ESKAPE Bacteria and Extended-Spectrum-β-Lactamase-Producing Escherichia coli Isolated from Wastewater and Process Water from German Poultry Slaughterhouses

Bacteria from livestock may be opportunistic pathogens and carriers of clinically relevant resistance genes, as many antimicrobials are used in both veterinary and human medicine. They may be released into the environment from wastewater treatment plants (WWTPs), which are influenced by wastewater from slaughterhouses, thereby endangering public health. Moreover, process water that accumulates during the slaughtering of poultry is an important reservoir for livestock-associated multidrug-resistant bacteria and may serve as a vector of transmission to occupationally exposed slaughterhouse employees. Mitigation solutions aimed at the reduction of the bacterial discharge into the production water circuit as well as interventions against their further transmission and dissemination need to be elaborated. Furthermore, the efficacy of in-house WWTPs needs to be questioned. Reliable data on the occurrence and diversity of clinically relevant bacteria within the slaughtering production chain and in the WWTP effluents in Germany will help to assess their impact on public and environmental health.

IMPORTANCE Bacteria from livestock may be opportunistic pathogens and carriers of clinically relevant resistance genes, as many antimicrobials are used in both veterinary and human medicine. They may be released into the environment from wastewater treatment plants (WWTPs), which are influenced by wastewater from slaughterhouses, thereby endangering public health. Moreover, process water that accumulates during the slaughtering of poultry is an important reservoir for livestock-associated multidrug-resistant bacteria and may serve as a vector of transmission to occupationally exposed slaughterhouse employees. Mitigation solutions aimed at the reduction of the bacterial discharge into the production water circuit as well as interventions against their further transmission and dissemination need to be elaborated.
The objective of the present study was to evaluate the occurrence and diversity of ESKAPE bacteria and E. coli along different slaughtering steps in two German poultry slaughterhouses. For this purpose, samples of process waters from the washing of poultry transport trucks and vehicles, the stunning facilities, scalders, and eviscerators, as well as the wastewater effluents of the in-house wastewater treatment plants, were subjected to bacterium-specific isolation procedures. Besides species identification and antimicrobial resistance testing, the recovered bacteria were subjected to molecular epidemiological classification (phylogenetic typing and multilocus sequence typing [MLST] of E. coli and VRE, spa typing of MRSA) and determination of the genetic basis of the ␤-lactam, carbapenem, and mobilizable colistin resistance. Data were used for comparison of the content of the target bacteria in waters obtained from different slaughtering steps. Based on the results, the impact of clinically relevant bacteria released into the environment by insufficient wastewater management was assessed.

RESULTS
Clinically relevant resistant bacteria were detected in the majority of the investigated samples. Within this study, 41 samples were collected from two individual slaughterhouses (slaughterhouses S1 and S2) at seven sampling points: transport trucks (only S2, n ϭ 5), transport crates (n ϭ 5 for each slaughterhouse), stunning facilities (n ϭ 5 each), scalding water (n ϭ 5 each), eviscerators (n ϭ 5 each), production facilities (only S1, n ϭ 5), and the influent (n ϭ 8 each) and effluent (n ϭ 8 each) of the in-house WWTPs. Further information on the sampling campaign dates is summarized in Table S1 in the supplemental material.
Overall, 92.7% (n ϭ 38) and 80.5% (n ϭ 33) of the samples from S1 and S2, respectively, were positive for at least one of the ESKAPE target bacteria or ESBL-producing E. coli (Fig. 1). Detailed information about the proportion of samples positive for each species detected at each sampling point in S1 and S2 is given in Table S1.
The target bacteria exhibited a high diversity of antimicrobial resistance phenotypes. An overview on the antimicrobial resistance of the investigated target bacteria is presented in Fig. 3. It must be kept in mind, however, that the strains were isolated from selective agar plates containing cephalosporins, oxacillin, or vancomycin; therefore, susceptibility to the selective agents cannot be expected.
The single VRE isolate from S1 was identified to be an Enterococcus faecium isolate and was determined to be resistant to oxacillin, penicillin G, gentamicin, clindamycin, daptomycin, erythromycin, vancomycin, a combination of erythromycin and clindamycin, as well as cefoxitin.
Information on the antimicrobial resistance patterns of individual isolates is given in Table S2.
Enterobacteriaceae isolates from CHROMagar ESBL plates (meropenem resistance cutoff, Ͼ0.125 mg/liter; n ϭ 10) as well as from CHROMagar mSuperCarba plates (n ϭ 22) were negative for the carbapenemase genes tested for in the molecular screening. However, we found two isolates of the ACB complex (2.6%) from S1 that tested positive for bla PER and bla GES .
Similar to the findings for the E. coli isolates from S1, one of the most predominant phylogenetic groups among the S2 E. coli isolates was B1 (36.6%, n ϭ 34). Phylogroups A and C exhibited equal proportions of 17.2% (n ϭ 16), followed by groups E and F (14.0% each, n ϭ 13 each). One isolate recovered from the effluent of the in-house WWTP belonged to group D (1.1%), and no isolates of group B2 were detected.
MLST of the E. coli isolates from phylogroups B2 and D (S1, n ϭ 15) revealed that ST4994 was the most predominant sequence type among the group B2 isolates (84.6%, n ϭ 11). Other individual isolates were classified as ST135 (n ϭ 1) or belonged to a yet unknown sequence type (n ϭ 1). Among the isolates of phylogroup D (n ϭ 2, bla TEM-52c ), ST69 and ST648 were detected. Isolates recovered from the effluent of the WWTPs (n ϭ 6) were assigned to ST361 (n ϭ 2/6, group C, bla CTX-M-15 ), whereas the remaining isolates (n ϭ 4) exhibited yet unassigned types. Among the E. coli isolates carrying bla CTX-M genes with the 3MDRO phenotype (n ϭ 19), ST361 (78.9%, n ϭ 15) was the most predominant sequence type. Four isolates could not be allocated to previously reported STs.
Vancomycin-resistant enterococci. The vancomycin-resistant E. faecium isolate was allocated to ST1249 and carried the vanA gene.

DISCUSSION
In this study, we investigated (i) process waters from different stages of the poultry slaughtering process as well as (ii) the influents and effluents of in-house wastewater treatment plants of two German slaughterhouses. We are not aware of previous studies in Germany in which such samples have been taken directly from the slaughterhouses and their on-site WWTPs. Our results showed that bacteria resistant to highly and critically important antimicrobials pollute the receiving water bodies, as they survived passage through the in-house WWTPs. These results clearly demonstrate that additional or alternative treatment steps are necessary before slaughterhouse wastewater can be released into the environment. The inefficacy of conventional biological WWTPs in Germany was also already reported for the treatment of municipal (45) and hospital (20) sewage.
The presence of potential clinically relevant ESKAPE bacteria (i.e., A. baumannii, K. pneumoniae) and ESBL-producing E. coli in most of the investigated stages of the poultry slaughtering process is not surprising, as most of them are able to colonize the gastrointestinal tract of livestock (especially poultry), which was also described by other authors (40,46). Bustillo-Lecompte and Mehrvar verified that their release into the process waters and, subsequently, into the wastewater is a consequence of the excretion of organic matter from colonized poultry during delivery and slaughter (47). However, to reduce the spread of resistant bacteria into the environment, the implementation of several mitigation measures during poultry primary production (i.e., breeding farms, hatcheries, fattening farms), the slaughtering process, as well as the wastewater treatment process needs to be taken into consideration. This includes the improvement of poultry welfare conditions, the implementation of strict health and infection control programs, as well as reduction of the use of antimicrobials (48,49).
Furthermore, during poultry processing, various intervention options, depending on the production step, are conceivable but are often not realized on the basis of their estimated cost-benefit ratios. To reduce the release of potentially dangerous bacteria into the process water used for the cleaning of poultry crates, the use of predisinfection equipment prior to the washing step is conceivable (50). The process waters applied during CO 2 stunning and evisceration represent further important reservoirs for the dissemination of the bacteria targeted in this study, which were released by defecation, fecal leakages, and gastrointestinal disruptions. Another strategy to reduce dissemination is a general reduction of clinically relevant bacteria on the surface and in the gastrointestinal tract of the animals. This can be achieved by the inclusion of probiotics and/or prebiotics in the feed (51), the administration of oral bacteriophage cocktails (52), and the use of competitive exclusion cultures (53). Furthermore, the use of nonimmersion scalders combined with the decontamination of carcasses with hot water could decrease the level of cross-contamination with resistant bacteria (54). Besides, using moisturized hot air would decrease the amount of wastewater produced. Moreover, the use of advanced wastewater treatment technologies and wastewater disinfection needs to be considered. Hembach et al. proposed that oxidative, adsorptive, and membrane-based technologies be combined in order to prevent environmental contamination with antibiotic resistance determinants and facultative pathogenic bacteria (i.e., ESKAPE bacteria) (55).
Unlike hospital and urban effluents, where the occurrence of XDR and carbapenemaseproducing bacteria is frequently described (20,56,57), wastewater and process water from the investigated poultry slaughterhouses did not exhibit such high-risk bacteria. This emphasizes the importance of the restricted use of carbapenems in human medicine. Moreover, the use of aminopenicillins and their ␤-lactamase inhibitor combinations, (fluoro)quinolones, as well as 3rd-and 4th-generation cephalosporins in livestock should be limited, and these antimicrobials should be used only to the extent that is absolutely necessary (58). They have wider spectra of action and, thus, are more likely to select for multidrug-resistant organisms, thereby compromising the activity of these antimicrobials for the treatment of severe infections in human medicine (58).
Our results on the prevailing E. coli bacteria showed a strong congruence with data published in previous papers (59,60). Furthermore, in this study, E. coli isolates of phylogroups B2, D, and F, implicated as extraintestinal pathogens (ExPEC) (61), were recovered from all sampling locations in both slaughterhouses. This emphasizes the increased risk of transmission of such ExPEC clones to the personnel involved in particular operations in the delivery and unclean areas of the slaughtering process (62).
In general, the ESBL-producing E. coli isolates found in this study showed higher rates of resistance to fluoroquinolones than the isolates from retail chicken meat (20.8%) reported by Casella et al. (63). The increased fluoroquinolone resistance rate in the isolates of this study may be caused by the use of enrofloxacin in slaughtered broiler herds. However, reliable data on the use of fluoroquinolones in the flocks are lacking.
While the majority of the isolates could be epidemiologically linked to poultry, some of the determined sequence types/clonal lineages were also attributed to the high-risk clones (i.e., ST69, ST10, ST648 and ST117) emerging in human infections in different countries (39,64,65). Isolates belonging to ST69, ST10, and ST212 were detected in cleaning samples from transport crates and the effluents of the WWTPs. In general, E. coli isolates of these sequence types are high-risk clones that have been isolated from broilers and poultry meat (66), as well as from various patients with infections in different countries (65). Previously, E. coli ST212 isolates were identified to be enterotoxigenic Escherichia coli (ETEC) isolates that have been recovered from surface water, pigs, broilers, and humans (67,68).
In this study, the majority of the ESBL-producing E. coli isolates exhibited genes that code for CTX-M-1, TEM-116, TEM-52, and SHV-12 ␤-lactamases. These enzymes have already been reported in isolates from poultry and humans (19,69). Among our isolates, bla CTX-M-1 belongs to the most abundant resistance determinants in ESBL-producing E. coli, which correlates well with prevalence data for bla CTX-M-1 (18.0 to 69.0%) in isolates from chickens and chicken meat in Germany (70,71). Similar to the findings of other studies, E. coli isolates carrying bla SHV-2 , bla SHV-2a , and bla TEM-20 were only sporadically detected in chicken and retail chicken meat (72,73). In contrast to the findings of other studies from Germany and the Netherlands (70,74,75), where low percentages (0.0 to 5.2%) of bla CTX-M-15 -producing E. coli isolates in chickens and poultry products have been identified, 12.2% of the ESBL-producing E. coli isolates from the wastewater of slaughterhouse S1 carried this gene. This may have been due to the possible acquisition of bla CTX-M-15 plasmids from human strains, as has already been shown for animal E. coli strains in the United Kingdom (76). CTX-M-15 is one of the most frequently encountered ESBL enzymes in human clinical isolates from various countries (77). This is partially due to the clonal spread and predominance of a subset of ExPEC lineages in the human population that are commonly associated with bla CTX-M genes (particularly with bla CTX-M-15 ), e.g., ST131, ST69, and ST10 (39,78). However, the abundance of such ExPEC clones in poultry production in Germany is moderate, and often they are associated with pAmpC rather than bla CTX-M (59). In this context, the transfer of bla CTX-M-15 in E. coli by mobile genetic elements between humans, livestock, and the environment through the food chain (79) and occupational exposure (80) may play a primary role.
In previous studies, ESBL-producing K. pneumoniae isolates were only sporadically identified in German broilers during slaughter (81,82), whereas in this study, 63.4% of the samples in almost all sampling locations in slaughterhouse S2 tested positive for K. pneumoniae. In contrast to samples from S2, only 7.3% of the samples from S1 exhibited K. pneumoniae. These different proportions might be caused by the content of the colonized flocks that were supplied by different fattening farms. Taking into consideration that there are only a few breeding companies and hatcheries in Germany, the possibility of vertical transmission through the production chain cannot be excluded. Moreover, the majority of the K. pneumoniae isolates carried bla SHV-2 , which has also been frequently found in isolates from egg shells, broilers, and humans (46,83). The results of our study are consistent with the observations that resistance genes of the SHV ␤-lactamase family are ubiquitous in ESBL-producing K. pneumoniae strains (84,85). However, in contrast to the isolates occurring in environmental sources in rural areas (86) as well as those cultured from patient specimens (87), almost all K. pneumoniae isolates from this study lacked genes of the CTX-M/TEM families. This emphasizes the need for further studies applying high-resolution technologies, such as whole-genome sequencing, to better elucidate their epidemiology and clinical relevance.
To date, only limited data on Enterobacter spp. exhibiting resistance to 3rd-and 4th-generation cephalosporins in poultry are available. Overall, the occurrence of bacteria of the E. cloacae complex (1.23%) in retail poultry meat seems to be low, as previously reported for Germany (72). ESBL-producing bacteria of the E. cloacae complex (15.8%) exhibited only the ESBL enzyme SHV-12. These data are in good agreement with the observations of Towne et al. (88), who detected bla SHV-12 in 8.7% of clinical Enterobacter isolates. A steadily increasing abundance of ESBL-encoding isolates of the E. cloacae complex along with the inducible production of AmpC, as well as its constitutive derepression, could lead to nearly pan-resistance to ␤-lactam antibiotics, diminishing the already limited number of therapeutic options (89)(90)(91).
Bacteria of the ACB complex belong to the most important nosocomial pathogens and are able to survive in competitive and demanding environments (92). However, reliable data on the impact of livestock-associated isolates on human and animal health are lacking. Wilharm et al. indicated linkages between livestock isolates and human clinical isolates, suggesting that A. baumannii might have zoonotic potential (40). Despite the importance of these bacteria, data on the emergence and antimicrobial resistance of bacteria of the ACB complex in broilers are scarce (40,93). However, among the raw meats tested, poultry meat retailed in different countries showed the highest rates of contamination with species of the ACB complex (26.7% to 48.0%) (94).
As bacteria of the ACB complex were sporadically isolated from chickens, goslings, ducks, and wild birds (40,95), these animals might play an important role as natural reservoirs for these organisms. Given that Acinetobacter spp. are considered intrinsically resistant to cefotaxime, temocillin, fosfomycin, and chloramphenicol (96), the ACB complex isolates in our study contrast with human and animal clinical strains, which exhibited high levels of resistance to fluoroquinolones and carbapenems (97,98). Almost all tested isolates lacked acquired bla ESBL genes, like bla CTX-M , bla TEM , bla SHV , bla VEB , bla PER , and bla GES , which would be genetic evidence of acquired resistance to ␤-lactams (92). However, their resistance to 3rd-generation cephalosporins might be a consequence of the increased expression of the chromosomal bla ADC gene or other mechanisms (99).
The results of this study show that the majority of the detected MRSA lineages belonged to CC398 and ST9, which are the most common livestock-associated MRSA (LA-MRSA) lineages in Europe (100). spa types t034, t011, and t1430 have already been identified among isolates from chicken and meat products in countries with pronounced conventional farming, like Denmark (101), Germany, and the Netherlands (102). Furthermore, they were also detected in environmental samples from broiler barns (14), poultry slaughterhouses and their personnel (103), as well as human patients in Norway and different countries of the European Union/European Economic Area (38,104). Interestingly, isolates of spa types t034 and t011 represent the most frequent LA-MRSA isolates recovered from hospital inpatients and ambulatory patients in Germany in regions with high levels of livestock production (38). Another notable finding was the detection of spa type t13177 in MRSA isolates from the effluent of the WWTPs. Isolates of this type were sporadically detected in fresh broiler meat and retail chicken meat in Germany (105) and Switzerland (106), respectively. Unlike the other LA-MRSA isolates, these isolates carried genes coding for major staphylococcal enterotoxins, which may cause toxic shock-like syndromes and which are implicated in food poisoning (107).
The antibiotic resistance patterns of the MRSA isolates in this study are similar to those of the isolates described by Rosenberg Goldstein et al. (12). However, the observed differences between isolates from slaughterhouses S1 and S2 may be due to the distinct prevalent clonal lineages detected in the two slaughterhouses. In another study (103), 95.0% of t1430 MRSA isolates but only 11.5% of t034 MRSA isolates were resistant to ciprofloxacin, while these were the most predominant spa types in MRSA isolates from S1 and S2. It has been postulated that t1430 is a poultry-associated MRSA type (103) and that its high levels of resistance to moxifloxacin might be due to the usage of (fluoro)quinolones in the poultry industry.
Within this study, only one vancomycin-resistant E. faecium ST1249 isolate was recovered from cleaning samples from poultry transport crates. VRE ST1249 has been previously isolated from 3.7% of chicken products in the United Kingdom (108). The occurrence of VRE in livestock is related to the glycopeptide avoparcin (109,110), which was used for growth promotion in Germany between 1975 and 1996 (111). However, Johnsen et al. (112) and Andersson and Hughes (113) suggest that the reversibility of acquired glycopeptide resistance is slow and could last for Ͼ25 years. Our findings and other reports (114,115) reinforce this theory. Furthermore, it is presumed that without the selective pressure of avoparcin, coselection by macrolides, which are often used to treat poultry, can occur (116). Moreover, the copper added to the feed can also exert a selective effect on VRE (117).
Conclusion. Process water and wastewater from poultry slaughterhouses are important reservoirs for antimicrobial-resistant bacteria with clinical relevance. The ubiquitous distribution of enterobacteria and MRSA with resistances to highly and critically important antimicrobials in the process water and wastewater of poultry slaughterhouses is worrisome, as they (i) may colonize slaughterhouse workers and (ii) may be reintroduced into the food chain by cross-contamination during carcass processing. (iii) Furthermore, they are released into the environment via surface waters due to insufficient treatment within in-house WWTPs. The implementation of new measures to reduce the input of resistant bacteria into the slaughterhouses and their consequent excretion into process water and wastewater, as well as strategies for improvement of discharge water status and treatment processes, needs to be taken into consideration.

MATERIALS AND METHODS
Sampling and sample preparation. Two German poultry slaughterhouses (slaughterhouses S1 and S2) exhibiting different slaughtering capacities above 100,000 chickens per day were investigated. S1 and S2 produce daily 600 m 3 and 3,600 m 3 wastewater, respectively. S1 operates a wastewater treatment plant (WWTP) based on the membrane bioreactor (MBR) technology with immersed ultrafiltration membranes. S2 possesses a conventional biological WWTP. After treatment, effluents are discharged into the preflooder and further into surface water bodies.
The collected samples represent various waters that accumulate during delivery, in the unclean area of the poultry slaughtering process, as well as in their in-house WWTPs. Samples were taken at seven sampling sites: transport trucks (only S2), transport crates, stunning facilities, scalders, eviscerators, production facilities (only S1), and the influent and the effluent of the in-house WWTPs. Sampling of both slaughterhouses was conducted during five independent visits between December 2016 and September 2018. Three further visits were conducted to obtain additional samples from the in-house WWTPs of S1 and S2 in the same time period. A minimum time interval of 1 month was kept between two independent sampling visits to minimize the possible carryover of the targeted bacteria from poultry flocks originating from the same fattening farm and to ensure that the individual samplings would be representative of different poultry populations.
From each individual sample, 1 liter was collected using sterile Nalgene wide-mouth environmental sample bottles (Thermo Fisher Scientific, Waltham, MA, USA). Composite samples from the precleaning of five poultry trucks after unloading of birds (only S2) and samples of water applied for the precleaning of the stunning facilities were collected by catching runoffs. In general, all precleaning steps were conducted without using cleaning or disinfection agents. Water samples from the cleaning of the poultry transport crates and scalding water were taken by immersion of sterile sampling bottles into the sump of the crate washing facility and the scalder tank, respectively. Process water used during evisceration was collected as runoffs from eviscerators in operation. Aggregate wastewater from production facilities (only S1) was taken by immersion of sterile sampling bottles into mixing and homogenization containers after the wastewater had run through a mechanical deposition. The samples of influent and effluent of the in-house WWTPs were taken as qualified samples according to the German standard methods for the examination of water, wastewater, and sludge (DIN 38402-11:2009-02) (118). The samples were labeled and transported to the laboratory in a Styrofoam box cooled to 5 Ϯ 2°C. To get rid of coarse particles (e.g., bedding, feathers), the samples were manually filtered using stomacher strainer bags with a tissue filter (pore size, 0.5 mm; VWR, Radnor, PA, USA) and afterwards subjected to cultivation within 24 h after sampling.
Cultivation and identification of antimicrobial-resistant target bacteria. Water samples were screened for Gram-negative ESBL-producing bacteria of the Enterobacteriaceae, nonfermenting A. baumannii and P. aeruginosa, as well as methicillin-resistant S. aureus (MRSA), vancomycin-resistant enterococci (VRE), and carbapenemase-producing Enterobacteriaceae (CPE). To detect ESBL-producing target bacteria and VRE, 100-l aliquots of serial 10-fold dilutions or 1 ml of undiluted samples was applied to CHROMagar ESBL and CHROMagar VRE plates (Mast Diagnostica, Reinfeld, Germany) for cultivation. Furthermore, 10-and 100-ml aliquots of the in-house WWTP effluent were filtered through sterile 0.45-m-pore-size, 47-mm mixed cellulose nitrate filters (GE Healthcare, Chicago, IL, USA) and placed on selective agar plates. To inhibit the growth of accompanying bacteria, all agar plates were incubated at 42°C for 18 to 24 h (ESBL-producing target bacteria) and for 42 to 48 h (VRE).
MRSA isolates were recovered following the recommendations of the National Reference Laboratory for Staphylococci, with some modifications. For this purpose, 100 ml of the water samples was (i) filtered through sterile 0.45-m-pore-size, 47-mm mixed cellulose nitrate filters (only for the effluents of the in-house WWTPs) or (ii) centrifuged for 15 min at 5,000 ϫ g and 4°C. The filters or resulting pellets were transferred to 100 ml of Mueller-Hinton broth (MHB; Sigma-Aldrich, St. Louis, MO, USA) supplemented with 6.5% NaCl for preenrichment. After incubation at 37°C for 18 to 24 h under shaking (150 rpm), 1 ml of the preenrichment broth was transferred to 9 ml of tryptic soy broth (TSB; Sigma-Aldrich, St. Louis, MO, USA) supplemented with aztreonam (50 mg/liter) and cefoxitin (3.5 mg/liter). The inoculated selective preenrichment broth was cultivated for 18 to 24 h at 37°C. Afterwards, a 10-l loopful of culture was streaked out on CHROMagar MRSA (Mast Diagnostica, Reinfeld, Germany) screening agar and incubated at 42°C for 18 to 24 h.
For the isolation of CPE, a selective preenrichment was carried out. Therefore, 10-ml water samples were subjected to filtration through 0.45-m-pore-size membrane filters. The filters were incubated at 42°C for 18 to 24 h in Mossel broth (Sigma-Aldrich, St. Louis, MO, USA) under aerobic conditions to inhibit the growth of accompanying flora (i.e., Gram-positive microorganisms). Thereafter, 100 l of the selective preenrichment broth was plated on CHROMagar mSuperCarba (Mast Diagnostica, Reinfeld, Germany) plates and incubated at 42°C for 18 to 24 h. Whenever possible, up to four presumptive colonies per sampling site of E. coli, Klebsiella spp., Enterobacter spp., Citrobacter spp., Acinetobacter spp., Pseudomonas spp., MRSA, as well as VRE were picked from the selective plates and subcultured on Columbia agar with 5% sheep blood (Mast Diagnostica, Reinfeld, Germany) at 37°C for 18 to 24 h. Presumptive coliform bacteria were confirmed by streaking on Chromocult coliform agar (Merck, Darmstadt, Germany) and oxidase testing. The nonfermenting Acinetobacter spp. and Pseudomonas spp. were subcultured on CHROMagar Acinetobacter agar (Mast Diagnostica, Reinfeld, Germany) and cetrimide agar (Merck, Darmstadt, Germany), respectively, and confirmed by oxidase testing. Potential VRE colonies were streaked onto Slanetz Bartley agar (Merck, Darmstadt, Germany).
Species identification was conducted using matrix-assisted laser desorption ionization-time of flight mass spectrometry, employing a Vitek MS mass spectrometer (bioMérieux, Marcy l'Etoile, France) equipped with Myla software. All isolates were purified on Columbia agar with 5% sheep blood and preserved in cryotubes (Mast Diagnostics, Reinfeld, Germany) at Ϫ70°C.
Antimicrobial susceptibility testing. The activities of 17 antimicrobials or antimicrobial combinations against Gram-negative bacteria were tested by the microdilution method according to the protocols of the European Committee on Antimicrobial Susceptibility Testing (EUCAST) using a Micronaut-S MDR MRGN screening system (Merlin, Gesellschaft für mikrobiologische Diagnostika GmbH, Bornheim-Hersel, Germany). Resistance testing of Gram-positive bacteria was conducted using the Micronaut-S MRSA/GP testing panel. The results were interpreted according to clinical cutoff values (EUCAST, v.9.0) in order to determine the profiles of resistance of ESKAPE bacteria of livestock origin to antimicrobials medically important for humans and to assess their clinical relevance in human medicine. The 3MDRO classification of the isolates was done according to the recommendations of the Commission for Hospital Hygiene and Infection Control of 2012 (KRINKO) at the Robert Koch Institute, Berlin, Germany; i.e., intermediate was interpreted as resistant (29).
According to the recommendations of EUCAST (125), isolates of Enterobacteriaceae picked from the CHROMagar ESBL plates (meropenem resistance cutoff, Ͼ0.125 mg/liter) as well as from the CHROMagar mSuperCarba plates were screened for the presence of the carbapenemase genes bla NDM , bla IMI , bla VIM , bla OXA-48 , bla KPC , and bla GES by multiplex real-time TaqMan PCR assays (126,127). Acinetobacter isolates were investigated for the presence of bla PER , bla GES , and bla VEB by PCR and sequencing (128).
For spa typing of the MRSA isolates, the Staphylococcus protein A repeat region was amplified and sequenced as previously described (133). spa types were predicted using SpaServer Ridom software (http://www.spaserver.ridom.de). Vancomycin-resistant enterococci were screened for the vanA, vanB, vanC1, and vanC2 genes by a multiplex PCR assay as previously described (134) and MLST typed using the method of Homan et al. (135). Sequence data were analyzed using the PubMLST database (https:// pubmlst.org/efaecium).

SUPPLEMENTAL MATERIAL
Supplemental material is available online only. SUPPLEMENTAL FILE 1, PDF file, 0.5 MB. SUPPLEMENTAL FILE 2, XLSX file, 0.04 MB.

ACKNOWLEDGMENTS
This work was funded by the Federal Ministry of Education and Research (HyReKA, grant 02WRS1377). The scientific work of Jens Andre Hammerl (BfR) was supported by grants from the Bundesinstitut für Risikobewertung (grants 43-001 and 1322-648).
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.