The silent presence of Mycoplasma hominis in patients with prostate cancer.

BACKGROUND: Mycoplasma hominis, an opportunistic pathogen in human genitourinary tract, can cause chronic infection in the prostate. Intracellular survival of M. hominis leads to a prolonged presence in the host cells that can affect the cell's biological cycle. In the present study, we aimed to evaluate the frequency of M. hominis DNA in prostate tissue of Iranian patients with prostate cancer (PCa) in comparison to a control group with benign prostatic hyperplasia (BPH). METHODS: This research was a retrospective case-control study using 61 archived formalin-fixed paraffin-embedded (FFPE) blocks of prostate tissue from patients with PCa and 70 FFPE blocks of patients with BPH. Real-time PCR, targeting two different genes, 16S rRNA and yidC, in the M. hominis genome was performed for all specimens. RESULTS: Out of 61 blocks of prostate biopsy from patients with PCa, eight samples (13%) were positive for M. hominis, while the bacterium was not detected in any of the 70 blocks of patients with BPH (P value, 0.002). CONCLUSIONS: The high frequency of M. hominis in patients with PCa likely shows a hidden role of the organism in prostate cancer during its chronic, apparently silent and asymptomatic colonization in prostate.

Genomic characterisation of a multidrug-resistant Mycoplasma hominis isolate recovered from a synovial fluid sample in China.

OBJECTIVES: Mycoplasma hominis is one of the smallest free-living opportunistic human pathogens responsible for a diverse range of infections. However, knowledge regarding the genetic and pathogenic mechanisms of M. hominis is still very limited. This study aimed to investigate the genomic features of a multidrug-resistant M. hominis isolate recovered from a synovial fluid sample in China. METHODS: Antimicrobial susceptibility of M. hominis MH-1 was determined by broth microdilution. Genomic DNA was extracted and was sequenced using an Illumina HiSeq X Ten platform. De novo genome assembly was performed using SPAdes, and the draft genome was annotated using the NCBI Prokaryotic Genome Annotation Pipeline (PGAP). Core genome single nucleotide polymorphism (cgSNP) analysis between M. hominis MH-1 and all 25 M. hominis strains retrieved from the NCBI GenBank database was performed using BacWGSTdb server. RESULTS: Antimicrobial susceptibility testing showed that M. hominis MH-1 was resistant to macrolides and fluoroquinolones. The genome size was calculated as 720 262 bp, with 608 protein-coding sequences and a G + C content of 26.8%. Several antimicrobial resistance genes, virulence genes, genomic islands and insertion sequences were identified in the genome. Phylogenetic analysis showed that the strains retrieved from NCBI as well as M. hominis MH-1 were not epidemiologically related. The closest relative of M. hominis MH-1 was recovered from the USA, which differed by 5898 SNPs. CONCLUSION: This study reports the first genome sequence of a multidrug-resistant M. hominis isolate in China. These data may help to understand the genomic features and antimicrobial resistance mechanisms of this pathogen.

Prevalence of double-stranded RNA virus in Trichomonas vaginalis isolated in Italy and association with the symbiont Mycoplasma hominis.

The flagellated protozoon Trichomonas vaginalis, responsible for trichomoniasis, can establish a symbiotic relationship with the bacterium Mycoplasma hominis and can harbor double-stranded RNA Trichomonasvirus (TVV). In this study, we investigated by real-time PCR the prevalence of the four TVVs and of M. hominis among 48 T. vaginalis strains isolated in Italy, and we evaluated a possible association with metronidazole resistance. Fifty percent of the analyzed trichomonad strains tested positive for at least one TVV T. vaginalis, with TVV2 being the most prevalent, followed by TVV1 and TVV3. Two T. vaginalis strains were infected by TVV4, detected in Europe for the first time. Interestingly, we found more than one TVV species in 75% of positive trichomonad strains. M. hominis was present in 81.25% of T. vaginalis isolates tested, and no statistically significant association was observed with the infection by any TVV. Metronidazole sensitivity of T. vaginalis isolates was evaluated in vitro, and no correlation was observed between minimal lethal concentration and the presence of TVVs. This is the first report on TVV infection of T. vaginalis in Italy. Even if no association of TVV positive isolates with the presence of the symbiont M. hominis or with metronidazole resistance was observed, further studies are needed to shed light on the effective role of infecting microorganisms on the pathophysiology of T. vaginalis.

Random transposon insertion in the Mycoplasma hominis minimal genome.

Mycoplasma hominis is an opportunistic human pathogen associated with genital and neonatal infections. Until this study, the lack of a reliable transformation method for the genetic manipulation of M. hominis hindered the investigation of the pathogenicity and the peculiar arginine-based metabolism of this bacterium. A genomic analysis of 20 different M. hominis strains revealed a number of putative restriction-modification systems in this species. Despite the presence of these systems, a reproducible polyethylene glycol (PEG)-mediated transformation protocol was successfully developed in this study for three different strains: two clinical isolates and the M132 reference strain. Transformants were generated by transposon mutagenesis with an efficiency of approximately 10-9 transformants/cell/µg plasmid and were shown to carry single or multiple mini-transposons randomly inserted within their genomes. One M132-mutant was observed to carry a single-copy transposon inserted within the gene encoding P75, a protein potentially involved in adhesion. However, no difference in adhesion was observed in cell-assays between this mutant and the M132 parent strain. Whole genome sequencing of mutants carrying multiple copies of the transposon further revealed the occurrence of genomic rearrangements. Overall, this is the first time that genetically modified strains of M. hominis have been obtained by random mutagenesis using a mini-transposon conferring resistance to tetracycline.

Successful Whole Genome Sequencing-guided Treatment of Mycoplasma hominis Ventriculitis in a Preterm Infant.

We report a case of Mycoplasma hominis ventriculitis in a preterm neonate that was successfully identified with 16S ribosomal RNA sequencing and whole genome sequencing after failure to detect the pathogen with conventional diagnostic methods. The infant required doxycycline with subsequent clearance of the infection and no evidence of drug toxicity.

Phylogenetics of Mycoplasma hominis clinical strains associated with gynecological infections or infertility as disclosed by an expanded multilocus sequence typing scheme.

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.

Genomic determination of minimum multi-locus sequence typing schemas to represent the genomic phylogeny of Mycoplasma hominis.

BACKGROUND: Mycoplasma hominis is an opportunistic human pathogen, associated with clinically diverse disease. Currently, there is no standardised method for typing M. hominis, which would aid in understanding pathogen epidemiology and transmission. Due to availability and costs of whole genome sequencing and the challenges in obtaining adequate M. hominis DNA, the use of whole genome sequence analysis to provide clinical guidance is unpractical for this bacterial species as well as other fastidious organisms. RESULTS: This study identified pan-genome set of 700 genes found to be present in four published reference genomes. A subset of 417 genes was identified to be core genome for 18 isolates and 1 reference. Leave-one-out analysis of the core genes highlighted set of 48 genes that are required to recapture the original phylogenetic relationships observed using whole genome SNP analysis. Three 7-locus MLST schemas with high diversity index (97%) and low dN/dS ratios (0.1, 0.13, and 0.11) were derived that could be used to confer good discrimination between strains and could be of practical use in future studies direct on clinical specimens. CONCLUSIONS: The genes proposed in this study could be utilised to design a cost-effective and rapid PCR-based MLST assay that could be applied directly to clinical isolates, without prior isolation. This study includes additional genomic analysis revealing high levels of genetic heterogeneity among this species. This provides a novel and evidence based approach for the development of MLST schema that accurately represent genomic phylogeny for use in epidemiology and transmission studies.

Antibiotic Susceptibility and Sequence Type Distribution of Ureaplasma Species Isolated from Genital Samples in Switzerland.

Antibiotic resistance in Ureaplasma urealyticum/Ureaplasma parvum and Mycoplasma hominis is an issue of increasing importance. However, data regarding the susceptibility and, more importantly, the clonality of these organisms are limited. We analyzed 140 genital samples obtained in Bern, Switzerland, in 2014. Identification and antimicrobial susceptibility tests were performed by using the Mycoplasma IST 2 kit and sequencing of 16S rRNA genes. MICs for ciprofloxacin and azithromycin were obtained in broth microdilution assays. Clonality was analyzed with PCR-based subtyping and multilocus sequence typing (MLST), whereas quinolone resistance and macrolide resistance were studied by sequencing gyrA, gyrB, parC, and parE genes, as well as 23S rRNA genes and genes encoding L4/L22 ribosomal proteins. A total of 103 samples were confirmed as positive for U. urealyticum/U. parvum, whereas 21 were positive for both U. urealyticum/U. parvum and M. hominis. According to the IST 2 kit, the rates of nonsusceptibility were highest for ciprofloxacin (19.4%) and ofloxacin (9.7%), whereas low rates were observed for clarithromycin (4.9%), erythromycin (1.9%), and azithromycin (1%). However, inconsistent results between microdilution and IST 2 kit assays were recorded. Various sequence types (STs) observed previously in China (ST1, ST2, ST4, ST9, ST22, and ST47), as well as eight novel lineages, were detected. Only some quinolone-resistant isolates had amino acid substitutions in ParC (Ser83Leu in U. parvum of serovar 6) and ParE (Val417Thr in U. parvum of serovar 1 and the novel Thr417Val substitution in U. urealyticum). Isolates with mutations in 23S rRNA or substitutions in L4/L22 were not detected. This is the first study analyzing the susceptibility of U. urealyticum/U. parvum isolates in Switzerland and the clonality outside China. Resistance rates were low compared to those in other countries. We hypothesize that some hyperepidemic STs spread worldwide via sexual intercourse. Large combined microbiological and clinical studies should address this important issue.

Mycoplasma hominis, a Rare but True Cause of Infective Endocarditis.

Mycoplasma spp. are rarely recognized agents of infective endocarditis. We report a case of Mycoplasma hominis prosthetic valve endocarditis diagnosed by 16S ribosomal DNA (rDNA) PCR and culture of valves in a 74-year-old man. We reviewed the literature and found only 8 other cases reported.

Development of a microarray-based tool to characterize vaginal bacterial fluctuations and application to a novel antibiotic treatment for bacterial vaginosis.

The healthy vaginal microbiota is generally dominated by lactobacilli that confer antimicrobial protection and play a crucial role in health. Bacterial vaginosis (BV) is the most prevalent lower genital tract infection in women in reproductive age and is characterized by a shift in the relative abundances of Lactobacillus spp. to a greater abundance of strictly anaerobic bacteria. In this study, we designed a new phylogenetic microarray-based tool (VaginArray) that includes 17 probe sets specific for the most representative bacterial groups of the human vaginal ecosystem. This tool was implemented using the ligase detection reaction-universal array (LDR-UA) approach. The entire probe set properly recognized the specific targets and showed an overall sensitivity of 6 to 12 ng per probe. The VaginArray was applied to assess the efficacy of rifaximin vaginal tablets for the treatment of BV, analyzing the vaginal bacterial communities of 22 BV-affected women treated with rifaximin vaginal tablets at a dosage of 25 mg/day for 5 days. Our results showed the ability of rifaximin to reduce the growth of various BV-related bacteria (Atopobium vaginae, Prevotella, Megasphaera, Mobiluncus, and Sneathia spp.), with the highest antibiotic susceptibility for A. vaginae and Sneathia spp. Moreover, we observed an increase of Lactobacillus crispatus levels in the subset of women who maintained remission after 1 month of therapy, opening new perspectives for the treatment of BV.

Molecular investigation of menstrual tissue for the presence of Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis collected by women with a history of infertility.

AIM: At present, routine laboratory investigation of the infectious agents implicated in female genital infections is mainly based on culture/direct fluorescence antibody (DFA) (immunofluorescence antibody test) results of cervicovaginal secretions. In this study the use of the menstrual tissue is introduced for the molecular detection of pathogens which are implicated in female infertility. MATERIAL AND METHODS: Cervicovaginal secretions and menstrual tissue samples of 87 women (mean age 34.07 ± 5.17) experiencing infertility problems were screened for Chlamydia trachomatis, Ureaplasma urealyticum and Mycoplasma hominis presence using polymerase chain reaction (PCR, light cycler-PCR). Cervicovaginal secretions were also tested by the culture/DFA technique. The results were compared using the binomial test. RESULTS: In the overall study group, the prevalence of C. trachomatis was 25.3%, 18.3%, and 13.8%, the prevalence of U. urealyticum was 18.3%, 16.09% and 12.6% and the prevalence of M. hominis was 13.7%, 19.5% and 8.0% in the menstrual tissue, cervicovaginal secretions using PCR and cervicovaginal secretions culture/DFA, respectively. A statistically significant difference was revealed between the two methods for all three microbes and between menstrual tissue and cervicovaginal secretions PCR for chlamydia. CONCLUSIONS: The use of menstrual tissue along with the PCR method seems to be an effective and thus novel alternative for the investigation of the infectious agents lying in the genital tract. One of the main advantages of this technique compared to cervicovaginal secretions is that it is non-invasive and the sample can be collected at home, thus allowing the early detection and treatment of a condition that can otherwise lead to serious consequences, such as tubal obstruction, pelvic inflammatory disease, ectopic pregnancy, spontaneous abortions and unexplained infertility.

Diversity of Mycoplasma hominis clinical isolates from Bordeaux, France, as assessed by multiple-locus variable-number tandem repeat analysis.

BACKGROUND: Mycoplasma hominis is an opportunistic human mycoplasma species that can cause various urogenital infections and, less frequently, extragenital infections. The objective of this work was to study the genetic diversity of this species using a molecular typing method based on multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA). RESULTS: The genome content of M. hominis PG21 was analysed for tandem repeats (TRs), and five of the 130 TRs identified were selected for use in an MLVA assay. The method was based on GeneScan analysis of VNTR loci using multiplex PCR with fluorescent dyes and resolution by capillary electrophoresis. This approach was used on a collection of 210 urogenital and extragenital French clinical isolates collected between 1987 and 2009. Forty MLVA types were found. The discriminatory index of our MLVA scheme was 0.924. Using this new typing tool, persistent infection was suggested for six patients and new infection for one patient. Furthermore, mother-to-child transmission was confirmed in the two cases studied. Application of MLVA to a wide range of M. hominis isolates revealed high genotypic diversity and no obvious link between the MLVA type and the isolate year of collection, the patient's age or sex, the anatomical origin of the isolates or resistance to antibiotics was found. CONCLUSIONS: Our MLVA scheme highlights the high genetic heterogeneity of the M. hominis species. It seems too discriminatory to be used for large epidemiological studies but has proven its usefulness for molecular studies at the individual level.

Cluster or coincidence? An unusual cause of surgical site infections in adult trauma patients.

Mycoplasma hominis is a bacteria that colonizes and infects the genitourinary tract; extragenital infections are not common. Three surgical site infections because of M hominis in immunocompetent adult trauma patients were detected at our facility in a 6-month time period. We investigated this case series and hypothesize transmission based on common exposures.

Mycoplasma hominis meningitis in a neonate: case report and review.

Despite frequent colonization with Mycoplasma hominis, the invasive disease is rare in neonates. This study describes a neonatal case with meningitis in which M. hominis was isolated from a cerebrospinal fluid sample by culture and detected by PCR. The M. hominis infection was confirmed by elevated metabolic inhibition titers against the isolated M. hominis strain and anti-M. hominis antibodies in serum samples. Minocycline and moxifloxacin were effective against M. hominis, which caused meningitis in the patient. However, the patient exhibited left hemiplegia because of massive brain infarction. Based on data of the previously reported 28 cases in addition to our case, the high morbidity and mortality of the M. hominis central nervous system infection were confirmed; it was assumed to result from delayed diagnosis and ineffective initial therapy. Early diagnosis and prompt initiation of appropriate antimicrobial treatment are necessary for a favorable prognosis. Fourth-generation fluoroquinolones, especially moxifloxacin, deserve wider use in such cases.

The presence of Mycoplasma hominis in isolates of Trichomonas vaginalis impacts significantly on DNA fingerprinting results.

The genetic characterization of Trichomonas vaginalis (Protista: Trichomonadidae), the causative agent of trichomoniasis in humans, is central to understanding the epidemiology, treatment, drug resistance, and virulence as well as the diagnosis and control of this parasite. Various molecular approaches, including DNA fingerprinting, have been employed for this purpose, and random amplification of polymorphic DNA (RAPD) continues to be utilized. However, little attention has been paid to the fact that some T. vaginalis populations can harbor symbiotic Mycoplasma hominis and/or other agents, which could cause artifacts in the RAPD results. In the present study, we demonstrate clearly that the presence of M. hominis from T. vaginalis isolates impacts significantly on RAPD results and on the subsequent analyses and interpretation of data sets. Moreover, symbiotic M. hominis displays an isolate-to-isolate variability in RAPD profile before elimination, suggesting a variability of M. hominis infection.

Genetic variability of the P120' surface protein gene of Mycoplasma hominis isolates recovered from Tunisian patients with uro-genital and infertility disorders.

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. METHODS: All specimens were processed for culture and PCR amplification of the N-terminal surface exposed region of p120' gene. PCR products were sequenced to evaluate the genetic variability, to test for adaptive selection, and to infer the phylogenetic relationship of the M. hominis isolates. RESULTS: Sequence analysis showed a total of 25 single nucleotide polymorphisms distributed through 23 polymorphic sites, yielding 13 haplotypes. All but one mutation were confined within three distinct regions. Analysis of the amino acid-based phylogenetic tree showed a predominant group of 17 closely related isolates while the remaining appear to have significantly diverged. CONCLUSION: By analysing a larger sample of M. hominis recovered from patients with urogenital infections, we show here that the P120' protein undergoes substantial level of genetic variability at its surface exposed region.

Mycoplasma hominis osteitis in an immunocompetent man.

Mycoplasma hominis has been associated with pelvic inflammatory illness, postpartum and neonatal infections and respiratory tract diseases. It is rarely isolated from patients with other infections. Reported here is a case of tibial osteitis that occurred in a 16-year-old immunocompetent man. Clinical and laboratory findings improved under treatment with clindamycin and fluoroquinolones.

Genital mycoplasmas--morbidity and a potential influence on human fertility.

Mycoplasmas comprise a big group of organisms consisting of one hundred eighty species which are found in nature as parasites of humans, other mammals, reptiles, fishes and plants, or living as commensals. The group commonly referred to as genital mycoplasmas comprise species most often found in the genitourinary tract of sexually active adults as common commensal inhabitants, or pathogens which can cause many different infections. The species we are most interested in this work are called: Mycoplasma genitalium, Mycoplasma hominis and Ureaplasma urealyticum. There is no doubt that they can cause non-gonococcal urethritis (NGU) in both men and women and bacterial vaginosis (BV), cervicitis, endometritis. Infection can spread to the upper port of female genital tract and it can lead to pelvic inflammatory disease, or if it happens during pregnancy--to chorioamnionitis and further pregnancy complications. Even though mycoplasmas have been known and described since 1898, the problem of their morbidity and the possible influence they have on human fertility is still not clear. Similar to research from 30 years ago, connecting Chlamydia trachomatis with infertility, new scientific work as well as the dynamic development of diagnostics procedure, especially more common use of PCR method, may be helpful in discovering the potential role genital mycoplasmas play in infertility.