Allelic Polymorphism of the tet(M) Determinant in Mycoplasma hominis and Ureaplasma urealyticum Clinical Isolates Resistant to Tetracyclines

Of the 130 clinical isolates of Mycoplasma hominisfrom patients with nonspecific inflammatory diseases of the urogenital tract (UGT), approximately 10% contained the tet(M) gene after the course of treatment with tetracyclines. This gene was found in nine (25%) of the 36 Ureaplasma urealyticum clinical isolates. The nucleotide sequence of 13 tet(M) genes in Tc^R clinical isolates ofM. hominis and five genes in U. urealyticum Tc^R clinical isolates was determined. A comparison of nucleotide sequences of eight tetM genes of different origin and tet(M) genes ofGardnerella vaginalis and M. hominis and U. urealyticumclinical isolates showed that the mosaic structure of thetet(M) gene is completely identical in 11 of 13 M. hominis Tc^Risolates but belongs to an unidentified allele different from those described earlier. Another new allelic variant oftet(M) was found in two isolates. In three of five Tc^R clinical isolates of U. urealyticum, a tet(M) gene, whose mosaic structure was identical to that of tet(M) reported previously for ureaplasmas, and also two new allelic variants, which have not been described so far, were found.     

Rapid detection of tetM in Mycoplasma hominis and Ureaplasma urealyticum by PCR: tetM confers resistance to tetracycline but not necessarily to doxycycline

Tetracycline resistance in Mycoplasma hominis and Ureaplasma urealyticum has been associated with the tetM determinant and has recently been increasing in incidence. We report here a rapid method for detection of the tetM determinant based on the use of the polymerase chain reaction (PCR) to amplify a 397-bp DNA fragment from the tetM gene and verification of specificity using the restriction enzyme TaqI. Analysis of 42 U. urealyticum and 49 M. hominis isolates indicates that the PCR method may be clinically useful for determination of tetracycline sensitivity, as tetM is presently the only known determinant associated with tetracycline resistance in these two organisms. All of the tetM-positive M. hominis isolates were sensitive to doxycycline, indicating that tetM does not necessarily confer resistance to this antibiotic.     

泌尿生殖道支原体感染检测及药敏试验

目的了解本地区泌尿生殖道解脲脲原体（Uu）和人型支原体（Mh）感染状况及药物敏感性,指导临床医生合理应用抗生素。方法应用法国生物梅里埃公司提供的IST试剂盒进行支原体鉴定及9种药物敏感检测,并对结果进行统计分析。结果1210例门诊患者检出支原体阳性683例,总感染率为56.4%,其中Uu单独感染占628例（占51.9%）,Mh单独感染14例（占1.2%）,Uu和Mh混合感染41例（占3.4%）。交沙霉素和原始霉素敏感率最高,对Uu分别为98.5%和97.0%,对Uu和Mh混合感染率都为100%;氧氟沙星敏感率最低,分别为1.5%和0.0%。结论泌尿生殖道系统感染主要由解脲支原体引起,交沙霉素和原始霉素敏感率最高,氧氟沙星敏感率最低。临床应选用培养敏感的抗菌药物,提高治疗效果。     

Prevalence and molecular characterization of tetracycline resistance in Enterococcus isolates from food

In the present study, a collection of 187 Enterococcus food isolates mainly originating from European cheeses were studied for the phenotypic and genotypic assessment of tetracycline (TC) resistance. A total of 45 isolates (24%) encompassing the species Enterococcus faecalis (n = 33), E. durans (n = 7), E. faecium (n = 3), E. casseliflavus (n = 1), and E. gallinarum (n = 1) displayed phenotypic resistance to TC with MIC ranges of 16 to 256 microg/ml. Eight of these strains exhibited multiresistance to TC, erythromycin, and chloramphenicol. By PCR detection, TC resistance could be linked to the presence of the tet(M) (n = 43), tet(L) (n = 16), and tet(S) (n = 1) genes. In 15 isolates, including all of those for which the MIC was 256 micro g/ml, both tet(M) and tet(L) were found. Furthermore, all tet(M)-containing enterococci also harbored a member of the Tn916-Tn1545 conjugative transposon family, of which 12 erythromycin-resistant isolates also contained the erm(B) gene. Filter mating experiments revealed that 10 E. faecalis isolates, 3 E. durans isolates, and 1 E. faecium isolate could transfer either tet(M), tet(L), or both of these genes to E. faecalis recipient strain JH2-2. In most cases in which only tet(M) was transferred, no detectable plasmids were acquired by JH2-2 but instead all transconjugants contained a member of the Tn916-Tn1545 family. Sequencing analysis of PCR amplicons and evolutionary modeling showed that a subset of the transferable tet(M) genes belonged to four sequence homology groups (SHGs) showing an internal homology of > or = 99.6%. Two of these SHGs contained tet(M) mosaic structures previously found in Tn916 elements and on Lactobacillus and Neisseria plasmids, respectively, whereas the other two SHGs probably represent new phylogenetic lineages of this gene.     

Comparison of polymerase chain reaction assay and Mycoplasma IST 2 test with culture for detection of infections caused by Ureaplasma urealyticum and Mycoplasma hominis

The polymerase chain reaction (PCR) technique and commercial Mycoplasma IST 2 test were compared with culture for the detection of U. urealyticum and M. hominis in 173 clinical samples obtained from patients without clinical symptoms from genito-urinary tract. The presence of U. urealyticum was diagnosed by culture in 24 samples, by PCR in 33 samples and by Mycoplasma IST 2 test in 39 samples. The presence of M. hominis was diagnosed in 26 samples only by Mycoplasma IST 2 test--culture and PCR were negative. The study showed the excellent sensitivity (100%) and good specificity (appropriately 94.0% and 90.0%) for U. urealyticum in PCR and Mycoplasma IST 2 test. The discrepancy of results obtained in Mycoplasma IST 2 test and culture as well as in PCR may suggest the over sensitivity of the commercial test for detection of M. hominis.     

Characterization of the variability of a 75-kDa membrane protein in Mycoplasma hominis

The gene p75 encoding a 75-kDa surface-exposed membrane protein P75 was cloned and sequenced from Mycoplasma hominis type strain PG21T. To investigate the intraspecies variability, sequences were obtained from an additional two isolates 7488 and 183, and the three sequences were compared. The nucleotide and amino acid differences were not confined to specific regions of the gene/protein, but when comparing the three sequences, differences were present as single site substitutions or small insertions or deletions of nucleotides/amino acids. The intraspecies variability was further investigated by restriction enzyme analysis with two restriction enzymes (Alul and MboII) of PCR products amplified from p75 from 28 M. hominis isolates. On the basis of band patterns produced by the two restriction enzymes, the isolates could be divided into five and six groups. These groups neither matched categories of the M. hominis vaa gene nor the M. hominis p120 gene classes, indicating that the three genes vary by different mechanisms and possibly indicating horizontal gene transfer. Federation of European Microbiological Societies.     

Microbial causative agents of male urethritis.

The incidence of Chlamydia trachomatis, Ureaplasma urealyticum, Mycoplasma hominis, Neisseria gonorrhoeae and Trichomonas vaginalis was studied in men with urethritis. Out of 150 examined men 48.7% had the positive isolation of U. urealyticum, 26.0% C. trachomatis, 22.7% N. gonorrhoeae, 18.7% M. hominis and in one (0.7%) patient T. vaginalis was found. None of the above mentioned microorganisms was detected in 24.7% of examined men. In 43.3% cases only one agent was isolated. In 23.3% of the men the combination of two agents, in 8.0% the combination of three and in 0.7% even the combination of four studied microorganisms was observed. C. trachomatis was most frequently observed in combination with N. gonorrhoeae (15 cases) and U. urealyticum (14 cases). M. hominis and U. urealyticum occurred simultaneously in 22 examined men.N.gonorrhoeae was most frequently found together with U. urealyticum (16 cases). Concerning the occurrence of other bacteria and yeasts, no significant difference was found between the groups positive and negative for the above mentioned microorganisms.     

Tetracycline resistance mediated by tet(W), tet(M), and tet(O) genes of Bifidobacterium isolates from humans

MICs of tetracyclines were determined for 86 human Bifidobacterium isolates and three environmental strains. The tet(O) gene was found to be absent in these isolates. tet(W) and tet(M) were found in 26 and 7%, respectively, of the Bifidobacterium isolates, and one isolate contained both genes. Chromosomal DNA hybridization showed that there was one chromosomal copy of tet(W) and/or tet(M).     

The prevalence of tetracycline resistance genes in clinical and commensal strains of Enterococcus faecalis isolated in Gdańsk

The common use of antibiotics is responsible for selecting of drug resistance not only in pathogenic, clinical bacteria but also in commensal, not pathogenic strains which could cause the rapid dissemination of the resistance to these antibacterial agents. However, information regarding the antibiotic resistance of commensal bacteria is very scarce, and the data is based mostly on phenotypical research. Therefore the use of genotyping methods for detection of tetracycline resistance genes, in commensal and medical isolates of bacteria, is essential, for understanding the spread of antibiotic resistance. In this study 24 commensal and 27 clinical isolates of Enterococcus faecalis has been screened by PCR methods for tet(M), tet(S) genes and Tn916 and Tn5397 transpozons. Subsequently, the tet(M) gene amplicones were sequenced and phylogenetic analysis was performed. We have found that the prevalence of tet(S) gene varied significantly between commensal and clinical strains. Moreover, the frequency of transpozons in clinical isolates was much higher comparing to strains isolated from healthy individuals. The phylogenetic analysis did not show significant differences between clinical and commensal strains but it could suggest that the genetic similarity between these two groups could be favourable factor for broad range spread of tet(M) gene.     

Occurrence and diversity of tetracycline resistance genes in lagoons and groundwater underlying two swine production facilities

In this study, we used PCR typing methods to assess the presence of tetracycline resistance determinants conferring ribosomal protection in waste lagoons and in groundwater underlying two swine farms. All eight classes of genes encoding this mechanism of resistance [tet(O), tet(Q), tet(W), tet(M), tetB(P), tet(S), tet(T), and otrA] were found in total DNA extracted from water of two lagoons. These determinants were found to be seeping into the underlying groundwater and could be detected as far as 250 m downstream from the lagoons. The identities and origin of these genes in groundwater were confirmed by PCR-denaturing gradient gel electrophoresis and sequence analyses. Tetracycline-resistant bacterial isolates from groundwater harbored the tet(M) gene, which was not predominant in the environmental samples and was identical to tet(M) from the lagoons. The presence of this gene in some typical soil inhabitants suggests that the vector of antibiotic resistance gene dissemination is not limited to strains of gastrointestinal origin carrying the gene but can be mobilized into the indigenous soil microbiota. This study demonstrated that tet genes occur in the environment as a direct result of agriculture and suggested that groundwater may be a potential source of antibiotic resistance in the food chain.     

Host and pathogen interaction during vaginal infection by Trichomonas vaginalis and Mycoplasma hominis or Ureaplasma urealyticum

Vaginal infections by Trichomonas vaginalis and Mycoplasma hominis have been shown to be associated. Since M. hominis and Ureaplasma urealyticum are similar pathogens, both belonging to the class of the mycoplasmata, we describe here a molecular study into the interdependence of U. urealyticum and T. vaginalis during infection. Susceptibility towards infection by U. urealyticum depends on genetic polymorphism in the interleukin-1 receptor antagonist (IL-1RA) gene. Now, we defined the relation between IL-1RA genotypes and infection by M. hominis and T. vaginalis. Finally, we also developed a restriction fragment length polymorphism (RFLP) tool for mapping variation in the T. vaginalis AP33 adhesin in order to define putative associations between parasite subtype and mycoplasmata or host. Studies using crudepellets from T. vaginalis culture broth clearly confirm the association between T. vaginalis and M. hominis infection. The association between IL-1RA genotype 2,2 and lack of U. urealyticum infection is corroborated as well. U. urealyticum infection and infection by T. vaginalis are independent. Furthermore, T. vaginalis and M. hominis infection are not depending on IL-1RA genotypes. Interestingly, one of the three AP33 RFLP types identified appeared to be associated with the absence of U. urealyticum infection. In conclusion, the complex interaction between bacterial and parasitic pathogens and the infected host is determined by genetic characteristics of host and microorganisms involved.     

Identification and sequence of a tet(M) tetracycline resistance determinant homologue in clinical isolates of Escherichia coli

The presence of the tetracycline resistance determinant tet(M) in human clinical isolates of Escherichia coli is described for the first time in this report. The homologue was >99% identical to the tet(M) genes reported to occur in Lactobacillus plantarum, Neisseria meningitidis, and Streptococcus agalactiae, and 3% of the residues in its deduced amino acid sequence diverge from tet(M) of Staphylococcus aureus. Sequence analysis of the regions immediately flanking the gene revealed that sequences upstream of tet(M) in E. coli have homology to Tn916; however, a complete IS26 insertion element was present immediately upstream of the promoter element. Downstream from the termination codon is an insertion sequence that was homologous to the ISVs1 element reported to occur in a plasmid from Vibrio salmonicida that has been associated with another tetracycline resistance determinant, tet(E). Results of mating experiments demonstrated that the E. coli tet(M) gene was on a mobile element so that resistance to tetracycline and minocycline could be transferred to a susceptible strain by conjugation. Expression of the cloned tet(M) gene, under the control of its own promoter, provided tetracycline and minocycline resistance to the E. coli host.     

Diverse tetracycline resistance genotypes of Megasphaera elsdenii strains selectively cultured from swine feces

A total of 30 Megasphaera elsdenii strains, selectively isolated from the feces of organically raised swine by using Me109 M medium, and one bovine strain were analyzed for tetracycline resistance genotypic and phenotypic traits. Tetracycline-resistant strains carried tet(O), tet(W), or a tet gene mosaic of tet(O) and tet(W). M. elsdenii strains carrying tet(OWO) genes exhibited the highest tetracycline MICs (128 to >256 microg/ml), suggesting that tet(O)-tet(W) mosaic genes provide the selective advantage of greater tetracycline resistance for this species. Seven tet genotypes are now known for M. elsdenii, an archetype commensal anaerobe and model for tet gene evolution in the mammalian intestinal tract.     

The identification of a tetracycline resistance gene tet(M), on a Tn916-like transposon,in the Bacillus cereus group

In order to investigate whether resistance genes present in bacteria in manure could transfer to indigenous soil bacteria, resistant isolates belonging to the Bacillus cereus group (Bacillus cereus, Bacillus anthracis and Bacillus thuringiensis) were isolated from farm soil (72 isolates) and manure (12 isolates) samples. These isolates were screened for tetracycline resistance genes (tet(K), tet(L), tet(M), tet(O), tet(S) and tet(T)). Of 88 isolates examined, three (3.4%) isolates carried both tet(M) and tet(L) genes, while four (4.5%) isolates carried the tet(L) gene. Eighty-one (92.1%) isolates did not contain any of the tested genes. All tet(M) positive isolates carried transposon Tn916 and could transfer this mobile DNA element to other Gram-positive bacteria.     

PCR-based detection of Mycoplasma species

In this study, we describe our newly-developed sensitive two-stage PCR procedure for the detection of 13 common mycoplasmal contaminants (M. arthritidis, M. bovis, M. fermentans, M. genitalium, M. hominis, M. hyorhinis, M. neurolyticum, M. orale, M. pirum, M. pneumoniae, M. pulmonis, M. salivarium, U. urealyticum). For primary amplification, the DNA regions encompassing the 16S and 23S rRNA genes of 13 species were targeted using general mycoplasma primers. The primary PCR products were then subjected to secondary nested PCR, using two different primer pair sets, designed via the multiple alignment of nucleotide sequences obtained from the 13 mycoplasmal species. The nested PCR, which generated DNA fragments of 165-353 bp, was found to be able to detect 1-2 copies of the target DNA, and evidenced no cross-reactivity with the genomic DNA of related microorganisms or of human cell lines, thereby confirming the sensitivity and specificity of the primers used. The identification of contaminated species was achieved via the performance of restriction fragment length polymorphism (RFLP) coupled with Sau3AI digestion. The results obtained in this study furnish evidence suggesting that the employed assay system constitutes an effective tool for the diagnosis of mycoplasmal contamination in cell culture systems.     

Prevalence of Chlamydia trachomatis and genital mycoplasmas in asymptomatic women.

To establish the prevalence of Chlamydia trachomatis, Mycoplasma hominis and Ureaplasma urealyticum in women attending a family planning and a prenatal clinic in Halifax, cervical swabs were obtained at the time of the first visit from 491 women who had no symptoms of genital infection. Among the women attending the family planning clinic M. hominis occurred in combination with C. trachomatis more frequently than expected (p less than 0.05). It occurred in the absence of U. urealyticum in only a few cases (13% of the occurrences in the family planning clinic and 6% of those in the prenatal clinic). C. trachomatis was significantly more prevalent in women under 25 years of age (p less than 0.04). However, mycoplasmas were as prevalent in women over 30 years as in those under 30. There were no significant differences in the infection rates of the organisms by trimester among pregnant women. More research is necessary for a proper understanding of the role of M. hominis and U. urealyticum in genitourinary infections and pregnancy outcomes.     

Isolation of tetracycline-resistant Megasphaera elsdenii strains with novel mosaic gene combinations of tet(O) and tet(W) from swine

Anaerobic bacteria insensitive to chlortetracycline (64 to 256 microg/ml) were isolated from cecal contents and cecal tissues of swine fed or not fed chlortetracycline. A nutritionally complex, rumen fluid-based medium was used for culturing the bacteria. Eight of 84 isolates from seven different animals were identified as Megasphaera elsdenii strains based on their large-coccus morphology, rapid growth on lactate, and 16S ribosomal DNA sequence similarities with M. elsdenii LC-1(T). All eight strains had tetracycline MICs of between 128 and 256 microg/ml. Based on PCR assays differentiating 14 tet classes, the strains gave a positive reaction for the tet(O) gene. By contrast, three ruminant M. elsdenii strains recovered from 30-year-old culture stocks had tetracycline MICs of 4 microg/ml and did not contain tet genes. The tet genes of two tetracycline-resistant M. elsdenii strains were amplified and cloned. Both genes bestowed tetracycline resistance (MIC = 32 to 64 microg/ml) on recombinant Escherichia coli strains. Sequence analysis revealed that the M. elsdenii genes represent two different mosaic genes formed by interclass (double-crossover) recombination events involving tet(O) and tet(W). One or the other genotype was present in each of the eight tetracycline-resistant M. elsdenii strains isolated in these studies. These findings suggest a role for commensal bacteria not only in the preservation and dissemination of antibiotic resistance in the intestinal tract but also in the evolution of resistance.     

Quantitative studies on the role of Ureaplasma urealyticum in non-gonococcal urethritis and chronic prostatitis

Quantitative determinations of U. urealyticum and M. hominis have been performed in 164 men with non-gonococcal urethritis (NGU) and 597 patients with chronic prostatitis. Evidence is provided that U. urealyticum plays an etiologic role in 29.3 percent of patients with non-gonococcal urethritis. Mixed infections of C. trachomatis and U. urealyticum, in high numbers, do occur in 11 percent of NGU cases. A constellation suggesting ureaplasma-associated disease could be observed in 13.7 to 15.2 percent of 597 patients with chronic prostatitis. M. hominis does not appear to be a causative agent of NGU or chronic prostatitis.     

Antibiotic resistance genes in multidrug-resistant Enterococcus spp. and Streptococcus spp. recovered from the indoor air of a large-scale swine-feeding operation

AIMS: In this study, multidrug-resistant bacteria previously recovered from the indoor air of a large-scale swine-feeding operation were tested for the presence of five macrolide, lincosamide and streptogramin (MLS) resistance genes and five tetracycline (tet) resistance genes. METHODS AND RESULTS: Enterococcus spp. (n = 16) and Streptococcus spp. (n =16) were analysed using DNA-DNA hybridization, polymerase chain reaction (PCR) and oligoprobing of PCR products. All isolates carried multiple MLS resistance genes, while 50% of the Enterococcus spp. and 44% of the Streptococcus spp. also carried multiple tet resistance genes. All Enterococcus spp. carried erm(A) and erm(B), 69% carried erm(F), 44% carried mef(A), 75% carried tet(M), 69% carried tet(L) and 19% carried tet(K). All Streptococcus spp. carried erm(B), 94% carried erm(F), 75% carried erm(A), 38% carried mef(A), 50% carried tet(M), 81% carried tet(L) and 13% carried tet(K). CONCLUSIONS: Multidrug resistance among airborne bacteria recovered from a swine operation is encoded by multiple MLS and tet resistance genes. These are the first data regarding resistance gene carriage among airborne bacteria from swine-feeding operations. SIGNIFICANCE AND IMPACT OF THE STUDY: The high prevalence of multiple resistance genes reported here suggests that airborne Gram-positive bacteria from swine operations may be important contributors to environmental reservoirs of resistance genes.