The wastewater of livestock slaughterhouses is considered a source of antimicrobial-resistant bacteria with clinical relevance and may thus be important for their dissemination into the environment. To get an overview of their occurrence and characteristics, we investigated process water (n = 50) from delivery and unclean areas as well as wastewater (n = 32) from the in-house wastewater treatment plants (WWTPs) of two German poultry slaughterhouses (slaughterhouses S1 and S2). The samples were screened for ESKAPE bacteria (Enterococcus spp., Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.) and Escherichia coli. Their antimicrobial resistance phenotypes and the presence of extended-spectrum-β-lactamase (ESBL), carbapenemase, and mobilizable colistin resistance genes were determined. Selected ESKAPE bacteria were epidemiologically classified using different molecular typing techniques. At least one of the target species was detected in 87.5% (n = 28/32) of the wastewater samples and 86.0% (n = 43/50) of the process water samples. The vast majority of the recovered isolates (94.9%, n = 448/472) was represented by E. coli (39.4%), the A. calcoaceticus-A. baumannii (ACB) complex (32.4%), S. aureus (12.3%), and K. pneumoniae (10.8%), which were widely distributed in the delivery and unclean areas of the individual slaughterhouses, including their wastewater effluents. Enterobacter spp., Enterococcus spp., and P. aeruginosa were less abundant and made up 5.1% of the isolates. Phenotypic and genotypic analyses revealed that the recovered isolates exhibited diverse resistance phenotypes and β-lactamase genes. In conclusion, wastewater effluents from the investigated poultry slaughterhouses exhibited clinically relevant bacteria (E. coli, methicillin-resistant S. aureus, K. pneumoniae, and species of the ACB and Enterobacter cloacae complexes) that contribute to the dissemination of clinically relevant resistances (i.e., bla_CTX-M or bla_SHV and mcr-1) in the environment. 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. 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.

Slaughterhouse wastewater is considered a reservoir for antibiotic-resistant bacteria and antibiotic residues, which are not sufficiently removed by conventional treatment processes. This study focuses on the occurrence of ESKAPE bacteria (Enterococcus spp., S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp.), ESBL (extended-spectrum β-lactamase)-producing E. coli, antibiotic resistance genes (ARGs) and antibiotic residues in wastewater from a poultry slaughterhouse. The efficacy of conventional and advanced treatments (i.e., ozonation) of the in-house wastewater treatment plant regarding their removal was also evaluated. Target culturable bacteria were detected only in the influent and effluent after conventional treatment. High abundances of genes (e.g., bla_TEM, bla_CTX-M-15, bla_CTX-M-32, bla_OXA-48, bla_CMY and mcr-1) of up to 1.48 × 10⁶ copies/100 mL were detected in raw influent. All of them were already significantly reduced by 1–4.2 log units after conventional treatment. Following ozonation, mcr-1 and bla_CTX-M-32 were further reduced below the limit of detection. Antibiotic residues were detected in 55.6% (n = 10/18) of the wastewater samples. Despite the significant reduction through conventional and advanced treatments, effluents still exhibited high concentrations of some ARGs (e.g., sul1, ermB and bla_OXA-48), ranging from 1.75 × 10² to 3.44 × 10³ copies/100 mL. Thus, a combination of oxidative, adsorptive and membrane-based technologies should be considered.

Due to the high prevalence of colistin-resistant Enterobacteriaceae in poultry and pigs, process waters and wastewater from slaughterhouses were considered as a hotspot for isolates carrying plasmid-encoded, mobilizable colistin resistances (mcr genes). Thus, questions on the effectiveness of wastewater treatment in in-house and municipal wastewater treatment plants (WWTPs) as well as on the diversity of the prevailing isolates, plasmid types, and their transmissibility arise. Process waters and wastewater accruing in the delivery and unclean areas of two poultry and two pig slaughterhouses were screened for the presence of target colistin-resistant bacteria (i.e., Escherichia coli, Klebsiella spp., Enterobacter cloacae complex). In-house and municipal WWTPs (mWWTPs) including receiving waterbodies were investigated as well. Samples taken in the poultry slaughterhouses yielded the highest occurrence of target colistin-resistant Enterobacteriaceae (40.2%, 33/82), followed by mWWTPs (25.0%, 9/36) and pig slaughterhouses (14.9%, 10/67). Recovered isolates exhibited various resistance patterns. The resistance rates using epidemiological cut-off values were higher in comparison to those obtained with clinical breakpoints. Noteworthy, MCR-1-producing Klebsiella pneumoniae and E. coli were detected in scalding waters and preflooders of mWWTPs. A total of 70.8% (46/65) of E. coli and 20.6% (7/34) of K. pneumoniae isolates carried mcr-1 on a variety of transferable plasmids with incompatibility groups IncI1, IncHI2, IncX4, IncF, and IncI2 ranging between 30 and 360 kb. The analyzed isolates carrying mcr-1 on transferable plasmids (n = 53) exhibited a broad diversity, as they were assigned to 25 different XbaI profiles. Interestingly, in the majority of colistin-resistant mcr-negative E. coli and K. pneumoniae isolates non-synonymous polymorphisms in pmrAB were detected. Our findings demonstrated high occurrence of colistin-resistant E. coli and K. pneumoniae carrying mcr-1 on transferrable plasmids in poultry and pig slaughterhouses and indicate their dissemination into surface water.

Currently, human and veterinary medicine are threatened worldwide by an increasing resistance to carbapenems, particularly present in opportunistic Enterobacterales pathogens (e.g., Klebsiella spp.). However, there is a lack of comprehensive and comparable data on their occurrence in wastewater, as well as on the phenotypic and genotypic characteristics for various countries including Germany. Thus, this study aims to characterize carbapenem-resistant Klebsiella spp. isolated from municipal wastewater treatment plants (mWWTPs) and their receiving water bodies, as well as from wastewater and process waters from poultry and pig slaughterhouses. After isolation using selective media and determination of carbapenem (i.e., ertapenem) resistance using broth microdilution to apply epidemiological breakpoints, the selected isolates (n = 30) were subjected to WGS. The vast majority of the isolates (80.0%) originated from the mWWTPs and their receiving water bodies. In addition to ertapenem, Klebsiella spp. isolates exhibited resistance to meropenem (40.0%) and imipenem (16.7%), as well as to piperacillin-tazobactam (50.0%) and ceftolozan-tazobactam (50.0%). A high diversity of antibiotic-resistance genes (n = 68), in particular those encoding β-lactamases, was revealed. However, with the exception of bla_GES-5-like, no acquired carbapenemase-resistance genes were detected. Virulence factors such as siderophores (e.g., enterobactin) and fimbriae type 1 were present in almost all isolates. A wide genetic diversity was indicated by assigning 66.7% of the isolates to 12 different sequence types (STs), including clinically relevant ones (e.g., ST16, ST252, ST219, ST268, ST307, ST789, ST873, and ST2459). Our study provides information on the occurrence of carbapenem-resistant, ESBL-producing Klebsiella spp., which is of clinical importance in wastewater and surface water in Germany. These findings indicate their possible dissemination in the environment and the potential risk of colonization and/or infection of humans, livestock and wildlife associated with exposure to contaminated water sources.

Acinetobacter baumannii is an important human pathogen usually associated with severe hospital-acquired infections. Here, we announce the draft genome sequences of two livestock-associated isolates recovered from sewage water from a poultry slaughterhouse in Germany. Short-read whole-genome sequencing was conducted to determine the genetic basis of their antimicrobial resistance phenotype.