Lymphogranuloma venereum (LGV), the invasive infection of the sexually transmissible infection (STI) Chlamydia trachomatis, is caused by strains from the LGV biovar, most commonly represented by ompA-genotypes L2b and L2. We investigated the diversity in LGV samples across an international collection over seven years using typing and genome sequencing. LGV-positive samples (n=321) from eight countries collected between 2011 and 2017 (Spain n=97, Netherlands n=67, Switzerland n=64, Australia n=53, Sweden n=37, Hungary n=31, Czechia n=30, Slovenia n=10) were genotyped for pmpH and ompA variants. All were found to contain the 9 bp insertion in the pmpH gene, previously associated with ompA-genotype L2b. However, analysis of the ompA gene shows ompA-genotype L2b (n=83), ompA-genotype L2 (n=180) and several variants of these (n=52; 12 variant types), as well as other/mixed ompA-genotypes (n=6). To elucidate the genomic diversity, whole genome sequencing (WGS) was performed from selected samples using SureSelect target enrichment, resulting in 42 genomes, covering a diversity of ompA-genotypes and representing most of the countries sampled. A phylogeny of these data clearly shows that these ompA-genotypes derive from an ompA-genotype L2b ancestor, carrying up to eight SNPs per isolate. SNPs within ompA are overrepresented among genomic changes in these samples, each of which results in an amino acid change in the variable domains of OmpA (major outer membrane protein, MOMP). A reversion to ompA-genotype L2 with the L2b genomic backbone is commonly seen. The wide diversity of ompA-genotypes found in these recent LGV samples indicates that this gene is under immunological selection. Our results suggest that the ompA-genotype L2b genomic backbone is the dominant strain circulating and evolving particularly in men who have sex with men (MSM) populations.

Amsterdam UMC University of Amsterdam Amsterdam Institute for Infection and Immunity Location Academic Medical Centre Amsterdam The Netherlands

Applied Microbiology Research Department of Biomedicine University of Basel Basel Switzerland

Bacterial STI Reference Laboratory National Public Health Center Budapest Hungary

Centre for Infectious Diseases and Microbiology Laboratory Services Institute of Clinical Pathology and Medical Research Westmead Hospital Westmead New South Wales Australia

CHU Bordeaux Department of Bacteriology French National Reference Center for bacterial STIs Bordeaux France

Clinical Bacteriology and Mycology University Hospital Basel University of Basel Switzerland

Clinical Research Department London School of Hygiene and Tropical Medicine London UK

Department of Dermatovenereology 2nd Faculty of Medicine Charles University and Hospital Bulovka Budinova 2 180 81 Prague 8 Czech Republic

Department of Infectious Diseases GGD Public Health Service of Amsterdam Amsterdam The Netherlands

Institute for Public Health Genomics Department of Genetics and Cell Biology Research Institute GROW University of Maastricht Maastricht The Netherlands

Laboratory of Immunogenetics Department of Medical Microbiology and Infection Control VU University Medical Center Amsterdam Amsterdam The Netherlands

Marie Bashir Institute for Infectious Diseases and Biosecurity and Westmead Clinical School University of Sydney Sydney New South Wales Australia

National Reference Laboratory for Diagnostics of Syphilis and Chlamydia Infections National Institute of Public Health Srobarova 48 100 42 Prague 10 Czech Republic

Present address Healthcare Systems Research Group VHIR Universitat Autònoma de Barcelona Passeig de la Vall d'Hebron 119 129 08035 Barcelona Spain

Section of Clinical Bacteriology Department of Medical Sciences Uppsala University Uppsala Sweden

Servicio de Microbiología Hospital Universitario Ramón y Cajal Instituto Ramón y Cajal de Investigación Sanitaria

SIB Swiss Institute of Bioinformatics Basel Switzerland

Unidad de ITS VIH Centro Sanitario Sandoval Hospital Clínico San Carlos Instituto de Investigación Sanitaria San Carlos Madrid Spain

University of Ljubljana Faculty of Medicine Institute of Microbiology and Immunology Ljubljana Slovenia

Wellcome Trust Sanger Institute Cambridge UK

Western Sydney Sexual Health Centre Western Sydney Local Health District Parramatta New South Wales Australia

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