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.

Impact of air humidity on the tenacity of different agents in bioaerosols.

Despite the variety of pathogens that are transmitted via the airborne route, few data are available on factors that influence the tenacity of airborne pathogens. In order to better understand and thus control airborne infections, knowledge of these factors is important. In this study, three agents, S. aureus, G. stearothermophilus spores and the MS2 bacteriophage, were aerosolized at relative humidities (RH) varying between 30% and 70%. Air samples were then analyzed to determine the concentration of the agents. S. aureus was found to have significantly lower survival rate in the aerosol at RH above 60%. It showed the lowest recovery rates of the three agents, ranging from 0.13% at approximately 70% RH to 4.39% at 30% RH. G. stearothermophilus spores showed the highest tenacity with recovery rates ranging from 41.85% to 61.73% with little effect of RH. For the MS2 bacteriophage, a significantly lower tenacity in the aerosol was observed with a recovery rate of 4.24% for intermediate RH of approximately 50%. The results of this study confirm the significant influence of the RH on the tenacity of airborne microorganisms depending on the specific agent. These data show that the behavior of microorganism in bioaerosols is varies under different environmental conditions.

Stability of Feline Coronavirus in aerosols and dried in organic matrices on surfaces at various environmental conditions.

Enveloped respiratory viruses, including the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), can be transmitted through aerosols and contact with contaminated surfaces. The stability of these viruses outside the host significantly impacts their transmission dynamics and the spread of diseases. In this study, we investigated the tenacity of Feline Coronavirus (FCoV) in aerosols and on surfaces under varying environmental conditions. We found that airborne FCoV showed different stability depending on relative humidity (RH), with higher stability observed at low and high RH. Medium RH conditions (50-60%) were associated with increased loss of infectivity. Furthermore, FCoV remained infectious in the airborne state over 7 h. On stainless-steel surfaces, FCoV remained infectious for several months, with stability influenced by organic material and temperature. The presence of yeast extract and a temperature of 4 °C resulted in the longest maintenance of infectivity, with a 5 log10 reduction of the initial concentration after 167 days. At 20 °C, this reduction was achieved after 19 days. These findings highlight the potential risk of aerosol and contact transmission of respiratory viruses, especially in enclosed environments, over extended periods. Studying surrogate viruses like FCoV provides important insights into the behavior of zoonotic viruses like SARS-CoV-2 in the environment.

Detection of Viable but Non-Culturable (VBNC)-Campylobacter in the Environment of Broiler Farms: Innovative Insights Delivered by Propidium Monoazide (PMA)-v-qPCR Analysis.

Campylobacteriosis cases in humans are of global concern, with high prevalence rates in the poultry reservoir considered the most important source of infection. Research findings show Campylobacters' ability to enter a viable but non-culturable (VBNC) state, remaining "viable" but unable to grow on culture media. We explored the persistence of VBNC states in specific environments, particularly at broiler farms, as this state may lead to an underestimation of the present Campylobacter prevalence. For VBNC detection, a propidium monoazide PMA-dye viability qPCR (v-qPCR) was used in combination with cultivation methods. We examined samples collected from broiler farm barns and their surroundings, as well as chicken manure from experimental pens. In addition, the tenacity of culturable and VBNC-Campylobacter was studied in vitro in soil and water. In a total of three visits, Campylobacter was not detected either culturally or by v-qPCR (no Campylobacter DNA) in the environment of the broiler farms. In four visits, however, VBNC-Campylobacter were detected both inside and outside the barns. The overall prevalence in environmental samples was 15.9% for VBNC-Campylobacter, 62.2% for Campylobacter DNA, and 1.2% for culturable C. jejuni. In the experimental pens, no cultivable C. jejuni was detected in chicken manure after 24 h. Strikingly, "VBNC-Campylobacter" persisted even after 72 h. "VBNC-Campylobacter" were confirmed in barn surroundings and naturally contaminated chicken manure. Laboratory studies revealed that VBNC-Campylobacter can remain intact in soil for up to 28 days and in water for at least 63 days, depending on environmental conditions.

Bacteriophage cocktail application for Campylobacter mitigation - from in vitro to in vivo.

BACKGROUND: Effective strategies are urgently needed to control Campylobacteriosis, one of the most important foodborne gastrointestinal diseases worldwide. Administering bacteriophages (phages) is under evaluation as a possible intervention strategy in primary poultry production to reduce the public health risk of human infection. A major challenge is the translation of results from small-scale animal studies to large broiler flocks. In this study, the in vitro lytic activity of 18 Campylobacter-specific group II phages and 19 group III phages were examined singly, and in different combinations from the same group and from both groups using a planktonic killing assay. Based on these results, a combination of phage NCTC 12,673 (group III) and vB_CcM-LmqsCPL1/1 (group II) was selected for in vivo application in a seeder bird model to study its effectiveness under conditions as close as possible to field conditions. One hundred eighty Ross 308 broiler chickens were divided into a control and a treatment group. Ten days post hatch, seeder birds were orally inoculated with the C. jejuni target strain. Phages were administered via drinking water at a total concentration of 107 PFU/mL four, three, and two days before necropsy. RESULTS: Combining group II and group III phages resulted in significantly higher in vitro growth inhibition against the C. jejuni target strain BfR-CA-14,430 than single application or combinations of phages from the same group. The results of the animal trial showed that the application of the two phages significantly reduced Campylobacter counts in cloacal swabs. At necropsy, Campylobacter counts in colonic content of the treatment group were significantly reduced by 2 log10 units compared to the control group. CONCLUSIONS: We demonstrated that combining phages of groups II and III results in significantly increased lytic activities. The in vitro results were successfully translated into practical application in a study design close to field conditions, providing new data to apply phages in conventional broiler flocks in the future. Phage application reduced the fecal Campylobacter excretion and Campylobacter concentrations in the colon of broilers.

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.

Antimicrobial effect of a drinking water additive comprising four organic acids on Campylobacter load in broilers and monitoring of bacterial susceptibility.

Application of organic acids via feed or drinking water is under discussion as a possible intervention strategy to reduce Campylobacter (C.) load in primary poultry production. A previous in vitro study showed that reduced concentrations of sorbic acid, benzoic acid, propionic acid, and acetic acid were required for antibacterial activity against Campylobacter when using a mixture of these 4 acids compared to when using the single acids. The present study aimed at determining the antibacterial efficiency of this combination in vivo as a drinking water additive for reducing shedding and intestinal C. jejuni colonization in broilers. Furthermore, we assessed whether the inoculated C. jejuni strain BfR-CA-14430 adapted in vivo to the applied organic acids. Results of this study showed that adding the organic acids consistently reduced Campylobacter loads in cloacal swabs. While significant reductions were observed within the entire study period, a maximum 2 log reduction occurred at an age of 18 d. However, after dissection at the end of the trial, no significant differences were detected in Campylobacter loads of cecal and colon contents compared to the control group. Susceptibility testing of re-isolates from cloacal swabs and cecal content revealed equal minimum inhibitory concentration (MIC) values compared to the inoculated test strain, suggesting that C. jejuni remained susceptible throughout the trial.

Transmission pathways of campylobacter spp. at broiler farms and their environment in Brandenburg, Germany.

Broiler meat is widely known as an important source of foodborne Campylobacter jejuni and Campylobacter coli infections in humans. In this study, we thoroughly investigated transmission pathways that may contribute to possible Campylobacter contamination inside and outside broiler houses. For this purpose we carried out a comprehensive longitudinal sampling approach, using a semi-quantitative cultivation method to identify and quantify transmissions and reservoirs of Campylobacter spp.. Three german broiler farms in Brandenburg and their surrounding areas were intensively sampled, from April 2018 until September 2020. Consecutive fattening cycles and intervening downtimes after cleaning and disinfection were systematically sampled in summer and winter. To display the potential phylogeny of barn and environmental isolates, whole genome sequencing (WGS) and bioinformatic analyses were performed. Results obtained in this study showed very high Campylobacter prevalence in 51/76 pooled feces (67.1%) and 49/76 boot swabs (64.5%). Average counts between 6.4 to 8.36 log10MPN/g were detected in pooled feces. In addition, levels of 4.7 and 4.1 log10MPN/g were detected in boot swabs and litter, respectively. Samples from the barn interior showed mean Campyloacter values in swabs from drinkers 2.6 log10MPN/g, walls 2.0 log10MPN/g, troughs 1.7 log10MPN/g, boards 1.6 log10MPN/g, ventilations 0.9 log10MPN/g and 0.7 log10MPN/g for air samples. However, Campylobacter was detected only in 7/456 (1.5%) of the environmental samples (water bodies, puddles or water-filled wheel tracks; average of 0.6 log10MPN/g). Furthermore, WGS showed recurring Campylobacter genotypes over several consecutive fattening periods, indicating that Campylobacter genotypes persist in the environment during downtime periods. However, after cleaning and disinfection of the barns, we were unable to identify potential sources in the broiler houses. Interestingly, alternating Campylobacter genotypes were observed after each fattening period, also indicating sources of contamination from the wider environment outside the farm. Therefore, the results of this study suggest that a potential risk of Campylobacter transmission may originate from present environmental sources (litter and water reservoirs). However, the sources of Campylobacter transmission may vary depending on the operation and farm environmental conditions.

A Complex Competitive Exclusion Culture Reduces Campylobacter jejuni Colonization in Broiler Chickens at Slaughter Age In Vivo.

Diminishing Campylobacter prevalence in poultry flocks has proven to be extremely challenging. To date, efficacious control measures to reduce Campylobacter prevalence are still missing. A potential approach to control Campylobacter in modern poultry productions is to occupy its niche in the mucosal layer by administering live intestinal microbiota from adult chickens to dayold-chicks (competitive exclusion (CE)). Therefore, this in vivo study investigates the efficacy of a complex CE culture to reduce Campylobacter (C.) jejuni colonization in broiler chickens. For this purpose, the complex CE culture was applied twice: once by spray application to day-old chicks immediately after hatching (on the 1st day of life) and subsequently by an additional application via drinking water on the 25th day of life. We observed a consistent and statistically significant reduction of C. jejuni counts in cloacal swabs throughout the entire fattening period. At the end of the trial after necropsy (at 33 days of age), C. jejuni cecal counts also showed a statistically significant decrease of 1 log10 MPN/g compared to the control group. Likewise, colon counts were reduced by 2.0 log10 MPN/g. These results suggest that CE cultures can be considered a practically relevant control strategy to reduce C. jejuni colonization in broiler chickens on poultry farms.

Innovative Textiles Used in Face Masks: Filtration Efficiency and Self-Disinfecting Properties against Coronaviruses.

Within the current SARS-CoV-2 pandemic, personal protective equipment, including face masks, is one important tool to interrupt virus transmission chains within the community. In this context, the quality of different face masks is frequently discussed and should, therefore, be evaluated. In this study, nanofleece textiles with a particle filtering effect and textiles with a self-disinfecting treatment were examined, which may be combined in face masks. Firstly, newly developed nanofleece textiles were tested regarding their filtration efficiency against airborne coronavirus, using feline coronavirus (FCoV) as a surrogate for SARS-CoV-2. The tested nanofleece textiles showed filtration efficiencies of over 95% against FCoV when used as a double layer and were, therefore, almost on par with the FFP-2 mask material, which was used as a reference. Secondly, eight treated, self-disinfecting textiles, which may increase the safety in the handling of potentially contaminated masks, were tested against SARS-CoV-2. Three out of eight treated textiles showed significant activity against SARS-CoV-2 and achieved about three LOG10 (99.9%) of virus titer reduction after twelve hours of incubation. Since all possible transmission paths of SARS-CoV-2, as well as the minimal infection doses, remain unknown, both investigated approaches seem to be useful tools to lower the virus spread within the community.

Emission Sources of Campylobacter from Agricultural Farms, Impact on Environmental Contamination and Intervention Strategies.

Although extensive research has been carried out to describe the transmission pathways of Campylobacter entering livestock farms, the role of livestock farms as source of Campylobacter contamination of the environment is still poorly investigated. It is assumed that Campylobacter-positive livestock farms contribute to an environmental contamination, depending on the animal species on the farm, their Campylobacter status, the housing system, manure management as well as their general farm hygienic and biosecurity management. Different emission sources, like manure, air, water, insects and rodents as well as personnel, including equipment and vehicles, contribute to Campylobacter emission into the environment. Even though Campylobacter are rather fastidious bacteria, they are able to survive in the environment for even a longer period of time, when environmental conditions enable survival in specific niches. We conclude that a significant reduction of Campylobacter emission in the environment can be successfully achieved if various intervention strategies, depending on the farm type, are applied simultaneously, including proper general and personal hygiene, establishing of hygienic barriers, insect controls, manure management and hygienization of stables, barns and exhaust air.

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 ß-lactamase- (ESBL-) and plasmid-mediated AmpC ß-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/m2, 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/m2, 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 102 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/m2. 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.

Influence of Immune Status on the Airborne Colonization of Piglets with Methicillin-Resistant Staphylococcus aureus (MRSA) Clonal Complex (CC) 398.

Colonized vertebrates including humans and pigs are to date the main reservoirs of livestock-associated Methicillin-resistant Staphylococcus aureus (LA-MRSA). Currently, the mechanisms underlying colonization of pigs are not fully understood. We investigated the influence of piglet pre-immune status on airborne MRSA colonization. Three groups of MRSA-negative piglets were primed and exposed to airborne LA-MRSA (104 colony forming units (cfu)/m3) in an aerosol chamber for 24 h. One group was treated intramuscularly with dexamethasone (1 mg/kg body weight) to imitate weaning stress. The second group was exposed to bacterial endotoxin containing MRSA aerosol. Both conditions play a role in the development of multifactorial diseases and may promote MRSA colonization success. The third group served as control. The piglets' MRSA status was monitored for 21 days via swab samples. At necropsy, specific tissues and organs were analyzed. Blood was collected to examine specific immunological parameters. The duration of MRSA colonization was not extended in both treated groups compared to the control group, indicating the two immune-status influencing factors do not promote MRSA colonization. Blood sample analysis confirmed a mild dexamethasone-induced immune suppression and typical endotoxin-related changes in peripheral blood. Of note, the low-dose dexamethasone treatment showed a trend of increased MRSA clearance.

Bacteriophage amplification - A comparison of selected methods.

The bactericidal properties of bacteriophages have been used almost since the moment of the discovery of bacterial viruses. In the light of the rapidly growing number of antibiotic-resistant bacteria, phage therapy is considered one of the most promising alternatives to classical treatment. Phage amplification is one of the most common procedures of working with phages, and high-titer preparations are beneficial at the experimental stage of studies as well as in practice. The objective of this study was to compare five commonly applied methods of phage amplification: (i) pooled plaques method, (ii) the plate wash method, (iii) the agar culture method, (iv) the two-stage culture method, and (v) in liquid culture. All methods were tested for fifteen different phages. The results described herein indicate that there is no optimal, universal method for phage amplification, and the most effective method has to be established individually for each phage.

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.

ESBL-Producing Klebsiella pneumoniae in the Broiler Production Chain and the First Description of ST3128.

ESBL-producing Klebsiella pneumoniae (K. pneumoniae) represent an increasing problem both in human and veterinary medicine. As SHV-2 - encoding K. pneumoniae were recently detected in the broiler production we were interested in investigating a possible transmission along the broiler production chain and furthermore, in evaluating their possible impact on human health. Therefore, 41 ESBL-producing K. pneumoniae originating from a parent flock, from the hatcherys' environment during the hatching of that parent flocks' chickens, and from an associated fattening flock were investigated on an Illumina Miseq. Whole genome sequences were analyzed concerning their MLST-type, cgMLST-type, genotypic and phenotypic resistance, plasmid profiles and virulence genes. Irrespective of the origin of isolation all investigated isolates were multi-drug resistant, harbored the same ESBL-gene bla SHV-2, shared the same sequence type (ST3128) and displayed 100% similarity in core genome multilocus sequence typing (cgMLST). In addition, in silico plasmid typing found several Inc/Rep types associated with ESBL-plasmids. Summarizing, identical clones of SHV-2-producing K. pneumoniae were detected in different stages of the industrial broiler production in one out of seven investigated broiler chains. This proves the possibility of pseudo-vertical transmission of multi-resistant human pathogens from parent flocks to hatcheries and fattening flocks. Furthermore, the importance of cross-contamination along the production chain was shown. Although the ESBL-producing K. pneumoniae clone detected here in the broiler production has not been associated with clinical settings so far, our findings present a potential public health threat.

Association of farm-related factors with characteristics profiles of extended-spectrum ß-lactamase- / plasmid-mediated AmpC ß-lactamase-producing Escherichia coli isolates from German livestock farms.

Resistance to ß-lactam antibiotics, including third-generation cephalosporins, is of major concern for animal and human health. In this study, extended-spectrum ß-lactamase (ESBL) / plasmid-mediated AmpC (pAmpC) ß-lactamase -producing Escherichia coli isolates from German livestock farms were characterised and associations of these isolate characteristics with farm-related factors were investigated across different types of livestock. A total of 469 isolates originating from 150 farms (34 broiler farms, 38 fattening pig farms, 43 dairy cattle farms, 35 beef cattle farms) was included in the analyses. ESBL-gene family, phylogroup and phenotypic antimicrobial susceptibility for several antimicrobial agents were determined. This data was used to define different profiles characterising the isolates. Multivariate analyses using a distance-based non-parametric approach were performed to investigate associations between the profiles of the isolates and farm-related factors (e.g. management, husbandry, and environment of the farms). Co-occurrence of ESBL-gene families were not found in any of the isolates analysed. Sixty-eight percent of the isolates carried blaCTX-M variant genes. The frequency of phylogroups was as follows: A (55%), B1 (35%), D (17%) and B2 (3%). The most frequent phenotypic non-wildtype profile was non-wildtype status of solely cefepime (27%). Profiles of isolates from broilers differed substantially from those of other isolates. Associations between farm-related factors and characteristics profiles differed, depending on the isolate characteristics included in the analyses. Some factors describing the farm environment, like waterfowl in the surrounding of the farm, were associated with all tested profiles. The epidemiological method applied defines distances between isolates on basis of isolate characteristics data and is capable of analysing associations between isolate characteristics and epidemiological factors. As additional data, such as plasmid characteristics, gene type, or sequence information could be included in future studies, the method is suitable to identify points of action to reduce the occurrence of antimicrobial resistant bacteria.

Persistent and Transient Airborne MRSA Colonization of Piglets in a Newly Established Animal Model.

Livestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) was first found in 2005 and is up to date widespread in animal husbandry reservoir - focusing on pig farming. The regular detectability of MRSA in the air of pigsties as well as in exhaust air of pig farms (mean count: 102 cfu/m3) poses the question whether an airborne spread and, therefore, a MRSA colonization of animals via the airborne route exists. To answer this question, we exposed three groups of nine MRSA-negative tested piglets each to a defined airborne MRSA concentration (102, 104, and 106 cfu/m3) in our aerosol chamber for 24 h. In the following observation period of 21 days, the MRSA status of the piglets was monitored by taking different swab samples (nasal, pharyngeal, skin, conjunctival, and rectal swab). At the end of the experiment, we euthanized the piglets and investigated different tissues and organs for the spread of MRSA. The data of our study imply the presence of an airborne MRSA colonization route: the animals exposed to 106 cfu/m3 MRSA in the air were persistent colonized. The piglets exposed to an airborne MRSA concentration of 104 cfu/m3 were transient, and the piglets exposed to an airborne MRSA concentration of 102 cfu/m3 were not colonized. Consequently, a colonization via the airborne route was proven.

Prevalence and quantitative analysis of ESBL and AmpC beta-lactamase producing Enterobacteriaceae in broiler chicken during slaughter in Germany.

Food producing animals are considered a reservoir for Extended-spectrum beta-lactamase (ESBL) and AmpC beta-lactamase (AmpC) producing Enterobacteriaceae. Therefore, meat is discussed to be a potential source for the transmission of these resistant bacteria to humans. There is only limited information about the quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in different sample matrices during slaughter and their distribution in the slaughterhouse environment. Therefore, the aim of this study was to determine the prevalence as well as quantitative load of ESBL-/AmpC-producing Enterobacteriaceae in caecum, skin and filet samples of different broiler chicken flocks during slaughter in Germany. In addition, environmental samples were taken during slaughter of the respective flocks. To gain insights into possible transmission routes of ESBL-/AmpC-producing Enterobacteriaceae, the corresponding phylogroup and beta-lactamase genes were determined for selected isolates. ESBL-/AmpC-producing Enterobacteriaceae were detected during slaughter of all seven investigated flocks. On average, 47% (83/175) of caecum, 55% (96/175) of skin, 28% (49/175) of filet and 28% (25/89) of environmental samples harboured ESBL-/AmpC-producing Enterobacteriaceae. Prevalence varied widely between the flocks as well as between the different sample matrices. In about half of the caecum (23/40) and skin (19/40) samples as well as 85% (17/20) of the filet samples, the number of putative ESBL-/AmpC-producing Enterobacteriaceae (cefotaxime resistant Enterobacteriaceae) was below quantification limit. The median of cefotaxime resistant Enterobacteriaceae was 2.5 × 103 cfu/g in caecum, 1.5 × 103 cfu/g in skin and 1.5 × 102 cfu/g in filet samples. The median of cefotaxime resistant Enterobacteriaceae was, depending on the sample matrix, 1-4 log units below the median of total Enterobacteriaceae. Using real-time PCR, in 82% (629/767) of the cefotaxime resistant Enterobacteriaceae at least one of the investigated beta-lactamase genes blaCTX-M, blaSHV, blaTEM, blaAmpC-CIT was detected. The respective resistance genes of 322 isolates were further sequenced. The predominant bla-gene was blaCMY-2 (48%), followed by blaSHV-12 (23%). A contamination from the broiler chicken to the slaughterhouse environment and vice versa seems probable as isolates of the same species and phylogroup, encoding the same resistance genes were detected in all matrices during slaughter of the respective flock as well as in the slaughterhouse environment.

Environmental adaptation and vertical dissemination of ESBL-/pAmpC-producing Escherichia coli in an integrated broiler production chain in the absence of an antibiotic treatment.

High prevalence numbers of extended-spectrum beta-lactamase- (ESBL-)/plasmid-mediated AmpC beta-lactamase- (pAmpC-) producing Escherichia coli in broiler chicken and their distribution along the broiler production chain is an ongoing problem in food production. We, therefore, investigated resistant isolates along the broiler production chain to determine whether there is a constantly occurring direct vertical transmission of the ESBL-/pAmpC-producing E. coli from the parent flocks to their offspring or not. We, furthermore, analysed the isolates concerning the occurrence of virulence factors and their ability to form biofilms to estimate their potential to effectively colonize broiler chickens and/or persist and survive in the environment of the broiler production facilities. Using whole genome sequencing, we could show that ESBL-/pAmpC-producing E. coli were likely transferred in a step-wise process along the broiler production chain but not directly from the parent flock to the fattening flock with every single batch of offspring chickens. Additionally, resistant E. coli strains showing an extraintestinal pathogenic genotype as well as high numbers of virulence-associated genes including the production of curli fibres and cellulose have high capabilities to persist and spread in the broiler production chain.