Conjugative Transposon Research Articles

Technological characterisation, antibiotic susceptibility and antimicrobial activity of wild-typeLeuconostocstrains isolated from north Italian traditional cheeses

Genotypic and technological properties, antibiotic susceptibility and antimicrobial activity of 35 Leuconostoc strains, isolated from different Italian raw milk cheeses, were investigated. RAPD-PCR was used to study genetic variability and to distinguish closely related strains. The results showed a high degree of heterogeneity among isolates. All the strains had weak acidifying activity and showed low proteolytic and lipolytic activities. Reduction activity, was generally low. All the Leuconostoc were susceptible to ampicillin, mupirocin, erythromycin, quinupristin/dalfopristin and tetracycline. Many strains were classified as resistant to oxacillin, ciprofloxacin and nitrofurantonin, while all isolates were found resistant to vancomycin. PCR-based detection did not identify any of the common genetic determinants for vancomycin (vanA, vanB, vanC1, vanC2, vanC3, vanD, vanE, vanG) or erythromycin (ermB and ermC). Tetracycline resistance genes were detected in 25 tetracycline susceptible strains, the most frequent one being tetM. One strain, belonging to Ln. pseudomesenteroides species, was positive for the presence of the int gene of the Tn916/Tn1545 trasposon family. This is the first time the conjugative transposon Tn916 has been detected inside the Leuconostoc species. All strains showed antimicrobial activity against Enterococcus faecalis and Ent. faecium. The presence of genes encoding amino-acid decarboxylases (hdc and tdc) was not detected. Some strains are interesting in view of their use in cheese production as starter and non starter cultures.

Structures of a Bifunctional Cell Wall Hydrolase CwlT Containing a Novel Bacterial Lysozyme and an NlpC/P60 dl-Endopeptidase

Tn916-like conjugative transposons carrying antibiotic resistance genes are found in a diverse range of bacteria. Orf14 within the conjugation module encodes a bifunctional cell wall hydrolase CwlT that consists of an N-terminal bacterial lysozyme domain (N-acetylmuramidase, bLysG) and a C-terminal NlpC/P60 domain (γ-d-glutamyl-l-diamino acid endopeptidase) and is expected to play an important role in the spread of the transposons. We determined the crystal structures of CwlT from two pathogens, Staphylococcus aureus Mu50 (SaCwlT) and Clostridium difficile 630 (CdCwlT). These structures reveal that NlpC/P60 and LysG domains are compact and conserved modules, connected by a short flexible linker. The LysG domain represents a novel family of widely distributed bacterial lysozymes. The overall structure and the active site of bLysG bear significant similarity to other members of the glycoside hydrolase family 23 (GH23), such as the g-type lysozyme (LysG) and Escherichia coli lytic transglycosylase MltE. The active site of bLysG contains a unique structural and sequence signature (DxxQSSES+S) that is important for coordinating a catalytic water. Molecular modeling suggests that the bLysG domain may recognize glycan in a similar manner to MltE. The C-terminal NlpC/P60 domain contains a conserved active site (Cys-His-His-Tyr) that appears to be specific to murein tetrapeptide. Access to the active site is likely regulated by isomerism of a side chain atop the catalytic cysteine, allowing substrate entry or product release (open state), or catalysis (closed state).

Mutations of the domain forming the dimeric interface of theArdAprotein affect dimerization and antimodification activity but not antirestriction activity

ArdA antirestriction proteins are encoded by genes present in many conjugative plasmids and transposons within bacterial genomes. Antirestriction is the ability to prevent cleavage of foreign incoming DNA by restriction-modification (RM) systems. Antimodification, the ability to inhibit modification by the RM system, can also be observed with some antirestriction proteins. As these mobile genetic elements can transfer antibiotic resistance genes, the ArdA proteins assist their spread. The consequence of antirestriction is therefore the enhanced dissemination of mobile genetic elements. ArdA proteins cause antirestriction by mimicking the DNA structure bound by Type I RM enzymes. The crystal structure of ArdA showed it to be a dimeric protein with a highly elongated curved cylindrical shape [McMahon SA et al. (2009) Nucleic Acids Res37, 4887–4897]. Each monomer has three domains covered with negatively charged side chains and a very small interface with the other monomer. We investigated the role of the domain forming the dimer interface for ArdA activity via site-directed mutagenesis. The antirestriction activity of ArdA was maintained when up to seven mutations per monomer were made or the interface was disrupted such that the protein could only exist as a monomer. The antimodification activity of ArdA was lost upon mutation of this domain. The ability of the monomeric form of ArdA to function in antirestriction suggests, first, that it can bind independently to the restriction subunit or the modification subunits of the RM enzyme, and second, that the many ArdA homologues with long amino acid extensions, present in sequence databases, may be active in antirestriction.Structured digital abstractArdA and ArdA bind by molecular sieving (1, 2)ArdA and ArdA bind by cosedimentation in solution (1, 2)

Metagenomic Insights into Metabolic Capacities of the Gut Microbiota in a Fungus-Cultivating Termite (Odontotermes yunnanensis)

Macrotermitinae (fungus-cultivating termites) are major decomposers in tropical and subtropical areas of Asia and Africa. They have specifically evolved mutualistic associations with both a Termitomyces fungi on the nest and a gut microbiota, providing a model system for probing host-microbe interactions. Yet the symbiotic roles of gut microbes residing in its major feeding caste remain largely undefined. Here, by pyrosequencing the whole gut metagenome of adult workers of a fungus-cultivating termite (Odontotermes yunnanensis), we showed that it did harbor a broad set of genes or gene modules encoding carbohydrate-active enzymes (CAZymes) relevant to plant fiber degradation, particularly debranching enzymes and oligosaccharide-processing enzymes. Besides, it also contained a considerable number of genes encoding chitinases and glycoprotein oligosaccharide-processing enzymes for fungal cell wall degradation. To investigate the metabolic divergence of higher termites of different feeding guilds, a SEED subsystem-based gene-centric comparative analysis of the data with that of a previously sequenced wood-feeding Nasutitermes hindgut microbiome was also attempted, revealing that SEED classifications of nitrogen metabolism, and motility and chemotaxis were significantly overrepresented in the wood-feeder hindgut metagenome, while Bacteroidales conjugative transposons and subsystems related to central aromatic compounds metabolism were apparently overrepresented here. This work fills up our gaps in understanding the functional capacities of fungus-cultivating termite gut microbiota, especially their roles in the symbiotic digestion of lignocelluloses and utilization of fungal biomass, both of which greatly add to existing understandings of this peculiar symbiosis.

Fitness costs of various mobile genetic elements in Enterococcus faecium and Enterococcus faecalis

ObjectivesTo determine the fitness effects of various mobile genetic elements (MGEs) in Enterococcus faecium and Enterococcus faecalis when newly acquired. We also tested the hypothesis that the biological cost of vancomycin resistance plasmids could be mitigated during continuous growth in the laboratory.MethodsDifferent MGEs, including two conjugative transposons (CTns) of the Tn916 family (18 and 33 kb), a pathogenicity island (PAI) of 200 kb and vancomycin-resistance (vanA) plasmids (80–200 kb) of various origins and classes, were transferred into common ancestral E. faecium and E. faecalis strains by conjugation assays and experimentally evolved (vanA plasmids only). Transconjugants were characterized by PFGE, S1 nuclease assays and Southern blotting hybridization analyses. Single specific primer PCR was performed to determine the target sites for the insertion of the CTns. The fitness costs of various MGEs in E. faecium and E. faecalis were estimated in head-to-head competition experiments, and evolved populations were generated in serial transfer assays.ResultsThe biological cost of a newly acquired PAI and two CTns were both host- and insertion-locus-dependent. Newly acquired vanA plasmids may severely reduce host fitness (25%–27%), but these costs were rapidly mitigated after only 400 generations of continuous growth in the absence of antibiotic selection.ConclusionsNewly acquired MGEs may impose an immediate biological cost in E. faecium. However, as demonstrated for vanA plasmids, the initial costs of MGE carriage may be mitigated during growth and beneficial plasmid–host association can rapidly emerge.

Occurrence and analysis of rare cfiA–bft doubly positive Bacteroides fragilis strains

We detected four cfiA–bft1 doubly positive Bacteroides fragilis strains out of 486 B. fragilis isolates analyzed for antibiotic susceptibilities and antibiotic resistance genes from a recent pan-European survey. The prevalence of the enterotoxin bft genes was roughly equal among cfiA-negative and -positive B. fragilis strains. We also demonstrated that the cfiA–bft doubly positive strains had the most common B. fragilis genomic pattern (I.1.). Thus we concluded that the bft-carrying CTn86 conjugative transposons are mobile accounting for this unexpected simultaneous occurrence of the cfiA and bft genes.

Two novel PIWI families: roles in inter-genomic conflicts in bacteria and Mediator-dependent modulation of transcription in eukaryotes

BackgroundThe PIWI module, found in the PIWI/AGO superfamily of proteins, is a critical component of several cellular pathways including germline maintenance, chromatin organization, regulation of splicing, RNA interference, and virus suppression. It binds a guide strand which helps it target complementary nucleic strands.ResultsHere we report the discovery of two divergent, novel families of PIWI modules, the first such to be described since the initial discovery of the PIWI/AGO superfamily over a decade ago. Both families display conservation patterns consistent with the binding of oligonucleotide guide strands. The first family is bacterial in distribution and is typically encoded by a distinctive three-gene operon alongside genes for a restriction endonuclease fold enzyme and a helicase of the DinG family. The second family is found only in eukaryotes. It is the core conserved module of the Med13 protein, a subunit of the CDK8 subcomplex of the transcription regulatory Mediator complex.ConclusionsBased on the presence of the DinG family helicase, which specifically acts on R-loops, we infer that the first family of PIWI modules is part of a novel RNA-dependent restriction system which could target invasive DNA from phages, plasmids or conjugative transposons. It is predicted to facilitate restriction of actively transcribed invading DNA by utilizing RNA guides. The PIWI family found in the eukaryotic Med13 proteins throws new light on the regulatory switch through which the CDK8 subcomplex modulates transcription at Mediator-bound promoters of highly transcribed genes. We propose that this involves recognition of small RNAs by the PIWI module in Med13 resulting in a conformational switch that propagates through the Mediator complex.ReviewersThis article was reviewed by Sandor Pongor, Frank Eisenhaber and Balaji Santhanam.

Clostridium difficile erm(B)-containing elements and the burden on the in vitro fitness

In Clostridium difficile, resistance to the macrolide-lincosamide-streptogramin B group of antibiotics generally relies on erm(B) genes. In this study, we investigated elements with a genetic organization different from Tn5398, the mobilizable non-conjugative element identified in C. difficile strain 630. Our results suggested that the elements most frequently found in strains isolated during the European surveillance study in 2005 were related to Tn6194, the conjugative transposon recently detected in different C. difficile types, including PCR-ribotype 027. We characterized a Tn6194-like and a novel element rarely found in clinical isolates. A burden on the in vitro fitness of C. difficile was observed after the acquisition of these elements as well as of Tn5398.

Interactions of the excision proteins of CTnDOT in the attR intasome

Excision of the conjugative transposon CTnDOT from the chromosome of Bacteroides spp. involves four CTnDOT-encoded proteins: IntDOT, Xis2c, Xis2d, and Exc along with a host factor. These proteins form excisive intasomes on the attR and attL sites which undergo synapsis and recombination to form the attDOT and attB sites. We recently developed an in vitro intramolecular excision reaction where the attL and attR sites are on the same plasmid. This reaction requires IntDOT, Xis2c, Xis2d, and is stimulated by Exc. We used this reaction to identify the binding sites of the IntDOT, Xis2c, and Xis2d. In this paper, we show that three of the six arm-type sites are absolutely required for excision. Furthermore, we also identified two binding sites for Xis2d and two possible binding sites for Xis2c on the attR site. We also showed that IntDOT interacts cooperatively with the Xis2c and Xis2d proteins on the attR site.

Scarce detection of mobile erm genes associated with tetQ in Bacteroides and Parabacteroides from Costa Rica

The frequency of finding of clindamycin-resistant anaerobic bacteria in clinical samples has doubled from 2008 to 2010 in Costa Rica. To determine whether this increase is due to dissemination of erm genes aided by tetQ elements, we analyzed 100 isolates of Bacteroides or Parabacteroides from a regional hospital, a national hospital, and the community. Antimicrobial susceptibilities were recorded with a broth micro-dilution method and erm genes were detected by PCR and Southern blotting. In addition, plasmid isolation and mating experiments were performed to clarify the location and mobility of the detected erm genes. Resistance to clindamycin was by far more frequent in the regional hospital (72%) than in the national hospital (29%) and the community (26%). Resistance to tetracycline was even more common, with the community (85%) outweighing the hospitals (71–72%). While MIC of clindamycin were higher in the hospitals than in the community (P < 0.05), the opposite was seen for tetracycline (P < 0.0001). Of the sought-after genes, only ermG (n = 2), ermA (n = 1), and ermF (n = 1) were detected in the hospitals and ermF in the community (n = 2). In opposition to the low frequency of finding of erm genes, 71% of the isolates were positive for tetQ. None of the detected genes were encoded on plasmids. Only three isolates from the hospitals transferred their erm genes laterally. By contrast, 13 hospital isolates and two community isolates transferred tetQ. Despite the widespread finding of tetracycline-resistant tetQ-positive bacteria, mobile erm genes were rare in our bacterial collection. We conclude that the detected erm genes are likely not included in typical conjugative transposons of Bacteroides and Parabacteroides.

The prevalence of antibiotic resistance genes in Bacteroides fragilis group strains isolated in different European countries

From the 2008–2009 European Bacteroides antibiotic resistance survey, we selected 161 strains for detection of antibiotic resistance genes (cepA, cfxA, cfiA, nim, ermB, ermF, ermG, linA, mefA, msrSA, tetM, tetQ, tetX, tetX1, tet36 and bexA). To facilitate the throughput, the genes were detected by Real-Time PCR. The presence of the genes was correlated with the known MIC data of the strains for the appropriate antibiotics. For the β-lactams, the cepA gene was found in 70.8% of the tested strains (all resistant to ampicillin), but its presence did not correlate with the ampicillin MIC values. The cepA gene occurred at different frequencies among Bacteroides fragilis and non-fragilis Bacteroides strains. The cfxA gene was not a major factor in determining cefoxitin resistance and it was found with higher prevalence in non-fragilis Bacteroides strains than in B. fragilis. Among the five possible clindamycin resistance genes, ermF was the most common and had the highest effect on clindamycin resistance after linA. The ermG-mefA-msrSA combination was found in a set of strains and their linked occurrence implied that they were harbored by the conjugative transposon CTnGERM1. All strains tested were susceptible to metronidazole and none of them harbored nim genes. TetQ was prevalent among both the B. fragilis and non-fragilis Bacteroides strains (78.9 and 84.8%, respectively) and no gene could be clearly linked to tigecycline resistance other than tetQ. BexA, which codes for the fluoroquinolone efflux pump, was found in 7.5% of strains and occurred at different frequencies among B. fragilis and non-fragilis Bacteroides strains, but was represented only in a minor proportion of moxifloxacin-resistant strains.

Tn6198, a novel transposon containing the trimethoprim resistance gene dfrG embedded into a Tn916 element in Listeria monocytogenes

To characterize Tn6198, a novel conjugative transposon from the clinical Listeria monocytogenes strain TTH-2007, which contains the tetracycline and trimethoprim resistance genes tet(M) and dfrG, respectively, and to assess its transferability in vitro and in situ. The complete sequence of Tn6198 was determined using a primer walking strategy. Horizontal gene transfer studies were performed by filter matings, as well as on the surface of smear-ripened cheese and smoked salmon. The presence of Tn916-like circular intermediates was determined by PCR. Antibiotic resistance was determined by the broth microdilution method and microarray hybridization. Sequencing of Tn6198 revealed that a 3.3 kb fragment containing dfrG was integrated between open reading frames 23 and 24 of Tn916. Furthermore, an additional copy of Tn916 was present in L. monocytogenes TTH-2007. Both elements were transferred simultaneously and separately in vitro to recipients L. monocytogenes 10403S and Enterococcus faecalis JH2-2 by conjugation, resulting in either tetracycline- and trimethoprim-resistant or solely tetracycline-resistant transconjugants. On the surface of cheese and salmon, only L. monocytogenes 10403S transconjugants were detected. This study reports the first Tn916-like element associated with a trimethoprim resistance gene, as well as the first fully characterized transposon conferring multidrug resistance in L. monocytogenes. This is of concern, as trimethoprim is administered to listeriosis patients with β-lactam allergy and as Tn6198 has a large potential for dissemination, indicated by both intra-species and inter-genus transfer.

IntDOT Interactions with Core Sites during Integrative Recombination

Integrative and conjugative elements (ICEs), formerly called conjugative transposons, have been implicated in the proliferation of antibiotic resistance genes. CTnDOT is an extensively studied ICE found in Bacteroides spp. In addition to carrying resistance genes to both erythromycin and tetracycline, CTnDOT carries a gene that encodes a tyrosine recombinase called IntDOT that catalyzes integration into and excision out of the bacterial host chromosome. CTnDOT integrates into one of several known attB sites in the bacterial chromosome that consists of a pair of inverted repeat core sites called B and B' in attB. The attDOT site contains the core sites and D and D'. These sites flank the overlap regions where strand exchanges occur. A notable feature of all known attB sites is the conservation of the B core site sequence, which is also found in the D core site of attDOT. In this study, we used a mutational analysis to establish the importance of this conserved sequence for integration and characterize the interaction of IntDOT with individual base pairs. We identified important T-A base pairs at position -5 in the B and D core sites and position +5 in the poorly conserved B' core site that are important for integrative recombination. Base analog studies suggest that IntDOT may make specific contacts with the A residues in the major groove at positions -5 and +5. IntDOT interaction with the A at position -5 in the B core site is required for the first strand exchange.

The excision proteins of CTnDOT positively regulate the transfer operon

The Bacteroides conjugative transposon, CTnDOT, is an integrated conjugative element (ICE), found in many human colonic Bacteroides spp. strains. It has a complex regulatory system for both excision from the chromosome and transfer and mobilization into a new host. It was previously shown that a cloned DNA segment encoding the xis2c, xis2d, orf3, and exc genes was required for tetracycline dependent activation of the Ptra promoter. The Xis2c and Xis2d proteins are required for excision while the Exc protein stimulates excision. We report here that neither the Orf3 nor the Exc proteins are involved in activation of the Ptra promoter. Deletion analysis and electromobility shift assays showed that the Xis2c and Xis2d proteins bind to the Ptra promoter to activate the tra operon. Thus, the recombination directionality factors of CTnDOT excision also function as activator proteins of the Ptra promoter.

Emergence and global spread of epidemic healthcare-associated Clostridium difficile

Epidemic Clostridium difficile (027/BI/NAP1) rapidly emerged in the past decade as the leading cause of antibiotic-associated diarrhea worldwide. However, the key moments in the evolutionary history leading to its emergence and subsequent patterns of global spread remain unknown. Here we define the global population structure of C. difficile 027/BI/NAP1 based on whole-genome sequencing and phylogenetic analysis. We demonstrate that two distinct epidemic lineages, FQR1 and FQR2, not one as previously thought, emerged in North America within a relatively short period after acquiring the same fluoroquinolone resistance mutation and a highly-related conjugative transposon. The two epidemic lineages displayed distinct patterns of global spread, and the FQR2 lineage spread more widely leading to healthcare outbreaks in the UK, continental Europe and Australia. Our analysis identifies key genetic changes linked to the rapid trans-continental dissemination of epidemic C. difficile 027/BI/NAP1 and highlights the routes by which it spreads through the global healthcare system.

Multiplex Bead Suspension Array for Screening Neisseria gonorrhoeae Antibiotic Resistance Genetic Determinants in Noncultured Clinical Samples

The increasing threat of antibiotic-resistant Neisseria gonorrhoeae highlights the need for new diagnostic options. A high-throughput multiplex bead suspension array assay was developed for profiling 29 N. gonorrhoeae genomic mutations and 2 plasmid genes conferring resistance to 6 antimicrobial agents: penicillin, ciprofloxacin, cefixime, tetracycline, azithromycin, and spectinomycin. The three steps of this assay include amplification of 12 N. gonorrhoeae chromosomal and plasmid loci, multiplex allele-specific primer extension reaction, and multiplex bead suspension array detection. Antibiotic resistance genetic determinants were identified successfully in 239 cervicovaginal N. gonorrhoeae-positive noncultured swab samples. This molecular assay can be used for detection of gonococci in clinical specimens, molecular typing, mutation profiling, and predictive assessment of N. gonorrhoeae susceptibility to antibiotics without the need for culture.

Comparative Transcriptional Analysis of Clinically Relevant Heat Stress Response in Clostridium difficile Strain 630

Clostridium difficile is considered to be one of the most important causes of health care-associated infections worldwide. In order to understand more fully the adaptive response of the organism to stressful conditions, we examined transcriptional changes resulting from a clinically relevant heat stress (41°C versus 37°C) in C. difficile strain 630 and identified 341 differentially expressed genes encompassing multiple cellular functional categories. While the transcriptome was relatively resilient to the applied heat stress, we noted upregulation of classical heat shock genes including the groEL and dnaK operons in addition to other stress-responsive genes. Interestingly, the flagellin gene (fliC) was downregulated, yet genes encoding the cell-wall associated flagellar components were upregulated suggesting that while motility may be reduced, adherence – to mucus or epithelial cells – could be enhanced during infection. We also observed that a number of phage associated genes were downregulated, as were genes associated with the conjugative transposon Tn5397 including a group II intron, thus highlighting a potential decrease in retromobility during heat stress. These data suggest that maintenance of lysogeny and genome wide stabilisation of mobile elements could be a global response to heat stress in this pathogen.

The highly dynamic CRISPR1 system of Streptococcus agalactiae controls the diversity of its mobilome

Clustered regularly interspaced short palindromic repeats (CRISPR) confer immunity against mobile genetic elements (MGEs) in prokaryotes. Streptococcus agalactiae, a leading cause of neonatal infections contains in its genome two CRISPR/Cas systems. We show that type 1-C CRISPR2 is present in few strains but type 2-A CRISPR1 is ubiquitous. Comparative sequence analysis of the CRISPR1 spacer content of 351 S. agalactiae strains revealed that it is extremely diverse due to the acquisition of new spacers, spacer duplications and spacer deletions that witness the dynamics of this system. The spacer content profile mirrors the S. agalactiae population structure. Transfer of a conjugative transposon targeted by CRISPR1 selected for spacer rearrangements, suggesting that deletions and duplications pre-exist in the population. The comparison of protospacers located within MGE or the core genome and protospacer-associated motif-shuffling demonstrated that the GG motif is sufficient to discriminate self and non-self and for spacer selection and integration. Strikingly more than 40% of the 949 different CRISPR1 spacers identified target MGEs found in S. agalactiae genomes. We thus propose that the S. agalactiae type II-A CRISPR1/Cas system modulates the cohabitation of the species with its mobilome, as such contributing to the diversity of MGEs in the population.

The Small RNA RteR Inhibits Transfer of the Bacteroides Conjugative Transposon CTnDOT

CTnDOT is a 65-kb conjugative transposon present in Bacteroides spp. that confers resistance to erythromycin [erm(F)] and tetracycline [tet(Q)]. An interesting feature of CTnDOT is that both excision from the chromosome and transfer of CTnDOT are stimulated by exposure to tetracycline. However, when no tetracycline is present, transfer of CTnDOT is not detectable. Previous studies suggested that a region containing a small RNA, RteR, appeared to mediate repression of CTnDOT transfer; however, virtually nothing was known about RteR. We have demonstrated that RteR is a 90-nucleotide transcript that is not further processed. RteR inhibits conjugative transfer of CTnDOT by targeting the transfer region, a 13-kb operon that encodes the tra genes required to assemble the mating apparatus. We report here that RteR interacts with the region downstream of traA. Levels of the downstream tra mRNA are dramatically reduced when RteR is present. Further, RteR does not appear to decrease the half-life of the tra mRNA transcript, suggesting that RteR does not bind to the transcript to initiate RNase-dependent decay, similar to other trans-acting small RNAs. We predict that RteR may act to enhance termination of the tra operon within traB, which could account for the decreased abundance of the tra transcript downstream of traA and explain why the tra mRNA has the same half-life whether or not RteR is present. RteR is the only small RNA that has been characterized so far within the Bacteroidetes phylum.

Evidence for the predominance of a single tet(M) gene sequence type in tetracycline-resistant Ureaplasma parvum and Mycoplasma hominis isolates from Tunisian patients

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.