an independent treatment method, particularly at critical control points, such as an on-site management of wastewater from hospitals or other healthcare facilities, or in series with other conventional methods to reduce total bacterial loads and concentrations of clinically-relevant ARB., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. The authors declare no conflict of interest., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Carbapenemase-producing bacteria (CPB) are rare, multidrug resistant organisms most commonly associated with hospitalized patients. Metropolitan wastewater treatment plants (WWTP) treat wastewater from large geographic areas which include hospitals and may serve as epidemiologic reservoirs for the maintenance or expansion of CPB that originate from hospitals and are ultimately discharged in treated effluent. However, little is known about the potential impact of these WWTP CPB on the local surface water and their risk to the public health. In addition, CPB that are present in surface water may ultimately disseminate to intensively-managed animal agriculture facilities where there is potential for amplification by extended-spectrum cephalosporins. To better understand the role of WWTPs in the dissemination of CPB in surface waters, we obtained samples of treated effluent, and both upstream and downstream nearby surface water from 50 WWTPs throughout the US. A total of 30 CPB with clinically-relevant genotypes were recovered from 15 WWTPs (30%) of which 13 (50%) serviced large metropolitan areas and 2 (8.3%) represented small rural populations (P < 0.05). Recovery of CPB was lowest among WWTPs that utilized ultraviolet radiation for primary disinfection (12%), and higher (P = 0.11) for WWTPs that used chlorination (42%) or that did not utilize disinfection (50%). We did not detect a difference in CPB recovery by sampling site, although fewer CPB were detected in upstream (8%) compared to effluent (20%) and downstream (18%) samples. Our results indicate that WWTP effluent and nearby surface waters in the US are routinely contaminated with CPB with clinically important genotypes including those producing Klebsiella pneumoniae carbapenemase (KPC) and New Delhi metallo-beta-lactamase (NDM). This is a concern for both public health and animal agriculture because introduction of CPB into intensively managed livestock populations could lead to their amplification and foodborne dissemination., Competing Interests: The authors have declared that no competing interests exist.