Modeling of interventions for reducing external Enterobacteriaceae contamination of broiler carcasses during processing

This article presents a mathematical model for the Enterobacteriaceae count on the surface of broiler chicken during slaughter and how it may be affected by different processing technologies. The model is based on a model originally developed for Campylobacter and has been adapted for Enterobacteriaceae using a Bayesian updating approach and hitherto unpublished data gathered from German abattoirs. The slaughter process in the model consists of five stages: input, scalding, defeathering, evisceration, washing, and chilling. The impact of various processing technologies along the broiler processing line on the Enterobacteriaceae count on the carcasses’ surface has been determined from literature data. The model is implemented in the software R and equipped with a graphical user interface which allows interactively to choose among different processing technologies for each stage along the processing line. Based on the choice of processing technologies the model estimates the Enterobacteriaceae count on the surface of each broiler chicken at each stage of processing. This result is then compared to a so-called baseline model which simulates a processing line with a fixed set of processing technologies. The model calculations showed how even very effective removal of bacteria on the exterior of the carcass in a previous step will be undone by the cross-contamination with leaked feces, if feces contain high concentrations of bacteria

Detection of VIM-1-producing Enterobacter cloacae and Salmonella enterica Serovars Infantis and Goldcoast at a breeding pig farm in Germany in 2017 and their molecular relationship to former VIM-1-producing S. Infantis Isolates in German livestock production

In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel electrophoresis (PFGE) hybridization experiments. WGS data of these isolates were compared with those for the previously isolated VIM-1-producing Salmonella Infantis isolates from pigs and poultry. Among 103 samples, one Salmonella Goldcoast isolate, one Salmonella Infantis isolate, and one Enterobacter cloacae isolate carrying the blaVIM-1 gene were detected. Comparative WGS analysis revealed that the blaVIM-1 gene was part of a particular Tn21-like transposable element in all isolates. It was located on IncHI2 (ST1) plasmids of ∼290 to 300 kb with a backbone highly similar (98 to 100%) to that of reference pSE15-SA01028. SNP analysis revealed a close relationship of all VIM-1-positive S. Infantis isolates described since 2011. The findings of this study demonstrate that the occurrence of the blaVIM-1 gene in German livestock is restricted neither to a certain bacterial species nor to a certain Salmonella serovar but is linked to a particular Tn21-like transposable element located on transferable pSE15-SA01028-like IncHI2 (ST1) plasmids, being present in all of the investigated isolates from 2011 to 2017. IMPORTANCE: Carbapenems are considered one of few remaining treatment options against multidrug-resistant Gram-negative pathogens in human clinical settings. The occurrence of carbapenemase-producing Enterobacteriaceae in livestock and food is a major public health concern. Particularly the occurrence of VIM-1-producing Salmonella Infantis in livestock farms is worrisome, as this zoonotic pathogen is one of the main causes for human salmonellosis in Europe. Investigations on the epidemiology of those carbapenemase-producing isolates and associated mobile genetic elements through an in-depth molecular characterization are indispensable to understand the transmission of carbapenemase-producing Enterobacteriaceae along the food chain and between different populations to develop strategies to prevent their further spread

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

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 blaSHV−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

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

Novel whole blood assay for phenotyping platelet reactivity in mice identifies ICAM-1 as a mediator of platelet-monocyte interaction

British Heart Foundation (PG/12/68/29779 and PG/14/48/30916) and the Wellcome Trust (101604/Z/13/Z to SN and TW, and 098291/Z/12/Z to S.N)

High Resolution Discrimination of Clinical Mycobacterium tuberculosis Complex Strains Based on Single Nucleotide Polymorphisms

Recently, the diversity of the Mycobacterium tuberculosis complex (MTBC) population structure has been described in detail. Based on geographical separation and specific host pathogen co-evolution shaping MTBC virulence traits, at least 20 major lineages/genotypes have evolved finally leading to a clear influence of strain genetic background on transmissibility, clinical presentation/outcome, and resistance development. Therefore, high resolution genotyping for characterization of strains in larger studies is mandatory for understanding mechanisms of host-pathogen-interaction and to improve tuberculosis (TB) control. Single nucleotide polymorphisms (SNPs) represent the most reliable markers for lineage classification of clinical isolates due to the low levels of homoplasy, however their use is hampered either by low discriminatory power or by the need to analyze a large number of genes to achieve higher resolution. Therefore, we carried out de novo sequencing of 26 genes (approx. 20000 bp per strain) in a reference collection of MTBC strains including all major genotypes to define a highly discriminatory gene set. Overall, 161 polymorphisms were detected of which 59 are genotype-specific, while 13 define deeper branches such as the Euro-American lineage. Unbiased investigation of the most variable set of 11 genes in a population based strain collection (one year, city of Hamburg, Germany) confirmed the validity of SNP analysis as all strains were classified with high accuracy. Taken together, we defined a diagnostic algorithm which allows the identification of 17 MTBC phylogenetic lineages with high confidence for the first time by sequencing analysis of just five genes. In conclusion, the diagnostic algorithm developed in our study is likely to open the door for a low cost high resolution sequence/SNP based differentiation of the MTBC with a very high specificity. High throughput assays can be established which will be needed for large association studies that are mandatory for detailed investigation of host-pathogen-interaction during TB infection

Impact of wet-lab protocols on quality of whole-genome short-read sequences from foodborne microbial pathogens

For successful elucidation of a food-borne infection chain, the availability of high-quality sequencing data from suspected microbial contaminants is a prerequisite. Commonly, those investigations are a joint effort undertaken by different laboratories and institutes. To analyze the extent of variability introduced by differing wet-lab procedures on the quality of the sequence data we conducted an interlaboratory study, involving four bacterial pathogens, which account for the majority of food-related bacterial infections: Campylobacter spp., Shiga toxin-producing Escherichia coli, Listeria monocytogenes, and Salmonella enterica. The participants, ranging from German federal research institutes, federal state laboratories to universities and companies, were asked to follow their routine in-house protocols for short-read sequencing of 10 cultures and one isolated bacterial DNA per species. Sequence and assembly quality were then analyzed centrally. Variations within isolate samples were detected with SNP and cgMLST calling. Overall, we found that the quality of Illumina raw sequence data was high with little overall variability, with one exception, attributed to a specific library preparation kit. The variability of Ion Torrent data was higher, independent of the investigated species. For cgMLST and SNP analysis results, we found that technological sequencing artefacts could be reduced by the use of filters, and that SNP analysis was more suited than cgMLST to compare data of different contributors. Regarding the four species, a minority of Campylobacter isolate data showed the in comparison highest divergence with regard to sequence type and cgMLST analysis. We additionally compared the assembler SPAdes and SKESA for their performance on the Illumina data sets of the different species and library preparation methods and found overall similar assembly quality metrics and cgMLST statistics

European 1 : a globally important clonal complex of Mycobacterium bovis

We have identified a globally important clonal complex of Mycobacterium bovis by deletion analysis of over one thousand strains from over 30 countries. We initially show that over 99% of the strains of M. bovis, the cause of bovine tuberculosis, isolated from cattle in the Republic of Ireland and the UK are closely related and are members of a single clonal complex marked by the deletion of chromosomal region RDEu1 and we named this clonal complex European 1 (Eu1). Eu1 strains were present at less than 14% of French, Portuguese and Spanish isolates of M. bovis but are rare in other mainland European countries and Iran. However, strains of the Eu1 clonal complex were found at high frequency in former trading partners of the UK (USA, South Africa, New Zealand, Australia and Canada). The Americas, with the exception of Brazil, are dominated by the Eu1 clonal complex which was at high frequency in Argentina, Chile, Ecuador and Mexico as well as North America. Eu1 was rare or absent in the African countries surveyed except South Africa. A small sample of strains from Taiwan were non-Eu1 but, surprisingly, isolates from Korea and Kazakhstan were members of the Eu1 clonal complex. The simplest explanation for much of the current distribution of the Eu1 clonal complex is that it was spread in infected cattle, such as Herefords, from the UK to former trading partners, although there is evidence of secondary dispersion since. This is the first identification of a globally dispersed clonal complex M. bovis and indicates that much of the current global distribution of this important veterinary pathogen has resulted from relatively recent International trade in cattle.This work was funded by: TBadapt project (LSHp-CT-2007-037919); B.M. received financial support from the Swiss National Science Foundation; Swedish Research Council, Swedish Heart-Lung foundation, Swedish International Development Agency; Department of Agriculture and Rural Development Northern Ireland (project DARD0407); EU project TB-STEP (KBBE-2007-1-3-04, no. 212414); Swiss National Science Foundation (Grant No. 107559); Damien Foundation, Belgium; Commission Universitaire pour le Développement (CUD), University of Liege (Project PIC); The Wellcome Trust Livestock for Life and Animal Health in the Developing World initiatives (075833/A/04/Z); Chilean National Livestock Service -FONDOSAGC5-100-10-23 and CONICYT-FIC-R-EQU18 and by the Department of Environment, Food and Rural Affairs, UK (project SB4020).http://www.elsevier.com/locate/meegidab2012ab2013 (Author correction

Molekulare Epidemiologie und vertikale Transmission von ESBL-/AmpCproduzierenden Enterobacteriaceae entlang der Masthähnchenproduktionskette

Enterobacteriaceae which demonstrate resistance against beta-lactam antibiotics are of major concern in public health. The occurrence of extended-spectrum beta-lactamase (ESBL)-/AmpC betalactamase-producing E. coli in humans and animals is a challenging problem in antibiotic treatment in human and veterinary medicine. Especially in broiler chickens these resistant bacteria were frequently detected and a certain relevance of the exposure of contaminated chicken meat to humans is controversially discussed during the last decades. However, is was found, that ESBL-/pAmpC-producing Enterobacteriaceae were detectable on all stages of the broiler production and that already one-day old (grand-/parent)chicks can be affected. During the RESETII-project we therefore investigated seven ESBL-/pAmpC-positive parent broiler flocks, their respective hatching eggs as well as the subsequent broiler fattening flocks at the fattening farms as well as on slaughterhouse level concerning the occurrence of the resistant bacteria (chain investigations A-G). This project was performed by the Institute for Animal Hygiene and Environmental Health as well as the Institute of Food Safety and Food Hygiene from the Freie Universität Berlin. The task of the presented PhD thesis was to investigate the collected samples and resistant strains with regard to possible vertical transmission events in the whole broiler production chain. Therefore, high resolution molecular methods like pulsed-field gel-electrophoresis (PFGE) as well as whole genome sequencing (WGS) was applied to the respective isolates to determine their phylogenetic relationship. Using these methods, it could be shown that the introduction of the ESBL-/pAmpCproducing Enterobacteriaceae from the parent flocks into the hatchery occurred via contaminated egg shells (pseudo-vertical transmission) and that these bacteria can also survive the disinfection procedure. The hatchery acts as a reservoir for the resistant bacteria and certain strains are stepwise transmitted to the recently hatched chicks via the hatchery environment. However, the colonization of the chicks can also occur later on at the fattening farms. This indicates that the transmission routes of the ESBL-/pAmpC-producing Enterobacteriaceae in the broiler production are highly complex. Once the broiler chickens are infected with the resistant bacteria they directly introduce them into the slaughterhouse and the processing steps of the chicken meat. Especially the scalding and the defeathering steps are critical for cross-contamination events as shown in this study. Furthermore, selected isolates from all stages of the broiler production where investigated concerning the occurrence of virulence associated genes (VAGs) and the ability to produce curli fibers and cellulose. These factors support the colonization of a respective host as well as the survival in unfavorable environmental conditions. Using a macrocolony assay it could be shown that most of the tested E. coli isolates were able to produce curli fibers and cellulose which are the major components of biofilms. Therefore, it is very likely that these ESBL-/pAmpC-producing E. coli strains are also highly resistant against environmental stress and for example might also better survive certain cleaning and disinfection procedures. In addition, the investigated E. coli strains also showed up to harbor high numbers of VAGs. However, the numbers and the distribution of certain genes and virulence gene classes varied among the strains. Therefore, no specific VAG could be identified for the enhanced survival in broiler chickens. Furthermore, only isolates of one chain transmission investigation could be assigned as extra-intestinal pathogenic E. coli strains (ExPEC). This indicates that further investigations on the impact of certain bacterial factors on the survival, spread and transmission of ESBL-/pAmpC-producing E. coli in the broiler production chain are needed. For the successful development and application of intervention strategies against these resistant bacteria in the whole broiler production chain the complex network of transmission routes as well as supporting bacterial factors should be considered. Therefore, further detailed studies using an appropriate high number of samples and isolates as well as respective high resolution molecular methods need to be carried out.Enterobacteriaceae, welche eine Resistenz gegenüber Beta-Laktam Antibiotika aufzeigen, sind ein großes Problem für die öffentliche Gesundheit. Das Vorkommen von Extended-Spektrum Beta-Laktamase (ESBL)-/AmpC Beta-Laktamase-produzierenden E. coli in Mensch und Tier ist eine Herausforderung für die Behandlungsstrategien in der Human- und Veterinärmedizin. Besonders in Masthähnchen finden sich diese resistenten Bakterien und daher wird auch dem kontaminierten Hühnchenfleisch eine große Bedeutung zugemessen bei einer möglichen Übertragung auf den Menschen. Dieses Thema wird in den letzten Jahren jedoch auch sehr kontrovers diskutiert. Es konnte gezeigt werden, dass ESBL-/AmpC-produzierende Enterobacteriaceae sich in allen Stufen der Masthähnchenproduktionskette nachweisen lassen und dass auch bereits Eintagsküken kolonisiert sein können. Innerhalb des RESETII-Projektes wurden daher sieben ESBL-/pAmpC-positive Elterntierherden, deren Bruteier und die zugehörigen Masttierherden auf den Farmen und im Schlachthof bezüglich des Vorkommens dieser resistenten Bakterien untersucht (Kettenuntersuchungen A-G). Das Projekt wurde in den Instituten für Tier- und Umwelthygiene sowie für Lebensmittelsicherheit und –hygiene der Freien Universität Berlin durchgeführt. Im Rahmen dieser Doktorarbeit wurden die gesammelten Proben und resistenten Stämme insbesondere hinsichtlich möglicher vertikaler Übertragungswege innerhalb der Masthähnchenproduktionskette untersucht. Dabei wurden hochauflösende molekulare Methoden wie pulsed-field Gelelektrophorese (PFGE) und Ganzgenomsequenzierung (WGS) verwendet um die phylogenetische Verwandtschaft der untersuchten Isolate zu bestimmen. Es konnte gezeigt werden, dass der Eintrag der ESBL-/AmpC produzierende Enterobacteriaceae von den Elterntierherden in die Brüterei über kontaminierte Bruteioberflächen erfolgt (pseudovertikale Übertragung) und dass diese Bakterien auch die Desinfektionsmaßnahmen in der Brüterei überleben können. Die Brüterei fungiert dann als Reservoir für die resistenten Bakterien und einzelne Stämme werden nach und nach durch die Umgebung auf die geschlüpften Küken übertragen. Die Kolonisierung der frisch geschlüpften Küken kann aber auch erst im späteren Verlauf direkt auf der Mastfarm erfolgen. Diese Ergebnisse zeigen, dass die Möglichkeiten der Übertragungen von ESBL-/AmpC-produzierenden Enterobacteriaceae in der Masthähnchenproduktionskette sehr komplex sind. Sind die Masthähnchen jedoch einmal mit den resistenten Keimen kolonisiert, erfolgt ein direkter Eintrag in den Schlachthof und in die einzelnen Verarbeitungsschritte von Hähnchenfleisch. Insbesondere der Brühprozess und der Rupfvorgang sind kritische Schritte in Bezug auf mögliche Kreuzkontaminationen, wie in der vorliegenden Studie gezeigt werden konnte. Zusätzlich wurden ausgewählte Isolate aus allen Produktionsstufen der Masthähnchenkette auf das Vorkommen von Virulenz-assoziierten Genen (VAGs) hin untersucht sowie auf die Fähigkeiten Curli-Fimbrien beziehungsweise Zellulose zu produzieren. Diese Faktoren unterstützen die Kolonisierung eines entsprechenden Wirts und das Überleben unter ungünstigen Umweltbedingungen. Mit Hilfe eines Makrokolonieassays konnte gezeigt werden, dass die Mehrheit der untersuchten Isolate die Fähigkeit zur Bildung von Curli-Fimbrien und Zellulose besitzen, welche die Hauptkomponenten von bakteriellen Biofilmen darstellen. Daher ist es sehr wahrscheinlich, dass diese ESBL-/AmpC-produzierenden Enterobacteriaceae eine hohe Widerstandskraft gegen Umweltstress und beispielsweise auch gegen die Reinigungs- und Desinfektionsvorgänge aufweisen. Zusätzlich besaßen die untersuchten Isolate eine hohe Anzahl an VAGs in ihren Genomen. Die Gesamtzahl dieser VAGs sowie die Verteilung der VAGs unterschiedlicher Genkategorien variierten jedoch zum Teil zwischen den Stämmen. Es konnte daher kein spezifischer Faktor identifiziert werden für die Kolonisierung von Masthähnchen. Außerdem konnten nur Isolate aus einer einzigen Kettenuntersuchung als sogenannte extra-intestinal pathogene E. coli (ExPEC) ausgemacht werden. Das zeigt, dass weitere Untersuchungen benötigt werden, bezüglich der Überlebensmöglichkeiten, der Verbreitung und der Übertragung von ESBL-/AmpC-produzierenden E. coli innerhalb der Masthähnchenproduktionskette. Für die Entwicklung und erfolgreiche Anwendung von Interventionsmaßnahmen gegen diese resistenten Bakterien in der Masthähnchenproduktionskette sollten das komplexe Übertragungsnetzwerk sowie bestimmte bakterielle Faktoren mit in Betracht gezogen werden. Dazu bedarf es weiterer detaillierter Studien mit einer entsprechenden Anzahl an Proben und Isolaten und die Anwendung von hochauflösenden molekularen Methoden