Lucia Manso-Silvan

Mycoplasma leachii sp. nov. as a new species designation for Mycoplasma sp. bovine group 7 of Leach, and reclassification of Mycoplasma mycoides subsp. mycoides LC as a serovar of Mycoplasma mycoides subsp. capri

The Mycoplasma mycoides cluster consists of six pathogenic mycoplasmas causing disease in ruminants, which share many genotypic and phenotypic traits. The M. mycoides cluster comprises five recognized taxa: Mycoplasma mycoides subsp. mycoides Small Colony (MmmSC), M. mycoides subsp. mycoides Large Colony (MmmLC), M. mycoides subsp. capri (Mmc), Mycoplasma capricolum subsp. capricolum (Mcc) and M. capricolum subsp. capripneumoniae (Mccp). The group of strains known as Mycoplasma sp. bovine group 7 of Leach (MBG7) has remained unassigned, due to conflicting data obtained by different classification methods. In the present paper, all available data, including recent phylogenetic analyses, have been reviewed, resulting in a proposal for an emended taxonomy of this cluster: (i) the MBG7 strains, although related phylogenetically to M. capricolum, hold sufficient characteristic traits to be assigned as a separate species, i.e. Mycoplasma leachii sp. nov. (type strain, PG50(T) = N29(T) = NCTC 10133(T) = DSM 21131(T)); (ii) MmmLC and Mmc, which can only be distinguished by serological methods and are related more distantly to MmmSC, should be combined into a single subspecies, i.e. Mycoplasma mycoides subsp. capri, leaving M. mycoides subsp. mycoides (MmmSC) as the exclusive designation for the agent of contagious bovine pleuropneumonia. A taxonomic description of M. leachii sp. nov. and emended descriptions of M. mycoides subsp. mycoides and M. mycoides subsp. capri are presented. As a result of these emendments, the M. mycoides cluster will hereafter be composed of five taxa comprising three subclusters, which correspond to the M. mycoides subspecies, the M. capricolum subspecies and the novel species M. leachii.

Mycoplasma mycoides, from "mycoides Small Colony" to "capri". A microevolutionary perspective

BackgroundThe Mycoplasma mycoides cluster consists of five species or subspecies that are ruminant pathogens. One subspecies, Mycoplasma mycoides subspecies mycoides Small Colony (MmmSC), is the causative agent of contagious bovine pleuropneumonia. Its very close relative, Mycoplasma mycoides subsp. capri (Mmc), is a more ubiquitous pathogen in small ruminants causing mastitis, arthritis, keratitis, pneumonia and septicaemia and is also found as saprophyte in the ear canal. To understand the genetics underlying these phenotypic differences, we compared the MmmSC PG1 type strain genome, which was already available, with the genome of an Mmc field strain (95010) that was sequenced in this study. We also compared the 95010 genome with the recently published genome of another Mmc strain (GM12) to evaluate Mmc strain diversity.ResultsThe MmmSC PG1 genome is 1,212 kbp and that of Mmc 95010 is ca. 58 kbp shorter. Most of the sequences present in PG1 but not 95010 are highly repeated Insertion Sequences (three types of IS) and large duplicated DNA fragments. The 95010 genome contains five types of IS, present in fewer copies than in PG1, and two copies of an integrative conjugative element. These mobile genetic elements have played a key role in genome plasticity, leading to inversions of large DNA fragments. Comparison of the two genomes suggested a marked decay of the PG1 genome that seems to be correlated with a greater number of IS. The repertoire of gene families encoding surface proteins is smaller in PG1. Several genes involved in polysaccharide metabolism and protein degradation are also absent from, or degraded in, PG1.ConclusionsThe genome of MmmSC PG1 is larger than that of Mmc 95010, its very close relative, but has less coding capacity. This is the result of large genetic rearrangements due to mobile elements that have also led to marked gene decay. This is consistent with a non-adaptative genomic complexity theory, allowing duplications or pseudogenes to be maintained in the absence of adaptive selection that would lead to purifying selection and genome streamlining over longer evolutionary times. These findings also suggest that MmmSC only recently adapted to its bovine host.

Phylogeny and molecular typing of Mycoplasma agalactiae and Mycoplasma bovis by multilocus sequencing

Mycoplasma agalactiae and Mycoplasma bovis are important pathogens producing similar pathologies in small ruminants and cattle, respectively. They share many phenotypic and genotypic traits and comparison of their 16S rDNA sequences lacks sufficient resolution for phylogenetic analysis. The aim of this study was to develop a multilocus sequence typing (MLST) scheme to analyse the phylogenetic relationships between M. agalactiae and M. bovis and to assess its use for unequivocal strain characterisation and molecular typing. An MLST based on fusA, gyrB, lepA and rpoB was applied to a sample of strains from both species, some of which could not be classified by serology or PCR. A robust phylogenetic tree was inferred, where the two species were clearly resolved. The use of this tool for the molecular typing of M. agalactiae strains was further evaluated on 19 presumably unrelated isolates, resulting in the discrimination of 14 sequence types (ST). The discriminatory power was increased (17 ST) by including an alternative target located in a more variable region. The diversity of M. agalactiae in Turkey (9 strains) and Israel (15 strains) was also assessed. Five closely related ST were evidenced in Turkey and 6 in Israel, with one ST common to both countries. Each country showed a predominant type that persisted over years. The MLST scheme developed here constitutes a universal tool for unequivocal strain characterisation and global, long-term screening of dissemination of M. agalactiae and M. bovis, whereas addition of more variable, non-housekeeping gene targets allows precise epidemiological investigations.

Characterization of in vivo-acquired resistance to macrolides of Mycoplasma gallisepticum strains isolated from poultry

The macrolide class of antibiotics, including tylosin and tilmicosin, is widely used in the veterinary field for prophylaxis and treatment of mycoplasmosis. In vitro susceptibility testing of 50 strains of M. gallisepticum isolated in Israel during the period 1997-2010 revealed that acquired resistance to tylosin as well as to tilmicosin was present in 50% of them. Moreover, 72% (13/18) of the strains isolated from clinical samples since 2006 showed acquired resistance to enrofloxacin, tylosin and tilmicosin. Molecular typing of the field isolates, performed by gene-target sequencing (GTS), detected 13 molecular types (I-XIII). Type II was the predominant type prior to 2006 whereas type X, first detected in 2008, is currently prevalent. All ten type X strains were resistant to both fluoroquinolones and macrolides, suggesting selective pressure leading to clonal dissemination of resistance. However, this was not a unique event since resistant strains with other GTS molecular types were also found. Concurrently, the molecular basis for macrolide resistance in M. gallisepticum was identified. Our results revealed a clear-cut correlation between single point mutations A2058G or A2059G in domain V of the gene encoding 23S rRNA (rrnA, MGA_01) and acquired macrolide resistance in M. gallisepticum. Indeed, all isolates with MIC ≥ 0.63 μg/mL to tylosin and with MIC ≥ 1.25 μg/mL to tilmicosin possess one of these mutations, suggesting an essential role in decreased susceptibility of M. gallisepticum to 16-membered macrolides.

Emergence of Atypical Mycoplasma agalactiae Strains Harboring a New Prophage and Associated with an Alpine Wild Ungulate Mortality Episode

ABSTRACT The bacterium Mycoplasma agalactiae is responsible for contagious agalactia (CA) in small domestic ruminants, a syndrome listed by the World Organization for Animal Health and responsible for severe damage to the dairy industry. Recently, we frequently isolated this pathogen from lung lesions of ibexes during a mortality episode in the French Alps. This situation was unusual in terms of host specificity and tissue tropism, raising the question of M. agalactiae emergence in wildlife. To address this issue, the ibex isolates were characterized using a combination of approaches that included antigenic profiles, molecular typing, optical mapping, and whole-genome sequencing. Genome analyses showed the presence of a new, large prophage containing 35 coding sequences (CDS) that was detected in most but not all ibex strains and has a homolog in Mycoplasma conjunctivae, a species causing keratoconjunctivitis in wild ungulates. This and the presence in all strains of large integrated conjugative elements suggested highly dynamic genomes. Nevertheless, M. agalactiae strains circulating in the ibex population were shown to be highly related, most likely originating from a single parental clone that has also spread to another wild ungulate species of the same geographical area, the chamois. These strains clearly differ from strains described in Europe so far, including those found nearby, before CA eradication a few years ago. While M. agalactiae pathogenicity in ibexes remains unclear, our data showed the emergence of atypical strains in Alpine wild ungulates, raising the question of a role for the wild fauna as a potential reservoir of pathogenic mycoplasmas.

Multi-locus sequence analysis of Mycoplasma capricolum subsp. capripneumoniae for the molecular epidemiology of contagious caprine pleuropneumonia.

Mycoplasma capricolum subsp. capripneumoniae (Mccp) is the causative agent of contagious caprine pleuropneumonia (CCPP), a devastating disease of domestic goats. The exact distribution of CCPP is not known but it is present in Africa and the Middle East and represents a significant threat to many disease-free areas including Europe. Furthermore, CCPP has been recently identified in Tajikistan and China. A typing method with an improved resolution based on Multi-Locus Sequence Analysis (MLSA) has been developed to trace new epidemics and to elucidate whether the recently identified cases in continental Asia were due to recent importation of Mccp. The H2 locus, a polymorphic region already in use as a molecular marker for Mccp evolution, was complemented with seven new loci selected according to the analysis of polymorphisms observed among the genome sequences of three Mccp strains. A total of 25 strains, including the two new strains from Asia, were analysed by MLSA resulting in the discrimination of 15 sequence types based on 53 polymorphic positions. A distance tree inferred from the concatenated sequences of the eight selected loci revealed two evolutionary lineages comprising five groups, which showed good correlation with geographic origins. The presence of a distinct Asian cluster strongly indicates that CCPP was not recently imported to continental Asia. It is more likely that the disease has been endemic in the area for a long time, as supported by historical clinical descriptions. In conclusion, this MLSA strategy constitutes a highly discriminative tool for the molecular epidemiology of CCPP.

Genotyping of Mycoplasma mycoides subsp. mycoides SC by multilocus sequence analysis allows molecular epidemiology of contagious bovine pleuropneumonia

Mycoplasma mycoides subsp. mycoides SC (MmmSC) is the etiological agent of contagious bovine pleuropneumonia (CBPP). Although eradicated in most developed countries, the disease reappeared in Europe in the 1990s. This reappearance may have been caused either by importation from sub-Saharan Africa, where CBPP is still endemic, or by the reemergence of virulent strains in Europe, as suggested by earlier studies. A multilocus sequence analysis scheme has been developed to address this issue and, most importantly, to be able to monitor new epidemics. The alignment of the full genome sequence of the reference strain PG1 and the partial genome sequence of a pathogenic strain allowed the identification of polymorphic sites. Nineteen initial loci were selected within housekeeping genes, genes of unknown function and non coding sequences. The suitability of these loci for genotyping MmmSC strains was first tested on six strains of diverse geographic origin. The analyses showed that the published PG1 sequence contained a number of specific polymorphisms that were therefore of no use for molecular typing. Among the eight informative polymorphic loci finally selected, only one (ftsY) was positioned within a housekeeping gene. Three main groups and 31 different allelic profiles were identified among 51 strains and strain variants examined. Cluster analysis confirmed that European strains from the 1990s did not originate from Africa. It also showed a genetic link between a European strain isolated in 1967 and those found in southern Africa and Australia. This was in agreement with historical data showing that CBPP was introduced in these regions during colonisation in the 19th century.

Evolutionary History of Contagious Bovine Pleuropneumonia Using Next Generation Sequencing of Mycoplasma mycoides Subsp. mycoides “Small Colony”

Mycoplasma mycoides subsp. mycoides “Small Colony” (MmmSC) is responsible for contagious bovine pleuropneumonia (CBPP) in bovidae, a notifiable disease to the World Organization for Animal Health (OIE). Although its origin is not documented, the disease was known in Europe in 1773. It reached nearly world-wide distribution in the 19th century through the cattle trade and was eradicated from most continents by stamping-out policies. During the 20th century it persisted in Africa, and it reappeared sporadically in Southern Europe. Yet, classical epidemiology studies failed to explain the re-occurrence of the disease in Europe in the 1990s. The objectives of this study were to obtain a precise phylogeny of this pathogen, reconstruct its evolutionary history, estimate the date of its emergence, and determine the origin of the most recent European outbreaks. A large-scale genomic approach based on next-generation sequencing technologies was applied to construct a robust phylogeny of this extremely monomorphic pathogen by using 20 representative strains of various geographical origins. Sixty two polymorphic genes of the MmmSC core genome were selected, representing 83601 bp in total and resulting in 139 SNPs within the 20 strains. A robust phylogeny was obtained that identified a lineage specific to European strains; African strains were scattered in various branches. Bayesian analysis allowed dating the most recent common ancestor for MmmSC around 1700. The strains circulating in Sub-Saharan Africa today, however, were shown to descend from a strain that existed around 1810. MmmSC emerged recently, about 300 years ago, and was most probably exported from Europe to other continents, including Africa, during the 19th century. Its diversity is now greater in Africa, where CBPP is enzootic, than in Europe, where outbreaks occurred sporadically until 1999 and where CBPP may now be considered eradicated unless MmmSC remains undetected.

Characterization of Free Exopolysaccharides Secreted by Mycoplasma mycoides Subsp. mycoides

Contagious bovine pleuropneumonia is a severe respiratory disease of cattle that is caused by a bacterium of the Mycoplasma genus, namely Mycoplasma mycoides subsp. mycoides (Mmm). In the absence of classical virulence determinants, the pathogenicity of Mmm is thought to rely on intrinsic metabolic functions and specific components of the outer cell surface. One of these latter, the capsular polysaccharide galactan has been notably demonstrated to play a role in Mmm persistence and dissemination. The free exopolysaccharides (EPS), also produced by Mmm and shown to circulate in the blood stream of infected cattle, have received little attention so far. Indeed, their characterization has been hindered by the presence of polysaccharide contaminants in the complex mycoplasma culture medium. In this study, we developed a method to produce large quantities of EPS by transfer of mycoplasma cells from their complex broth to a chemically defined medium and subsequent purification. NMR analyses revealed that the purified, free EPS had an identical β(1−>6)-galactofuranosyl structure to that of capsular galactan. We then analyzed intraclonal Mmm variants that produce opaque/translucent colonies on agar. First, we demonstrated that colony opacity was related to the production of a capsule, as observed by electron microscopy. We then compared the EPS extracts and showed that the non-capsulated, translucent colony variants produced higher amounts of free EPS than the capsulated, opaque colony variants. This phenotypic variation was associated with an antigenic variation of a specific glucose phosphotransferase permease. Finally, we conducted in silico analyses of candidate polysaccharide biosynthetic pathways in order to decipher the potential link between glucose phosphotransferase permease activity and attachment/release of galactan. The co-existence of variants producing alternative forms of galactan (capsular versus free extracellular galactan) and associated with an antigenic switch constitutes a finely tuned mechanism that may be involved in virulence.

Real-time polymerase chain reaction assays for the detection of memebers of the Mycoplasma mycoides cluster

Abstract AIM: To develop real-time PCR assays for the detection and differentiation of members of the Mycoplasma mycoides cluster. METHODS: Five real-time PCR assays were designed to allow differentiation of members of the M. mycoides cluster: an assay for detection of the M. mycoides subspecies, viz M. mycoides subsp mycoides large colony (MmmLC), M. mycoides subsp capri (Mmc), and M. mycoides subsp mycoides small colony (MmmSC); one for the detection of the M. capricolum subspecies, viz M. capricolum subsp capricolum (Mcc), M. capricolum subsp capripneumoniae (Mccp), and Mycoplasma sp bovine group 7 (BG7); and three for the specifi c detection of MmmSC, Mccp, and BG7. A panel of 74 Mycoplasma isolates from various geographical origins and a panel of 21 other bacterial isolates were used to evaluate the sensitivity and specificity of the assays. RESULTS: The assays displayed 100% analytical sensitivity in detecting all target Mycoplasma isolates. The analytical detection limit for the assays to detect the M. mycoides subspecies, M. capricolum subspecies, and MmmSC was determined to be 100 fg of genomic DNA, while the Mccp and BG7 assays had a detection limit of 100 fg and 10 fg of genomic DNA, respectively. The M. mycoides subspecies assay had a detection limit of 103 (SD 102) cfu/ml milk, 104 (SD 104) cfu per swab, and 103 (SD 103) cfu/g lung in inoculated samples. The assays displayed 100% specificity when applied to non-target bacterial isolates and to 110 culture-negative milk samples. CONCLUSIONS: The assays were highly sensitive and specific, and provide accurate detection and differentiation of the members of the M. mycoides cluster.