Use of whole-genome sequencing to identify clusters of Shigella flexneri associated with sexual transmission in men who have sex with men in England: a validation study using linked behavioural data

Since the 1970s, shigellosis has been reported as a sexually transmissible infection, and in recent years, genomic data have revealed the breadth of Shigella spp. transmission among global networks of men who have sex with men (MSM). In 2015, Public Health England (PHE) introduced routine whole-genome sequencing (WGS) of Shigella spp. to identify transmission clusters. However, limited behavioural information for the cases hampers interpretation. We investigated whether WGS can distinguish between clusters representing sexual transmission in MSM and clusters representing community (non-sexual) transmission to inform infection control. WGS data for Shigella flexneri from August 2015 to July 2017 were aggregated into single linkage clusters based on SNP typing using a range of SNP distances (the standard for Shigella surveillance at PHE is 10 SNPs). Clusters were classified as 'adult male', 'household', 'travel-associated' or 'community' using routine demographic data submitted alongside laboratory cultures. From August 2015 to March 2017, PHE contacted those with shigellosis as part of routine public-health follow-up and collected exposure data on a structured questionnaire, which for the first time included questions about sexual identity and behaviour. The questionnaire data were used to determine whether clusters classified as 'adult male' represented likely sexual transmission between men, thereby validating the use of the SNP clustering tool for informing appropriate public-health responses. Overall, 1006 S. flexneri cases were reported, of which 563 clustered with at least one other case (10-SNP threshold). Linked questionnaire data were available for 106 clustered cases, of which 84.0 % belonged to an 'adult male' cluster. At the 10-SNP threshold, 95.1 % [95 % confidence interval (CI) 88.0-98.1%] of MSM belonged to an 'adult male' cluster, while 73.2 % (95 % CI 49.1-87.5%) of non-MSM belonged to a 'community' or 'travel-associated' cluster. At the 25-SNP threshold, all MSM (95 % CI 96.0-100%) belonged to an 'adult male' cluster and 77.8 % (95 % CI 59.2-89.4%) of non-MSM belonged to a 'community' or 'travel-associated' cluster. Within one phylogenetic clade of S. flexneri, 9 clusters were identified (7 'adult male'; 2 'community') using a 10-SNP threshold, while a single 'adult male' cluster was identified using a 25-SNP threshold. Genotypic markers of azithromycin resistance were detected in 84.5 % (294/348) of 'adult male' cases and 20.9 % (9/43) of cases in other clusters (10-SNP threshold), the latter of which contained gay-identifying men who reported recent same-sex sexual contact. Our study suggests that SNP clustering can be used to identify Shigella clusters representing likely sexual transmission in MSM to inform infection control. Defining clusters requires a flexible approach in terms of genetic relatedness to ensure a clear understanding of underlying transmission networks

Listeria monocytogenes : the silent assassin

Graphical abstract Listeria monocytogenes is ubiquitous in both plant and animal reservoirs. It can persist in food production environments due to its capacity to grow at refrigerated temperatures and its resistance to biocides. The source of most human infections is contaminated food. Healthy individuals present with mild gastrointestinal symptoms. However, in immunocompromised individuals the infection is more severe, causing bacteraemia, meningitis and, in pregnancy-associated listeriosis, miscarriage and stillbirth. In vulnerable groups, including the elderly, pregnant women and their infants, listeriosis has a 20–30% mortality rate

Phylogenomic analysis of gastroenteritis-associated Clostridium perfringens in England and Wales over a 7-year period indicates distribution of clonal toxigenic strains in multiple outbreaks and extensive involvement of enterotoxin-encoding (CPE) plasmids

Clostridium perfringens is a major enteric pathogen known to cause gastroenteritis in human adults. Although major outbreak cases are frequently reported, only limited whole-genome sequencing (WGS) based studies have been performed to understand the genomic epidemiology and virulence gene content of outbreak-associated C. perfringens strains. We performed phylogenomic analysis on 109 C. perfringens isolates (human and food) obtained from disease cases in England and Wales between 2011 and 2017. Initial findings highlighted the enhanced discriminatory power of WGS in profiling outbreak C. perfringens strains, when compared to the current Public Health England referencing laboratory technique of fluorescent amplified fragment length polymorphism analysis. Further analysis identified that isogenic C. perfringens strains were associated with nine distinct care-home-associated outbreaks over the course of a 5-year interval, indicating a potential common source linked to these outbreaks or transmission over time and space. As expected, the enterotoxin cpe gene was encoded in all but 4 isolates (96.3 %; 105/109), with virulence plasmids encoding cpe (particularly pCPF5603 and pCPF4969 plasmids) extensively distributed (82.6 %; 90/109). Genes encoding accessory virulence factors, such as beta-2 toxin, were commonly detected (46.7 %; 51/109), and genes encoding phage proteins were also frequently identified. Overall, this large-scale genomic study of gastroenteritis-associated C. perfringens suggested that three major cpe-encoding (toxinotype F) genotypes underlie these outbreaks: strains carrying (1) pCPF5603 plasmid, (2) pCPF4969 plasmid and (3) chromosomal-cpe strains. Our findings substantially expanded our knowledge on type F C. perfringens involved in human-associated gastroenteritis, with further studies required to fully probe the dissemination and regional reservoirs of this enteric pathogen, which may help devise effective prevention strategies to reduce the food-poisoning disease burden in vulnerable patients, such as the elderly

WGS for surveillance of antimicrobial resistance:A pilot study to detect the prevalence and mechanism of resistance to azithromycin in a UK population of non-typhoidal Salmonella

Objectives: WGS and phenotypic methods were used to determine the prevalence of azithromycin resistance in Salmonella enterica isolates from the UK and to identify the underlying mechanisms of resistance.  Methods: WGS by Illumina HiSeq was carried out on 683 Salmonella spp. isolates. Known genes associated with azithromycin resistance were detected by WGS using a mapping-based approach. Macrolide resistance determinants were identified and the genomic context of these elements was assessed by various bioinformatics tools. Susceptibility testing was in accordance with EUCAST methodology (MIC ≤16 mg/L).  Results: Fifteen isolates of non-typhoidal Salmonella enterica belonging to serovars Salmonella Blockley, Salmonella Typhimurium, Salmonella Thompson, Salmonella Ridge and Salmonella Kentucky showed resistance or decreased susceptibility to azithromycin (from 6 to >16 mg/L) due to the presence of macrolide resistance genes mphA, mphB or mefB. These genes were either plasmid or chromosomally mediated. Azithromycin-resistant Salmonella Blockley isolates harboured a macrolide inactivation gene cluster, mphA-mrx-mphr(A), within a novel Salmonella azithromycin resistance genomic island (SARGI) determined by MinION sequencing. This is the first known chromosomally mediated mphA gene cluster described in salmonellae. Phylogenetic analysis and epidemiological information showed that mphA Salmonella Blockley isolates were not derived from a single epidemiologically related event. The azithromycin MICs of the 15 Salmonella spp. isolates showed that the presence of the mphA gene was associated with MIC ≥16 mg/L, while the presence of mefB or mphB was not.  Conclusions: Azithromycin resistance due to acquisition of known macrolide resistance genes was seen in four different Salmonella serovars and can be either plasmid-encoded or chromosomally encoded

Closing gaps for performing a risk assessment on Listeria monocytogenes in ready-to-eat (RTE) foods : activity 3, the comparison of isolates from different compartments along the food chain, and from humans using whole genome sequencing (WGS) analysis

We would like to thank all the persons and institutes that have provided the project with isolates and accompanying information. Without them, this project would not have been possible. Lin Cathrine T. Brandal, Norwegian Institute of Public Health, Norway Julio Vázquez Moreno and Raquel Abad Torreblanca, Instituto de Salud Carlos III, Spain Marc Lecuit, Institut Pasteur, France Alexandre Leclercq, Institut Pasteur, France Iva Hristova, National Center of Infectious and Parasitic Diseases, Bulgaria Marija Trkov, National Laboratory of Health, Environment and Food, Slovenia Cecilia Jernberg, Public Health Agency of Sweden, Sweden Ariane Pietzka, Austrian Agency for Health and Food Safety, Austria Eelco Franz and Ingrid Friesema, RIVM, The Netherlands Carlo Spanu, University of Sassari Sardinia Ifip, French Institute for Pig and Pork Industry, Maisons-Alfort, France All the NRLs for providing the isolates from the EU baseline study Special thanks to Sylvain Brisse and Alexandra Moura, Institut Pasteur, France, for providing cgMLST data. The authors would also like to thank the EFSA staff members: Maria Teresa da Silva Felicio, Beatriz Guerra, Ernesto Lìebana and Valentina Rizzi as well as the members of the Working Group on Listeria monocytogenes contamination of ready-to-eat foods: Kostas Koutsoumanis, Roland Lindqvist, Moez Sanaa, Panagiotis Skandamis, Niko Speybroek, Johanna Takkinen and Martin Wagner for the support, revisions and suggestions during the development of the present procurement activity and report.Publisher PD

LiSEQ – whole-genome sequencing of a cross-sectional survey of Listeria monocytogenes in ready-to-eat foods and human clinical cases in Europe

Funding information This work was funded by EFSA, contract number C/EFSA/BIOCONTAM/2014/01-CT 1 on “Closing gaps for performing a risk assessment on Listeria monocytogenes in ready-to-eat (RTE) foods: activity 3, the comparison of isolates from different compartments along the food chain, and from humans using whole genome sequencing (WGS) analysis’, EFSA-Q-2014-00 026. Acknowledgements A. P., T. D. and K. G. are affiliated to the National Institute for Health Research – Health Protection Research Unit (NIHR HPRU) in Gastrointestinal Infections at University of Liverpool in partnership with Public Health England, in collaboration with the University of East Anglia, the University of Oxford and the Quadram Institute. A. P., T. D. and K. G. are based at Public Health England. The views expressed are those of the author(s) and not necessarily those of the NHS, the NIHR, the Department of Health or Public Health England.Peer reviewedPublisher PD

Use of whole-genome sequencing to identify clusters of Shigella flexneri associated with sexual transmission in men who have sex with men in England: a validation study using linked behavioural data.

Since the 1970s, shigellosis has been reported as a sexually transmissible infection, and in recent years, genomic data have revealed the breadth of Shigella spp. transmission among global networks of men who have sex with men (MSM). In 2015, Public Health England (PHE) introduced routine whole-genome sequencing (WGS) of Shigella spp. to identify transmission clusters. However, limited behavioural information for the cases hampers interpretation. We investigated whether WGS can distinguish between clusters representing sexual transmission in MSM and clusters representing community (non-sexual) transmission to inform infection control. WGS data for Shigella flexneri from August 2015 to July 2017 were aggregated into single linkage clusters based on SNP typing using a range of SNP distances (the standard for Shigella surveillance at PHE is 10 SNPs). Clusters were classified as 'adult male', 'household', 'travel-associated' or 'community' using routine demographic data submitted alongside laboratory cultures. From August 2015 to March 2017, PHE contacted those with shigellosis as part of routine public-health follow-up and collected exposure data on a structured questionnaire, which for the first time included questions about sexual identity and behaviour. The questionnaire data were used to determine whether clusters classified as 'adult male' represented likely sexual transmission between men, thereby validating the use of the SNP clustering tool for informing appropriate public-health responses. Overall, 1006?S. flexneri cases were reported, of which 563 clustered with at least one other case (10-SNP threshold). Linked questionnaire data were available for 106 clustered cases, of which 84.0?% belonged to an 'adult male' cluster. At the 10-SNP threshold, 95.1?% [95?% confidence interval (CI) 88.0-98.1%] of MSM belonged to an 'adult male' cluster, while 73.2?% (95?% CI 49.1-87.5%) of non-MSM belonged to a 'community' or 'travel-associated' cluster. At the 25-SNP threshold, all MSM (95?%?CI 96.0-100%) belonged to an 'adult male' cluster and 77.8?% (95?% CI 59.2-89.4%) of non-MSM belonged to a 'community' or 'travel-associated' cluster. Within one phylogenetic clade of S. flexneri, 9 clusters were identified (7 'adult male'; 2 'community') using a 10-SNP threshold, while a single 'adult male' cluster was identified using a 25-SNP threshold. Genotypic markers of azithromycin resistance were detected in 84.5?% (294/348) of 'adult male' cases and 20.9?% (9/43) of cases in other clusters (10-SNP threshold), the latter of which contained gay-identifying men who reported recent same-sex sexual contact. Our study suggests that SNP clustering can be used to identify Shigella clusters representing likely sexual transmission in MSM to inform infection control. Defining clusters requires a flexible approach in terms of genetic relatedness to ensure a clear understanding of underlying transmission networks

Genomic epidemiology of the clinically dominant clonal complex 1 in the Listeria monocytogenes population in the UK

Listeria monocytogenes is a food-borne pathogen, typically affecting the elderly, immunocompromised patients and pregnant women. The aim of this study was to determine the population structure of L. monocytogenes clonal complex 1 (CC1) in the UK and describe the genomic epidemiology of this clinically significant CC. We interrogated a working dataset of 4073 sequences of L. monocytogenes isolated between January 2015 and December 2020 from human clinical specimens, food and/or food-production environments. A minimum spanning tree was reconstructed to determine the population structure of L. monocytogenes in the UK. Subsequent analysis focused on L. monocytogenes CC1, as the cause of the highest proportion of invasive listeriosis in humans. Sequencing data was integrated with metadata on food and environmental isolates, and information from patient questionnaires, including age, sex and clinical outcomes. All isolates either belonged to lineage I (n=1299/4073, 32%) or lineage II (n=2774/4073, 68%), with clinical isolates from human cases more likely to belong to lineage I (n=546/928, 59%) and food isolates more likely to belong to lineage II (n=2352/3067, 77%). Of the four largest CCs, CC1 (n=237) had the highest proportion of isolates from human cases of disease (CC1 n=160/237, 67.5 %; CC121 n=13/843, 2 %; CC9 n=53/360, 15 %; CC2 n=69/339, 20%). Within CC1, most cases were female (n=95/160, 59%, P=0.01771) and the highest proportion of cases were in people >60 years old (39/95, 41%, P=1.314×10−6) with a high number of them aged 20–39 years old (n=35/95, 37%) most linked to pregnancy-related listeriosis (n=29/35, 83%). Most of the male cases were in men aged over 60 years old (40/65, 62%), and most of the fatal cases in both males and females were identified in this age group (42/55, 76%). Phylogenetic analysis revealed 23 5 SNP single linkage clusters comprising 80/237 (34 %) isolates with cluster sizes ranging from 2 to 19. Five 5 SNP clusters comprised isolates from human cases and an implicated food item. Expanding the analysis to 25 SNP single linkage clusters resolved an additional two clusters linking human cases to a potential food vehicle. Analysis of demographic and clinical outcome data identified CC1 as a clinically significant cause of invasive listeriosis in the elderly population and in women of child-bearing age. Phylogenetic analysis revealed the population structure of CC1 in the UK comprised small, sparsely populated genomic clusters. Only clusters containing isolates from an implicated food vehicle, or food processing or farming environments, were resolved, emphasizing the need for clinical, food and animal-health agencies to share sequencing data in real time, and the importance of a One Health approach to public-health surveillance of listeriosis