Genome-wide phylodynamic approach reveals the epidemic dynamics of the main Mycoplasma bovis subtype circulating in France.

Mycoplasma bovis is a major aetiological agent of bovine respiratory disease worldwide. Genome-based analyses are increasingly being used to monitor the genetic diversity and global distribution of M. bovis, complementing existing subtyping schemes based on locus sequencing. However, these analyses have so far provided limited information on the spatiotemporal and population dynamics of circulating subtypes. Here we applied a genome-wide phylodynamic approach to explore the epidemic dynamics of 88 French M. bovis strains collected between 2000 and 2019 in France and belonging to the currently dominant polC subtype 2 (st2). A strong molecular clock signal detected in the genomic data enabled robust phylodynamic inferences, which estimated that the M. bovis st2 population in France is composed of two lineages that successively emerged from independent introductions of international strains. The first lineage appeared around 2000 and supplanted the previously established antimicrobial-susceptible polC subtype 1. The second lineage, which is likely more transmissible, progressively replaced the first M. bovis st2 lineage population from 2005 onward and became predominant after 2010. Analyses also showed a brief decline in this second M. bovis st2 lineage population in around 2011, possibly due to the challenge from the concurrent emergence of M. bovis polC subtype 3 in France. Finally, we identified non-synonymous mutations in genes associated with lineages, which raises prospects for identifying new surveillance molecular markers. A genome-wide phylodynamic approach provides valuable resources for monitoring the evolution and epidemic dynamics of circulating M. bovis subtypes, and may prove critical for developing more effective surveillance systems and disease control strategies.

Detection of Mycoplasma spp. in horses with respiratory disorders.

BACKGROUND: Bacteria belonging to the genus Mycoplasma are small-sized, have no cell walls and small genomes. They commonly cause respiratory disorders in their animal hosts. Three species have been found in the respiratory tract of horses worldwide, that is., Mycoplasma (M.) equirhinis, M. pulmonis and M. felis, but their role in clinical cases remains unclear. OBJECTIVES: The aim of this study was to i) develop and validate tools to detect, isolate and identify different Mycoplasma spp. strains in clinical equine respiratory-tract specimens and ii) subsequently define the prevalence of the three species in France depending on sample types and horse characteristics (age, breed, sex). STUDY DESIGN: Validation of a workflow for mycoplasma diagnosis and subsequent prevalence study. METHODS: Mycoplasma-free tracheal wash samples spiked with numerated strains and DNA dilutions were used to validate the culture methods and real-time PCR (rt-PCR) assay. Isolated strains were identified by 16S rRNA gene sequencing. Prevalences were determined on a population of 616 horses with respiratory disorders, sampled in France in 2020. RESULTS: In total, 104 horses (16.9%) were found to be positive for Mycoplasma spp. by at least one method. M. equirhinis was the predominant circulating species, accounting for 85% of the rt-PCR-positive samples and 98% of the 40 cultured strains. MAIN LIMITATION: The proposed pre-enrichment procedure improves the sensitivity of detection but hinders the quantification of the initial mycoplasma load in the clinical specimens. CONCLUSIONS: Prevalence of mycoplasma varied with age, breed, and type of sample.

Genomic features of Mycoplasma bovis subtypes currently circulating in France.

BACKGROUND: Mycoplasma (M.) bovis is a major etiological agent of bovine respiratory disease, which is the most economically costly disease of cattle worldwide. Cattle disease surveillance on M. bovis is increasingly using gene-based techniques, such as multilocus sequence typing (MLST), or genome-based techniques such as core genome MLST that both require only partial genomic data. However, accurate up-to-date surveillance also demands complete, circular genomes that can be used as reference to track the evolution of the different lineages. Yet, in France, two of the main subtypes currently circulating still have no representing genome in public databases. Here, to address this gap, we provide and compare three new complete M. bovis genomes obtained from recent clinical isolates that represent major subtypes circulating in France and Europe. RESULTS: Genomes were obtained using a hybrid assembly strategy (Illumina and Nanopore) with fine-tuning of settings and inputs used in the Unicycler assembly pipeline, such as size selection of reads and quality trimming of the FASTQ files. The main characteristics and synteny of the genomes were compared. The three genomes mainly differed by their content in terms of mobile genetic elements, i.e. integrative conjugative elements (ICE) and insertion sequences (IS), a feature that impacts their structure. For instance, strain L15527, representing subtype3 (st3), harbours an exceptionally high number of ICEs, which results in a bigger-sized genome than all those previously described and could be associated with the propensity of st3 to gain and fix mutations through chromosomal transfer mechanisms. In contrast, strain F9160, of st1, is very close to the PG45 type strain isolated in 1961 in the USA, and harbours a huge number of IS. These features may be associated with an evolution towards a host-restricted state or in a "closed" host or environment reservoir until a recent re-emergence. CONCLUSIONS: Whole-genome comparison of the three French M. bovis subtypes provides valuable resources for future studies combining epidemiology, phylogenetic data, and phylodynamic methods.

Addressing the Antimicrobial Resistance of Ruminant Mycoplasmas Using a Clinical Surveillance Network.

Antimicrobial resistance (AMR) surveillance of mycoplasmas of veterinary importance has been held back for years due to lack of harmonized methods for antimicrobial susceptibility testing (AST) and interpretative criteria, resulting in a crucial shortage of data. To address AMR in ruminant mycoplasmas, we mobilized a long-established clinical surveillance network called "Vigimyc." Here we describe our surveillance strategy and detail the results obtained during a 2-year monitoring period. We also assess how far our system complies with current guidelines on AMR surveillance and how it could serve to build epidemiological cut-off values (ECOFFs), as a first attainable criterion to help harmonize monitoring efforts and move forward to clinical breakpoints. Clinical surveillance through Vigimyc enables continuous collection, identification and preservation of Mycoplasma spp. isolates along with metadata. The most frequent pathogens, i.e., M. bovis and species belonging to M. mycoides group, show stable clinicoepidemiological trends and were included for annual AST. In the absence of interpretative criteria for ruminant mycoplasmas, we compared yearly minimum inhibitory concentration (MIC) results against reference datasets. We also ran a SWOT (Strengths, Weaknesses, Opportunities, Threats) analysis on the overall service provided by our AMR surveillance strategy. Results of the 2018-2019 surveillance campaign were consistent with the reference datasets, with M. bovis isolates showing high MIC values for all antimicrobial classes except fluoroquinolones, and species of the Mycoides group showing predominantly low MIC values. A few new AMR patterns were detected, such as M. bovis with lower spectinomycin MICs. Our reference dataset partially complied with European Committee on Antimicrobial Susceptibility Testing (EUCAST) requirements, and we were able to propose tentative epidemiological cut-off values (TECOFFs) for M. bovis with tilmicosin and spectinomycin and for M. mycoides group with tilmicosin and lincomycin. These TECOFFs were consistent with other published data and the clinical breakpoints of Pasteurellaceae, which are often used as surrogates for mycoplasmas. SWOT analysis highlighted the benefit of pairing clinical and antimicrobial resistance surveillance despite the AST method-related gaps that remain. The international community should now direct efforts toward AST method harmonization and clinical interpretation.

Mycoplasma bovis in Nordic European Countries: Emergence and Dominance of a New Clone.

Mycoplasma (M.) bovis is an important pathogen of cattle implicated in a broad range of clinical manifestations that adversely impacts livestock production worldwide. In the absence of a safe, effective commercial vaccine in Europe, the reported reduced susceptibility to antimicrobials for this organism has contributed to difficulties in controlling infection. Despite global presence, some countries have only recently experienced outbreaks of this pathogen. In the present study, M. bovis isolates collected in Denmark between 1981 and 2016 were characterized to determine (i) genetic diversity and phylogenetic relationships using whole genome sequencing and various sequence-based typing methods and (ii) patterns of antimicrobial resistance compared to other European isolates. The M. bovis population in Denmark was found to be highly homogeneous genomically and with respect to the antimicrobial resistance profile. Previously dominated by an old genotype shared by many other countries (ST17 in the PubMLST legacy scheme), a new predominant type represented by ST94-adh1 has emerged. The same clone is also found in Sweden and Finland, where M. bovis introduction is more recent. Although retrieved from the Netherlands, it appears absent from France, two countries with a long history of M. bovis infection where the M. bovis population is more diverse.

Population structure and antimicrobial susceptibility of Mycoplasma ovipneumoniae isolates in France.

Chronic non-progressive pneumonia in small ruminants caused by Mycoplasma (M.) ovipneumoniae is mainly controlled by chemotherapy. In France, during the last decade, a rise in M. ovipneumoniae cases was recorded in both sheep and goats, suggesting a possible emergence. Whether this rise is associated with antimicrobial resistance, as observed in other ruminant Mycoplasma species, has yet to be examined. The aim of the study was to characterize the diversity of M. ovipneumoniae strains circulating in France and assess their antimicrobial resistance, together with the underlying mechanisms, to help find an explanation for the increase in reported cases. The genetic diversity of 56 strains isolated between 2007 and 2018 from sheep and goats was assessed using different subtyping methods. Their susceptibility to six antimicrobial classes was profiled by estimating Minimum Inhibitory Concentrations (MICs) using an optimised agar dilution method. Resistance mechanisms were explored by sequence analysis of rRNA targets. A high genetic diversity of strains was evidenced, with consistent, marked animal-host clustering in the Hsp70 gene and whole genome sequence phylogeny. No clonal evolution could thus account for putative emergence. Apart from florfenicol, MICs were low except for a few isolates with increased values for tetracyclines, macrolides and lincosamides. Hotspot mutations in the target ribosomal gene could explain increased tetracycline MICs. Other mechanisms are suspected for macrolide-lincosamide and florfenicol resistance. The emergence of M. ovipneumoniae is thus not related to any increase in resistance or to a clonal spread. Explanations may lie in breeding practices.

Monitoring Mycoplasma bovis Diversity and Antimicrobial Susceptibility in Calf Feedlots Undergoing a Respiratory Disease Outbreak.

Bovine respiratory diseases (BRD) are widespread in veal calf feedlots. Several pathogens are implicated, both viruses and bacteria, one of which, Mycoplasma bovis, is under-researched. This worldwide-distributed bacterium has been shown to be highly resistant in vitro to the main antimicrobials used to treat BRD. Our objective was to monitor the relative prevalence of M. bovis during BRD episodes, its diversity, and its resistance phenotype in relation to antimicrobial use. For this purpose, a two-year longitudinal follow-up of 25 feedlots was organized and 537 nasal swabs were collected on 358 veal calves at their arrival in the lot, at the BRD peak and 4 weeks after collective antimicrobial treatments. The presence of M. bovis was assessed by real-time PCR and culture. The clones isolated were then subtyped (polC subtyping and PFGE analysis), and their susceptibility to five antimicrobials was determined. The course of the disease and the antimicrobials used had no influence on the genetic diversity of the M. bovis strains: The subtype distribution was the same throughout the BRD episode and similar to that already described in France, with a major narrowly-variable subtype circulating, st2. The same conclusion holds for antimicrobial resistance (AMR) phenotypes: All the clones were already multiresistant to the main antimicrobials used (except for fluoroquinolones) prior to any treatments. By contrast, changes of AMR phenotypes could be suspected for Pasteurellaceae in two cases in relation to the treatments registered.