Abstract
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 × 10
6
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 × 10
2
to 3.44 × 10
3
copies/100 mL. Thus, a combination of oxidative, adsorptive and membrane-based technologies should be considered.
Display omitted
•High diversity of inherent blaOXA-51-like and blaADC genes detected.•Acquired carbapenemases and biocide resistances found only in clinical isolates.•Unlike livestock and ...environmental isolates, most clinical isolates had plasmids.•Clinical isolates exhibited the highest transformation frequencies.•As-yet-unassigned sequence types found mainly in livestock isolates.
Acinetobacter baumannii has become a prominent nosocomial pathogen, primarily owing to its remarkable ability to rapidly acquire resistance to a wide range of antimicrobial agents and its ability to persist in diverse environments. However, there is a lack of data on the molecular epidemiology and its potential implications for public health of A. baumannii strains exhibiting clinically significant resistances that originate from non-clinical environments.
Therefore, the genetic characteristics and resistance mechanisms of 80 A. baumannii-calcoaceticus (ABC) complex isolates, sourced from environments associated with poultry and pig production, municipal wastewater treatment plants (WWTPs), and clinical settings, were investigated.
In total, our study classified 54 isolates into 29 previously described sequence types (STs), while 26 isolates exhibited as-yet-unassigned STs. We identified a broad range of A. baumannii STs originating from poultry and pig production environments (e.g., ST10, ST238, ST240, ST267, ST345, ST370, ST372, ST1112 according to Pasteur scheme). These STs have also been documented in clinical settings worldwide, highlighting their clinical significance. These findings also raise concerns about the potential zoonotic transmission of certain STs associated with livestock environments. Furthermore, we observed that clinical isolates exhibited the highest diversity of antimicrobial resistance genes (ARGs). In contrast to non-clinical isolates, clinical isolates typically carried a significantly higher number of ARGs, ranging from 10 to 15. They were also the exclusive carriers of biocide resistance genes and acquired carbapenemases (blaOXA-23, blaOXA-58, blaOXA-72, blaGIM-1, blaNDM-1). Additionally, we observed that clinical strains displayed an increased capacity for carrying plasmids and undergoing genetic transformation. This heightened capability could be linked to the intense selective pressures commonly found within clinical settings.
Our study provides comprehensive insights into essential aspects of ABC isolates originating from livestock-associated environments and clinical settings. We explored their resistance mechanisms and potential implications for public health, providing valuable knowledge for addressing these critical issues.
On a commercial broiler farm with substantial health problems, shown by a reported loss rate of approx. 10% and regular antibiotic use, samples were taken at different locations in 2 barns, with the ...aim of analyzing possible entry routes and persistence of pathogens and antibiotic-resistant bacteria as well as revealing weak points in sanitation. Therefore, swab samples for biofilm and water samples from animal drinking water lines and the spray cooling system were taken twice immediately before restocking. In addition, swab samples from drain holes and air samples were collected. At restocking, hatchlings that died during transportation and chick paper were sampled. All samples were analyzed for the occurrence of Pseudomonas aeruginosa, total coliform count, and antibiotic-resistant bacteria, namely, methicillin-resistant Staphylococcus aureus (MRSA), Escherichia coli, Klebsiella spp., Citrobacter spp., Enterobacter spp., Acinetobacter baumannii, P. aeruginosa, and vancomycin-resistant Enterococci (VRE). No MRSA or VRE were detectable. In all samples from drinking water and sprinkler system pipes, P. aeruginosa was detectable; in most cases, antibiotic-resistant P. aeruginosa was also detected, with varying resistance profiles. Samples from the hatchlings and chick paper were contaminated with antibiotic-resistant Enterobacter spp., with resistance to piperacillin, fosfomycin, and the third-generation cephalosporins cefotaxime and ceftazidime. Therefore, the initial entry of antibiotic-resistant Enterobacteriaceae likely occurred via exposure at the hatchery, resulting in colonization of the chicks. Animals on the fattening farm were treated with colistin, amoxicillin, and lincomycin in the 3 production cycles before sampling. Owing to the frequent administration of several antibiotic classes during the fattening period via piped water in both barns, resistance of isolates from water pipes accumulated, showing additional resistance to chloramphenicol and frequently to ciprofloxacin and levofloxacin. To prevent the development of secondary diseases caused by the facultative pathogen P. aeruginosa in chicks with a weak immune status, the hygiene management for drinking water lines and the spray cooling system was changed. These changes resulted in an improvement in water line sanitation, shown by the absence of antibiotic-resistant bacteria and rare detection of P. aeruginosa.
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
Xba
I 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
pathogens (e.g.,
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
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 (
= 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,
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 (
= 68), in particular those encoding β-lactamases, was revealed. However, with the exception of
, 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
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.
is frequently associated with multiple antimicrobial resistances and a major cause of bacterial extraintestinal infections in livestock and humans. However, data on the epidemiology of (i) ...multidrug-resistant (MDR) and (ii) extraintestinal pathogenic
(ExPEC) in poultry and pig slaughterhouses in Germany is currently lacking. Selected
isolates (
= 71) with phenotypic resistance to cephalosporins from two poultry and two pig slaughterhouses expressing high MDR rates (combined resistance to piperacillin, cefotaxime and/or ceftazidime, and ciprofloxacin) of 51.4% and 58.3%, respectively, were analyzed by whole-genome sequencing. They constituted a reservoir for 53 different antimicrobial resistance determinants and were assigned various sequence types, including high-risk clones involved in human infections worldwide. An ExPEC pathotype was detected in 17.1% and 5.6% of the isolates from poultry and pig slaughterhouses, respectively. Worryingly, they were recovered from scalding water and eviscerators, indicating an increased risk for cross-contaminations. Uropathogenic
(UPEC) were detected in the effluent of an in-house wastewater treatment plant (WWTP) of a poultry slaughterhouse, facilitating their further dissemination into surface waters. Our study provides important information on the molecular characteristics of (i) MDR, as well as (ii) ExPEC and UPEC regarding their clonal structure, antimicrobial resistance and virulence factors. Based on their clinical importance and pathogenic potential, the risk of slaughterhouse employees' exposure cannot be ruled out. Through cross-contamination, these MDR
pathotypes may be introduced into the food chain. Moreover, inadequate wastewater treatment may contribute to the dissemination of UPEC into surface waters, as shown for other WWTPs.
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 (
= 50) from delivery and unclean areas as well as wastewater (
= 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 (
spp.,
,
,
,
,
spp.) and
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% (
= 28/32) of the wastewater samples and 86.0% (
= 43/50) of the process water samples. The vast majority of the recovered isolates (94.9%,
= 448/472) was represented by
(39.4%), the
(ACB) complex (32.4%),
(12.3%), and
(10.8%), which were widely distributed in the delivery and unclean areas of the individual slaughterhouses, including their wastewater effluents.
spp.,
spp., and
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 (
, methicillin-resistant
,
, and species of the ACB and
complexes) that contribute to the dissemination of clinically relevant resistances (i.e.,
or
and
) in the environment.
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 process- and wastewater are considered as a hotspot for antibiotic-resistant bacteria and antimicrobial residues and may thus play an important role for their dissemination into the ...environment. In this study, we investigated occurrence and characteristics of ESKAPE bacteria (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp.) and ESBL (extended spectrum β-lactamase) -producing E. coli in water samples of different processing stages of two German pig slaughterhouses (S1/S2) as well as their municipal wastewater treatment plants (mWWTPs). Furthermore, residues of various antimicrobials were determined.
A total of 103 water samples were taken in delivery and dirty areas of the slaughterhouses S1/S2 (n = 37), their in-house WWTPs (n = 30) and mWWTPs including their receiving water bodies (n = 36). The recovered isolates (n = 886) were characterized for their antimicrobial resistance pattern and its genetic basis.
Targeted species were ubiquitous along the slaughtering and wastewater chains. Phenotypic and genotypic analyses revealed a broad variety of resistance phenotypes and β-lactamase genes. Carbapenemase-producing Enterobacteriaceae (CPE), vancomycin-resistant enterococci (VRE) and healthcare-associated (HA) MRSA were recovered only from mWWTPs and their preflooders. In contrast, the mcr-1 gene was exclusively detected in E. coli from S1/S2. Residues of five antimicrobials were detected in 14.9% (10/67) of S1/S2 samples in low range concentrations (≤1.30 μg/L), whereas 91.7% (33/36) of mWWTPs samples exhibited residues of 22 different antibiotics in concentrations of up to 4.20 μg/L.
Target bacteria from S1/S2 and mWWTPs exhibited differences in their abundances, resistance phenotypes and genotypes as well as clonal lineages. S1/S2 samples exhibited bacteria with zoonotic potential (e.g. MRSA of CC398, E. coli of significant clones), whereas ESKAPE bacteria exhibiting resistances of clinical importance were mainly detected in mWWTPs. Municipal WWTPs seem to fail to eliminate these bacteria leading to a discharge into the preflooder and a subsequent dissemination into the surface water.
Display omitted
•Livestock-associated wastewater is an important reservoir of zoonotic bacteria.•Pig slaughterhouses are negligible emitters of antibiotic residues.•CPE, VRE and HA-MRSA were detected only in municipal WWTPs.•ESKAPE bacteria are spread into surface water by inadequate wastewater treatment.
The frequencies of 6 different facultative pathogenic bacteria of the ESKAPE group (priority list WHO) and a total of 14 antibiotic resistance genes (ARGs) with different priorities for human ...medicine were quantified in wastewaters of poultry and pig slaughterhouses using molecular biological approaches. Raw sewage from poultry and pig slaughterhouses was found to be contaminated not only with facultative pathogenic bacteria but also with various categories of clinically relevant ARGs, including ARGs against the reserve antibiotics group. The concentration of the different gene targets decreased after on-site conventional biological or advanced oxidative wastewater treatments, but was not eliminated. Hence, the antimicrobial BlueLight (aBL) in combination with a porphyrin photo-sensitizer was studied with ESKAPE bacteria and real slaughterhouse wastewaters. The applied broad LED-based blue light (420–480 nm) resulted in groups of sensitive, intermediate, and non-sensitive ESKAPE bacteria. The killing effect of aBL was increased in the non-sensitive bacteria
Klebsiella pneumoniae
and
Enterococcus faecium
due to the addition of porphyrins in concentrations of 10
−6
M. Diluted slaughterhouse raw wastewater was treated with broad spectrum aBL and in combination with porphyrin. Here, the presence of the photo-sensitizer enhanced the aBL biocidal impact.
Klebsiella spp. are ubiquitous bacteria capable of colonizing humans and animals, and sometimes leading to severe infections in both. Due to their high adaptability against environmental/synthetic ...conditions as well as their potential in aquiring antimicrobial/metal/biocide resistance determinants, Klebsiella spp. are recognized as an emerging threat to public health, worldwide. Currently, scarce information on the impact of livestock for the spread of pathogenic Klebsiella spp. is available. Thus, the phenotypic and genotypic properties of extended-spectrum β-lactamase-producing, and colistin-resistant Klebsiella strains (n = 185) from process- and wastewater of two poultry and pig slaughterhouses as well as their receiving municipal wastewater treatment plants (mWWTPs) were studied to determine the diversity of isolates that might be introduced into the food-production chain or that are released into the environment by surviving the wastewater treatment.
Selectively-isolated Klebsiella spp. from slaughterhouses including effluents and receiving waterbodies of mWWTPs were assigned to various lineages, including high-risk clones involved in human outbreaks, and exhibited highly heterogeneous antibiotic-resistance patterns. While isolates originating from poultry slaughterhouses showed the highest rate of colistin resistance (32.4%, 23/71), carbapenem-resistant Klebsiella spp. were only detected in mWWTP samples (n = 76). The highest diversity of resistance genes (n = 77) was detected in Klebsiella spp. from mWWTPs, followed by isolates from pig (n = 56) and poultry slaughterhouses (n = 52). Interestingly, no carbapenemase-encoding genes were detected and mobile colistin resistance genes were only obeserved in isolates from poultry and pig slaughterhouses.
Our study provides in-depth information into the clonality of livestock-associated Klebsiella spp. and their determinants involved in antimicrobial resistance and virulence development. On the basis of their pathogenic potential and clinical importance there is a potential risk of colonization and/or infection of wildlife, livestock and humans exposed to contaminated food and/or surface waters.
Display omitted
•Study was conducted using culture-dependent and -independent approaches.•A broad overview on MDR klebsiellae in slaughterhouse wastewater is presented.•Isolates exhibit a broad heterogeneity including clinically-relevant clones.•Diverse antimicrobial resistances and virulence factors were detected.•MDR klebsiellae are spread into surface water by inadequate wastewater treatment.
PDF
