Comparison of approaches for source attribution of ESBL-producing Escherichia coli in Germany.

Extended-spectrum beta-lactamase (ESBL)-producing Escherichia (E.) coli have been widely described as the cause of treatment failures in humans around the world. The origin of human infections with these microorganisms is discussed controversially and in most cases hard to identify. Since they pose a relevant risk to human health, it becomes crucial to understand their sources and the transmission pathways. In this study, we analyzed data from different studies in Germany and grouped ESBL-producing E. coli from different sources and human cases into subtypes based on their phenotypic and genotypic characteristics (ESBL-genotype, E. coli phylogenetic group and phenotypic antimicrobial resistance pattern). Then, a source attribution model was developed in order to attribute the human cases to the considered sources. The sources were from different animal species (cattle, pig, chicken, dog and horse) and also from patients with nosocomial infections. The human isolates were gathered from community cases which showed to be colonized with ESBL-producing E. coli. We used the attribution model first with only the animal sources (Approach A) and then additionally with the nosocomial infections (Approach B). We observed that all sources contributed to the human cases, nevertheless, isolates from nosocomial infections were more related to those from human cases than any of the other sources. We identified subtypes that were only detected in the considered animal species and others that were observed only in the human population. Some subtypes from the human cases could not be allocated to any of the sources from this study and were attributed to an unknown source. Our study emphasizes the importance of human-to-human transmission of ESBL-producing E. coli and the different role that pets, livestock and healthcare facilities may play in the transmission of these resistant bacteria. The developed source attribution model can be further used to monitor future trends. A One Health approach is necessary to develop source attribution models further to integrate also wildlife, environmental as well as food sources in addition to human and animal data.

The role of parameterization in comparing source attribution models based on microbial subtyping for salmonellosis.

Source attribution methods attribute human cases of a zoonotic disease to a certain source putatively responsible for this disease. Identifying and quantifying the contribution of different sources to human infections is important for taking appropriate actions for reducing the exposure of the consumer to zoonotic pathogens. One widely used method is the microbial subtyping approach, whose principle is to compare the frequency of pathogen subtypes from different sources (e.g. animals or food) with the frequency of these subtypes in human cases. This paper studies the relationship between a Bayesian microbial subtyping approach described by Hald and coworkers subsequently modified by David and coworkers, here called the Hald model, and a frequentist approach known as the "Dutch model." The comparison between the Bayesian and frequentist model is done for two data sets on salmonellosis in Germany from different time periods (year 2004-2007 and 2010-2011). The results of both approaches are in good agreement with each other for the used data. It is shown here mathematically that a certain parameterization can be used to transform the probabilistic Hald model into a deterministic form, which is equivalent to the Dutch model. That certain parameterization secures independence of the model outcomes from the choice of so-called unique subtypes (which are unique in the sense that they are found exclusively in one of the sources). It is shown that deviating from that certain parameterization leads variations in the model outcome dependent on which unique subtypes are chosen in the process of modelling.

Is pseudarthrosis after spinal instrumentation caused by a chronic infection?

HYPOTHESIS: To assess whether a chronic bacterial infection is present in a subset of patients with pseudarthrosis after instrumented spinal fusion. METHODS: This was a prospective diagnostic study including adult patients with previous instrumented spinal fusion. Patients underwent revision surgery for either pseudarthrosis or other causes (e.g. implant removal, curve progression or junctional kyphosis) (control group). Five separate biopsies were randomly collected, intraoperatively, from the pseudarthrosis site and cultivated under both aerobic (5 days) and anaerobic (14 days) conditions. If cultivation was positive in at least 2/5 tissue samples, the biopsy was sectioned and stained using peptide nucleic acid fluorescence in situ hybridization (PNA-FISH). Confocal laser scanning microscopy was used to examine the sections and visualize bacterial aggregates. RESULTS: The study included 32 pseudarthrosis and 32 control patients. Cultivation yielded bacteria in at least 1/5 biopsies in 52% of patients with no difference between the groups (p = 1.0). Bacteria of the same species was found in at least 2/5 samples in seven pseudarthrosis patients and four controls (p = 0.509). Propionibacterium acnes was found in 8 of these 11 samples. Microscopy demonstrated tissue-embedded bacterial aggregates in two of these patients but with no inflammatory cells indicating an active infection. The presence of bacteria was not associated with the number of previous spinal procedures or the pre-revision fusion length (p ≥ 0.503). CONCLUSIONS: Pseudarthrosis after instrumented spinal surgery was not significantly associated with the presence of bacteria at the pseudarthrosis site. Positive cultivation results are common after spinal instrumentation, but our results indicate that they rarely represent an organized infection. These slides can be retrieved under Electronic Supplementary Material.

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.

Subgrouping of ESBL-producing Escherichia coli from animal and human sources: an approach to quantify the distribution of ESBL types between different reservoirs.

Escherichia (E.) coli producing extended-spectrum beta-lactamases (ESBLs) are an increasing problem for public health. The success of ESBLs may be due to spread of ESBL-producing bacterial clones, transfer of ESBL gene-carrying plasmids or exchange of ESBL encoding genes on mobile elements. This makes it difficult to identify transmission routes and sources for ESBL-producing bacteria. The objectives of this study were to compare the distribution of genotypic and phenotypic properties of E. coli isolates from different animal and human sources collected in studies in the scope of the national research project RESET. ESBL-producing E. coli from two longitudinal and four cross-sectional studies in broiler, swine and cattle farms, a cross-sectional and a case-control study in humans and diagnostic isolates from humans and animals were used. In the RESET consortium, all laboratories followed harmonized methodologies for antimicrobial susceptibility testing, confirmation of the ESBL phenotype, specific PCR assays for the detection of bla(TEM), bla(CTX), and bla(SHV) genes and sequence analysis of the complete ESBL gene as well as a multiplex PCR for the detection of the four major phylogenetic groups of E. coli. Most ESBL genes were found in both, human and non-human populations but quantitative differences for distinct ESBL-types were detectable. The enzymes CTX-M-1 (63.3% of all animal isolates, 29.3% of all human isolates), CTX-M-15 (17.7% vs. 48.0%) and CTX-M-14 (5.3% vs. 8.7%) were the most common ones. More than 70% of the animal isolates and more than 50% of the human isolates contained the broadly distributed ESBL genes bla(CTX-M-1), bla(CTX-M-15), or the combinations bla(SHV-12)+bla(TEM) or bla(CTX-M-1)+bla(TEM). While the majority of animal isolates carried bla(CTX-M-1) (37.5%) or the combination bla(CTX-M-1)+bla(TEM) (25.8%), this was the case for only 16.7% and 12.6%, respectively, of the human isolates. In contrast, 28.2% of the human isolates carried bla(CTX-M-15) compared to 10.8% of the animal isolates. When grouping data by ESBL types and phylogroups bla(CTX-M-1) genes, mostly combined with phylogroup A or B1, were detected frequently in all settings. In contrast, bla(CTX-M-15) genes common in human and animal populations were mainly combined with phylogroup A, but not with the more virulent phylogroup B2 with the exception of companion animals, where a few isolates were detectable. When E. coli subtype definition included ESBL types, phylogenetic grouping and antimicrobial susceptibility data, the proportion of isolates allocated to common clusters was markedly reduced. Nevertheless, relevant proportions of same subtypes were detected in isolates from the human and livestock and companion animal populations included in this study, suggesting exchange of bacteria or bacterial genes between these populations or a common reservoir. In addition, these results clearly showed that there is some similarity between ESBL genes, and bacterial properties in isolates from the different populations. Finally, our current approach provides good insight into common and population-specific clusters, which can be used as a basis for the selection of ESBL-producing isolates from interesting clusters for further detailed characterizations, e.g. by whole genome sequencing.

[Estimation of the transfer of ESBL-producing Escherichia coli to humans in Germany]. / Abschätzung des Transfers von ESBL-bildenden Escherichia coli zum Menschen für Deutschland.

In 2011 EFSA has evaluated the risk for the consumer caused by ESBL-/AmpC-producing bacteria in food of animal origin and in livestock animals. Human-to-human transfer in hospitals.and in the community was considered as the most relevant route of transmission for ESBL-producing E. coli. ESBL-/AmpC-producing E. coli are in Germany, as in many other Member States of the European Union, widely spread in food of animal origin and in livestock animals. In an assessment of the relevance of livestock animals as reservoir for ESBL-/AmpC-producing E. coli as well as for ESBL-coding resistance genes the heterogeneity of the resistance genes, plasmids and bacteria in animals, foods and humans needs to be considered. In this context, both, the clonal spread of bacteria as well as horizontal transfer of resistance genes, e. g. by plasmids, have to be analyzed. Whereas studies in The Netherlands identified poultry as the most relevant reservoir, the transfer of ESBL-gene carrying plasmids from pigs to the farmers was demonstrated in Denmark. First attempts to quantify the relevance of livestock animals as reservoir for ESBL-producing E. coli in Germany showed, that the proportions of the most frequent ESBL-resistance genes are quite different between animal and human derived E. coli isolates. If in addition properties of the bacterial cells, e.g. resistance to several antibiotic classes are considered, only a small proportion of human isolates showed the same patterns as animal isolates. The results achieved so far demonstrate that certain ESBL-types are prevalent in all livestock populations investigated. Currently, the majority of cases of colonizations with ESBL-producing E. coli among humans cannot be directly linked to livestock and food-producing animals as reservoirs. This reflects that transmission routes are more complex and other reservoirs and sources including human-human interactions have to be taken into consideration.