Resistance to tetracyclines in genital mycoplasmas is due mainly to acquisition of the tet(M) determinant, which is frequently associated with conjugative transposon elements of the Tn916/ Tn1545 family. The aim of the present work was to evaluate the prevalence of tet(M) in Tunisian isolates and to gain an insight into its origin and evolution. Twenty Ureaplasma parvum, two Ureaplasma urealyticum and 48 Mycoplasma hominis isolates, recovered from Tunisian patients with urogenital and infertility disorders, were evaluated for their resistance to tetracyclines and interrogated by PCR amplification for the presence of tet(M) and int-Tn, the gene encoding the integrase of Tn916/Tn1545-like transposons. The resistance rates to tetracyclines were 22.72 and 25.0 % among U. parvum and M. hominis isolates, respectively, with high-level resistance observed in 11 of the 12 resistant M. hominis isolates. All resistant isolates harboured both tet(M) and int-Tn sequences. Nucleotide sequence analysis of the tet(M) amplicon revealed a unique sequence shared by all tetracycline-resistant clinical isolates of both species. Molecular typing indicated that the tetracycline-resistant U. parvum and M. hominis isolates were not clonal. Taken together, these data indicate that a single tet(M) gene sequence type, most probably transmitted via a Tn916/Tn1545-like transposon, contributes to most of the tetracycline resistance in U. parvum and M. hominis isolates in Tunisia. Because this tet(M) gene sequence type was harboured by different Mycoplasma spp. and by phylogenetically distinct isolates within these species, one could reasonably argue that it may have benefited from an efficient horizontal transfer context, making it highly competent to spread.

To our knowledge, the phylodistribution of M. hominis clinical strains associated with various pathological conditions of the urogenital tract has not been explored hitherto. Here we analyzed the genetic diversity and phylogenetic relationships among 59 M. hominis Tunisian clinical isolates, categorized as gynecological infections- or infertility-associated pathotypes. For this purpose, we developed an expanded multilocus sequence typing (eMLST) scheme, combining the previously reported multilocus sequence typing (MLST) loci (gyrB, tuf, ftsY, uvrA, gap) with a new selected set of putative virulence genes (p120’, vaa, lmp1, lmp3, p60), referred herein to as multi-virulence-locus sequence typing (MVLST) loci. In doing so, M. hominis population was segregated into two distinct genetic lineages, which were differentially associated with each pathotype. Such a clear dichotomy was supported by several phylogenetic and population genetic analysis tools. Recombination was found to take place, but not sufficient enough to break down the overall clonal population structure of M. hominis, most likely as a result of purifying selection, which accommodated the most fit clones. In sum, and owing to the eMLST scheme described herein, we provide insightful data on the phylogenetics of M. hominis, arguing for the existence of genetically differentiable urogenital pathotypes.

We developed a duplex PCR assay targeting the hemagglutinin multigene families, vlhA and pMGA, of Mycoplasma synoviae and Mycoplasma gallisepticum, respectively. The assay proved to be specific and sensitive enough to justify its use for the simultaneous detection of the two major avian mycoplasma species from field isolates.Mycoplasma gallisepticum and M. synoviae are considered important in commercial poultry industries. M. gallisepticum causes chronic respiratory disease in chickens and sinusitis in turkeys (17), and M. synoviae is commonly involved in respiratory tract infection, synovitis in chickens, and poor growth (12). The antigenic relatedness (1, 2, 7, 11) of these organisms make them difficult to identify with conventional serological tests (5, 6). Attempts to differentiate between these two major avian mycoplasmas by using molecular methods, such as PCR tests, were mainly based on the 16S rRNA gene (4, 10, 13). However, it often requires additional steps, such as restriction fragment length analysis (9) or hybridization with species-specific probes (3, 10). In addition, these analyses resulted in concomitant amplification of unrelated bacterial DNA, making this approach useless for the testing of clinical material.We show here that PCR amplification targeting speciesspecific structural genes provides an efficient tool for the simultaneous detection and differentiation of M. gallisepticum and M. synoviae.The Mycoplasma species and walled bacteria used in the present study are listed in Table 1. All of the mycoplasma strains were propagated in Frey's medium (8). Unrelated bacterial species were cultured in brain heart infusion broth (Difco Laboratories, Detroit, Mich.). Forty tracheal swabs were immediately processed for culture and used as clinical mycoplasma samples to test the applicability of the duplex PCR assay.The template DNA of M. synoviae and M. gallisepticum was prepared from 200 l of culture, to which an equal volume of nonionic detergent mix solution (0.45% Nonidet P-40, 0.45% Tween 20, and 100 g of proteinase K/ml) was added. The sample was incubated at 56°C for 1 h, boiled for 10 min, and then centrifuged at 14,000 ϫ g for 5 min. Then, 10 l of the resulting supernatant was directly used for PCR.All of the primers used in the present study are listed in Table 2. They targeted pMGA1.2 gene encoding a hemagglutinin protein (pMGA) from M. gallisepticum (14) and M. synoviae2/12 DNA fragment (accession no. M. synoviaeU66314) (3) of the M. synoviae hemagglutinin vlhA gene (15, 16). To confirm the specificity of the amplification reaction, a nested PCR was performed by using inner primers (Tab.2). Additional avian Mycoplasma spp. and other common bacteria (Table 1) were tested to determine the specificity of the duplex PCR assay.The amplification reaction was performed in a total volume of 50 l containing 5 l of 10ϫ PCR buffer (Amersham Biosciences), 250 M concentrations of each deoxynucleoside triphosphate (Pharmacia), 50 pmol of each external or internal primers, 2.25 mM MgCl 2 , 2 U of Taq D...

Background: Among the surface antigens of Mycoplasma hominis, the P120' protein was previously shown to elicit a subtle antibody response and appears to be relatively conserved. To get better insight into the evolution of this protein, we analysed the genetic variability of its surface exposed region in 27 M. hominis isolates recovered from the genital tract of Tunisian patients with infertility disorders.