Low dose colonization of broiler chickens with ESBL-/AmpC- producing escherichia coli in a seeder-bird model independent of antimicrobial selection pressure

Extended-spectrum beta-lactamase- (ESBL-) and AmpC beta-lactamase- (AmpC-) producing Enterobacteriaceae pose a risk for both human and animal health. For livestock, highest prevalences have been reported in broiler chickens, which are therefore considered as a reservoir of multidrug-resistant bacteria. The possibility of transfer to humans either by a close contact to colonized broiler flocks or through contaminated retail meat results in the necessity to develop intervention measures for the entire broiler production chain. In this regard, a basic understanding of the colonization process is mandatory including the determination of the minimal bacterial load leading to a persistent colonization of broiler chickens. Therefore, we conducted a bivalent broiler colonization study close to real farming conditions without applying any antimicrobial selection pressure. ESBL- and AmpC- negative broiler chickens (Ross 308) were co- colonized on their third day of life with two strains: one CTX-M-15-producing Escherichia coli-ST410 and one CMY-2/mcr-1-positive E. coli-ST10. Colonization was assessed by cloacal swabs over the period of the trial, starting 24 h post inoculation. During the final necropsy, the contents of crop, jejunum, cecum, and colon were quantified for the occurrence of both bacterial strains. To define the minimal oral colonization dosage 104 to 101 colony forming units (cfu) were orally inoculated to four separately housed broiler groups (each n = 19, all animals inoculated) and a dosage of already 101 cfu E. coli led to a persistent colonization of all animals of the group after 3 days. To assure stable colonization, however, a dosage of 102 cfu E. coli was chosen for the subsequent seeder-bird trial. In the seeder-bird trial one fifth of the animals (seeder, n = 4) were orally inoculated and kept together with the non-inoculated animals (sentinel, n = 16) to mimic the route of natural infection. After 35 days of trial, all animals were colonized with both E. coli strains. Given the low colonization dosage and the low seeder/sentinel ratio, the rapid spread of ESBL- and AmpC- producing Enterobacteriaceae in conventional broiler farms currently seems inevitably resulting in an urgent need for the development of intervention strategies to reduce colonization of broilers during production

Impact of different management measures on the colonization of broiler chickens with ESBL- and pAmpC- producing Escherichia coli in an experimental seeder-bird model

The colonization of broilers with extended-spectrum beta-lactamase- (ESBL-) and plasmid-mediated AmpC beta-lactamase- (pAmpC-) producing Enterobacteriaceae has been extensively studied. However, only limited data on intervention strategies to reduce the colonization throughout the fattening period are available. To investigate practically relevant management measures for their potential to reduce colonization, a recently published seeder-bird colonization model was used. Groups of 90 broilers (breed Ross 308) were housed in pens under conventional conditions (stocking of 39 kg/m(2), no enrichment, water and feed ad libitum). Tested measures were investigated in separate trials and included (I) an increased amount of litter in the pen, (II) the reduction of stocking density to 25 kg/m(2), and (III) the use of an alternative broiler breed (Rowan x Ranger). One-fifth of ESBL- and pAmpC- negative broilers (n = 18) per group were orally co-inoculated with two E. coli strains on the third day of the trial (seeder). One CTX-M-15-positive E. coli strain (ST410) and one CMY-2 and mcr-1-positive E. coli strain (ST10) were simultaneously administered in a dosage of 10(2) cfu. Colonization of all seeders and 28 non-inoculated broilers (sentinel) was assessed via cloacal swabs during the trials and a final necropsy at a target weight of two kilograms (= d 36 (control, I-II), d 47 (III)). None of the applied intervention measures reduced the colonization of the broilers with both the ESBL- and the pAmpC- producing E. coli strains. A strain-dependent reduction of colonization for the ESBL- producing E. coli strain of ST410 by 2 log units was apparent by the reduction of stocking density to 25 kg/m(2). Consequently, the tested management measures had a negligible effect on the ESBL- and pAmpC- colonization of broilers. Therefore, intervention strategies should focus on the prevention of ESBL- and pAmpC- colonization, rather than an attempt to reduce an already existing colonization

Survival of highly related ESBL- and pAmpC- producing Escherichia coli in broiler farms identified before and after cleaning and disinfection using cgMLST

Background Broiler chickens are frequently colonized with Extended-Spectrum Beta-Lactamase- (ESBL-) and plasmid mediated AmpC Beta-Lactamase- (pAmpC-) producing Enterobacterales, and we are confronted with the potential spread of these resistant bacteria in the food chain, in the environment, and to humans. Research focused on identifying of transmission routes and investigating potential intervention measures against ESBL- and pAmpC- producing bacteria in the broiler production chain. However, few data are available on the effects of cleaning and disinfection (C&D) procedures in broiler stables on ESBL- and pAmpC- producing bacteria. Results We systematically investigated five broiler stables before and after C&D and identified potential ESBL- and pAmpC- colonization sites after C&D in the broiler stables, including the anteroom and the nearby surrounding environment of the broiler stables. Phenotypically resistant E. coli isolates grown on MacConkey agar with cefotaxime were further analyzed for their beta-lactam resistance genes and phylogenetic groups, as well as the relation of isolates from the investigated stables before and after C&D by whole genome sequencing. Survival of ESBL- and pAmpC- producing E. coli is highly likely at sites where C&D was not performed or where insufficient cleaning was performed prior to disinfection. For the first time, we showed highly related ESBL-/pAmpC- producing E. coli isolates detected before and after C&D in four of five broiler stables examined with cgMLST. Survival of resistant isolates in investigated broiler stables as well as transmission of resistant isolates from broiler stables to the anteroom and surrounding environment and between broiler farms was shown. In addition, enterococci (frequently utilized to detect fecal contamination and for C&D control) can be used as an indicator bacterium for the detection of ESBL-/pAmpC- E. coli after C&D. Conclusion We conclude that C&D can reduce ESBL-/pAmpC- producing E. coli in conventional broiler stables, but complete ESBL- and pAmpC- elimination does not seem to be possible in practice as several factors influence the C&D outcome (e.g. broiler stable condition, ESBL-/pAmpC- status prior to C&D, C&D procedures used, and biosecurity measures on the farm). A multifactorial approach, combining various hygiene- and management measures, is needed to reduce ESBL-/pAmpC- E. coli in broiler farms

The therapy frequency of antibiotics and phenotypical resistance of Escherichia coli in calf rearing sites in Germany

Introduction The association between antibiotic use and the occurrence of resistant bacteria is a global health problem and is subject to enormous efforts at national and international levels. Within the scope of the study “KAbMon”, the resistance situation as well as the use of antibiotics in calf rearing farms in Germany was investigated. We hypothesized that the levels of resistance are associated with certain calf keeping farm types, such as pre-weaned calf farms, animal groups, and therapy frequency. Methods In total, 95 calf keeping farms were visited between October 2019 and April 2021. At each farm, up to three pooled fecal samples (10 freshly released feces each) were collected. One sample was taken in the youngest calf group, another in the oldest calf group, and one in the hospital box, if available. Escherichia coli was isolated from non-selective MacConkey agar. The therapy frequency reflects the average number of treatment days per calf in a half-year, while the resistance score is the sum of the relative minimum inhibitory concentration per substance over all 10 tested substances. Results The 1781 isolates from 178 samples showed high resistance rates against sulfamethoxazole (82%), tetracycline (49%), and ampicillin (40%). High resistance scores were mainly found in pre-weaned calf farms (purchasing calves from 2  weeks of life) and in the youngest animals. The therapy frequency showed an almost linear relationship with the resistance scores, and the age at purchase was negatively related to the resistance score. Discussion The high use of antimicrobials of young calves might be associated with a high risk for infectious diseases and might indicate that the current system of crowding 14-day-old calves from different farms in one group is not optimal. Further efforts are necessary to educate and motivate the calf keepers to ensure highest levels of hygiene and management as well as animal welfare conditions and to increase animal health

Genetic but No Phenotypic Associations between Biocide Tolerance and Antibiotic Resistance in Escherichia coli from German Broiler Fattening Farms

Biocides are frequently applied as disinfectants in animal husbandry to prevent the transmission of drug-resistant bacteria and to control zoonotic diseases. Concerns have been raised, that their use may contribute to the selection and persistence of antimicrobial-resistant bacteria. Especially, extended-spectrum β-lactamase- and AmpC β-lactamase-producing Escherichia coli have become a global health threat. In our study, 29 ESBL-/AmpC-producing and 64 NON-ESBL-/AmpC-producing E.coli isolates from three German broiler fattening farms collected in 2016 following regular cleaning and disinfection were phylogenetically characterized by whole genome sequencing, analyzed for phylogenetic distribution of virulence-associated genes, and screened for determinants of and associations between biocide tolerance and antibiotic resistance. Of the 30 known and two unknown sequence types detected, ST117 and ST297 were the most common genotypes. These STs are recognized worldwide as pandemic lineages causing disease in humans and poultry. Virulence determinants associated with extraintestinal pathogenic E.coli showed variable phylogenetic distribution patterns. Isolates with reduced biocide susceptibility were rarely found on the tested farms. Nine isolates displayed elevated MICs and/or MBCs of formaldehyde, chlorocresol, peroxyacetic acid, or benzalkonium chloride. Antibiotic resistance to ampicillin, trimethoprim, and sulfamethoxazole was most prevalent. The majority of ESBL-/AmpC-producing isolates carried blaCTX-M (55%) or blaCMY-2 (24%) genes. Phenotypic biocide tolerance and antibiotic resistance were not interlinked. However, biocide and metal resistance determinants were found on mobile genetic elements together with antibiotic resistance genes raising concerns that biocides used in the food industry may lead to selection pressure for strains carrying acquired resistance determinants to different antimicrobials

Impact of On-Farm Interventions against CTX-Resistant Escherichia coli on the Contamination of Carcasses before and during an Experimental Slaughter

Cefotaxime (CTX)-resistant Enterobacteriaceae are still an ongoing challenge in human and veterinary health. High prevalence of these resistant bacteria is detected in broiler chickens and the prevention of their dissemination along the production pyramid is of major concern. The impact of certain on-farm interventions on the external bacterial contamination of broiler chickens, as well as their influence on single processing steps and (cross-) contamination, have not yet been evaluated. Therefore, we investigated breast skin swab samples of broiler chickens before and during slaughter at an experimental slaughter facility. Broiler chickens were previously challenged with CTX-resistant Escherichia coli strains in a seeder-bird model and subjected to none (control group (CG)) or four different on-farm interventions: drinking water supplementation based on organic acids (DW), slow growing breed Rowan × Ranger (RR), reduced stocking density (25 kg/sqm) and competitive exclusion with Enterobacteriales strain IHIT36098(CE). Chickens of RR, 25 kg/sqm, and CE showed significant reductions of the external contamination compared to CG. The evaluation of a visual scoring system indicated that wet and dirty broiler chickens are more likely a vehicle for the dissemination of CTX-resistant and total Enterobacteriaceae into the slaughterhouses and contribute to higher rates of (cross-) contamination during processing

Managementmaßnahmen zur Reduktion der Prävalenz ESBL-/pAmpC- bildender Enterobakterien beim Masthähnchen

Extended-spectrum beta-lactamase (ESBL) and plasmid-mediated AmpC beta-lactamase (pAmpC) producing bacteria are frequently detected in the broiler production chain. Investigations revealed a high prevalence of ESBL- and pAmpC- producing bacteria throughout the broiler fattening process up to the slaughterhouse level and consumer goods and revealed several transmission routes of these antibiotic-resistant bacteria. Insights into the colonization dynamics of broiler chickens and possible intervention measures are needed as the transmission of ESBL- and pAmpC- producing bacteria to humans via close contact with livestock or the consumption of contaminated meat is assumed. This thesis aimed to determine the minimal bacterial count necessary to colonize broiler chickens with ESBL- and pAmpC- producing Escherichia coli (E. coli) and to establish a broiler chicken colonization model (seeder-bird) close to real farming conditions without applying any antimicrobial selection pressure. Subsequently, we aimed to evaluate distinct intervention measures on their potential to reduce the colonization of broiler chickens with ESBL- and pAmpC- producing E. coli throughout the fattening process. For the determination of the minimal bacterial count, ESBL- and pAmpC- negative day-old broiler chickens (Ross 308) were kept under conventional conditions and were orally co-inoculated on day three of the trial with 10^4,10^3, 10^2, or 10^1 colony forming units (cfu) of one ESBL- (CTX-M-15) and one pAmpC- (CMY-2) producing E. coli strain in separate trials. All investigated bacterial counts led to the colonization of all broiler chickens in the trials, with all broiler chickens tested positive after 24 h (10^4-10^2 cfu) or 72 h (10^1 cfu) post inoculation (p.i.) up to the end of each trial. At necropsy (14 d p.i.), the cecal colonization with the ESBL- and pAmpC- producing E. coli strains of all investigated bacterial counts showed equivalence in the statistical analysis. To assure stable colonization, the bacterial count of 10^2 cfu ESBL- and pAmpC- producing E. coli was chosen to establish the seeder-bird model. An inoculation of one-fifth of the day-old broiler chickens with 10^2 cfu E. coli led to the colonization of all inoculated broiler chickens (seeder-birds) after 24 h p.i. and all non-inoculated broiler chickens (sentinel-birds) after 72 h p.i. up to the end of the trial. At necropsy (35 d p.i.) no significant differences in the cecal colonization with the ESBL- and pAmpC producing E. coli strains was apparent between the seeder-birds or sentinel-birds. Distinct intervention measures were subsequently investigated on their potential to reduce the colonization of broiler chickens with ESBL- and pAmpC- producing E. coli throughout the fattening process using the established seeder-bird model. Applying a complex, non-defined Competitive Exclusion-culture led to a significant reduction of both the ESBL- and pAmpC- producing E. coli strain in our trial. Applying a single CE-strain or reducing the stocking density to 25 kg/sqm led to a strain-dependent reduction of the ESBL- E. coli strain but had no impact on the colonization with the pAmpC- E. coli strain. No effect on the colonization of broiler chickens with ESBL- and pAmpC- producing E. coli was evident for the use of an alternative, slower-growing broiler breed (Rowan x Ranger). A negative effect on the colonization of broiler chickens with ESBL- and pAmpC- producing E. coli was shown for the application of a tripled amount of litter in the pen and the acidification of the drinking water with a commercially available product. The minimal bacterial counts of 10^1 to 10^2 cfu ESBL- and pAmpC- producing E. coli highlight the need for effective intervention measures to reduce the broiler chickens’ colonization with ESBL- and pAmpC- producing bacteria including improved biosecurity- and disinfection measures. A promising approach is the application of a Competitive Exclusion-culture, whereas other separately applied management measures were not capable of reducing the broiler chickens’ colonization with both the ESBL- and pAmpC- producing E. coli strains. It needs further investigations whether a combination of different measures can contribute to a reduced colonization of the broiler chickens’ with ESBL- and pAmpC- producing bacteria.Extended-Spektrum Beta-Laktamase (ESBL) und plasmid-vermittelte AmpC Beta-Laktamase (pAmpC) produzierende Bakterien können häufig in der Masthähnchenproduktionskette nachgewiesen werden. Untersuchungen zeigten hohe Prävalenzen und identifizierten verschiedenste Transmissionswege sowohl im Mastprozess und auf Schlachthofebene als auch auf Hähnchenfleisch-Produkten. Da ein Übertrag auf den Menschen durch einen engen Kontakt zu Nutztieren oder durch den Verzehr von kontaminiertem Fleisch angenommen wird, sind Erkenntnisse über die Kolonisierungsdynamiken von Masthähnchen und über mögliche Interventionsmaßnahmen erforderlich. Die vorliegende Dissertationsschrift hatte zum Ziel, die geringste erforderliche Bakterienzahl an ESBL- und pAmpC- produzierenden Escherichia coli (E. coli) für eine Kolonisierung von Masthähnchen zu ermitteln und ein praxisnahes Kolonisierungsmodell (Seeder-Bird) zu entwickeln, ohne dabei einen antimikrobiellen Selektionsdruck auszuüben. Ein weiteres Ziel der vorliegenden Arbeit war die Evaluation verschiedener Interventionsmaßnahmen, um die Kolonisierung von Masthähnchen mit ESBL- und pAmpC- produzierenden E. coli während des Mastprozesses zu reduzieren. Für die Ermittlung der geringsten erforderlichen Bakterienzahl wurden ESBL- und pAmpC- negative Eintagsküken (Ross 308) konventionell aufgestallt und in separaten Versuchen jeweils am dritten Versuchstag mit einem ESBL- (CTX-M-15) und einem pAmpC- (CMY-2) produzierenden E. coli Stamm bivalent kolonisiert. Alle untersuchten Keimzahlen von 10^4,10^3, 10^2 und 10^1 koloniebildenden Einheiten (KbE) führten zu einer Kolonisierung aller Masthähnchen in den einzelnen Versuchen. Eine Kolonisierung aller Tiere war mittels Kloakentupfer 24 h (10^4-10^2 KbE E. coli) oder 72 h (10^1 KbE E. coli) nach Inokulation bis zum Versuchsende nachweisbar. Die am Versuchsende durchgeführte Sektion (14 d nach Inokulation) zeigte eine äquivalente zäkale Kolonisierung der ESBL- und pAmpC- produzierenden E. coli Stämme in allen Versuchsgruppen. Für eine sichere experimentelle Kolonisierung der Masthähnchen wurde bei der Etablierung des Seeder-Bird Kolonisierungsmodells eine Bakterienzahl von 10^2 KbE ESBL- und pAmpC- produzierende E. coli genutzt. Im Seeder-Bird Modell führte eine Inokulation von einem Fünftel der Eintagsküken mit 10^2 KbE E. coli nach 24 h zu einer Kolonisierung aller inokulierten Tiere (Seeder-Birds) und nach 72 h zu einer Kolonisierung aller nicht inokulierten Tiere (Sentinel-Birds). Im Folgenden war die Kolonisierung über den gesamten Versuchszeitraum nachweisbar. Zu Versuchsende (35 d nach Inokulation) war kein statistisch signifikanter Unterschied der zäkalen Kolonisierung von Seeder-Birds und Sentinel-Birds mit den ESBL- und pAmpC- produzierenden E. coli Stämmen nachweisbar. Das etablierte Seeder-Bird Modell wurden nachfolgend genutzt, um unterschiedliche Interventionsmaßnahmen auf ihr Potenzial einer Reduktion der ESBL- und pAmpC- Kolonisierung von Masthähnchen während des Mastprozessen hin zu untersuchen. Eine signifikante Reduktion beider eingesetzter E. coli Stämme war unter Anwendung einer nicht definierten, komplexen Competitive Exclusion-Kultur ersichtlich. Eine stammabhängige Reduktion des ESBL- E. coli Stammes, ohne einen Effekt auf die Kolonisierung der Masthähnchen mit dem pAmpC- E. coli Stamm, war sowohl unter der Applikation eines einzelnen CE-Stammes als auch bei der Reduktion der Besatzdichte auf 25 kg/qm nachweisbar. Keinen Effekt auf die Kolonisierung von Masthähnchen mit ESBL- und pAmpC- produzierenden E. coli zeigte sich beim Einsatz einer alternativen, langsam-wachsenden Rasse (Rowan x Ranger). Eine stärkere Kolonisierung von Masthähnchen mit ESBL- und pAmpC- produzierenden E. coli lag bei der Verdreifachung der Einstreumenge im Stall und bei der Ansäuerung des Tränkwassers mit einem kommerziell erwerblichen Produkt vor. Die sehr geringe Keimzahl von 10^1-10^2 KbE ESBL- und pAmpC- produzierender E. coli zeigt die Notwendigkeit von effektiven Interventionsmaßnahmen zur Reduktion der Kolonisierung von Masthähnchen mit ESBL- und pAmpC- produzierenden Bakterien, einschließlich verbesserter Biosicherheits- und Desinfektionsmaßnahmen. Ein vielversprechender Ansatz ist die Applikation von Competitive Exklusion-Kulturen. Alle anderen einzeln untersuchten Maßnahmen konnten die Kolonisierung mit jeweils beiden eingesetzten ESBL- und pAmpC- produzierenden E. coli Stämmen nicht reduzieren. Weitere Untersuchungen sind in diesem Kontext notwendig, um den Einfluss einer Kombination von Maßnahmen auf die Reduktion der ESBL- und pAmpC- Kolonisierung von Masthähnchen zu evaluieren

Survival of highly related ESBL- and pAmpC- producing Escherichia coli in broiler farms identified before and after cleaning and disinfection using cgMLST

Abstract Background Broiler chickens are frequently colonized with Extended-Spectrum Beta-Lactamase- (ESBL-) and plasmid mediated AmpC Beta-Lactamase- (pAmpC-) producing Enterobacterales, and we are confronted with the potential spread of these resistant bacteria in the food chain, in the environment, and to humans. Research focused on identifying of transmission routes and investigating potential intervention measures against ESBL- and pAmpC- producing bacteria in the broiler production chain. However, few data are available on the effects of cleaning and disinfection (C&D) procedures in broiler stables on ESBL- and pAmpC- producing bacteria. Results We systematically investigated five broiler stables before and after C&D and identified potential ESBL- and pAmpC- colonization sites after C&D in the broiler stables, including the anteroom and the nearby surrounding environment of the broiler stables. Phenotypically resistant E. coli isolates grown on MacConkey agar with cefotaxime were further analyzed for their beta-lactam resistance genes and phylogenetic groups, as well as the relation of isolates from the investigated stables before and after C&D by whole genome sequencing. Survival of ESBL- and pAmpC- producing E. coli is highly likely at sites where C&D was not performed or where insufficient cleaning was performed prior to disinfection. For the first time, we showed highly related ESBL-/pAmpC- producing E. coli isolates detected before and after C&D in four of five broiler stables examined with cgMLST. Survival of resistant isolates in investigated broiler stables as well as transmission of resistant isolates from broiler stables to the anteroom and surrounding environment and between broiler farms was shown. In addition, enterococci (frequently utilized to detect fecal contamination and for C&D control) can be used as an indicator bacterium for the detection of ESBL-/pAmpC- E. coli after C&D. Conclusion We conclude that C&D can reduce ESBL-/pAmpC- producing E. coli in conventional broiler stables, but complete ESBL- and pAmpC- elimination does not seem to be possible in practice as several factors influence the C&D outcome (e.g. broiler stable condition, ESBL-/pAmpC- status prior to C&D, C&D procedures used, and biosecurity measures on the farm). A multifactorial approach, combining various hygiene- and management measures, is needed to reduce ESBL-/pAmpC- E. coli in broiler farms

The therapy frequency of antibiotics and phenotypical resistance of Escherichia coli in calf rearing sites in Germany

IntroductionThe association between antibiotic use and the occurrence of resistant bacteria is a global health problem and is subject to enormous efforts at national and international levels. Within the scope of the study “KAbMon”, the resistance situation as well as the use of antibiotics in calf rearing farms in Germany was investigated. We hypothesized that the levels of resistance are associated with certain calf keeping farm types, such as pre-weaned calf farms, animal groups, and therapy frequency.MethodsIn total, 95 calf keeping farms were visited between October 2019 and April 2021. At each farm, up to three pooled fecal samples (10 freshly released feces each) were collected. One sample was taken in the youngest calf group, another in the oldest calf group, and one in the hospital box, if available. Escherichia coli was isolated from non-selective MacConkey agar. The therapy frequency reflects the average number of treatment days per calf in a half-year, while the resistance score is the sum of the relative minimum inhibitory concentration per substance over all 10 tested substances.ResultsThe 1781 isolates from 178 samples showed high resistance rates against sulfamethoxazole (82%), tetracycline (49%), and ampicillin (40%). High resistance scores were mainly found in pre-weaned calf farms (purchasing calves from 2  weeks of life) and in the youngest animals. The therapy frequency showed an almost linear relationship with the resistance scores, and the age at purchase was negatively related to the resistance score.DiscussionThe high use of antimicrobials of young calves might be associated with a high risk for infectious diseases and might indicate that the current system of crowding 14-day-old calves from different farms in one group is not optimal. Further efforts are necessary to educate and motivate the calf keepers to ensure highest levels of hygiene and management as well as animal welfare conditions and to increase animal health

Impact of On-Farm Interventions against CTX-Resistant Escherichia coli on the Contamination of Carcasses before and during an Experimental Slaughter

Cefotaxime (CTX)-resistant Enterobacteriaceae are still an ongoing challenge in human and veterinary health. High prevalence of these resistant bacteria is detected in broiler chickens and the prevention of their dissemination along the production pyramid is of major concern. The impact of certain on-farm interventions on the external bacterial contamination of broiler chickens, as well as their influence on single processing steps and (cross-) contamination, have not yet been evaluated. Therefore, we investigated breast skin swab samples of broiler chickens before and during slaughter at an experimental slaughter facility. Broiler chickens were previously challenged with CTX-resistant Escherichia coli strains in a seeder-bird model and subjected to none (control group (CG)) or four different on-farm interventions: drinking water supplementation based on organic acids (DW), slow growing breed Rowan × Ranger (RR), reduced stocking density (25 kg/sqm) and competitive exclusion with Enterobacteriales strain IHIT36098(CE). Chickens of RR, 25 kg/sqm, and CE showed significant reductions of the external contamination compared to CG. The evaluation of a visual scoring system indicated that wet and dirty broiler chickens are more likely a vehicle for the dissemination of CTX-resistant and total Enterobacteriaceae into the slaughterhouses and contribute to higher rates of (cross-) contamination during processing