Draft genome sequences of a blaCTX-M-15-harboring and an atypical enteropathogenic Escherichia coli isolate recovered from slaughterhouses in Nigeria.

We present the draft genome sequences of two Escherichia coli strains isolated from slaughterhouses in Edo State, Nigeria, in 2019. The isolates were identified as blaCTX-M-15-harboring (19-47-58) and atypical enteropathogenic E. coli (aEPEC) (19-47-66), belonging to multilocus sequence types (MLST) ST46 and ST2089, respectively.

Molecular and serological diagnosis of multiple bacterial zoonoses in febrile outpatients in Garissa County, north-eastern Kenya.

Bacterial zoonoses are diseases caused by bacterial pathogens that can be naturally transmitted between humans and vertebrate animals. They are important causes of non-malarial fevers in Kenya, yet their epidemiology remains unclear. We investigated brucellosis, Q-fever and leptospirosis in the venous blood of 216 malaria-negative febrile patients recruited in two health centres (98 from Ijara and 118 from Sangailu health centres) in Garissa County in north-eastern Kenya. We determined exposure to the three zoonoses using serological (Rose Bengal test for Brucella spp., ELISA for C. burnetti and microscopic agglutination test for Leptospira spp.) and real-time PCR testing and identified risk factors for exposure. We also used non-targeted metagenomic sequencing on nine selected patients to assess the presence of other possible bacterial causes of non-malarial fevers. Considerable PCR positivity was found for Brucella (19.4%, 95% confidence intervals [CI] 14.2-25.5) and Leptospira spp. (1.7%, 95% CI 0.4-4.9), and high endpoint titres were observed against leptospiral serovar Grippotyphosa from the serological testing. Patients aged 5-17 years old had 4.02 (95% CI 1.18-13.70, p-value = 0.03) and 2.42 (95% CI 1.09-5.34, p-value = 0.03) times higher odds of infection with Brucella spp. and Coxiella burnetii than those of ages 35-80. Additionally, patients who sourced water from dams/springs, and other sources (protected wells, boreholes, bottled water, and water pans) had 2.39 (95% CI 1.22-4.68, p-value = 0.01) and 2.24 (1.15-4.35, p-value = 0.02) times higher odds of exposure to C. burnetii than those who used unprotected wells. Streptococcus and Moraxella spp. were determined using metagenomic sequencing. Brucellosis, leptospirosis, Streptococcus and Moraxella infections are potentially important causes of non-malarial fevers in Garissa. This knowledge can guide routine diagnosis, thus helping lower the disease burden and ensure better health outcomes, especially in younger populations.

Brucella spp. are facultative anaerobic bacteria under denitrifying conditions.

IMPORTANCE: Respiration is a fundamental and complex process that bacteria use to produce energy. Despite aerobic respiration being the most common, some bacteria make use of a mode of respiration in the absence of oxygen, called anaerobic respiration, which can yield advantages in adaptation to various environmental conditions. Denitrification is part of this respiratory process ensuring higher respiratory flexibility under oxygen depletion. Here, we report for the first time the evidence of anaerobic growth of Brucella spp. under denitrifying conditions, which implies that this genus should be reconsidered as facultative anaerobic. Our study further describes that efficient denitrification is not equally found within the Brucella genus, with atypical species showing a greater ability to denitrify, correlated with higher expression of the genes involved, as compared to classical species.

Survey on microbial contamination of opened skin care products used for tattooing.

AIMS: Inadequate hygiene measures as well as the use of contaminated inks or non-sterile needles are considered as important infection sources in the process of tattooing. In tattoo parlors and at conventions, it is common practice to apply cosmetic products from bulk packs as lubricants during tattooing and as ointments for tattoo aftercare. The objective of our study was to assess the microbial load of opened skin care products used during tattooing or for tattoo aftercare. METHODS AND RESULTS: First, we established a homogenization method suitable for the microbiological examination of water-immiscible products. To this end, we compared the efficiency of FastPrepTM and Stomacher® homogenizers on artificially contaminated petroleum jelly. FastPrep homogenates revealed significantly higher detection rates (≥97%) compared to Stomacher ones (31%-64%). Second, we investigated 106 cosmetic bulk pack products collected from tattoo artists. After FastPrep homogenization for 30 seconds, total aerobic mesophilic bacteria and the presence of Pseudomonas aeruginosa, Staphylococcus aureus, and Candida albicans were determined through culture. We also tested for Mycobacteria spp. by qPCR. In total, 4.7% of the cosmetic products under study turned out to be contaminated. CONCLUSION: The observed microbial contamination of opened skin care bulk packs can hold a risk to introduce bacteria into the fresh skin wound resulting from tattooing and may be a risk factor for post-tattoo infections.

If You're Not Confused, You're Not Paying Attention: Ochrobactrum Is Not Brucella.

Bacteria of the genus Brucella are facultative intracellular parasites that cause brucellosis, a severe animal and human disease. Recently, a group of taxonomists merged the brucellae with the primarily free-living, phylogenetically related Ochrobactrum spp. in the genus Brucella. This change, founded only on global genomic analysis and the fortuitous isolation of some opportunistic Ochrobactrum spp. from medically compromised patients, has been automatically included in culture collections and databases. We argue that clinical and environmental microbiologists should not accept this nomenclature, and we advise against its use because (i) it was presented without in-depth phylogenetic analyses and did not consider alternative taxonomic solutions; (ii) it was launched without the input of experts in brucellosis or Ochrobactrum; (iii) it applies a non-consensus genus concept that disregards taxonomically relevant differences in structure, physiology, population structure, core-pangenome assemblies, genome structure, genomic traits, clinical features, treatment, prevention, diagnosis, genus description rules, and, above all, pathogenicity; and (iv) placing these two bacterial groups in the same genus creates risks for veterinarians, medical doctors, clinical laboratories, health authorities, and legislators who deal with brucellosis, a disease that is particularly relevant in low- and middle-income countries. Based on all this information, we urge microbiologists, bacterial collections, genomic databases, journals, and public health boards to keep the Brucella and Ochrobactrum genera separate to avoid further bewilderment and harm.

Biocide Susceptibility and Antimicrobial Resistance of Escherichia coli Isolated from Swine Feces, Pork Meat and Humans in Germany.

Phenotypic susceptibility testing of Escherichia (E.) coli is an essential tool to gain a better understanding of the potential impact of biocide selection pressure on antimicrobial resistance. We, therefore, determined the biocide and antimicrobial susceptibility of 216 extended-spectrum ß-lactamase-producing (ESBL) and 177 non-ESBL E. coli isolated from swine feces, pork meat, voluntary donors and inpatients and evaluated associations between their susceptibilities. Minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs) of benzalkonium chloride, chlorhexidine digluconate (CHG), chlorocresol (PCMC), glutaraldehyde (GDA), isopropanol (IPA), octenidine dihydrochloride and sodium hypochlorite (NaOCl) showed unimodal distributions, indicating the absence of bacterial adaptation to biocides due to the acquisition of resistance mechanisms. Although MIC95 and MBC95 did not vary more than one doubling dilution step between isolates of porcine and human origin, significant differences in MIC and/or MBC distributions were identified for GDA, CHG, IPA, PCMC and NaOCl. Comparing non-ESBL and ESBL E. coli, significantly different MIC and/or MBC distributions were found for PCMC, CHG and GDA. Antimicrobial susceptibility testing revealed the highest frequency of resistant E. coli in the subpopulation isolated from inpatients. We observed significant but weakly positive correlations between biocide MICs and/or MBCs and antimicrobial MICs. In summary, our data indicate a rather moderate effect of biocide use on the susceptibility of E. coli to biocides and antimicrobials.

Large Multicountry Outbreak of Invasive Listeriosis by a Listeria monocytogenes ST394 Clone Linked to Smoked Rainbow Trout, 2020 to 2021.

Whole-genome sequencing (WGS) has revolutionized surveillance of infectious diseases. Disease outbreaks can now be detected with high precision, and correct attribution of infection sources has been improved. Listeriosis, caused by the bacterium Listeria monocytogenes, is a foodborne disease with a high case fatality rate and a large proportion of outbreak-related cases. Timely recognition of listeriosis outbreaks and precise allocation of food sources are important to prevent further infections and to promote public health. We report the WGS-based identification of a large multinational listeriosis outbreak with 55 cases that affected Germany, Austria, Denmark, and Switzerland during 2020 and 2021. Clinical isolates formed a highly clonal cluster (called Ny9) based on core genome multilocus sequence typing (cgMLST). Routine and ad hoc investigations of food samples identified L. monocytogenes isolates from smoked rainbow trout filets from a Danish producer grouping with the Ny9 cluster. Patient interviews confirmed consumption of rainbow trout as the most likely infection source. The Ny9 cluster was caused by a MLST sequence type (ST) ST394 clone belonging to molecular serogroup IIa, forming a distinct clade within molecular serogroup IIa strains. Analysis of the Ny9 genome revealed clpY, dgcB, and recQ inactivating mutations, but phenotypic characterization of several virulence-associated traits of a representative Ny9 isolate showed that the outbreak strain had the same pathogenic potential as other serogroup IIa strains. Our report demonstrates that international food trade can cause multicountry outbreaks that necessitate cross-border outbreak collaboration. It also corroborates the relevance of ready-to-eat smoked fish products as causes for listeriosis. IMPORTANCE Listeriosis is a severe infectious disease in humans and characterized by an exceptionally high case fatality rate. The disease is transmitted through consumption of food contaminated by the bacterium Listeria monocytogenes. Outbreaks of listeriosis often occur but can be recognized and stopped through implementation of whole-genome sequencing-based pathogen surveillance systems. We here describe the detection and management of a large listeriosis outbreak in Germany and three neighboring countries. This outbreak was caused by rainbow trout filet, which was contaminated by a L. monocytogenes clone belonging to sequence type ST394. This work further expands our knowledge on the genetic diversity and transmission routes of an important foodborne pathogen.

Towards a Harmonized Terminology: A Glossary for Biocide Susceptibility Testing.

Disinfection is a key strategy to reduce the burden of infections. The contact of bacteria to biocides-the active substances of disinfectants-has been linked to bacterial adaptation and the development of antimicrobial resistance. Currently, there is no scientific consensus on whether the excessive use of biocides contributes to the emergence and spread of multidrug resistant bacteria. The comprehensive analysis of available data remains a challenge because neither uniform test procedures nor standardized interpretive criteria nor harmonized terms are available to describe altered bacterial susceptibility to biocides. In our review, we investigated the variety of criteria and the diversity of terms applied to interpret findings in original studies performing biocide susceptibility testing (BST) of field isolates. An additional analysis of reviews summarizing the knowledge of individual studies on altered biocide susceptibility provided insights into currently available broader concepts for data interpretation. Both approaches pointed out the urgent need for standardization. We, therefore, propose that the well-established and approved concepts for interpretation of antimicrobial susceptibility testing data should serve as a role model to evaluate biocide resistance mechanisms on a single cell level. Furthermore, we emphasize the adaptations necessary to acknowledge the specific needs for the evaluation of BST data. Our approach might help to increase scientific awareness and acceptance.

Longitudinal Study of Selected Bacterial Zoonoses in Small Ruminants in Tana River County, Kenya.

Brucellosis, Q fever, and leptospirosis are priority zoonoses worldwide, yet their epidemiology is understudied, and studies investigating multiple pathogens are scarce. Therefore, we selected 316 small ruminants in irrigated, pastoral, and riverine settings in Tana River County and conducted repeated sampling for animals that were initially seronegative between September 2014 and June 2015. We carried out serological and polymerase chain reaction tests and determined risk factors for exposure. The survey-weighted serological incidence rates were 1.8 (95% confidence intervals [CI]: 1.3-2.5) and 1.3 (95% CI: 0.7-2.3) cases per 100 animal-months at risk for Leptospira spp. and C. burnetii, respectively. We observed no seroconversions for Brucella spp. Animals from the irrigated setting had 6.83 (95% CI: 2.58-18.06, p-value = 0.01) higher odds of seropositivity to C. burnetii than those from riverine settings. Considerable co-exposure of animals to more than one zoonosis was also observed, with animals exposed to one zoonosis generally having 2.5 times higher odds of exposure to a second zoonosis. The higher incidence of C. burnetii and Leptospira spp. infections, which are understudied zoonoses in Kenya compared to Brucella spp., demonstrate the need for systematic prioritization of animal diseases to enable the appropriate allocation of resources.

The Retrospective on Atypical Brucella Species Leads to Novel Definitions.

The genus Brucella currently comprises twelve species of facultative intracellular bacteria with variable zoonotic potential. Six of them have been considered as classical, causing brucellosis in terrestrial mammalian hosts, with two species originated from marine mammals. In the past fifteen years, field research as well as improved pathogen detection and typing have allowed the identification of four new species, namely Brucella microti, Brucella inopinata, Brucella papionis, Brucella vulpis, and of numerous strains, isolated from a wide range of hosts, including for the first time cold-blooded animals. While their genome sequences are still highly similar to those of classical strains, some of them are characterized by atypical phenotypes such as higher growth rate, increased resistance to acid stress, motility, and lethality in the murine infection model. In our review, we provide an overview of state-of-the-art knowledge about these novel Brucella sp., with emphasis on their phylogenetic positions in the genus, their metabolic characteristics, acid stress resistance mechanisms, and their behavior in well-established in cellulo and in vivo infection models. Comparison of phylogenetic classification and phenotypical properties between classical and novel Brucella species and strains finally lead us to propose a more adapted terminology, distinguishing between core and non-core, and typical versus atypical brucellae, respectively.

Invasive listeriosis outbreaks and salmon products: a genomic, epidemiological study.

Invasive listeriosis, caused by Listeria (L.) monocytogenes, is a severe foodborne infection, especially for immunocompromised individuals. The aim of our investigation was the identification and analysis of listeriosis outbreaks in Germany with smoked and graved salmon products as the most likely source of infection using whole-genome sequencing (WGS) and patient interviews. In a national surveillance programme, WGS was used for subtyping and core genome multi locus sequence typing (cgMLST) for cluster detection of L. monocytogenes isolates from listeriosis cases as well as food and environmental samples in Germany. Patient interviews were conducted to complement the molecular typing. We identified 22 independent listeriosis outbreaks occurring between 2010 and 2021 that were most likely associated with the consumption of smoked and graved salmon products. In Germany, 228 cases were identified, of 50 deaths (22%) reported 17 were confirmed to have died from listeriosis. Many of these 22 outbreaks were cross-border outbreaks with further cases in other countries. This report shows that smoked and graved salmon products contaminated with L. monocytogenes pose a serious risk for listeriosis infection in Germany. Interdisciplinary efforts including WGS and epidemiological investigations were essential to identifying the source of infection. Uncooked salmon products are high-risk foods frequently contaminated with L. monocytogenes. In order to minimize the risk of infection for consumers, food producers need to improve hygiene measures and reduce the entry of pathogens into food processing. Furthermore, susceptible individuals should be better informed of the risk of acquiring listeriosis from consuming smoked and graved salmon products.

A systematic review and meta-analysis of the aetiological agents of non-malarial febrile illnesses in Africa.

BACKGROUND: The awareness of non-malarial febrile illnesses (NMFIs) has been on the rise over the last decades. Therefore, we undertook a systematic literature review and meta-analysis of causative agents of non-malarial fevers on the African continent. METHODOLOGY: We searched for literature in African Journals Online, EMBASE, PubMed, Scopus, and Web of Science databases to identify aetiologic agents that had been reported and to determine summary estimates of the proportional morbidity rates (PMr) associated with these pathogens among fever patients. FINDINGS: A total of 133 studies comprising 391,835 patients from 25 of the 54 African countries were eligible. A wide array of aetiologic agents were described with considerable regional differences among the leading agents. Overall, bacterial pathogens tested from blood samples accounted for the largest proportion. The summary estimates from the meta-analysis were low for most of the agents. This may have resulted from a true low prevalence of the agents, the failure to test for many agents or the low sensitivity of the diagnostic methods applied. Our meta-regression analysis of study and population variables showed that diagnostic methods determined the PMr estimates of typhoidal Salmonella and Dengue virus. An increase in the PMr of Klebsiella spp. infections was observed over time. Furthermore, the status of patients as either inpatient or outpatient predicted the PMr of Haemophilus spp. infections. CONCLUSION: The small number of epidemiological studies and the variety of NMFI agents on the African continent emphasizes the need for harmonized studies with larger sample sizes. In particular, diagnostic procedures for NMFIs should be standardized to facilitate comparability of study results and to improve future meta-analyses. Reliable NMFI burden estimates will inform regional public health strategies.

Comparative Genome-Wide Transcriptome Analysis of Brucella suis and Brucella microti Under Acid Stress at pH 4.5: Cold Shock Protein CspA and Dps Are Associated With Acid Resistance of B. microti.

Brucellae are facultative intracellular coccobacilli causing brucellosis, one of the most widespread bacterial zoonosis affecting wildlife animals, livestock and humans. The genus Brucella comprises classical and atypical species, such as Brucella suis and Brucella microti, respectively. The latter is characterized by increased metabolic activity, fast growth rates, and extreme acid resistance at pH 2.5, suggesting an advantage for environmental survival. In addition, B. microti is more acid-tolerant than B. suis at the intermediate pH of 4.5. This acid-resistant phenotype of B. microti may have major implications for fitness in soil, food products and macrophages. Our study focused on the identification and characterization of acid resistance determinants of B. suis and B. microti in Gerhardt's minimal medium at pH 4.5 and 7.0 for 20 min and 2 h by comparative RNA-Seq-based transcriptome analysis, validated by RT-qPCR. Results yielded a common core response in both species with a total of 150 differentially expressed genes, and acidic pH-dependent genes regulated specifically in each species. The identified core response mechanisms comprise proton neutralization or extrusion from the cytosol, participating in maintaining physiological intracellular pH values. Differential expression of 441 genes revealed species-specific mechanisms in B. microti with rapid physiological adaptation to acid stress, anticipating potential damage to cellular components and critical energy conditions. Acid stress-induced genes encoding cold shock protein CspA, pseudogene in B. suis, and stress protein Dps were associated with survival of B. microti at pH 4.5. B. suis response with 284 specifically regulated genes suggested increased acid stress-mediated protein misfolding or damaging, triggering the set-up of repair strategies countering the consequences rather than the origin of acid stress and leading to subsequent loss of viability. In conclusion, our work supports the hypothesis that increased acid stress resistance of B. microti is based on selective pressure for the maintenance of functionality of critical genes, and on specific differential gene expression, resulting in rapid adaptation.

Protein Biomarker Identification for the Discrimination of Brucella melitensis Field Isolates From the Brucella melitensis Rev.1 Vaccine Strain by MALDI-TOF MS.

Brucella melitensis Rev.1 is a live attenuated vaccine strain that is widely used to control brucellosis in small ruminants. For successful surveillance and control programs, rapid identification and characterization of Brucella isolates and reliable differentiation of vaccinated and naturally infected animals are essential prerequisites. Although MALDI-TOF MS is increasingly applied in clinical microbiology laboratories for the diagnosis of brucellosis, species or even strain differentiation by this method remains a challenge. To detect biomarkers, which enable to distinguish the B. melitensis Rev.1 vaccine strain from B. melitensis field isolates, we initially searched for unique marker proteins by in silico comparison of the B. melitensis Rev.1 and 16M proteomes. We found 113 protein sequences of B. melitensis 16M that revealed a homologous sequence in the B. melitensis Rev.1 annotation and 17 of these sequences yielded potential biomarker pairs. MALDI-TOF MS spectra of 18 B. melitensis Rev.1 vaccine and 183 Israeli B. melitensis field isolates were subsequently analyzed to validate the identified marker candidates. This approach detected two genus-wide unique biomarkers with properties most similar to the ribosomal proteins L24 and S12. These two proteins clearly discriminated B. melitensis Rev.1 from the closely related B. melitensis 16M and the Israeli B. melitensis field isolates. In addition, we verified their discriminatory power using a set of B. melitensis strains from various origins and of different MLVA types. Based on our results, we propose MALDI-TOF MS profiling as a rapid, cost-effective alternative to the traditional, time-consuming approach to differentiate certain B. melitensis isolates on strain level.

Testing assembly strategies of Francisella tularensis genomes to infer an evolutionary conservation analysis of genomic structures.

BACKGROUND: We benchmarked sequencing technology and assembly strategies for short-read, long-read, and hybrid assemblers in respect to correctness, contiguity, and completeness of assemblies in genomes of Francisella tularensis. Benchmarking allowed in-depth analyses of genomic structures of the Francisella pathogenicity islands and insertion sequences. Five major high-throughput sequencing technologies were applied, including next-generation "short-read" and third-generation "long-read" sequencing methods. RESULTS: We focused on short-read assemblers, hybrid assemblers, and analysis of the genomic structure with particular emphasis on insertion sequences and the Francisella pathogenicity island. The A5-miseq pipeline performed best for MiSeq data, Mira for Ion Torrent data, and ABySS for HiSeq data from eight short-read assembly methods. Two approaches were applied to benchmark long-read and hybrid assembly strategies: long-read-first assembly followed by correction with short reads (Canu/Pilon, Flye/Pilon) and short-read-first assembly along with scaffolding based on long reads (Unicyler, SPAdes). Hybrid assembly can resolve large repetitive regions best with a "long-read first" approach. CONCLUSIONS: Genomic structures of the Francisella pathogenicity islands frequently showed misassembly. Insertion sequences (IS) could be used to perform an evolutionary conservation analysis. A phylogenetic structure of insertion sequences and the evolution within the clades elucidated the clade structure of the highly conservative F. tularensis.

Direct identification and molecular characterization of zoonotic hazards in raw milk by metagenomics using Brucella as a model pathogen.

Metagenomics is a valuable diagnostic tool for enhancing microbial food safety because (i) it enables the untargeted detection of pathogens, (ii) it is fast since primary isolation of micro-organisms is not required, and (iii) it has high discriminatory power allowing for a detailed molecular characterization of pathogens. For shotgun metagenomics, total nucleic acids (NAs) are isolated from complex samples such as foodstuff. Along with microbial NAs, high amounts of matrix NAs are extracted that might outcompete microbial NAs during next-generation sequencing and compromise sensitivity for the detection of low abundance micro-organisms. Sensitive laboratory methods are indispensable for detecting highly pathogenic foodborne bacteria like Brucella spp., because a low infectious dose is sufficient to cause human disease through the consumption of contaminated dairy or meat products. In our study, we applied shotgun metagenomic sequencing for the identification and characterization of Brucella spp. in artificially and naturally contaminated raw milk from various ruminant species. With the depletion of eukaryotic cells prior to DNA extraction, Brucella was detectable at 10 bacterial cells ml-1, while at the same time microbiological culture and isolation of the fastidious bacteria commonly failed. Moreover, we were able to retrieve the genotype of a Brucella isolate from a metagenomic dataset, indicating the potential of metagenomics for outbreak investigations using SNPs and core-genome multilocus sequence typing (cgMLST). To improve diagnostic applications, we developed a new bioinformatics approach for strain prediction based on SNPs to identify the correct species and define a certain strain with only low numbers of genus-specific reads per sample. This pipeline turned out to be more sensitive and specific than Mash Screen. In raw milk samples, we simultaneously detected numerous other zoonotic pathogens, antimicrobial resistance genes and virulence factors. Our study showed that metagenomics is a highly sensitive tool for biological risk assessment of foodstuffs, particularly when pathogen isolation is hazardous or challenging.

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 ß-lactamase- and AmpC ß-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.

Translatability of WGS typing results can simplify data exchange for surveillance and control of Listeria monocytogenes.

Where classical epidemiology has proven to be inadequate for surveillance and control of foodborne pathogens, molecular epidemiology, using genomic typing methods, can add value. However, the analysis of whole genome sequencing (WGS) data varies widely and is not yet fully harmonised. We used genomic data on 494 Listeria monocytogenes isolates from ready-to-eat food products and food processing environments deposited in the strain collection of the German National Reference Laboratory to compare various procedures for WGS data analysis and to evaluate compatibility of results. Two different core genome multilocus sequence typing (cgMLST) schemes, different reference genomes in single nucleotide polymorphism (SNP) analysis and commercial as well as open-source software were compared. Correlation of allele distances from the different cgMLST approaches was high, ranging from 0.97 to 1, and unified thresholds yielded higher clustering concordance than scheme-specific thresholds. The number of detected SNP differences could be increased up to a factor of 3.9 using a specific reference genome compared with a general one. Additionally, specific reference genomes improved comparability of SNP analysis results obtained using different software tools. The use of a closed or a draft specific reference genome did not make a difference. The harmonisation of WGS data analysis will finally guarantee seamless data exchange, but, in the meantime, knowledge on threshold values that lead to comparable clustering of isolates by different methods may improve communication between laboratories. We therefore established a translation code between commonly applied cgMLST and SNP methods based on optimised clustering concordances. This code can work as a first filter to identify WGS-based typing matches resulting from different methods, which opens up a new perspective for data exchange and thereby accelerates time-critical analyses, such as in 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.