Association between antimicrobial usage in livestock and antimicrobial resistance in Escherichia coli isolates from human urinary tract infections in the Netherlands, 2009-2020.

BACKGROUND: In the last decade, veterinary antimicrobial usage (AMU) and antimicrobial resistance (AMR) among indicator bacteria in livestock have decreased substantially in the Netherlands. The extent to which this decrease has affected AMR levels among human infections remains unclear. OBJECTIVES: To assess the association between AMU in livestock and AMR in Escherichia coli isolates from human urinary tract infections (UTIs). METHODS: Data on AMR and AMU between 2009 and 2020 from Dutch national surveillance programmes for humans and livestock were used. Associations between AMU in four major livestock sectors and AMR in humans were assessed for 10 antimicrobial classes and the ESBL resistance profile, using logistic regression analysis. Associations between AMU and AMR in livestock, between AMR in livestock and in humans, and between AMU and AMR in humans were also assessed. RESULTS: Statistical significance was reached for 16/31 of the tested associations between AMU in livestock and AMR in human E. coli UTIs. Of the significant associations, 11 were positive (OR 1.01-1.24), whereas 5 were negative (OR 0.96-0.99). All associations between human AMU and AMR in E. coli isolates from UTIs were positive and statistically significant. Weak but significant positive correlations were also observed between livestock AMR and human AMR. CONCLUSIONS: Although several significant associations between AMU in livestock and AMR in human UTIs caused by E. coli were observed, the associations between AMU and AMR were generally stronger within the human and animal populations. This indicates that potential zoonotic spread of AMR in E. coli causing human UTIs from livestock sources is limited.

Discovery of Vibrio cholerae in Urban Sewage in Copenhagen, Denmark.

We report the discovery of a persistent presence of Vibrio cholerae at very low abundance in the inlet of a single wastewater treatment plant in Copenhagen, Denmark at least since 2015. Remarkably, no environmental or locally transmitted clinical case of V. cholerae has been reported in Denmark for more than 100 years. We, however, have recovered a near-complete genome out of 115 metagenomic sewage samples taken over the past 8 years, despite the extremely low relative abundance of one V. cholerae read out of 500,000 sequenced reads. Due to the very low relative abundance, routine screening of the individual samples did not reveal V. cholerae. The recovered genome lacks the gene responsible for cholerae toxin production, but although this strain may not pose an immediate public health risk, our finding illustrates the importance, challenges, and effectiveness of wastewater-based pathogen surveillance.

Impact of non-pharmaceutical interventions during the COVID-19 pandemic on pathogens transmitted via food in the Netherlands.

The COVID-19 pandemic impacted the transmission of many pathogens. The aim was to determine the effect of non-pharmaceutical interventions on the incidence of diseases transmitted via food. Weekly incidence rates for nine foodborne pathogens were collected from national surveillance registries. Weekly pathogen incidence during lockdown weeks of 2020 and 2021 were compared with corresponding weeks in 2015-2019. The same analyses were performed to determine the effect of self-defined expected impact levels of measures (low, intermediate and high). Eight out of 9 diseases showed a significant decrease in case number in 2020, except for listeriosis, which remained unchanged. The largest decrease was observed for rotavirus gastronteritis A (-81%), norovirus gastroenteritis (-78%), hepatitis A (-75%) and shigellosis (-72). In 2021, lower case numbers were observed for 6 out of 9 diseases compared with 2015-2019, with the largest decrease for shigellosis (-5/%) and hepatitis E (-47%). No significant change was observed for listeriosis, STEC infection and rotavirus gastroenteritis. Overall, measures with increased expected impact level did not result in a larger decrease in number of cases, except for Campylobacter, and norovirus and rotavirus gastroenteritis. Disease transmitted via food significantly decreased during the COVID-19 pandemic, with a more pronounced effect during 2020 than 2021.

Genomic analysis of Vibrio cholerae O1 isolates from cholera cases, Europe, 2022.

BackgroundThe number of cholera cases reported to the World Health Organization (WHO) in 2022 was more than double that of 2021. Nine countries of the WHO European Region reported 51 cases of cholera in 2022 vs five reported cases in 2021.AimWe aimed to confirm that the Vibrio cholerae O1 isolates reported by WHO European Region countries in 2022 belonged to the seventh pandemic El Tor lineage (7PET). We also studied their virulence, antimicrobial resistance (AMR) determinants and phylogenetic relationships.MethodsWe used microbial genomics to study the 49 V. cholerae O1 isolates recovered from the 51 European cases. We also used > 1,450 publicly available 7PET genomes to provide a global phylogenetic context for these 49 isolates.ResultsAll 46 good-quality genomes obtained belonged to the 7PET lineage. All but two isolates belonged to genomic Wave 3 and were grouped within three sub-lineages, one of which, Pre-AFR15, predominated (34/44). This sub-lineage, corresponding to isolates from several countries in Southern Asia, the Middle East and Eastern or Southern Africa, was probably a major contributor to the global upsurge of cholera cases in 2022. No unusual AMR profiles were inferred from analysis of the AMR gene content of the 46 genomes.ConclusionReference laboratories in high-income countries should use whole genome sequencing to assign V. cholerae O1 isolates formally to the 7PET or non-epidemic lineages. Periodic collaborative genomic studies based on isolates from travellers can provide useful information on the circulating strains and their evolution, particularly as concerns AMR.

Large outbreak of typhoid fever on a river cruise ship used as accommodation for asylum seekers, the Netherlands, 2022.

On 6 April 2022, the Public Health Service of Kennemerland, the Netherlands, was notified about an outbreak of fever and abdominal complaints on a retired river cruise ship, used as shelter for asylum seekers. The diagnosis typhoid fever was confirmed on 7 April. An extensive outbreak investigation was performed. Within 47 days, 72 typhoid fever cases were identified among asylum seekers (n = 52) and staff (n = 20), of which 25 were hospitalised. All recovered after treatment. Consumption of food and tap water on the ship was associated with developing typhoid fever. The freshwater and wastewater tanks shared a common wall with severe corrosion and perforations, enabling wastewater to leak into the freshwater tank at high filling levels. Salmonella Typhi was cultured from the wastewater tank, matching the patient isolates. In the freshwater tank, Salmonella species DNA was detected by PCR, suggesting the presence of the bacterium and supporting the conclusion of contaminated freshwater as the probable source of the outbreak. Outbreaks of uncommon infections may occur if persons from endemic countries are accommodated in crowded conditions. Especially when accommodating migrants on ships, strict supervision on water quality and technical installations are indispensable to guarantee the health and safety of the residents.

Sporadic Occurrence of Enteroaggregative Shiga Toxin-Producing Escherichia coli O104:H4 Similar to 2011 Outbreak Strain.

We describe the recent detection of 3 Shiga toxin-producing enteroaggregative Escherichia coli O104:H4 isolates from patients and 1 from pork in the Netherlands that were genetically highly similar to isolates from the 2011 large-scale outbreak in Europe. Our findings stress the importance of safeguarding food supply production chains to prevent future outbreaks.

Changing epidemiology of Salmonella Enteritidis human infections in the Netherlands and Belgium, 2006 to 2019: a registry-based population study.

BackgroundSalmonellosis remains the second most common zoonosis in the European Union despite a long-term decreasing trend. However, this trend has been reported to have stagnated in recent years, particularly for Salmonella enterica serotype Enteritidis (SE).AimTo describe temporal changes in the incidence of SE human infections, and in its associated factors between 2006 and 2019. In addition, we aim to determine which factors influenced the stagnated trend seen in recent years.MethodsData on culture-confirmed SE human infections from national surveillance registries in the Netherlands and Belgium between 2006 and 2019 were analysed using multivariable negative-binomial regression models with restricted cubic splines.ResultsSE incidence was significantly higher in summer and autumn than winter, in persons aged 0-4 years and 5-14 years than in persons ≥ 60 years, and increased with increasing proportions of travel-related and resistant SE infections. SE incidence decreased significantly in both countries until 2015, followed by an increasing trend, which was particularly pronounced in the Netherlands. Potential SE outbreaks in both countries and invasive infections in the Netherlands also increased after 2015.ConclusionThe increase in potential outbreaks and invasive infections since 2015 may partially explain the observed reversal of the decreasing trend. While these results provide insights into the possible causes of this trend reversal, attention should also be given to factors known to influence SE epidemiology at primary (animal) production and pathogen genomic levels.

Investigation of an international outbreak of multidrug-resistant monophasic Salmonella Typhimurium associated with chocolate products, EU/EEA and United Kingdom, February to April 2022.

An extensive multi-country outbreak of multidrug-resistant monophasic Salmonella Typhimurium infection in 10 countries with 150 reported cases, predominantly affecting young children, has been linked to chocolate products produced by a large multinational company. Extensive withdrawals and recalls of multiple product lines have been undertaken. With Easter approaching, widespread product distribution and the vulnerability of the affected population, early and effective real-time sharing of microbiological and epidemiological information has been of critical importance in effectively managing this serious food-borne incident.

Increasing the Efficiency of a National Laboratory Response to COVID-19: a Nationwide Multicenter Evaluation of 47 Commercial SARS-CoV-2 Immunoassays by 41 Laboratories.

In response to the worldwide pandemic of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and the subsequent antibody tests that flooded the market, a nationwide collaborative approach in the Netherlands was employed. Forty-one Dutch laboratories joined forces and shared their evaluation data to allow for the evaluation of a quantity of serological assays for SARS-CoV-2 that exceeds the capacity of each individual laboratory. As of April 2020, these performance data had been aggregated and shared in regularly updated reports with other laboratories, Dutch government, public health organizations, and the public. This frequently updated overview of assay performance increased the efficiency of our national laboratory response, supporting laboratories in their choice and implementation of assays. Aggregated performance data for 47 immunoassays for SARS-CoV-2 showed that none of the evaluated immunoassays that detect only IgM or IgA met the diagnostic criteria, indicating that they are not suitable for diagnosing acute infections. For the detection of IgG, only the Biozek Corona virus COVID rapid test, Euroimmun SARS-CoV-2 IgG, and Wantai SARS-CoV-2 antibody (Ab) ELISA met predefined performance criteria in hospitalized patients where samples were collected 14 days post-onset of symptoms (DPO), while for patients with mild or asymptomatic infections, only the Wantai SARS-CoV-2 Ab ELISA met the predefined performance criteria if samples were collected 14 days postonset. Here, we describe this unique nationwide collaboration during the onset of the COVID-19 pandemic; the collected data and their results are an example of what can be accomplished when forces are joined during a public health crisis.

Outbreak of Central American born Shigella sonnei in two youth camps in Belgium in the summer of 2019.

In 2019, an outbreak of Shigella sonnei occurred during two youth camps in Belgium. The clustering of isolates from both camps was confirmed by next-generation sequencing, as well as a secondary infection of a technician. The outbreak strain clustered with internationally isolated strains from patients with recent travel history to Central America. This report exemplifies enhanced surveillance and international collaboration between public health institutes by enabling to link local outbreaks to region-specific sublineages circulating abroad.

Impact of the COVID-19 pandemic on human salmonellosis in the Netherlands.

The public health measures implemented to control coronavirus disease 2019 (COVID-19) may influence also other infectious diseases. Using national laboratory surveillance data, we assessed the impact of the COVID-19 pandemic on human salmonellosis in the Netherlands until March 2021. Salmonellosis incidence decreased significantly after March 2020: in the second, third and fourth quarters of 2020, and in the first quarter of 2021, the incidence decreased by 55%, 57%, 47% and 37%, respectively, compared to the same quarters of 2016-2019. The decrease was strongest among travel-related cases (94%, 84%, 79% and 93% in the aforementioned quarters, respectively). Other significant changes were: increased proportion of cases among older adults and increased proportion of invasive infections, decreased proportion of trimethoprim resistance and increased proportion of serovar Typhimurium monophasic variant vs. Enteritidis. This led to decreased contributions of laying hens and increased contributions of pigs and cattle as sources of human infections. The observed changes probably reflect a combination of reduced exposure to Salmonella due to restrictions on international travels and gatherings, closure of dine-in restaurants, catering and hospitality sectors at large and changes in healthcare-seeking and diagnostic behaviours.

Genome-wide association studies of Shigella spp. and Enteroinvasive Escherichia coli isolates demonstrate an absence of genetic markers for prediction of disease severity.

BACKGROUND: We investigated the association of symptoms and disease severity of shigellosis patients with genetic determinants of infecting Shigella and entero-invasive Escherichia coli (EIEC), because determinants that predict disease outcome per individual patient could be used to prioritize control measures. For this purpose, genome wide association studies (GWAS) were performed using presence or absence of single genes, combinations of genes, and k-mers. All genetic variants were derived from draft genome sequences of isolates from a multicenter cross-sectional study conducted in the Netherlands during 2016 and 2017. Clinical data of patients consisting of binary/dichotomous representation of symptoms and their calculated severity scores were also available from this study. To verify the suitability of the methods used, the genetic differences between the genera Shigella and Escherichia were used as control. RESULTS: The isolates obtained were representative of the population structure encountered in other Western European countries. No association was found between single genes or combinations of genes and separate symptoms or disease severity scores. Our benchmark characteristic, genus, resulted in eight associated genes and > 3,000,000 k-mers, indicating adequate performance of the algorithms used. CONCLUSIONS: To conclude, using several microbial GWAS methods, genetic variants in Shigella spp. and EIEC that can predict specific symptoms or a more severe course of disease were not identified, suggesting that disease severity of shigellosis is dependent on other factors than the genetic variation of the infecting bacteria. Specific genes or gene fragments of isolates from patients are unsuitable to predict outcomes and cannot be used for development, prioritization and optimization of guidelines for control measures of shigellosis or infections with EIEC.

Incidence, clinical implications and impact on public health of infections with Shigella spp. and entero-invasive Escherichia coli (EIEC): results of a multicenter cross-sectional study in the Netherlands during 2016-2017.

BACKGROUND: Shigella spp. and entero-invasive E. coli (EIEC) use the same invasive mechanism to cause diarrheal diseases. Public health regulations apply only to Shigella spp. infections, but are hampered by the lack of simple methods to distinguish them from EIEC. In the last decades, molecular methods for detecting Shigella spp. and EIEC were implemented in medical microbiological laboratories (MMLs). However, shigellosis cases identified with molecular techniques alone are not notifiable in most countries. Our study investigates the impact of EIEC versus Shigella spp. infections and molecular diagnosed shigellosis versus culture confirmed shigellosis for re-examination of the rationale for the current public health regulations. METHODS: In this multicenter cross-sectional study, fecal samples of patients suspected for gastro-enteritis, referred to 15 MMLs in the Netherlands, were screened by PCR for Shigella spp. or EIEC. Samples were cultured to discriminate between the two pathogens. We compared risk factors, symptoms, severity of disease, secondary infections and socio-economic consequences for (i) culture-confirmed Shigella spp. versus culture-confirmed EIEC cases (ii) culture positive versus PCR positive only shigellosis cases. RESULTS: In 2016-2017, 777 PCR positive fecal samples with patient data were included, 254 of these were culture-confirmed shigellosis cases and 32 were culture-confirmed EIEC cases. EIEC cases were more likely to report ingestion of contaminated food and were less likely to be men who have sex with men (MSM). Both pathogens were shown to cause serious disease although differences in specific symptoms were observed. Culture-negative but PCR positive cases were more likely report travel or ingestion of contaminated food and were less likely to be MSM than culture-positive cases. Culture-negative cases were more likely to suffer from multiple symptoms. No differences in degree of secondary infections were observed between Shigella spp. and EIEC, and culture-negative and culture-positive cases. CONCLUSIONS: No convincing evidence was found to support the current guidelines that employs different measures based on species or detection method. Therefore, culture and molecular detection methods for Shigella spp. and EIEC should be considered equivalent for case definition and public health regulations regarding shigellosis. Differences were found regarding risks factors, indicating that different prevention strategies may be required.

Evaluation of a Culture-Dependent Algorithm and a Molecular Algorithm for Identification of Shigella spp., Escherichia coli, and Enteroinvasive E. coli.

Identification of Shigella spp., Escherichia coli, and enteroinvasive E. coli (EIEC) is challenging because of their close relatedness. Distinction is vital, as infections with Shigella spp. are under surveillance of health authorities, in contrast to EIEC infections. In this study, a culture-dependent identification algorithm and a molecular identification algorithm were evaluated. Discrepancies between the two algorithms and original identification were assessed using whole-genome sequencing (WGS). After discrepancy analysis with the molecular algorithm, 100% of the evaluated isolates were identified in concordance with the original identification. However, the resolution for certain serotypes was lower than that of previously described methods and lower than that of the culture-dependent algorithm. Although the resolution of the culture-dependent algorithm is high, 100% of noninvasive E. coli, Shigella sonnei, and Shigella dysenteriae, 93% of Shigella boydii and EIEC, and 85% of Shigella flexneri isolates were identified in concordance with the original identification. Discrepancy analysis using WGS was able to confirm one of the used algorithms in four discrepant results. However, it failed to clarify three other discrepant results, as it added yet another identification. Both proposed algorithms performed well for the identification of Shigella spp. and EIEC isolates and are applicable in low-resource settings, in contrast to previously described methods that require WGS for daily diagnostics. Evaluation of the algorithms showed that both algorithms are capable of identifying Shigella species and EIEC isolates. The molecular algorithm is more applicable in clinical diagnostics for fast and accurate screening, while the culture-dependent algorithm is more suitable for reference laboratories to identify Shigella spp. and EIEC up to the serotype level.

Possible misidentification of species in the Pseudomonas fluorescens lineage as Burkholderia pseudomallei and Francisella tularensis, and emended descriptions of Pseudomonas brenneri,Pseudomonas gessardii and Pseudomonas proteolytica.

Bacteria were isolated from an industrial water circuit in the Netherlands. These strains were identified using API 20 NE as possible, or likely, Burkholderia pseudomallei. With VITEK 2 some of these strains scored 'low discrimination' for Francisella tularensis, amongst others. A total of twenty-six strains were assigned to the species Pseudomonas brenneri, Pseudomonas gessardii or Pseudomonas proteolytica. Because of the possibility of misidentification of these environmental species as medical- and public-health relevant B. pseudomallei and F. tularensis, an emended description, based on tests results more customarily used in clinical laboratories, was suitable. For this reason, the strains in this study, including the type strains DSM 15294T, DSM 17152T and DSM 15321T, were subjected to a polyphasic identification procedure. This procedure consisted of multiple phenotypic tests, fatty acid analysis, 16S rDNA sequence analysis, matrix-assisted laser desorption ionization time-of-flight mass spectronomy and various species-specific molecular tests. Based on the results of the polyphasic procedures, the species descriptions of P. brenneri, P. gessardii and P. proteolytica have been emended.

Use of whole genome sequencing for surveillance and control of foodborne diseases: status quo and quo vadis.

Improvements in sequencing quality, availability, speed and costs results in an increased presence of genomics in infectious disease applications. Nevertheless, there are still hurdles in regard to the optimal use of WGS for public health purposes. Here, we discuss the current state ("status quo") and future directions ("quo vadis") based on literature regarding the use of genomics in surveillance, hazard characterization and source attribution of foodborne pathogens. The future directions include the application of new techniques, such as machine learning and network approaches that may overcome the current shortcomings. These include the use of fixed genomic distances in cluster delineation, disentangling similarity or lack thereof in source attribution, and difficulties ascertaining function in hazard characterization. Although, the aforementioned methods can relatively easily be applied technically, an overarching challenge is the inference and biological/epidemiological interpretation of these large amounts of high-resolution data. Understanding the context in terms of bacterial isolate and host diversity allows to assess the level of representativeness in regard to sources and isolates in the dataset, which in turn defines the level of certainty associated with defining clusters, sources and risks. This also marks the importance of metadata (clinical, epidemiological, and biological) when using genomics for public health purposes.

Temporal association of antimicrobial use in livestock with antimicrobial resistance in non-typhoid Salmonella human infections in the Netherlands, 2008-2019.

Background: Antimicrobial use (AMU) in livestock contributes to antimicrobial resistance (AMR) among zoonotic pathogens, such as non-typhoid Salmonella (NTS). Since 2009, the Netherlands has made substantial efforts to reduce AMU in livestock. Objectives: To assess the association between AMU in livestock and AMR in NTS human isolates. Additionally, associations between AMU in broilers/pigs and AMR in NTS broiler/pig isolates, and between AMR in broilers/pigs and in human NTS isolates were assessed. The focus was on Salmonella Enteritidis (SE) and Salmonella Typhimurium including its monophasic variant (ST/STM). Methods: A national population registry-based study was conducted in the Netherlands from 2008 to 2019. Multivariable logistic regression models were used to assess the associations between livestock AMU and NTS resistance proportion in humans and broilers/pigs, overall as well as per class-specific antimicrobials. Correlation analysis was performed to relate AMR proportions between human and broiler/pig NTS isolates. Results: For SE, only a positive association between penicillins use in broilers and resistance to ampicillin among human isolates was significant. For ST/STM, most associations between AMU in livestock and AMR among human isolates were significantly positive, overall and per class-specific antimicrobials, namely for penicillins-ampicillin, tetracyclines-tetracycline and sulfonamides/trimethoprim-sulfamethoxazole/trimethoprim. Significantly positive associations between AMU in broilers/pigs and AMR in broiler/pig ST/STM isolates were also observed, but not between broiler/pig and human AMR levels. Conclusions: Significant associations were generally found between livestock AMU and AMR in human and broiler/pig ST/STM isolates. However, confounding factors, such as imported meat and travel are of concern. To fully comprehend the impact of livestock AMU on resistance in human NTS isolates, it is imperative to enhance AMR surveillance of NTS.

Whole-genome sequencing of Shigella for surveillance purposes shows (inter)national relatedness and multidrug resistance in isolates from men who have sex with men.

In the Netherlands, more than half of domestic shigellosis cases are among men who have sex with men (MSM), particularly in the Amsterdam region. However, there is limited insight into which Shigella strains circulate in the Netherlands. Our objective was to assess the added value of whole-genome sequencing (WGS)-based surveillance for Shigella. To this end, we determined the relatedness among Shigella spp. isolates from patients in the Amsterdam region, as well as in an international context, including antimicrobial resistance markers, using WGS. The following criteria were used: it should provide insight into (1) clustering of shigellosis cases and the affected population, (2) the extent of admixture of MSM-associated isolates with those from the broader population and (3) the presence of antimicrobial resistance. It should then lead to more opportunities for targeted control measures. For this study, Shigella isolates from three laboratories in the Amsterdam region obtained between February 2019 and October 2021 were subjected to Illumina WGS at the National Institute for Public Health and the Environment (RIVM). Raw data were quality-checked and assembled, the Shigella serotype was determined with ShigaTyper, and antimicrobial resistance markers were detected using ResFinder and PointFinder. For Shigella sonnei, subclades were determined using Mykrobe. Relatedness of isolates, including 21 international reference genomes, was assessed with core genome multilocus sequence typing. In total, 109 isolates were included, of which 27 were from females (25 %) and 66 were from males (61 %), with which the majority (n=48, 73 %) being from MSM. No information on sex was available for the remaining 16 cases. The WGS data for all isolates, comprising 55 S. sonnei, 52 Shigella flexneri, 1 Shigella boydii and 1 Shigella dysenteriae, met the quality criteria. In total, 14 clusters containing 51 isolates (49 %) were identified, with a median cluster size of 2.5 cases (range: 2-15). Nine out of 14 clusters were MSM-associated, and 8 clusters (57 %) were travel-related. Six of the MSM clusters were related to international reference genomes. The prevalence of antimicrobial resistance markers was higher among isolates from MSM than non-MSM patients, particularly for ciprofloxacin (89 vs 33 %) and azithromycin (58 vs 17 %). In conclusion, about half of Shigella spp. patients were part of a cluster, of which a substantial part were related to international reference genomes, particularly among MSM, and a high prevalence of antimicrobial resistance markers was found. These findings indicate widespread international circulation of Shigella spp., particularly among MSM, with multidrug resistance that hampers treatment of patients. Moreover, the results of this study led to the implementation of a national WGS-based laboratory surveillance programme for Shigella spp. that started in April 2022.

Identification of Francisella tularensis Subspecies in a Clinical Setting Using MALDI-TOF MS: An In-House Francisella Library and Biomarkers.

Francisella tularensis is a zoonotic bacterium that is endemic in large parts of the world. It is absent in the standard library of the most applied matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) systems: the Vitek MS and the Bruker Biotyper system. The additional Bruker MALDI Biotyper Security library contains F. tularensis without subspecies differentiation. The virulence of F. tularensis differs between the subspecies. The F. tularensis subspecies (ssp.) tularensis is highly pathogenic, whereas the subspecies holarctica displays lower virulence and subspecies novicida and F. tularensis ssp. mediasiatica are hardly virulent. To differentiate the Francisellaceae and the F. tularensis-subspecies, an in-house Francisella library was built with the Bruker Biotyper system and validated together with the existing Bruker databases. In addition, specific biomarkers were defined based on the main spectra of the Francisella strains supplemented with in silico genome data. Our in-house Francisella library accurately differentiates the F. tularensis subspecies and the other Francisellaceae. The biomarkers correctly differentiate the various species within the genus Francisella and the F. tularensis subspecies. These MALDI-TOF MS strategies can successfully be applied in a clinical laboratory setting as a fast and specific method to identify F. tularensis to subspecies level.

New insights into the epidemiology of Listeria monocytogenes - A cross-sectoral retrospective genomic analysis in the Netherlands (2010-2020).

Introduction: Listeriosis, caused by infection with Listeria monocytogenes (Lm), is a relatively rare but severe disease with one of the highest mortality rates among bacterial foodborne illnesses. A better understanding on the degree of Lm clustering, the temporal distribution of the clusters, and their association with the various food sources is expected to lead to improved source tracing and risk-based sampling. Methods: We investigated the genomic epidemiology of Lm in the Netherlands between 2010 and 2020 by analyzing whole-genome-sequencing (WGS) data of isolates from listerioss patients and food sources from nationwide integrated surveillance and monitoring. WGS data of 756 patient and 770 food/environmental isolates was assessed using core-genome multi-locus sequence typing (cgMLST) with Hamming distance as measure for pairwise distances. Associations of genotype with the epidemiological variables such as patient's age and gender, and systematic use of specific drugs were tested by multinomial logistic regressions. Genetic differentiation of the Lm within and between food categories was calculated based on allele frequencies at the 1701 cgMLST loci in each food category. Results: We confirmed previous results that some clonal complexes (CCs) are overrepresented among clinical isolates but could not identify any epidemiological risk factors. The main findings of this study include the observation of a very weak attribution of Lm types to food categories and a much better attribution to the producer level. In addition, we identified a high degree of temporal persistence of food, patient and mixed clusters, with more than half of the clusters spanning over more than 1 year and up to 10 years. Discussion: Taken together this would indicate that identifying persistent contamination in food production settings, and producers that process a wide variety of raw food produce, could significantly contribute to lowering the Lm disease burden.