Annemarie Kaesbohrer

Emerging antimicrobial resistance in commensal Escherichia coli with public health relevance.

In 2009, 1462 Escherichia coli isolates were collected in a systematic resistance monitoring approach from primary production, slaughterhouses and at retail and evaluated on the basis of epidemiological cut‐off values. Besides resistance to antimicrobial classes that have been extensively used for a long time (e.g. sulphonamides and tetracyclines), resistance to (fluoro)quinolones and third‐generation cephalosporins was observed. While in the poultry production chain the majority (60%) of isolates from laying hens was susceptible to all antimicrobials tested, most isolates from broilers, chicken meat and turkey meat showed resistance to at least one (85–93%) but frequently even to several antimicrobial classes (73–84%). In the cattle and pig production chain, the share of isolates showing resistance to at least one antimicrobial was lowest (16%) in dairy cows, whereas resistance to at least one antimicrobial ranged between 43% and 73% in veal calves, veal and pork. Resistance rates to ciprofloxacin and nalidixic acid in isolates from broilers were 41.1% and 43.1%, respectively. Likewise, high resistance rates to (fluoro)quinolones were observed in isolates from chicken meat and turkey meat. In contrast, ciprofloxacin resistance was less frequent in E. coli isolates from the cattle and pig production chain with highest rate in veal calves (13.3%). Highest resistance rates to cephalosporins were observed in broilers and chicken meat, with 5.9% and 6.2% of the isolates showing resistance. In dairy cattle and veal, no isolates with cephalosporin resistance were detected, whereas 3.3% of the isolates from veal calves showed resistance to ceftazidime. Resistance to (fluoro)quinolones and cephalosporins in E. coli isolates is of special concern because they are critically important antimicrobials in human antimicrobial therapy. The emergence of this resistance warrants increased monitoring. Together with continuous monitoring of antimicrobial usage, management strategies should be regularly assessed and adapted.

Factors associated with the occurrence of MRSA CC398 in herds of fattening pigs in Germany

BackgroundThe purpose of this study was to investigate the prevalence of MRSA in herds of fattening pigs in different regions of Germany, and to determine factors associated with the occurrence of this pathogen. For this purpose pooled dust samples were collected, and a questionnaire covered information regarding herd characteristics and management practices. Samples were pre-enriched in high-salt medium followed by selective enrichment containing cefoxitin/aztreonam, and culturing. Presumptive colonies were confirmed by multiplex-PCR targeting nuc-, mecA- and 16S rRNA-genes. Isolates were spa- and SCCmec-, and in selected cases, multilocus sequence-typed. Susceptibilities to 13 antimicrobials were determined by broth microdilution. Statistical analysis was carried out using backward stepwise logistic regression to calculate odds ratios with the MRSA test result as the outcome and herd characteristics as categorical covariates.ResultsOverall, 152 of 290 (52%) fattening pig farms tested positive for MRSA. The prevalence in the east, north- and south-west of Germany ranged from 39 to 59%.t011 (66%) and t034 (23%) were the most commonly identified spa-types, and 85% of isolates carried SCCmec Type V. Identified spa-types were all associated with clonal complex CC398. Susceptibility testing revealed that all isolates were resistant to tetracycline. High resistance rates were also found for sulfamethoxazole/trimethoprim (40%), and quinupristin/dalfopristin (32%). In addition, 83% of strains displayed multidrug resistant (> 3 substance classes) phenotypes.Logistic regression revealed herd size (large farms OR: 5.4; CI: 2.7-11.2; p < 0.05), and production type (wean-to-finish OR: 4.0; CI: 1.6-10.4; p < 0.05) as risk factors associated with a positive MRSA finding in fattening pig operations.ConclusionsMRSA CC398 is widely distributed among herds of fattening pigs in Germany. Farm management plays a crucial role in the dissemination of MRSA with herd size, and production type representing potential major indicators.

blaCTX-M-15-carrying Escherichia coli and Salmonella isolates from livestock and food in Germany

OBJECTIVES The characterization of CTX-M-₁₅ β-lactamase-producing Escherichia coli and Salmonella isolates originating mainly from German livestock and food. METHODS E. coli (526, mainly commensals) and Salmonella (151) non-human isolates resistant to third-generation cephalosporins, originating from routine and monitoring submissions (2003-12) to the Federal Institute for Risk Assessment and different national targeted studies (2011-12), were examined for the presence of blaCTX-M-₁₅ genes by PCR amplification/sequencing. Additional resistance and virulence genes were screened by DNA microarray and PCR amplification. E. coli isolates with blaCTX-M-₁₅ were characterized by phylogenetic grouping, PFGE and multilocus sequence typing (MLST). The blaCTX-M-15 plasmids were analysed by replicon typing, plasmid MLST, S1 nuclease PFGE and Southern blot hybridization experiments. RESULTS Twenty-one E. coli (livestock, food and a toy; 4.0%) and two Salmonella (horse and swine; 1.3%) isolates were CTX-M-₁₅ producers. E. coli isolates were mainly ascribed to three clonal lineages of sequence types ST678 (German outbreak with enteroaggregative Shiga-toxin-producing E. coli O104:H4; salmon, cucumber and a toy), ST410 (poultry, swine and cattle farms) and ST167/617 (swine farms and turkey meat). The blaCTX-M-₁₅ genes were located on IncI1 and multireplicon IncF plasmids or on the chromosome of E. coli ST410 isolates. CONCLUSIONS The prevalence of CTX-M-₁₅-producing isolates from non-human sources in Germany is still low. The blaCTX-M-₁₅ gene is, however, present in multidrug-resistant E. coli clones with pathogenic potential in livestock and food. The maintenance of the blaCTX-M-₁₅ gene due to chromosomal carriage is noteworthy. The possibility of an exchange of CTX-M-₁₅-producing isolates or plasmids between livestock and humans (in both directions) deserves continuous surveillance.

A strategy to establish Food Safety Model Repositories.

Transferring the knowledge of predictive microbiology into real world food manufacturing applications is still a major challenge for the whole food safety modelling community. To facilitate this process, a strategy for creating open, community driven and web-based predictive microbial model repositories is proposed. These collaborative model resources could significantly improve the transfer of knowledge from research into commercial and governmental applications and also increase efficiency, transparency and usability of predictive models. To demonstrate the feasibility, predictive models of Salmonella in beef previously published in the scientific literature were re-implemented using an open source software tool called PMM-Lab. The models were made publicly available in a Food Safety Model Repository within the OpenML for Predictive Modelling in Food community project. Three different approaches were used to create new models in the model repositories: (1) all information relevant for model re-implementation is available in a scientific publication, (2) model parameters can be imported from tabular parameter collections and (3) models have to be generated from experimental data or primary model parameters. All three approaches were demonstrated in the paper. The sample Food Safety Model Repository is available via: http://sourceforge.net/projects/microbialmodelingexchange/files/models and the PMM-Lab software can be downloaded from http://sourceforge.net/projects/pmmlab/. This work also illustrates that a standardized information exchange format for predictive microbial models, as the key component of this strategy, could be established by adoption of resources from the Systems Biology domain.

Prevalence of carbapenemase producing Enterobacteriaceae isolated from German pig-fattening farms during the years 2011-2013.

Since recently Enterobacteriaceae carrying blaVIM-1 genes have been isolated in German animal husbandries, the monitoring of carbapenemase producing Enterobacteriaceae (CPE) in livestock became a major topic within the European Union. Nevertheless, due to missing surveillance studies the worldwide situation in livestock and livestock associated surroundings might still be underestimated. The here described study provides an overview of the CPE-prevalence in German pig-fattening farms during the years 2011-2013 (period when previously described blaVIM-1 findings occurred on pig-fattening farms (Efsa, 2011; Fischer et al., 2012, 2013a)). Therefore, a collection of 238 bacterial anacultures derived from pooled faeces and boot swab samples, collected in a cross-sectional study including 58 pig-fattening farms throughout Germany, were investigated. The bacteria were selected on MacConkey agar plates containing 0.125μg/ml meropenem. Enterobacteriaceae which were able to grow on these plates were further investigated for the presence of carbapenemase genes. Out of eight CPE-suspicious strains, two Escherichia (E.) coli strains-deriving from the same farm-contained the carbapenemase gene blaVIM-1. For the remaining six Enterobacteriaceae it seems to be likely that they possess other resistance mechanisms, leading to reduced carbapenem susceptibility. Based on the obtained results, the overall CPE prevalence for German pig-fattening farms, sampled during the years 2011-2013 was 1.7%; 95% CI: 0-10. However, as it is of great importance to prevent a further spread of these bacteria between farms and livestock populations as well as their introduction into the food chain, an understanding of their routes of introduction and spread in combination with intensified monitoring programs are considered necessary.

VIM-1-producing Salmonella Infantis isolated from swine and minced pork meat in Germany

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In Vivo Transfer and Microevolution of Avian Native IncA/C2 blaNDM-1-Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study

ABSTRACT The emergence and spread of carbapenemase-producing Enterobacteriaceae (CPE) in wildlife and livestock animals pose an important safety concern for public health. With our in vivo broiler chicken infection study, we investigated the transfer and experimental microevolution of the blaNDM-1-carrying IncA/C2 plasmid (pRH-1238) introduced by avian native Salmonella enterica subsp. enterica serovar Corvallis without inducing antibiotic selection pressure. We evaluated the dependency of the time point of inoculation on donor (S. Corvallis [12-SA01738]) and plasmid-free Salmonella recipient [d-tartrate-fermenting (d-Ta+) S. Paratyphi B (13-SA01617), referred to here as S. Paratyphi B (d-Ta+)] excretion by quantifying their excretion dynamics. Using plasmid profiling by S1 nuclease-restricted pulsed-field gel electrophoresis, we gained insight into the variability of the native plasmid content among S. Corvallis reisolates as well as plasmid acquisition in S. Paratyphi B (d-Ta+) and the enterobacterial gut microflora. Whole-genome sequencing enabled us to gain an in-depth insight into the microevolution of plasmid pRH-1238 in S. Corvallis and enterobacterial recipient isolates. Our study revealed that the fecal excretion of avian native carbapenemase-producing S. Corvallis is significantly higher than that of S. Paratyphi (d-Ta+) and is not hampered by S. Paratyphi (d-Ta+). Acquisition of pRH-1238 in other Enterobacteriaceae and several events of plasmid pRH-1238 transfer to different Escherichia coli sequence types and Klebsiella pneumoniae demonstrated an interspecies broad host range. Regardless of the microevolutionary structural deletions in pRH-1238, the single carbapenem resistance marker blaNDM-1 was maintained on pRH-1238 throughout the trial. Furthermore, we showed the importance of the gut E. coli population as a vector of pRH-1238. In a potential scenario of the introduction of NDM-1-producing S. Corvallis into a broiler flock, the pRH-1238 plasmid could persist and spread to a broad host range even in the absence of antibiotic pressure.

Discussing State-of-the-Art Spatial Visualization Techniques Applicable for the Epidemiological Surveillance Data on the Example of Campylobacter spp. in Raw Chicken Meat

Within the European activities for the ‘Monitoring and Collection of Information on Zoonoses’, annually EFSA publishes a European report, including information related to the prevalence of Campylobacter spp. in Germany. Spatial epidemiology becomes here a fundamental tool for the generation of these reports, including the representation of prevalence as an essential element. Until now, choropleth maps are the default visualization technique applied in epidemiological monitoring and surveillance reports made by EFSA and German authorities. However, due to its limitations, it seems to be reasonable to explore alternative chart type. Four maps including choropleth, cartogram, graduated symbols and dot‐density maps were created to visualize real‐world sample data on the prevalence of Campylobacter spp. in raw chicken meat samples in Germany in 2011. In addition, adjacent and coincident maps were created to visualize also the associated uncertainty. As an outcome, we found that there is not a single data visualization technique that encompasses all the necessary features to visualize prevalence data alone or prevalence data together with their associated uncertainty. All the visualization techniques contemplated in this study demonstrated to have both advantages and disadvantages. To determine which visualization technique should be used for future reports, we recommend to create a dialogue between end‐users and epidemiologists on the basis of sample data and charts. The final decision should also consider the knowledge and experience of end‐users as well as the specific objective to be achieved with the charts.

Reviewing Interventions against Enterobacteriaceae in Broiler Processing: Using Old Techniques for Meeting the New Challenges of ESBL E. coli?

Extended-spectrum beta-lactamase- (ESBL-) producing Enterobacteriaceae are frequently detected in poultry and fresh chicken meat. Due to the high prevalence, an impact on human colonization and the spread of antibiotic resistance into the environment is assumed. ESBL-producing Enterobacteriaceae can be transmitted along the broiler production chain but also their persistence is reported because of insufficient cleaning and disinfection. Processing of broiler chickens leads to a reduction of microbiological counts on the carcasses. However, processing steps like scalding, defeathering, and evisceration are critical concerning fecal contamination and, therefore, cross-contamination with bacterial strains. Respective intervention measures along the slaughter processing line aim at reducing the microbiological load on broiler carcasses as well as preventing cross-contamination. Published data on the impact of possible intervention measures against ESBL-producing Enterobacteriaceae are missing and, therefore, we focused on processing measures concerning Enterobacteriaceae, in particular E. coli or coliform counts, during processing of broiler chickens to identify possible hints for effective strategies to reduce these resistant bacteria. In total, 73 publications were analyzed and data on the quantitative reductions were extracted. Most investigations concentrated on scalding, postdefeathering washes, and improvements in the chilling process and were already published in and before 2008 (n=42, 58%). Therefore, certain measures may be already installed in slaughterhouse facilities today. The effect on eliminating ESBL-producing Enterobacteriaceae is questionable as there are still positive chicken meat samples found. A huge number of studies dealt with different applications of chlorine substances which are not approved in the European Union and the reduction level did not exceed 3 log10 values. None of the measures was able to totally eradicate Enterobacteriaceae from the broiler carcasses indicating the need to develop intervention measures to prevent contamination with ESBL-producing Enterobacteriaceae and, therefore, the exposure of humans and the further release of antibiotic resistances into the environment.

Antimicrobial susceptibility in faecal Escherichia coli from pigs after enrofloxacin administration in an experimental environment

The study objective was to evaluate the effect of oral (OT) and parenteral (PT) administration of enrofloxacin to weaners on untreated contact animals. We assessed a) fluoroquinolone occurrence in the blood serum of untreated contact animals (COT, CPT); b) resistance to (fluoro)quinolones in commensal Escherichia coli (E. coli) in OT, PT, COT and CPT compared to the control (CON), and c) resistance to other antimicrobials in E. coli in OT, PT, COT and CPT compared to the initial situation before the treatment in these groups. Five groups of 14 weaners each were housed in three separate rooms (OT with COT, PT with CPT, CON alone). OT and PT were treated with enrofloxacin for five days. Rectal swabs and blood samples were taken before, during and until 51 days after treatment. Enro- and ciprofloxacin were detected in all treated, all COT and half of the CPT pigs. Neither through selective isolation nor by susceptibility testing of one random non-selectively isolated faecal E. coli per sample, resistance to ciprofloxacin (metabolite of enrofloxacin) and nalidixic acid was detected in both treatment and contact groups during and short after treatment. However, a transient increase of E. coli resistant to antimicrobials other than quinolones followed the treatment in isolates from OT and COT (e.g. ampicillin p < 0.05). In conclusion, animals in contact with treated animals are exposed to and can intake antimicrobials. Animals in contact with orally treated animals show occurrence of antibiotic resistant E. coli. Further studies are needed to show whether these preliminary findings can be confirmed under different conditions and with more sensitive detection methods.