Tracing clinically-relevant antimicrobial resistances in Acinetobacter baumannii-calcoaceticus complex across diverse environments: A study spanning clinical, livestock, and wastewater treatment settings.

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.

Effect of Climatic Condition, Type of Trough and Water Cleanliness on Drinking Behavior in Dairy Cows.

Increasing ambient temperatures lead to higher water intake and higher risks of microbial growth in cattle troughs. This study aims to analyze drinking water quality and dairy cows' drinking behavior (n = 8081 drinking episodes) on a commercial farm with 135 and 144 lactating cows in two climatic conditions, considering trough type and cleanliness, respectively. Daily video recording was conducted at two trough types (two open troughs, 70 L; two-valve troughs, variable volume of 5-15 L) in the first two hours after feeding (n = 60 days in total) under cold (December 2019-February 2020) and warm ambient temperatures (September 2021). The trough cleaning scheme allowed cows to access either cleaned or uncleaned troughs in each system. Water quality was tested daily and analyzed at the beginning and end of the trials. In warmer ambient temperatures, fewer and-at uncleaned troughs and open troughs-shorter drinking episodes were recorded, with longer but fewer water intake periods, longer drinking breaks, and fewer sips (p < 0.0001). Considering the drinking episodes, respectively, water intake and drinking breaks in number and duration, the number of sips and the number of agonistic behaviors might optimize dairy cow water supply and hygiene management.

Exposure of a single wild boar population in North Rhine-Westphalia (Germany) to perfluoroalkyl acids.

Perfluoroalkyl acids (PFAA) are among the leading chemical pollutants in the twenty-first century. Of these, perfluorooctanoic acid (PFOA) and perfluorooctanesulfonic acid (PFOS) have been widely detected in a large number of animal and environmental samples. Wild boars accumulate PFAA in their livers, but it has not yet been clarified to what extent wild boars of the same population accumulate different PFAA in their livers or whether any conclusions can be drawn from any differences found in regard to environmental contamination. In this study, liver samples from wild boars killed during driven hunts in 2019 and 2020 from a defined forest area in North Rhine-Westfalia, Germany were analyzed for 13 different PFAA. A mean load of 493 µg/kg (± 168 µg/kg) PFAA was measured in 2020. Perfluorosulfonic acids accounted for 87% of the total load in both years, with PFOS dominating this group. These results were similar to those of 14 liver samples collected from other regions of Germany for comparison. In addition, the livers of hunted pregnant sows and fetuses were examined. The load of short-chain perfluorocarboxylic acids (< C8) in the fetus liver was as high as that of the sows, whereas the concentrations of long-chain perfluorocarboxylic acids (≥ C8) were lower than in the dams. This result shows for the first time that fetuses take up PFAA from their mothers in utero. Our study shows that PFAA content in wild boar livers is comparably high in all animals in a local population and indicates a need for further research regarding a nationwide background exposure to PFAA in wild boars and their surrounding environment.

A risk-oriented evaluation of biofilm and other influencing factors on biological quality of drinking water for dairy cows.

Despite the importance of livestock drinking water quality on animal physiology, welfare, and performance, influences such as biofilm formation on trough surfaces on microbial water quality are rarely researched. The objective of this study was to assess the microbial quality of water offered to lactating dairy cows and identify risk factors for poor water quality. We further aimed to determine the impact of biofilm formation on water quality and evaluate rapid test systems to score the hygiene status of dairy troughs on the farm. A total of 105 troughs located on 24 typical Western German dairy farms were sampled. Samples of livestock drinking water and biofilm were analyzed for aerobic total viable count (TVC), coliform count (CC), Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), and other bacteria resistant to 3rd generation cephalosporins (CRB). Surface protein- and adenosine triphosphate (ATP)-rapid tests were evaluated to detect biofilm formation. The influence of 22 selected fixed and variable trough characteristics on impaired livestock drinking water quality was evaluated by calculating odds ratios. The average TVC, CC, and E. coli counts were 4.4 ±â€…0.06 (mean ±â€…SD), 1.7 ±â€…0.1, and 0.6 ±â€…0.1 log10 cfu per mL, respectively. CC was detectable in 94.3% of all water samples and E. coli in 48.6%. MRSA was found in pooled livestock drinking water samples of a single farm and CRB on three farms, suggesting that troughs might function as a reservoir of antibiotic-resistant bacteria, thereby contributing to an exchange of antibiotic-resistant bacteria between animals. Risk factors for the impairment of at least one microbial quality criteria (TVC, CC, or E. coli) increased significantly (P < 0.05) when using high-volume troughs, other trough materials than stainless steel, a lower distance to the milking parlor, heavy visible soiling, biofilm formation, and high ambient and high water temperatures. CC (r = 0.46; P < 0.001) and E. coli (r = 0.31; P < 0.01) of water samples correlated with their equivalent in biofilm and with the results of rapid tests on trough surfaces (0.31 > r > 0.19; P < 0.05). Addressing the identified risk factors could be an approach to ensure sufficient biological quality of livestock drinking water.

Although water is recognized as one of the most important nutrients for dairy cows, only limited research on its quality and potential risk factors for quality impairments is available. This study aimed to evaluate biological water quality on Western German dairy farms, identify potential risk factors for impairment, and evaluate rapid test systems to score the hygienic status of troughs. Water, biofilm, and trough surfaces of 105 water troughs on 24 dairy farms were sampled and analyzed. The bacterial load of sampled livestock drinking water was relatively high. Nearly half of all water samples were contaminated with Escherichia coli, and antibiotic-resistant bacteria were detectable on four farms. Identified risk factors for poor biological water quality that could be used to ensure sufficient water quality on-farm were: high-volume troughs, plastic or cast iron as trough material, a lower distance to the milking parlor, heavy visible soiling, biofilm formation, and high ambient and high water temperatures. Rapid test systems to evaluate trough surfaces correlated with biofilm and water analysis results and could be a useful tool for farmers to check water quality on-farm.

Slaughterhouse wastewater as a reservoir for extended-spectrum ß-lactamase (ESBL)-producing, and colistin-resistant Klebsiella spp. and their impact in a "One Health" perspective.

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 ß-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.

Influences on the assessment of resource- and animal-based welfare indicators in unweaned dairy calves for usage by farmers.

Consumers, industrial stakeholders, and the legislature demand a stronger focus on animal welfare of all livestock at the farm level by using suitable indicators in self-assessments. In order to deduce farms' animal welfare status reliably, factors that influence indicators' results need to be identified. Hence, this study aimed to apply possible animal welfare indicators for unweaned dairy calves on conventional dairy farms with early cow-calf separation and evaluate influencing factors such as age and sex of calves or climatic conditions on the applied indicators' results. An animal welfare assessment using 7 resource-based and 14 animal-based indicators was conducted at 42 typical Western German dairy farms (844 calves) in 2018 and 2019 by two observers. The effect of influencing factors was calculated by binary and ordinal logistic regressions and expressed as odds ratios. Although every unweaned calf was assessed during the farm visits, most farms had relatively few unweaned calves (average number of calves ± standard deviation = 20.1 ± 6.7 calves), with six farms having not more than 10 calves. The small sample sizes question the usage of those indicators to compare between farms and to set thresholds at the farm level. Only one assessed indicator (cleanliness core body) was not statistically affected by the evaluated influencing factors. Calf age was identified as the most decisive factor, as it affected 16 of 21 evaluated indicators, and calf age distribution on-farm varied greatly. Climatic conditions (ambient temperature and rainfall) influenced resource-based indicators such as access to concentrate and water or the cleanliness of feeding implements and bedding as well as animal-based cleanliness indicators and the occurrence of health-related impairments such as coughing and diarrhea. The authors found differences between calves on farms assessed by the different observers not only in resource-based hygiene indicators but also in animal-based indicators such as hyperthermia or hypothermia, highlighting the need for further evaluation of quality criteria in dairy calf welfare assessments. Nevertheless, animal welfare assessments by farmers themselves could be useful tools to sensitize farmers to animal welfare and thereby improve calves' welfare.

Survey on storage, application and incorporation practices for organic fertilizers in Germany.

Organic fertilizers from animal production might contain undesirable components, such as veterinary medical product (VMP) residues, that are released into the environment during application. In addition to measures to reduce the use of VMPs through animal health measures, manure management could be an expedient strategy to prevent VMPs from entering the environment. The quantity applied is mainly determined by the nitrogen content. In addition, the depth of incorporation into the soil plays a major role in the environmental risk assessment of VMPs. The new regulations of the German fertilizer ordinance (DüV, 2020), which came into force at the beginning of 2020, as well as the changes that have not yet been fully implemented, will result in adjustments to the storage, application and incorporation practices for organic fertilizer. The aim of this study was to gain more information about the practice of storage, application and incorporation and the challenges for farmers in Germany. An online survey among farmers was conducted to determine the status quo. Almost all of the 125 participants kept livestock, predominantly cattle (68%) and pigs (33%). A third of participants (30%) needed a temporary storage site, for example at neighboring farms. Of the participants, 81% (n = 125) owned cropland and/or grassland. On cropland, manure was mostly incorporated at a depth of 3-15 cm, whereas on grassland, it was mostly applied superficially. On grassland lower-emission application techniques such as slot drill or injector procedures have so far rarely been used. The survey results provided important insights about storage, application and incorporation practices for organic fertilizers in Germany which could be used for the calculation of predicted environmental concentrations (PEC) as part of the environmental risk assessments of veterinary pharmaceuticals.

Implementation of management recommendations in unweaned dairy calves in western Germany and associated challenges.

To improve the welfare of livestock, it is important to assess management practices on farms and to identify areas where current scientific recommendations are rarely implemented. Differences in the implementation of recommendations might be explained by the individual farm as well as the characteristics of survey respondents and their attitude toward animal welfare. Hence, the aim of this study was to assess dairy calf management practices, compare them with current scientific recommendations, and to explore factors that influence implementation of the recommended management practices. A 1.5-h interview was performed with stockpersons on 42 dairy farms (mean herd size ± SD = 149.9 ± 16.6 cows) distributed across western Germany in 2018 to 2019. We observed that the management of unweaned calves varied greatly from farm to farm in aspects such as milk-feeding protocols; timing of grouping and disbudding; and access to water, roughage, and concentrate. Major deviations from management recommendations were (1) cleaning calving pen only by removal of bedding without a following disinfection before restocking on 23.8% farms, cleaning of teat buckets without detergents and disinfection (23.8 and 11.9% of farms, respectively), and failure to disinfect navels (29.3% of farms); (2) separating calf and dam after only 5 to 8 h postpartum for calving at night in 97.6% farms and unchecked colostrum quality by 23.8% of survey respondents; (3) feeding waste milk by 72.4% of the farms surveyed; and (4) removing supernumerary teats and disbudding without local anesthesia (90 and 80% farms surveyed, respectively). The number of implemented recommendations on the farms surveyed correlated with who was responsible for calf rearing, and whether prioritizing animal welfare was considered important by the respondents. This study indicated that limitations of individual housing systems, time management, the stockperson's knowledge, and the stockperson's ability to relate to animals posed challenges in implementation of the recommendations. Further research on challenges in calf management and how to overcome them would be helpful to improve calves' welfare in current husbandry systems.

Impact of tearing spermatic cords during castration in live and dead piglets and consequences on welfare.

BACKGROUND: Although the tearing of tissues during castration is forbidden in the EU, it is still routinely applied in many countries. The goal of this study was to evaluate vocalizations and movements of male piglets undergoing castration by applying different techniques and pain treatments based on scores under practical conditions (Trial 1, n = 50) and to investigate anatomical features after castration of dead piglets with different techniques (Trial 2, n = 28). RESULTS: In Trial 1, treatment groups did not significantly influence the duration of castration. Both the duration of vocalization and the scores for vocalizations and movements were lower in piglets castrated under general anesthesia (P < 0.05). Behavior scores in conscious piglets did not differ. The incision and extraction caused lower vocalizations and movements than the pulling and severing of spermatic cords (P < 0.01). Movements were more intense during tearing of the spermatic cords than during cutting at the first and second severing (P < 0.01). In both trials, the remains of spermatic cords protruded tendentially more often from castration wounds after severing by tearing (P < 0.09). In Trial 2, the minimum, mean and maximum lengths of the testicles and spermatic cords were extended when severing was realized by tearing (P < 0.01). The mean relative testicle weight of 1.05‰ in dead piglets castrated by tearing was larger than that in dead piglets castrated by cutting (0.91‰) (P < 0.05). CONCLUSIONS: The trials uncovered significant differences between behavior expressed by piglets castrated by tearing or cutting, indicating a higher pain level in the tearing group. It was found that the castration technique tearing increased the amount of removed tissues and might cause intraabdominal damage to the remaining tissues and vessels in a yet unknown dimension. These findings should be considered for implementation and stricter enforcement of the ban on tearing for castration.

Hygiene management in newborn individually housed dairy calves focusing on housing and feeding practices.

In calf rearing, the first weeks of life are critical and associated with the highest mortality due to enteric and respiratory diseases. A well-implemented hygiene management can help to protect calves' health preventively by reducing the load of pathogenic bacteria and interrupting infection chains. The aim of this study was to identify deficiencies in hygiene management of individually housed dairy calves by surveying current practice and examining feeding and housing equipment with different hygiene indicators. On 11 farms, different locations in 2 pens or hutches for individual calf rearing prepared for restocking and 2 feeding buckets per farm, including the inner and outer surfaces of artificial teats, were visually scored for cleanliness and sampled with swabs (housing equipment: n = 167; feeding equipment: n = 120). The sanitation of floors was tested with sock samples (n = 41). A total of 328 samples were analyzed for adenosine triphosphate (ATP) and protein residues, aerobic total viable count (TVC), total coliform count (TCC), Escherichia coli, methicillin-resistant Staphylococcus aureus (MRSA), extended-spectrum ß-lactamase-producing bacteria (ESBL), and Salmonella spp. After evaluation of these results, the farmers were informed about the findings and trained on improvement in hygiene management personally. The sampling was repeated after 1 year to detect possible changes in hygiene management. The highest bacterial loads (TVC, TCC, and E. coli) were observed in feeding equipment, especially the inner teat of milk feeding buckets. Environmental samples, primarily the sidewalls and back walls of tested pens and hutches, exhibited the lowest bacterial counts and ATP and protein residues. All samples were negative for MRSA and Salmonella spp. In 10.5% of all samples, ESBL was detected, and in 6.8%, ESBL E. coli was detected, predominately in sock samples, followed by feeding equipment samples. Training in hygiene management showed only limited effects. In conclusion, there is still great potential to improve the implementation of hygiene measures in individual calf housing. In particular, more attention should be paid to the cleaning of feeding buckets and artificial teats, as this is a simple means of interrupting the possible spread of pathogens among calves.

Colistin-Resistant Enterobacteriaceae Isolated From Process Waters and Wastewater From German Poultry and Pig Slaughterhouses.

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.

Research Note: Tracing pathways of entry and persistence of facultative pathogenic and antibiotic-resistant bacteria in a commercial broiler farm with substantial health problems.

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.

Antibiotic-resistant bacteria and antimicrobial residues in wastewater and process water from German pig slaughterhouses and their receiving municipal wastewater treatment plants.

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 ß-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 ß-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.

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

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-ß-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 ß-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., blaCTX-M or blaSHV 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.

Individual training for farmers based on results from protein and ATP rapid tests and microbiological conventional cultural methods improves hygiene in pig fattening pens.

Optimal hygiene management is an essential part of maintaining a high standard of health in conventional pig production systems and for the successful interruption of infection chains. Currently, efficiency assessments on cleaning and disinfection are only performed by visual inspection or are neglected completely. The aim of this study was to evaluate the available methods for on farm monitoring of hygiene, identify critical points in pig pens and use the data obtained for training purposes. In addition to visual inspection by assessing the cleanliness, microbiological swab samples, i.e., aerobic total viable count (TVC), total coliform count, methicillin-resistant Staphylococcus aureus (MRSA), and extended-spectrum ß-lactamases-producing bacteria (ESBL), swab samples for ATP as well as protein residues and agar contact plates combined with 3 different culture media, were applied and ranked according to their suitability for livestock farming. Samples were collected on at least 15 critical points from one representative pen on 6 pig fattening farms with various hygiene management practices after cleaning and disinfection. After the first sampling, farmers were trained with their individual results, and sampling was repeated 6 mo after training. Nipple drinkers, feeding tubes (external and inner surface), and troughs (external and inner surface) showed the greatest bacterial loads (TVC: 4.5-6.7 log10 cfu cm-2) and values for ATP and protein residues; therefore, these surfaces could be identified as the most important critical points. Spearman rank correlations (P < 0.01) were found between the different assessment methods, especially for the TVC and ATP (r = 0.82, P < 0.001). For rapid assessment on farms, ATP tests represented an accurate and cost-efficient alternative to microbiological techniques. Training improved cleaning performance as indicated by a lower rating for visual inspection, TVC, ATP, MRSA, and ESBL in the second assessment. The monitoring of cleaning efficiency in pig pens followed by training of the staff constitutes a valuable strategy to limit the spread of infectious diseases and antibiotic-resistant bacteria. Special attention should be paid to the sufficient hygiene of drinkers and feeders.

Effect of methionine supplementation in chicken feed on the quality and shelf life of fresh poultry meat.

The aim of this study was to investigate the influence of different methionine sources and concentrations on the quality and spoilage process of broiler meat. The trial was comprised of 7 treatment groups: one basal group (suboptimal in Methionine+Cysteine; i.e., 0.89, 0.74, 0.69% in DM SID Met+Cys in starter, grower, and finisher diets, respectively) and 3 doses (0.10, 0.25, and 0.40%) of either DL-Methionine (DLM) or DL-2-hydroxy-4-methylthio butanoic acid (DL-HMTBA) on an equimolar basis of the DLM-supplemented groups. The broilers were fed the diets for 35 d, then slaughtered and processed. The filets were aerobically packed and stored under temperature controlled conditions at 4°C. Meat quality investigations were comprised of microbial investigations (total viable count and Pseudomonas spp.), pH and drip loss measurements of the filets. The shelf life of the meat samples was determined based on sensory parameters. After slaughtering, all supplemented meat samples showed a high quality, whereby no differences between the 2 methionine sources could be detected for the microbial load, pH, and drip loss. In comparison to the control group, the supplemented samples showed a higher sensory quality, characterized by a fresh smell and fresh red color. Methionine supplementation had a significant influence on meat quality parameters during storage. The microbial load, pH and drip loss of the chicken filets were positively correlated to the methionine concentration. Additionally, the microbial load at the end of storage was positively correlated to pH and drip loss values. Nevertheless, the microbial parameters were in a normal range and the positive correlation to methionine concentration did not affect the sensory shelf life. The mean sensory shelf life of the broiler filets varied between 7 to 9 d. During storage, no difference in the development of sensory parameters was observed between the supplemented groups, while the spoilage process of the basal group occurred slightly faster. In conclusion, methionine concentration, but not methionine source, effected meat quality parameters in breast muscles of broilers.