Isolation Of Chlamydia Trachomatis Research Articles

Comparative analysis of two genomes of Chlamydia pecorum isolates from an Alpine chamois and a water buffalo

BackgroundTo date, whole genome sequencing has been performed mainly for isolates of Chlamydia trachomatis, C. pneumoniae, C. psittaci and C. abortus, but only a few isolates of C. pecorum have been entirely sequenced and this makes it difficult to understand its diversity and population structure. In this study the genome of two C. pecorum strains isolated from the lung of an Alpine chamois affected with pneumonia (isolate PV7855) and the brain of a water buffalo affected with meningoencephalomyelitis (isolate PV6959), were completely sequenced with MiSeq system (Illumina) and analyzed in their most polymorphic regions.ResultsThe genome length and GC content of the two isolates were found to be consistent with other C. pecorum isolates and the gene content of polymorphic membrane proteins and plasticity zone was found to be very similar. Some differences were observed in the phospholipase genes for both isolates and in the number of genes in the plasticity zone, such as the presence of some hypothetical proteins in PV6959, not present in any other genomes analyzed in this study. Interestingly, PV6959 possesses an extra pmp and has an incomplete tryptophan biosynthesis operon. Plasmids were detected in both isolates.ConclusionsGenome sequencing of the two C. pecorum strains did not reveal differences in length and GC content despite the origin from different animal species with different clinical disease. In the plasticity zone, the differences in the genes pattern might be related to the onset of specific symptoms or infection of specific hosts. The absence of a tryptophan biosynthesis pathway in PV6959 may suggest a strict relationship between C. pecorum and its host.

Whole-genome sequencing of ocular Chlamydia trachomatis isolates from Gadarif State, Sudan

BackgroundTrachoma, caused by ocular Chlamydia trachomatis, is the leading infectious cause of blindness worldwide. Sudan first reported trachoma in the 1930s and has since been consistently endemic. Ocular C. trachomatis previously isolated from trachoma patients in Sudan in 1963 was antigenically identical to an isolate from Saudi Arabia (A/SA1). No contemporary ocular C. trachomatis whole genome sequences have been reported from Sudan.MethodsThis study sequenced twenty ocular C. trachomatis isolates to improve understanding of pathogen diversity in North-East Africa and examine for genomic variation specific to Sudan, possibly related to the persistence of trachoma in surveyed communities. High quality, whole genome sequences were obtained from 12/20 isolates.ResultsAll isolates were serovar A and had tarP and trpA sequences typical of classical, ocular C. trachomatis isolates. The Sudanese isolates formed a closely related subclade within the T2-trachoma clade of C. trachomatis phylogeny distinct from geographically disparate ocular isolates, with little intra-population diversity. We found 333 SNPs that were conserved in Sudanese ocular isolates but rare compared to other ocular C. trachomatis populations, which were focused in two genomic loci (CTA0172-CTA0173 and CTA0482).ConclusionsLimited intra-population diversity and geographical clustering of ocular C. trachomatis suggests minimal transmission between and slow diversification within trachoma-endemic communities. However, diversity may have been higher pre-treatment in these communities. Over-representation of Sudan-specific SNPs in three genes suggests they may have an impact on C. trachomatis growth and transmission in this population.

Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates

A striking difference between genital and ocular clinical isolates of Chlamydia trachomatis is that only the former express a functional tryptophan synthase and therefore can synthesize tryptophan by indole salvage. Ocular isolates uniformly cannot use indole due to inactivating mutations within tryptophan synthase, indicating a selection against maintaining this enzyme in the ocular environment. Here, we demonstrate that this selection occurs in two steps. First, specific indole derivatives, produced by the human gut microbiome and present in serum, rapidly induce expression of C. trachomatis tryptophan synthase, even under conditions of tryptophan sufficiency. We demonstrate that these indole derivatives function by acting as de-repressors of C. trachomatis TrpR. Second, trp operon de-repression is profoundly deleterious when infected cells are in an indole-deficient environment, because in the absence of indole, tryptophan synthase deaminates serine to pyruvate and ammonia. We have used biochemical and genetic approaches to demonstrate that expression of wild-type tryptophan synthase is required for the bactericidal production of ammonia. Pertinently, although these indole derivatives de-repress the trpRBA operon of C. trachomatis strains with trpA or trpB mutations, no ammonia is produced, and no deleterious effects are observed. Our studies demonstrate that tryptophan synthase can catalyze the ammonia-generating β-elimination reaction within any live bacterium. Our results also likely explain previous observations demonstrating that the same indole derivatives inhibit the growth of other pathogenic bacterial species, and why high serum levels of these indole derivatives are favorable for the prognosis of diseased conditions associated with bacterial dysbiosis.

Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates

Ammonia generation by tryptophan synthase drives a key genetic difference between genital and ocular Chlamydia trachomatis isolates

Detection and Isolation of Chlamydia trachomatis from Urine and Its Association with Preterm Labor

Detection and Isolation of Chlamydia trachomatis from Urine and Its Association with Preterm Labor

Delayed diagnosis of colorectal sexually transmitted diseases due to their resemblance to inflammatory bowel diseases

ObjectiveSexually transmitted diseases (STDs), mainly lymphogranuloma venereum (LGV), induce colorectal symptoms that may be misdiagnosed as inflammatory bowel disease (IBD). This study describes patients who presented with STDs masquerading as IBD in order to improve understanding of missed diagnosis of colorectal STDs and their association with LGV in Israel. MethodsThis retrospective, descriptive study characterized the clinical, endoscopic, and pathological findings of 16 patients who were diagnosed with a colorectal STD after erroneously being diagnosed with IBD. Molecular genotyping was used to characterize some of the Chlamydia trachomatis isolates. ResultsAll patients were men who have sex with men (MSM), mostly HIV-1-positive, and had clinical and endoscopic findings compatible with IBD. The STD was diagnosed 1–36 months after the initial diagnosis: 14 were positive for Chlamydia trachomatis, of which three were of the LGV2b (ST58) serotype and one was ST 108 serotype. Five were positive for gonorrhea and four were positive for syphilis. Several pathogens were diagnosed in six episodes. ConclusionsColorectal STDs may resemble IBD and therefore their diagnosis may be delayed. IBD symptoms in MSM who engage in non-protected anal sex should prompt at least syphilis and anal PCR for STD testing. If C. trachomatis is diagnosed but LGV subtyping cannot be done, doxycycline 100mg twice daily for 21days should be recommended.

Phenotypic antimicrobial susceptibility testing of Chlamydia trachomatis isolates from patients with persistent or successfully treated infections.

Antimicrobial susceptibility data for Chlamydia trachomatis are lacking. Methodologies for susceptibility testing in C. trachomatis are not well-defined, standardized or performed routinely owing to its intracellular growth requirements. We sought to develop an assay for the in vitro susceptibility testing of C. trachomatis isolates from two patient cohorts with different clinical outcomes. Twenty-four clinical isolates (11 from persistently infected and 13 from successfully treated patients) were overlaid with media containing two-fold serial dilutions of azithromycin or doxycycline. After incubation, aliquots were removed from the stock inoculum (SI) and each antimicrobial concentration for total RNA extraction, complementary DNA generation and real-time PCR. The MIC was defined as the lowest antimicrobial concentration where a 95% reduction in transcription was evident in comparison with the SI for each isolate. MICs of azithromycin were comparable for isolates from the two patient groups (82% ≤ 0.25 mg/L for persistently infected and 100% ≤ 0.25 mg/L for successfully treated patients). Doxycycline MICs were at least two-fold lower for isolates from the successfully treated patients (53.9% ≤ 0.064 mg/L) than for the persistently infected patients (100% ≥ 0.125 mg/L) (P = 0.006, Fisher's exact test). Overall, 96% of isolates gave reproducible MICs when re-tested. A reproducible assay was developed for antimicrobial susceptibility testing of C. trachomatis. MICs of azithromycin were generally comparable for the two different patient groups. MICs of doxycycline were significantly higher in the persistently infected patients. However, interpretation of elevated MICs in C. trachomatis is extremely challenging in the absence of breakpoints, or wild-type and treatment failure MIC distribution data.

2017: beginning of a new era for Chlamydia research in China and the rest of the world

The First Chinese Chlamydia Research Meeting was held in Lanzhou, China in May 2017, 60 years after the disclosure of reproducible isolation of Chlamydia trachomatis by 汤飞凡 (Fei-fan Tang). We report current state of the Chlamydia research community in China, and briefly review recent progress in Chlamydia vaccinology. The meeting represents a new milestone for Chlamydia research in the country. The Chinese Chlamydia Research Society (CCRS) was formed during the meeting. Future meetings will be held biennially and should facilitate collaboration of Chinese researchers with their domestic and international colleagues.

In vitro activity of rifampin against and rpoB mutations in Chlamydia trachomatis clinical isolates

Objective To evaluate the susceptibility of Chlamydia trachomatis clinical isolates to rifampin, and assess the relationship between rpoB mutations and antibiotic resistance in them. Methods A microculture method was used to determine the minimal inhibitory concentration (MIC) of rifampin in 52 Chlamydia trachomatis clinical isolates. The rpoB gene was amplified from all the clinical isolates and a standard strain of Chlamydia trachomatis followed by single-strand conformation polymorphism (SSCP) analysis. Sequencing of PCR products was carried out for two clinical isolates. Results No rifampin-resistant strain was found among these clinical isolates. The MIC of rifampin varied from 0.004 to 0.030 mg/L. Neither SSCP analysis nor sequencing showed rpoB mutations. Conclusions No rpoB mutations were found in Chlamydia trachomatis isolates from patients unresponsive to rifampin. The unresponsiveness to rifampin may be attributed to multiple factors. Key words: Chlamydia trachomatis; Rifampin; Microbial sensitivity tests; Drug resistance, microbial; DNA mutational analysis; Genes, rpoB

Differences in 23S ribosomal RNA mutations between wild-type and mutant macrolide-resistant Chlamydia trachomatis isolates.

The aim of the present study was to determine the in vitro susceptibility of wild-type and mutant clinical isolates of Chlamydia (C.) trachomatis strains to erythromycin, azithromycin and josamycin, and to identify the resistance-conferring 23S ribosomal (r)RNA mutations in the isolates. The wild-type resistant isolates were defined as those with minimum inhibitory concentration values above the tissue concentration of the antibiotic in the urogenital system. Furthermore, all resistant C. trachomatis isolates were exposed to sub-inhibitory concentrations of macrolides, and 13 resistant mutants were selected following serial passages. Among the 8 wild-type isolates that were resistant to erythromycin, 3 isolates had a mutation at T2611C in the 23S rRNA gene while the others did not show any 23S rRNA mutations. The selected mutant isolates showed a 4- to 16-fold reduction in in vitro sensitivities. With regard to the mutant strains, the T2611C mutation was found in 10 isolates, A2057G mutation in 6 isolates, and A2059G mutation in 1 isolate. Thus, the macrolide-resistant isolates of the wild-type strain had different mutations from those selected by exposure to sub-inhibitory concentrations of macrolides. Also, since 23S rRNA mutations were not identified in certain isolates, it was considered that other molecular mechanisms may also be responsible for the macrolide resistance of C. trachomatis.

Association of the in vitro susceptibility of clinical isolates of chlamydia trachomatis with serovar and duration of antibiotic exposure.

The presence of persistent Chlamydia trachomatis infection after treatment does not always correlate with in vitro susceptibility testing. The in vitro minimum inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of azithromycin, clarithromycin, roxithromycin, doxycycline, tetracycline, ofloxacin, and penicillin were tested against 61 clinical isolates of C. trachomatis on 6 serovars, and the MIC/MBC of azithromycin and ofloxacin at different points in time after antibiotic administration to infected cultures. Of the 7 antibiotics tested, clarithromycin showed the greatest activity against C. trachomatis isolates with MIC90 of 0.032 μg/mL and MBC90 of 0.064 μg/mL, followed by doxycycline with MIC90 0.064 μg/mL and MBC90 0.064 μg/mL, and azithromycin with MIC90 0.160 μg/mL and MBC90 0.320 μg/mL. Azithromycin had roughly the same MIC50 values (0.08 μg/mL) as the other serovars isolates tested, and other antibiotics showed a 2- to 4-fold difference in MICs50 between serovars. In addition, an increase in the azithromyin MIC was observed by 8 hours and the ofloxacin MIC by 16 hours. At 24 hours, the azithromycin MICs were greater than 40 μg/mL and ofloxacin MICs were greater than 64 μg/mL. The current data demonstrated that the antimicrobial susceptibility of C. trachomatis was influenced by both the serovar type and the duration of exposure to antibiotics in infected cultures.

Is There Evidence for Resistance of Ocular Chlamydia trachomatis to Azithromycin After Mass Treatment for Trachoma Control?

Trachoma, caused by repeated infections with ocular Chlamydia trachomatis, is targeted for elimination using multiple annual rounds of mass drug administration (MDA) in endemic communities. Infection rates do not decline as expected in some communities, leading to concerns about azithromycin resistance. After 3 yearly MDAs in 32 communities in Tanzania, 107 children were identified 1 year later with infection. All were provided MDA again, and 90 were seen again at 2 months, of whom 30 had infection. Chlamydia trachomatis isolates were obtained before and after MDA in 15 paired samples and were tested for antimicrobial susceptibility. The infectious load of C. trachomatis before MDA was determined in 30 children who had infection at both times and 60 whose infection cleared. The median load was 8.6 genome copies per polymerase chain reaction in the consistently infected, and 8.4 in those whose infection cleared (P = .86). For the consistently infected, the average minimum inhibitory concentration was 0.26 µg/mL for azithromycin before and 0.20 µg/mL after MDA. All isolates had minimum inhibitory concentration ≤0.50 µg/mL. There is no evidence that continued infection after MDA was due either to resistance to azithromycin or to a heavier load of organism before treatment. Other potential causes of persistent infection need to be evaluated.

Plasmid deficiency in urogenital isolates ofChlamydia trachomatisreduces infectivity and virulence in a mouse model

We hypothesized that the plasmid of urogenital isolates of Chlamydia trachomatis would modulate infectivity and virulence in a mouse model. To test this hypothesis, we infected female mice in the respiratory or urogenital tract with graded doses of a human urogenital isolate of C. trachomatis, serovar F, possessing the cognate plasmid. For comparison, we inoculated mice with a plasmid-free serovar F isolate. Following urogenital inoculation, the plasmid-free isolate displayed significantly reduced infectivity compared with the wild-type strain with the latter yielding a 17-fold lower infectious dose to yield 50% infection. When inoculated via the respiratory tract, the plasmid-free isolate exhibited reduced infectivity and virulence (as measured by weight change) when compared to the wild-type isolate. Further, differences in infectivity, but not in virulence were observed in a C. trachomatis, serovar E isolate with a deletion within the plasmid coding sequence 1 when compared to a serovar E isolate with no mutations in the plasmid. We conclude that plasmid loss reduces virulence and infectivity in this mouse model. These findings further support a role for the chlamydial plasmid in infectivity and virulence in vivo.

Genetic Transformation of a Clinical (Genital Tract), Plasmid-Free Isolate of Chlamydia trachomatis: Engineering the Plasmid as a Cloning Vector

Our study had three objectives: to extend the plasmid-based transformation protocol to a clinical isolate of C. trachomatis belonging to the trachoma biovar, to provide “proof of principle” that it is possible to “knock out” selected plasmid genes (retaining a replication competent plasmid) and to investigate the plasticity of the plasmid. A recently developed, plasmid-based transformation protocol for LGV isolates of C. trachomatis was modified and a plasmid-free, genital tract C. trachomatis isolate from Sweden (SWFP-) was genetically transformed. Transformation of this non-LGV C. trachomatis host required a centrifugation step, but the absence of the natural plasmid removed the need for plaque purification of transformants. Transformants expressed GFP, were penicillin resistant and iodine stain positive for accumulated glycogen. The transforming plasmid did not recombine with the host chromosome. A derivative of pGFP::SW2 carrying a deletion of the plasmid CDS5 gene was engineered. CDS5 encodes pgp3, a protein secreted from the inclusion into the cell cytoplasm. This plasmid (pCDS5KO) was used to transform C. trachomatis SWFP-, and established that pgp3 is dispensable for plasmid function. The work shows it is possible to selectively delete segments of the chlamydial plasmid, and this is the first step towards a detailed molecular dissection of the role of the plasmid. The 3.6 kb β-galactosidase cassette was inserted into the deletion site of CDS5 to produce plasmid placZ-CDS5KO. Transformants were penicillin resistant, expressed GFP and stained for glycogen. In addition, they expressed β-galactosidase showing that the lacZ cassette was functional in C. trachomatis. An assay was developed that allowed the visualisation of individual inclusions by X-gal staining. The ability to express active β-galactosidase within chlamydial inclusions is an important advance as it allows simple, rapid assays to measure directly chlamydial infectivity without the need for plaquing, fluorescence or antibody staining.

Transformation of a plasmid-free, genital tract isolate of Chlamydia trachomatis with a plasmid vector carrying a deletion in CDS6 revealed that this gene regulates inclusion phenotype.

The development of a plasmid-based genetic transformation protocol for Chlamydia trachomatis provides the basis for the detailed investigation of the function of the chlamydial plasmid and its individual genes or coding sequences (CDS). In this study we constructed a plasmid vector with CDS6 deleted (pCDS6KO) from the original Escherichia coli/C. trachomatis shuttle vector pGFP::SW2. pCDS6KO was transformed into a clinical isolate of C. trachomatis from Sweden that is plasmid-free (C. trachomatis SWFP–). Penicillin-resistant transformants expressing the green fluorescent protein were selected. These transformants did not stain with iodine, indicating that this property is regulated by CDS6 or its gene product. In addition, mature inclusions of C. trachomatis SWFP– transformed by pCDS6KO displayed an identical morphological phenotype to the untransformed plasmid-free recipient host. In this phenotype the morphology of inclusions was altered with the chlamydiae lining the periphery of the inclusion leaving a ‘hole’ in the centre. These green fluorescent inclusions appear ‘doughnut-shaped’ with an empty centre when examined under blue light, giving rise to a characteristic ‘black hole’ phenotype. Our study demonstrates the power of the new genetic system for investigating chlamydial gene function using gene deletion technology.

Isolation of Chlamydia trachomatis and membrane vesicles derived from host and bacteria

The study of intracellular bacteria and nanometer-size membrane vesicles within infected host cells poses an important challenge as it is difficult to identify each distinct population in the context of the complex populations generated from active host-pathogen interactions. Here, suspension cultures of L929 cells infected with the prevalent obligate intracellular bacterium Chlamydia trachomatis strain F/Cal-IC-13 are utilized for the large scale preparation and isolation of natural membrane vesicles and bacterial forms. Cell lysis with nitrogen cavitation in combination with differential centrifugation, OptiPrep™ density gradient separation, and immunoenrichment using anti-chlamydial lipopolysaccharide antibodies and MagnaBind beads allows for the isolation of both productive and persistent bacterial forms, as well as membrane vesicles derived from the host and pathogen. We have evaluated these populations by electron microscopy and Western blot analysis for identification of biomarkers. In addition, purified persistent forms of C. trachomatis induced by ampicillin display adenosine-5′-triphosphate (ATP) transport activity, suggesting that ampicillin-induced persistent C. trachomatis organisms, at least in part, rely upon host ATP as an energy source. Importantly, several chlamydial cytotoxic and/or secreted proteins are demonstrated to be associated with these vesicles, supporting the idea that membrane vesicles are generated by Chlamydia as a means of carrying and delivering virulence factors necessary for pathogenesis. The ability to produce large-scale infections and generate distinct bacteria and host-derived populations for biochemical analysis, while reducing the burdens of time and cost have implications in all areas of chlamydiology. These protocols can be applied to other strains of C. trachomatis or other intracellular bacteria.

Decreased Susceptibility to Azithromycin and Doxycycline in Clinical Isolates of Chlamydia trachomatis Obtained from Recurrently Infected Female Patients in India

Background: Recurrent genital Chlamydia trachomatis infection often results in serious sequelae and has a major impact on reproductive health. Materials and Methods: Recurrent infections were determined in symptomatic female patients. In vitro susceptibility assay was performed for azithromycin and doxycycline using the cell culture technique against 21 clinical isolates obtained from C. trachomatis-positive patients including those who were recurrently infected. Results: Thirteen isolates (61.9%) were found to be susceptible to azithromycin and doxycycline with minimum inhibitory concentration (MIC) values ≤0.125 and ≤0.25 µg/ml, respectively. Eight isolates (38%) were found to be less susceptible to the drugs. Two of them had MICs of 8 µg/ml for both the drugs and could not be completely eradicated as observed by minimum bactericidal concentration assay. Conclusions: Decreased antibiotic susceptibility to the current first-line drugs (azithromycin and doxycycline) for chlamydial infection treatment was observed in isolates obtained from recurrently infected patients.

In vitro susceptibilities of urogenital Chlamydia trachomatis clinical isolates to azithromycin alone and in combination with other antimicrobial agents

Objective To test the in vitro activity of azithromycin against recent clinical isolates of urogenital Chlamydia trachomatis, and combined activity of azithromycin with moxifloxacin, rifampicin, doxycycline and minocyline. Methods When more than 90% McCoy cells were infected, the 41 strains tested were collected to investigate minimal inhibitory concentrations (MICs) of 5 antimicrobials alone. Checkerboard array was used to calculate the fractional inhibitory concentrations(FICs) and then detected the interactions among the various combinations. Results In vitro, synergism or additivity effect of 51.22%,53.66% and 58.54% strains was found in azithromycin-moxifloxacin, azithromycin-doxycycline and asithromycin-rifampicin combinations, respectively. No difference was observed in all of the combinations ( P ＞0.05). However, antagonism effect of 90.24% strains was observed in azithromycin-minocyline combination. Conclusion This study indicates that the combination of azithromycin with moxifloxacin, doxycycline or rifampicin is more effective against Chlamydia trachomatis than individual antimicrobials. Therefore, these antimicrobials combinations might be recommended against Chlamydia trachomatis recurrent or persisitent infection. However, the combination of azithromycin with minocyline exhibited a markedly antagonism activity against Chlamydia trachomatis. Combined sensitivity test to a certain extent can compensate for some shortcomings of individual susceptibility test. Key words: Chlamydia trachomatis; Azithromycin; Minimal inhibitory concentration; Fraction inhibitory concentration; Drug interactions

Comparative evaluation of new typing schemes for urogenitalChlamydia trachomatisisolates

Thirty urogenital Chlamydia trachomatis isolates collected in Moscow in 2005 were typed using newly developed molecular typing approaches: (1) multilocus sequence typing (MLST(7)) based on sequences of seven housekeeping genes (http://pubmlst.org/chlamydiales/), (2) MLST(5) based on the investigation of five target regions of the chlamydial genome and (3) ompA gene sequencing supplemented with three variable number tandem repeat (VNTR) loci of the genome. ompA typing divided all isolates into 11 groups with E serotype dominating, while MLST7, MLST5 and VNTR analysis divided them into eight, 20 and 18 groups, respectively. The discriminatory power of each method calculated using the Hunter-Gaston discriminatory index was found to be 0.83 for the ompA typing scheme, 0.82 for MLST(7) and 0.95 for MLST(5). A novel sequence type combining 13% of all strains was discovered, as well as new alleles of genes. This is the first study characterizing the genetic diversity of the urogenital C. trachomatis population in Central Russia using MLST. We conclude that the MLST(7) scheme is the best possible choice for global epidemiological purposes, whereas MLST(5) is more appropriate for tracing local outbreaks.

Genital Mycoplasmas and Chlamydiae in Infertility and Abortion

The isolation of ureaplasma, mycoplasma hominis and chlamydia trachomatis from the genital tract was studied in 90 infertile women (24 primary infertile and 66 secondary infertile) and in 100 fertile pregnant women. From each subject, specimens from vagina, cervix, endometrium and urine were taken. Genital mycoplasmas were isolated from 75% of the primary infertility group 63.6% of the secondary infertility group and from 82% of the fertile pregnant group. Isolation rates of ureaplasma were significantly higher than M. hominis isolation rates. In specimens from vagina, cervix and urine genital mycoplasma isolation rates were higher in fertile women than these recorded in infertile women. In endometrium ureaplasma was recovered in 30% of the infertile women and in 20% of the fertile group; Mycoplasma hominis was recovered in 25.5% of the infertile and in 14% of the fertile women, associated or not with vagina or cervix isolation. Mycoplasma isolation from endometrium alone was successful only in infertile cases. Chlamydia trachomatis was isolated in only 5 infertile and in only 3 fertile women and even then associated with mycoplasma isolation. Twenty-five mycoplasma positive infertile women were treated in separate groups by tetracycline, erythromycin or vibramycine. Preliminary data show that from 11 endometrial positive mycoplasma cases, 6 became negative and 2 were pregnant after antibiotic therapy. The treated cases are under subsequent control and final results will be communicated later.