[Assessment of available and currently applied typing methods including genome-based methods for zoonotic pathogens with a focus on Salmonella enterica]. / Bestandsaufnahme der verfügbaren und aktuell eingesetzten Typisierungsmethoden einschließlich genombasierter Verfahren von Zoonoseerregern am Beispiel von Salmonella enterica.

BACKGROUND: In recent years, whole genome sequencing (WGS) in combination with bioinformatic analyses has become state of the art in evaluating the pathogenicity/resistance potential and relatedness of bacteria. WGS analysis thus represents a central tool in the investigation of the resistance and virulence potential of pathogens, as well as their dissemination via outbreak clusters and transmission chains within the framework of molecular epidemiology. In order to gain an overview of the available genotypic and phenotypic methods used for pathogen typing of Salmonella and Shiga toxin-producing and enterohemorrhagic Escherichia coli (STEC/EHEC) in Germany at state and federal level, along with the availability of WGS-based typing and corresponding analytical methods, a survey of laboratories was conducted. METHODS: An electronic survey of laboratories working for public health protection and consumer health protection was conducted from February to June 2020. RESULTS AND CONCLUSION: The results of the survey showed that many of the participating laboratories provide a wide range of phenotypic and molecular methods. Molecular typing is most commonly used for species identification of Salmonella. In many cases, WGS-based methods have already been established at federal and state institutions or are in the process of being established. The Illumina sequencing technology is the most widely used technology. The survey confirms the importance of molecular biology and whole genome typing technologies for laboratories in the diagnosis of bacterial zoonotic pathogens.

Toward an Integrated Genome-Based Surveillance of Salmonella enterica in Germany.

Despite extensive monitoring programs and preventative measures, Salmonella spp. continue to cause tens of thousands human infections per year, as well as many regional and international food-borne outbreaks, that are of great importance for public health and cause significant socio-economic costs. In Germany, salmonellosis is the second most common cause of bacterial diarrhea in humans and is associated with high hospitalization rates. Whole-genome sequencing (WGS) combined with data analysis is a high throughput technology with an unprecedented discriminatory power, which is particularly well suited for targeted pathogen monitoring, rapid cluster detection and assignment of possible infection sources. However, an effective implementation of WGS methods for large-scale microbial pathogen detection and surveillance has been hampered by the lack of standardized methods, uniform quality criteria and strategies for data sharing, all of which are essential for a successful interpretation of sequencing data from different sources. To overcome these challenges, the national GenoSalmSurv project aims to establish a working model for an integrated genome-based surveillance system of Salmonella spp. in Germany, based on a decentralized data analysis. Backbone of the model is the harmonization of laboratory procedures and sequencing protocols, the implementation of open-source bioinformatics tools for data analysis at each institution and the establishment of routine practices for cross-sectoral data sharing for a uniform result interpretation. With this model, we present a working solution for cross-sector interpretation of sequencing data from different sources (such as human, veterinarian, food, feed and environmental) and outline how a decentralized data analysis can contribute to a uniform cluster detection and facilitate outbreak investigations.

Nationwide outbreak of invasive listeriosis associated with consumption of meat products in health care facilities, Germany, 2014-2019.

OBJECTIVES: Invasive listeriosis is a severe foodborne infection caused by Listeria(L.)monocytogenes. The aim of this investigation was to verify and describe a molecular cluster of listeriosis patients and identify factors leading to this outbreak. METHODS: Whole genome sequencing and core genome multilocus sequence typing were used for subtyping L. monocytogenes isolates from listeriosis cases and food samples in Germany. Patient interviews and investigational tracing of foodstuffs offered in health-care facilities (HCF), where some of the cases occurred, were conducted. RESULTS: We identified a German-wide listeriosis outbreak with 39 genetically related cases occurring between 2014 and 2019. Three patients died as a result of listeriosis. After identification of HCF in different regions of Germany for at least 13 cases as places of exposure, investigational tracing of food supplies in six prioritized HCF revealed meat products from one company (X) as a commonality. Subsequently the outbreak strain was analysed in six isolates from ready-to-eat meat products and one isolate from the production environment of company X. No further Sigma1 cases were detected after recall of the meat products from the market and closure of company X (as of August 2020). CONCLUSIONS: Interdisciplinary efforts including whole genome sequencing, epidemiological investigations in patients and investigational tracing of foods were essential to identify the source of infections, and thereby prevent further illnesses and deaths. This outbreak underlines the vulnerability of hospitalized patients for foodborne diseases, such as listeriosis. Food producers and HCF should minimize the risk of microbiological hazards when producing, selecting and preparing food for patients.

Salmonellosis outbreak with novel Salmonella enterica subspecies enterica serotype (11:z41:e,n,z15) attributable to sesame products in five European countries, 2016 to 2017.

In spring 2016, Greece reported an outbreak caused by a previously undescribed Salmonella enterica subsp. enterica serotype (antigenic formula 11:z41:e,n,z15) via the Epidemic Intelligence Information System for Food- and Waterborne Diseases and Zoonoses (EPIS-FWD), with epidemiological evidence for sesame products as presumptive vehicle. Subsequently, Germany, Czech Republic, Luxembourg and the United Kingdom (UK) reported infections with this novel serotype via EPIS-FWD. Concerned countries in collaboration with the European Centre for Disease Prevention and Control (ECDC) and European Food Safety Authority (EFSA) adopted a common outbreak case definition. An outbreak case was defined as a laboratory-confirmed notification of the novel Salmonella serotype. Between March 2016 and April 2017, 47 outbreak cases were notified (Greece: n = 22; Germany: n = 13; Czech Republic: n = 5; Luxembourg: n = 4; UK: n = 3). Whole genome sequencing revealed the very close genetic relatedness of isolates from all affected countries. Interviews focusing on sesame product consumption, suspicious food item testing and trace-back analysis following Salmonella spp. detection in food products identified a company in Greece where sesame seeds from different countries were processed. Through European collaboration, it was possible to identify and recall sesame spread as one contaminated food item serving as vehicle of infection and trace it back to its origin.

Impact of lunch provision on anthropometry, hemoglobin, and micronutrient status of female Cambodian garment workers: exploratory randomized controlled trial.

BACKGROUND: Lunch provision is expected to improve the nutritional status of Cambodian garment workers. The objective of this study is to evaluate the effects of a model lunch provision through a canteen on anthropometry, hemoglobin, and micronutrient status in female garment workers in Cambodia. METHODS: This exploratory randomized controlled trial was implemented at a garment factory in Phnom Penh, Cambodia. Female workers (nulliparous, non-pregnant) were recruited and randomly allocated into an intervention arm (workday's lunch provision) and a control arm. Served lunch sets (~ 700 kcal on average) included diverse local dishes. Anthropometry (body mass index, weight, triceps skinfold thickness, and mid-upper arm muscle circumference), as well as hemoglobin, serum ferritin and soluble transferrin receptor, serum retinol binding protein, and serum folate concentrations were assessed at baseline and after 5 months of lunch provision. A general linear model with adjustments for baseline values was used to estimate intervention effects for each outcome variable. RESULTS: Two hundred twenty-three women were recruited (n = 112 control and n = 111 intervention). 172 (n = 86 in each arm) completed the study. Baseline prevalence of underweight, anemia, depleted iron stores, and marginal iron stores, were 31, 24, 21, and 50%, respectively. Subjects were not affected by frank vitamin A or folate deficiency, whereas 30% showed a marginal folate status. Overall, mean changes in anthropometric variables, hemoglobin, and retinol binding protein were marginal and not significant among intervention subjects. Mean folate concentration increased insignificantly by + 1.1 ng/mL (- 0.02, 2.2) (p = 0.054). On the other hand, mean ferritin decreased by - 6.6 µg/L (- 11.9, - 1.3) (p = 0.015). Subgroup analysis prompts that effects are differently pronounced according to the baseline status of workers. CONCLUSIONS: Findings indicate that model lunch sets provided a beneficial amount of dietary folate, but need to be revisited for iron content and/or iron bioavailability. It is believed that distinct positive effects on anthropometry, hemoglobin, and micronutrient status can solely be expected in malnourished individuals. The authors suggest that similar larger trials, which include sets adapted to the concrete needs of workers affected by underweight, anemia and/or definite micronutrient deficiencies, should be performed. TRIAL REGISTRATION: The trial was registered at the German Clinical Trials Register (9 January 2015, Identifier: DRKS00007666).

Changes in dominant groups of the gut microbiota do not explain cereal-fiber induced improvement of whole-body insulin sensitivity.

BACKGROUND: Diets high in cereal-fiber (HCF) have been shown to improve whole-body insulin sensitivity. In search for potential mechanisms we hypothesized that a supplemented HCF-diet influences the composition of the human gut microbiota and/or biomarkers of colonic carbohydrate fermentation. METHODS: We performed a randomized controlled 18-week intervention in group-matched overweight participants. Fecal samples of 69 participants receiving isoenergetic HCF (cereal-fiber 43 g/day), or control (cereal-fiber 14 g/day), or high-protein (HP, 28% of energy-intake, cereal-fiber 14 g/day), or moderately high cereal fiber/protein diets (MIX; protein 23% of energy-intake, cereal-fiber 26 g/day) with comparable fat contents were investigated for diet-induced changes of dominant groups of the gut microbiota, and of fecal short-chain fatty-acids (SCFA) including several of their proposed targets, after 0, 6, and 18-weeks of dietary intervention. In vitro fermentation of the cereal fiber extracts as used in the HCF and MIX diets was analyzed using gas chromatography. Diet-induced effects on whole-body insulin-sensitivity were measured using euglycaemic-hyperinsulinemic clamps and re-calculated in the here investigated subset of n = 69 participants that provided sufficient fecal samples on all study days. RESULTS: Gut microbiota groups and biomarkers of colonic fermentation were comparable between groups at baseline (week 0). No diet-induced differences were detected between groups during this isoenergetic intervention, neither in the full model nor in uncorrected subgroup-analyses. The cereal-fiber extract as used for preparation of the supplements in the HCF and MIX groups did not support in vitro fermentation. Fecal acetate, propionate, and butyrate concentrations remained unchanged, as well as potential targets of increased SCFA, whereas valerate increased after 6-weeks in the HP-group only (p = 0.037). Insulin-sensitivity significantly increased in the HCF-group from week-6 (baseline M-value 3.8 ± 0.4 vs 4.3 ± 0.4 mg·kg-1·min-1, p = 0.015; full model 0-18-weeks, treatment-x-time interaction, p = 0.046). CONCLUSIONS: Changes in the composition of the gut microbiota and/or markers of colonic carbohydrate fermentation did not contribute explaining the observed early onset and significant improvement of whole-body insulin sensitivity with the here investigated HCF-diet. TRIAL REGISTRATION: This trial was registered at http://www.clinicaltrials.gov as NCT00579657.

Human intestinal microbiota: characterization of a simplified and stable gnotobiotic rat model.

The study of host microbe interactions is hampered by the complexity and inter-individual variability of the human gut microbiota. Therefore, a simplified human intestinal microbiota (SIHUMI) consisting of seven bacterial species was introduced into germfree rats. Species selection was based on numerical importance and fermentative abilities in the human gut. Association of the rats with the SIHUMI (Anaerostipes caccae, Bacteroides thetaiotaomicron, Bifidobacterium longum, Blautia producta, Clostridium ramosum, Escherichia coli and Lactobacillus plantarum) resulted in increased faecal concentrations of short chain fatty acids compared to germfree animals. Since the faecal butyrate concentration was low (0.9 ± 0.5 µmol/g dry matter) the SIHUMI was complemented with Clostridium butyricum. This extended bacterial community (SIHUMIx) led to an increased faecal butyrate concentration of 1.5 ± 0.7 µmol/g dry matter. Besides forming SCFA, the SIHUMIx was capable of degrading mucins, ß-aspartylglycine and bilirubin. These features are characteristic of conventional animals but not observed in germfree animals. Dietary interventions with modifications in fibre and fat content led to changes in the proportion of community members. The relative increase of one member of this community in response to a high-fat diet reflects the situation reported for obese mice and human subjects. The strength of the model communities is their remarkable stability over time and their easy transfer to the offspring.