Antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) abundance in animal faeces, meat, production environments and human faeces in Europe.

BACKGROUND: Real-time quantitative PCR (qPCR) is an affordable method to quantify antimicrobial resistance gene (ARG) targets, allowing comparisons of ARG abundance along animal production chains. OBJECTIVES: We present a comparison of ARG abundance across various animal species, production environments and humans in Europe. AMR variation sources were quantified. The correlation of ARG abundance between qPCR data and previously published metagenomic data was assessed. METHODS: A cross-sectional study was conducted in nine European countries, comprising 9572 samples. qPCR was used to quantify abundance of ARGs [aph(3')-III, erm(B), sul2, tet(W)] and 16S rRNA. Variance component analysis was conducted to explore AMR variation sources. Spearman's rank correlation of ARG abundance values was evaluated between pooled qPCR data and earlier published pooled metagenomic data. RESULTS: ARG abundance varied strongly among animal species, environments and humans. This variation was dominated by between-farm variation (pigs) or within-farm variation (broilers, veal calves and turkeys). A decrease in ARG abundance along pig and broiler production chains ('farm to fork') was observed. ARG abundance was higher in farmers than in slaughterhouse workers, and lowest in control subjects. ARG abundance showed a high correlation (Spearman's ρ > 0.7) between qPCR data and metagenomic data of pooled samples. CONCLUSIONS: qPCR analysis is a valuable tool to assess ARG abundance in a large collection of livestock-associated samples. The between-country and between-farm variation of ARG abundance could partially be explained by antimicrobial use and farm biosecurity levels. ARG abundance in human faeces was related to livestock antimicrobial resistance exposure.

Risk factors for the abundance of antimicrobial resistance genes aph(3')-III, erm(B), sul2 and tet(W) in pig and broiler faeces in nine European countries.

OBJECTIVES: The occurrence and zoonotic potential of antimicrobial resistance (AMR) in pigs and broilers has been studied intensively in past decades. Here, we describe AMR levels of European pig and broiler farms and determine the potential risk factors. METHODS: We collected faeces from 181 pig farms and 181 broiler farms in nine European countries. Real-time quantitative PCR (qPCR) was used to quantify the relative abundance of four antimicrobial resistance genes (ARGs) [aph(3')-III, erm(B), sul2 and tet(W)] in these faeces samples. Information on antimicrobial use (AMU) and other farm characteristics was collected through a questionnaire. A mixed model using country and farm as random effects was performed to evaluate the relationship of AMR with AMU and other farm characteristics. The correlation between individual qPCR data and previously published pooled metagenomic data was evaluated. Variance component analysis was conducted to assess the variance contribution of all factors. RESULTS: The highest abundance of ARG was for tet(W) in pig faeces and erm(B) in broiler faeces. In addition to the significant positive association between corresponding ARG and AMU levels, we also found on-farm biosecurity measures were associated with relative ARG abundance in both pigs and broilers. Between-country and between-farm variation can partially be explained by AMU. Different ARG targets may have different sample size requirements to represent the overall farm level precisely. CONCLUSIONS: qPCR is an efficient tool for targeted assessment of AMR in livestock-related samples. The AMR variation between samples was mainly contributed to by between-country, between-farm and within-farm differences, and then by on-farm AMU.

Determinants for antimicrobial resistance genes in farm dust on 333 poultry and pig farms in nine European countries.

Livestock feces with antimicrobial resistant bacteria reaches the farm floor, manure pit, farm land and wider environment by run off and aerosolization. Little research has been done on the role of dust in the spread of antimicrobial resistance (AMR) in farms. Concentrations and potential determinants of antimicrobial resistance genes (ARGs) in farm dust are at present not known. Therefore in this study absolute ARG levels, representing the levels people and animals might be exposed to, and relative abundances of ARGs, representing the levels in the bacterial population, were quantified in airborne farm dust using qPCR. Four ARGs were determined in 947 freshly settled farm dust samples, captured with electrostatic dustfall collectors (EDCs), from 174 poultry (broiler) and 159 pig farms across nine European countries. By using linear mixed modeling, associations with fecal ARG levels, antimicrobial use (AMU) and farm and animal related parameters were determined. Results show similar relative abundances in farm dust as in feces and a significant positive association (ranging between 0.21 and 0.82) between the two reservoirs. AMU in pigs was positively associated with ARG abundances in dust from the same stable. Higher biosecurity standards were associated with lower relative ARG abundances in poultry and higher relative ARG abundances in pigs. Lower absolute ARG levels in dust were driven by, among others, summer season and certain bedding materials for poultry, and lower animal density and summer season for pigs. This study indicates different pathways that contribute to shaping the dust resistome in livestock farms, related to dust generation, or affecting the bacterial microbiome. Farm dust is a large reservoir of ARGs from which transmission to bacteria in other reservoirs can possibly occur. The identified determinants of ARG abundances in farm dust can guide future research and potentially farm management policy.

Performance and Accuracy of Four Open-Source Tools for In Silico Serotyping of Salmonella spp. Based on Whole-Genome Short-Read Sequencing Data.

We compared the performance of four open-source in silico Salmonella typing tools (SeqSero, SeqSero2, Salmonella In Silico Typing Resource [SISTR], and Metric Oriented Sequence Typer [MOST]) to assess their potential for replacing laboratory serological testing with serovar predictions from whole-genome sequencing data. We conducted a retrospective analysis of 1,624 Salmonella isolates of 72 serovars submitted to the German National Salmonella Reference Laboratory between 1999 and 2019. All isolates are derived from animal and foodstuff origins. We conducted Illumina short-read sequencing and compared the in silico serovar prediction results with the results of routine laboratory serotyping. We found the best-performing in silico serovar prediction tool to be SISTR, with 94% correctly typed isolates, followed by SeqSero2 (87%), SeqSero (81%), and MOST (79%). Furthermore, we found that mapping-based tools like SeqSero and SeqSero2 (allele mode) were more reliable for the prediction of monophasic variants, while sequence type and cluster-based methods like MOST and SISTR (core-genome multilocus sequence type [cgMLST]), showed greater resilience when confronted with GC-biased sequencing data. We showed that the choice of library preparation kit could substantially affect O antigen detection, due to the low GC content of the wzx and wzy genes. Although the accuracy of computational serovar predictions is still not quite on par with traditional serotyping by Salmonella reference laboratories, the command-line tools investigated in this study perform a rapid, efficient, inexpensive, and reproducible analysis, which can be integrated into in-house characterization pipelines. Based on our results, we find SISTR most suitable for automated, routine serotyping for public health surveillance of SalmonellaIMPORTANCESalmonella spp. are important foodborne pathogens. To reduce the number of infected patients, it is essential to understand which subtypes of the bacteria cause disease outbreaks. Traditionally, characterization of Salmonella requires serological testing, a laboratory method by which Salmonella isolates can be classified into over 2,600 distinct subtypes, called serovars. Due to recent advances in whole-genome sequencing, many tools have been developed to replace traditional testing methods with computational analysis of genome sequences. It is crucial to validate that these tools, many already in use for routine surveillance, deliver accurate and reliable serovar information. In this study, we set out to compare which of the currently available open-source command-line tools is most suitable to replace serological testing. A thorough evaluation of the differing computational approaches is highly important to ensure the backward compatibility of serotyping data and to maintain comparability between laboratories.

Characterization of mcr-5-Harboring Salmonella enterica subsp. enterica Serovar Typhimurium Isolates from Animal and Food Origin in Germany.

We characterized eight mcr-5-positive Salmonella enterica subsp. enterica serovar Typhimurium sequence type 34 (ST34) isolates obtained from pigs and meat in Germany. Five plasmid types were identified harboring mcr-5 on Tn6452 or putative mobile insertion cassettes. The mobility of mcr-5 was confirmed by integration of Tn6452 into the bacterial chromosomes of two strains and the detection of conjugative mcr-5 plasmids. The association with mobile genetic elements might further enhance mcr-5 distribution.

The blaNDM-1-Carrying IncA/C2 Plasmid Underlies Structural Alterations and Cointegrate Formation In Vivo.

In 2012, a carbapenemase-producing Salmonella enterica serovar Corvallis isolate carrying a blaNDM-1 multiresistance IncA/C2 plasmid, apart from IncHI2 and ColE-like plasmids, was detected in a wild bird in Germany. In a recent broiler chicken infection study, we observed transfer of this blaNDM-1-carrying IncA/C2 plasmid to other Enterobacteriaceae Here, we focused on the stability of this plasmid and gained insight into the type and frequency of its structural alterations after an in vivo passage in a broiler chicken infection study.

In Vivo Transfer and Microevolution of Avian Native IncA/C2blaNDM-1-Carrying Plasmid pRH-1238 during a Broiler Chicken Infection Study.

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.

Identification of a novel transposon-associated phosphoethanolamine transferase gene, mcr-5, conferring colistin resistance in d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B.

OBJECTIVES: Plasmid-mediated mobilized colistin resistance is currently known to be caused by phosphoethanolamine transferases termed MCR-1, MCR-2, MCR-3 and MCR-4. However, this study focuses on the dissection of a novel resistance mechanism in mcr-1-, mcr-2- and mcr-3-negative d-tartrate fermenting Salmonella enterica subsp. enterica serovar Paratyphi B (Salmonella Paratyphi B dTa+) isolates with colistin MIC values >2 mg/L. METHODS: A selected isolate from the strain collection of the German National Reference Laboratory for Salmonella was investigated by WGS and bioinformatical analysis to identify novel phosphoethanolamine transferase genes involved in colistin resistance. Subsequently PCR screening, S1-PFGE and DNA-DNA hybridization were performed to analyse the prevalence and location of the identified mcr-5 gene. Cloning and transformation experiments in Escherichia coli DH5α and Salmonella Paratyphi B dTa+ control strains were carried out and the activity of MCR-5 was determined in vitro by MIC testing. RESULTS: In this study, we identified a novel phosphoethanolamine transferase in 14 mcr-1-, mcr-2- and mcr-3-negative Salmonella Paratyphi B dTa+ isolates with colistin MIC values >2 mg/L that were received during 2011-13. The respective gene, further termed as mcr-5 (1644 bp), is part of a 7337 bp transposon of the Tn3 family and usually located on related multi-copy ColE-type plasmids. Interestingly, in one isolate an additional subclone with a chromosomal location of the mcr-5 transposon was observed. CONCLUSIONS: Our findings suggest that the transfer of colistin-resistance-mediating phosphoethanolamine transferase genes from bacterial chromosomes to mobile genetic elements has occurred in multiple independent events raising concern regarding their variety, prevalence and impact on public health.

VIM-1 carbapenemase-producing Escherichia coli isolated from retail seafood, Germany 2016.

Carbapenems belong to the group of last resort antibiotics in human medicine. Therefore, the emergence of growing numbers of carbapenemase-producing bacteria in food-producing animals or the environment is worrying and an important concern for the public health sector. In the present study, a set of 45 Enterobacteriaceae isolated from German retail seafood (clams and shrimps), sampled in 2016, were investigated by real-time PCR for the presence of carbapenemase-producing bacteria. One Escherichia coli (ST10), isolated from a Venus clam (Ruditapes philippinarum) harvested in the Mediterranean Sea (Italy), contained the carbapenemase gene blaVIM-1 as part of the variable region of a class I integron. Whole-genome sequencing indicated that the integron was embedded in a Tn3-like transposon that also contained the fluoroquinolone resistance gene qnrS1. Additional resistance genes such as the extended-spectrum beta-lactamase blaSHV-12 and the AmpC gene blaACC-1 were also present in this isolate. Except blaACC-1, all resistance genes were located on an IncY plasmid. These results confirm previous observations that carbapenemase-producing bacteria have reached the food chain and are of increasing concern for public health.

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

OBJECTIVES: The characterization of CTX-M-15 ß-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.

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.

From farm to fork: Spread of a multidrug resistant Salmonella Infantis clone encoding blaCTX-M-1 on pESI-like plasmids in Central Italy.

Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) is one of the "top five Salmonella serovars" of clinical significance in the European Union (EU). Antimicrobial resistant and extended spectrum ß-lactamase (ESBL) AmpC-producing S. Infantis have been described in food production systems and human clinical samples in Italy. Recently, an increase of MDR S. Infantis carrying blaCTX-M genes involved in 3rd generation cephalosporin resistance was noticed in the EU, including Italy, mainly due to the spread of S. Infantis harboring a pESI-like plasmid. The aim of this study was to investigate the occurrence of the S. Infantis pESI-like plasmid among antibiotic resistant S. Infantis strains isolated at different points of the food chain, and to provide a phylogenetic analysis to gain further insight on their transmission pathways from 'farm to fork'. MDR S. Infantis strains (n. 35) isolated from 2016 to 2021 at different stages of the food chain (animals, food, food-related environments, and humans) were investigated with in depth molecular characterization using real-time PCR, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and whole genome sequencing (WGS). Our study reported the occurrence of S. Infantis strains harboring pESI-like plasmids, carrying blaCTX-M-1 genes, in Central Italy, at different sampling points along the food chain. Results confirmed the presence of a plasmid with a molecular size around 224-310 kb, thus consistent with the pESI-like, in 97 % of the 35 samples investigated. Two variants of S. Infantis pESI-like IncFIB(K)_1_Kpn3 were detected, one associated with the European clone carrying blaCTX-M-1 (21 isolates) and the other associated with U.S. isolates carrying blaCTX-M-65 (2 isolates, pESI-like U.S. variant). The majority was resistant to 3rd generation cephalosporins but none of the strains tested positive for the carbapenemase encoding genes. A total of 118 virulence genes were identified in isolates harboring the pESI-like plasmid. cgMLST and SNP-based analysis revealed the presence of one main cluster, composed by strains isolated from the environment, animals, food and humans. The results of this investigation underline the importance of phylogenetic studies to monitor and understand pathogen and AMR spread in a One Health approach.

Salmonella enterica subsp. enterica serovar Paratyphi B from mute swan (Cygnus olor): complete genome sequence features point towards invasive variant potential.

A subgroup of Salmonella (S.) enterica subsp. enterica serovar Paratyphi B is significantly associated with invasive infections in humans. We report the complete genome sequence of a potentially invasive. S. Paratyphi B isolated from a mute swan (Cygnus olor) found dead at an urban recreation park in Berlin, Germany.

The nematode worm Caenorhabditis elegans as an animal experiment replacement for assessing the virulence of different Salmonella enterica strains.

Caenorhabditis (C.) elegans has become a popular toxicological and biological test organism in the last two decades. Furthermore, the role of C. elegans as an alternative for replacing or reducing animal experiments is continuously discussed and investigated. In the current study, we investigated whether C. elegans survival assays can help in determining differences in the virulence of Salmonella enterica strains and to what extent C. elegans assays could replace animal experiments for this purpose. We focused on three currently discussed examples where we compared the longevity of C. elegans when fed (i) with S. enterica serovar Enteritidis vaccination or wild-type strains, (ii) with lipopolysaccharide (LPS) deficient rough or LPS forming smooth S. enterica serovar Enteritidis, and (iii) with an S. enterica subsp. diarizonae strain in the presence or absence of the typical pSASd plasmid encoding a bundle of putative virulence factors. We found that the C. elegans survival assay could indicate differences in the longevity of C. elegans when fed with the compared strain pairs to a certain extent. Putatively higher virulent S. enterica strains reduced the lifespan of C. elegans to a greater extent than putatively less virulent strains. The C. elegans survival assay is an effective and relatively easy method for classifying the virulence of different bacterial isolates in vivo, but it has some limitations. The assay cannot replace animal experiments designed to determine differences in the virulence of Salmonella enterica strains. Instead, we recommend using the described method for pre-screening bacterial strains of interest to select the most promising candidates for further animal experiments. The C. elegans assay possesses the potential to reduce the number of animal experiments. Further development of the C. elegans assay in conjunction with omics technologies, such as transcriptomics, could refine results relating to the estimation of the virulent potential of test organisms.

Genetic characterization of a multidrug-resistant Salmonella enterica serovar Agona isolated from a dietary supplement in Germany.

Salmonella enterica subsp. enterica serovar Agona has a history of causing food-borne outbreaks and any emergence of multidrug-resistant (MDR) isolates in novel food products is of concern. Particularly, in food products frequently consumed without sufficient heating prior to consumption. Here, we report about the MDR isolate, 18-SA00377, which had been isolated from a dietary supplement in Germany in 2018 and submitted to the German National Reference Laboratory for Salmonella. WGS-based comparative genetic analyses were conducted to find a potential reservoir of the isolate itself or mobile genetic elements associated with MDR. As a phylogenetic analysis did not yield any closely related S. Agona isolates, either globally or from Germany, a detailed analysis of the largest plasmid (295,499 bp) was performed as it is the main carrier of resistances. A combined approach of long-read and short-read sequencing enabled the assembly of the isolate's chromosome and its four plasmids. Their characterization revealed the presence of 23 different antibiotic resistance genes (ARGs), conferring resistance to 12 different antibiotic drug classes, as well as genes conferring resistance to six different heavy metals. The largest plasmid, pSE18-SA00377-1, belongs to the IncHI2 plasmid family and carries 16 ARGs, that are organized as two distinct clusters, with each ARG associated with putative composite transposons. Through a two-pronged approach, highly similar plasmids to pSE18-SA00377-1 were identified in the NCBI database and a search for Salmonella isolates with a highly similar ARG resistance profile was conducted. Mapping and structural comparisons between pSE18-SA00377-1 and these plasmids and Salmonella isolates showed that both the plasmid backbone and identical or similar ARG clusters can be found not only in Salmonella isolates, originating mostly from a wide variety of livestock, but also in a diverse range of bacterial genera of varying geographical origins and isolation sources. Thus, it can be speculated that the host range of pSE18-SA00377-1 is not restricted to Salmonella and its spread already occurred in different bacterial populations. Overall, this hints at a complex history for pSE18-SA00377-1 and highlights the importance of surveilling multidrug-resistant S. enterica isolates, especially in novel food items that are not yet heavily regulated.

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.

Occurrence of selected bacterial pathogens in insect-based food products and in-depth characterisation of detected Bacillus cereus group isolates.

Insects are increasingly used as alternative protein sources and ingredients of foodstuffs produced in industrial scale. Previous studies on the microbial status of insect-based foods revealed that classical foodborne pathogens such as Salmonella spp., Campylobacter spp., Listeria monocytogenes or pathogenic Escherichia coli are rarely detected, whereas particularly spore-forming bacteria with pathogenic potential such as species of the Bacillus cereus group or Clostridium species may pose a food safety risk. However, detailed descriptions of the encountered pathogenic bacteria in insect foods are scarce. We investigated a variety of 73 food products with insect or other arthropod ingredients on the occurrence of potential bacterial pathogens. These included B. cereus (sensu lato (s.l.)), Clostridium perfringens and Clostridioides difficile as representatives of spore-formers and Salmonella spp. and Shiga toxin producing and enteropathogenic E. coli (STEC/EPEC) as representatives of non-spore-forming Enterobacteriaceae. Most of the investigated food products complied with food safety standards regarding the presence of pathogens considered. However, one cricket product contained two Salmonella enterica subspecies enterica serovars (S. Wandsworth and S. Stanley). B. cereus (s.l.) was found in 42 samples (58 %), of which six contained B. cereus (s.l.) at levels higher than 103 cfu/g. The highest B. cereus (s.l.) counts of 3.8 × 105 cfu/g were found in a product with boiled and dried scorpions. Clostridium perfringens was detected in twelve samples (16 %), whereas Clostridioides difficile and STEC/EPEC were not detected in any of the samples. Remarkably, five samples contained the B. cereus (s.l.) species B. cytotoxicus. Moreover, strikingly high numbers of B. cereus (s.l.) isolates carried the capsule syntheses genes capBCADE, which were presumably located on the B. cereus pBFI_2 plasmid. Whole genome sequencing-based phylogenetic analysis suggested a high relatedness for only very few of the B. cytotoxicus and cap-positive isolates, respectively.