New Diagnostic Real-Time PCR for Specific Detection of Mycoplasma hominis DNA.
ABSTRACT: Mycoplasma hominis is a fastidious micro-organism causing genital and extragenital infections. We developed a specific real-time PCR that exhibits high sensitivity and low intrarun and interrun variabilities. When applied to clinical samples, this quantitative PCR allowed to confirm the role of M. hominis in three patients with severe extragenital infections.
Project description:BACKGROUND: Mycoplasma hominis is a fastidious micro-organism causing systemic infections in the neonate and genital infections in the adult. It can also be the cause of serious extra-genital infections, mainly in immunosuppressed or predisposed subjects. CASE PRESENTATION: We describe a case of severe pneumonia and pericarditis due to Mycoplasma hominis in a previously healthy adolescent who did not respond to initial therapy. CONCLUSIONS: Mycoplasma hominis could be an underestimated cause of severe pneumonia in immunocompetent patients and should be particularly suspected in those not responding to standard therapy.
Project description:Mycoplasma hominis most frequently causes diseases of the genitourinary tract. Extragenital infections are uncommon, with almost all occurring in immunosuppressed persons or those predisposed due to trauma or surgery. We present the case of a previously well man who developed an M. hominis-associated parapharyngeal abscess following acute Epstein-Barr virus infection.
Project description: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.
Project description:Integrative and conjugative elements (ICEs) are modular mobile genetic elements that can disseminate through excision, circularization, and transfer. Mycoplasma ICEs have recently been found distributed among some mycoplasma species and there is accumulating evidence that they play a pivotal role in horizontal gene transfers. The occurrence of ICEs has not been documented in Mycoplasma hominis, a human urogenital pathogen responsible for urogenital infections, neonatal infections and extragenital infections. In this study, we searched for, characterized, and compared ICEs by genome analyses of 12 strains of M. hominis. ICEs of 27-30 kb were found in one or two copies in seven of the 12 M. hominis strains sequenced. Only five of these ICEs seemed to be functional, as assessed by detection of circular forms of extrachromosomal ICE. Moreover, the prevalence of ICEs in M. hominis was estimated to be 45% in a collection of 120 clinical isolates of M. hominis, including 27 tetracycline-resistant tet(M)-positive isolates. The proportion of ICEs was not higher in isolates carrying the tet(M) gene, suggesting that ICEs are not involved in tetracycline resistance. Notably, all M. hominis ICEs had a very similar structure, consisting of a 4.0-5.1 kb unusual module composed of five to six juxtaposed CDSs. All the genes forming this module were specific to M. hominis ICEs as they had no homologs in other mycoplasma ICEs. In each M. hominis ICE, one to three CDSs encode proteins that share common structural features with transcription activator-like (TAL) effectors involved in polynucleotide recognition and signal transduction in symbiotic plant pathogen bacteria. The conserved and specific structure of M. hominis ICEs and the high prevalence in clinical strains suggest that these ICEs may confer a selective advantage for the physiology or pathogenicity of this human pathogenic bacterium. These data open the way for further studies aiming at unraveling horizontal gene transfers and virulence factors in M. hominis.
Project description:BACKGROUND: Mycoplasma hominis is associated with pelvic inflammatory disease, bacterial vaginosis, post partum fever, sepsis and infections of the central nervous system often leading to serious conditions. Association with development of female infertility has also been suggested, but different publications present different results. We developed a sensitive and fast diagnostic real-time PCR to test clinical samples from women undergoing laparoscopic examination before fertility treatment. To develop a test for the detection and quantification of M. hominis we selected a housekeeping gene, glyceraldehyde-3-phosphate dehydrogenase (gap), as a target. RESULTS: Real-time PCR was optimized to detect 10 copies of M. hominis PG21 genomic DNA. A fluorescence signal was measured for all 20 other M. hominis isolates, and melting curves analysis showed variations in the melting temperature in agreement with sequence variation in the region of the probes. There was no amplification of other mycoplasmal DNA and human DNA. Eighty-three patient cervical swab samples from infertile women were cultured for M. hominis in the BEa medium. Two of the samples (2.4%) were positive after 48 hours of incubation. The real-time PCR detected the same two samples positive, and the DNA concentrations in the clinical specimens were calculated to 37.000 copies/ml and 88.500 copies/ml, respectively. CONCLUSION: The results demonstrate that real-time PCR may prove to be a rapid alternative to the traditional cultivation method. Information on bacterial load in genital swabs can be obtained. The assay allowed detection of M. hominis in a closed system reducing the risk of contamination by amplicon carry-over.
Project description:BACKGROUND AND OBJECTIVES:Mycoplasma hominis and Ureaplasma urealyticum are important opportunistic pathogens that cause urogenital infections and accelerated newborn delivery in pregnant women. Moreover genital mycoplasmas have been implicated in different neonatal diseases such as pneumonia, sepsis and meningitis. This study was conducted to find out the prevalence and transmission rate of these two organisms in pregnant women and their neonates. MATERIALS AND METHODS:Nasotracheal and pharyngeal specimens of 165 newborns hospitalized at Neonatal Intensive Care Unit (NICU) of Rasoul Akram Hospital (during 2010 - 2011) were assessed by PCR to detect M. hominis and U. urealyticum. Moreover, PCR of vaginal specimens from their mothers were obtained to determine the prevalence of these organisms in pregnant women and rate of transmission to their newborns. Data were analyzed using SPSS software. RESULTS:Totally, the results of PCR were positive in 33 newborns (20%). Vaginal colonization among the mothers was found to be 15% (25/165) for U. urealyticum and 15% (25/165) for M. hominis. The transmission rate to their infants was 72% and 60% for U. urealyticum and M. hominis, respectively. CONCLUSION:These data indicate that vertical transmission of mycoplasma and ureaplasma are prevalent in newborns. Since these organisms cause serious infections in neonates, it would be better to perform screening tests in pregnant women before the delivery in order to prevent transmission to neonates and consequent infections and morbidities among them.
Project description:The presence of more than one bacterial agent is relatively rare in infective endocarditis, although more common in prosthetic cases. Molecular diagnosis from a removed heart tissue is considered a quick and effective way to diagnose fastidious or intracellular agents.Here we describe the case of postpartum polymicrobial prosthetic valve endocarditis in a young woman. Sneathia sanguinegens and Mycoplasma hominis were simultaneously detected from the heart valve sample using broad range 16S rRNA polymerase chain reaction (PCR) followed by sequencing while culture remained negative. Results were confirmed by independent PCR combined with denaturing gradient gel electrophoresis. Before the final agent identification, the highly non-compliant patient left from the hospital against medical advice on empirical intravenous treatment with aminopenicillins, clavulanate and gentamicin switched to oral amoxycillin and clavulanate. Four months after surgery, no signs of inflammation were present despite new regurgitation and valve leaflet flail was detected. However, after another 5 months the patient died from sepsis and recurrent infective endocarditis of unclarified etiology.Mycoplasma hominis is a rare causative agent of infective endocarditis. To the best of our knowledge, presented case is the first report of Sneathia sanguinegens detected in this condition. Molecular techniques were shown to be useful even in polymicrobial infective endocarditis samples.
Project description:The features of Mycoplasma in human organ such lung and urinary tract are enigmatic. Here, the role of M. hominis in regard to biofilm formation of uropathogenic Escherichia coli (UPEC) strain CFT073 was investigated. Although M. hominis were inferred to not impact on UPEC bacterial fitness including growth and productions of signaling molecules as autoinducer-2 (AI-2) and indole, we found that the presence of M. hominis dramatically decreased biofilm formation of UPEC CFT073 as well as slightly repressed attachment and cytotoxicity of that. Importantly, this activity was observed on UPEC strain specifically, not enterohemorrhagic E. coli (EHEC) strain that exists on intestine. Whole-transcriptome profiling and quantitative real-time polymerase chain reaction (qRT-PCR) analysis revealed PhoPQ system and anti-termination protein (encoded by ybcQ) participates on the reduction of biofilm formation by M. hominis (corroborated by qRT-PCR). Furthermore, collaborating with previous report that toxin-antitoxin (TA) system involved in biofilm formation, M. hominis increased on the transcriptions of toxin genes including hha (toxin gene in Hha-TomB TA system) and pasT (toxin part in PasT-PasI TA system). Hence, we propose that one possible role of M. hominis is to influence bacterial biofilm formation in urinary tract. Only fourteen genes were induced (2.5-fold) by the presence of M. hominis in Uropathogenic Escherichia coli (UPEC) biofilm cells. Among upregulated genes, ybcQ (encodes anti-termination protein Q homolog) and phoP/phoQ (encode DNA-binding response regulators in two-component regulatory system), were induced by the presence of M. hominis. Two-condition experiment, UPEC CFT073 alone vs. UPEC CFT073 with Mycoplasma hominis PG21 (10^5 ccu/ml). For preparing the total RNA, UPEC CFT073 cells were grown at 37°C in biofilm cells on glass wool with or without M. hominis for 24 h.
Project description: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.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.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.
Project description:Objective. We compared laboratory developed real-time PCR assays for detection of Mycoplasma hominis and for detection and differentiation of Ureaplasma urealyticum and parvum to culture using genitourinary specimens submitted for M. hominis and Ureaplasma culture. Methods. 283 genitourinary specimens received in the clinical bacteriology laboratory for M. hominis and Ureaplasma species culture were evaluated. Nucleic acids were extracted using the Total Nucleic Acid Kit on the MagNA Pure 2.0. 5 μL of the extracts were combined with 15 μL of each of the two master mixes. Assays were performed on the LightCycler 480 II system. Culture was performed using routine methods. Results. M. hominis PCR detected 38/42 M. hominis culture-positive specimens, as well as 2 that were culture negative (sensitivity, 90.5%; specificity, 99.2%). Ureaplasma PCR detected 139/144 Ureaplasma culture-positive specimens, as well as 9 that were culture negative (sensitivity, 96.5%; specificity, 93.6%). Of the specimens that tested positive for Ureaplasma species, U. urealyticum alone was detected in 33, U. parvum alone in 109, and both in 6. Conclusion. The described PCR assays are rapid alternatives to culture for detection of M. hominis and Ureaplasma species, and, unlike culture, the Ureaplasma assay easily distinguishes U. urealyticum from parvum.