Detection of Putative Virulence Genes alr, goiB, and goiC in Mycoplasma hominis Isolates from Austrian Patients.

In Mycoplasma hominis, two genes (alr and goiB) have been found to be associated with the invasion of the amniotic cavity, and a single gene (goiC) to be associated with intra-amniotic infections and a high risk of preterm birth. The syntopic presence of Ureaplasma spp. in the same patient has been shown to correlate with the absence of goiC in M. hominis. The aim of our study was to investigate the presence of alr, goiB, and goiC genes in two groups of M. hominis isolates collected from symptomatic and asymptomatic male and non-pregnant female patients attending an Outpatients Centre. Group A consisted of 26 isolates from patients with only M. hominis confirmed; group B consisted of 24 isolates from patients with Ureaplasma spp. as the only co-infection. We extracted DNA from all M. hominis isolates and analysed the samples for the presence of alr, goiB, and goiC in a qPCR assay. Additionally, we determined their cytotoxicity against HeLa cells. We confirmed the presence of the alr gene in 85% of group A isolates and in 100% of group B isolates; goiB was detected in 46% of the samples in both groups, whereas goiC was found in 73% of group A and 79% of group B isolates, respectively. It was shown that co-colonisation with Ureaplasma spp. in the same patient had no effect on the presence of goiC in the respective M. hominis isolate. We did not observe any cytotoxic effect of the investigated isolates on human cells, regardless of the presence or absence of the investigated genes.

Assessing Acanthamoeba cytotoxicity: comparison of common cell viability assays.

Background: In vitro models for studying interactions between Acanthamoeba and host cells are crucial for understanding the pathomechanism of Acanthamoeba and assessing differences between strains and cell types. The virulence of Acanthamoeba strains is usually assessed and monitored by using cell cytotoxicity assays. The aim of the present study was to evaluate and compare the most widely used cytotoxicity assays for their suitability to assess Acanthamoeba cytopathogenicity. Methods: The viability of human corneal epithelial cells (HCECs) after co-culture with Acanthamoeba was evaluated in phase contrast microscopy. Results: It was shown that Acanthamoeba is unable to considerably reduce the tetrazolium salt and the NanoLuc® Luciferase prosubstrate to formazan and the luciferase substrate, respectively. This incapacity helped to generate a cell density-dependent signal allowing to accurately quantify Acanthamoeba cytotoxicity. The lactate dehydrogenase (LDH) assay led to an underestimation of the cytotoxic effect of Acanthamoeba on HCECs since their co-incubation negatively affected the lactate dehydrogenase activity. Discussion: Our findings demonstrate that cell-based assays using the aqueous soluble tetrazolium-formazan, and the NanoLuc® Luciferase prosubstrate products, in contrast to LDH, are excellent markers to monitor the interaction of Acanthamoeba with human cell lines and to determine and quantify effectively the cytotoxic effect induced by the amoebae. Furthermore, our data indicate that protease activity may have an impact on the outcome and thus the reliability of these tests.

The Prevalence of Genital Mycoplasmas and Coinfection with Trichomonas vaginalis in Female Patients in Vienna, Austria.

Trichomonas vaginalis causes trichomoniasis, the most recurrent sexually transmitted infection (STI) worldwide. Genital mycoplasmas, not considered STI agents, are frequently isolated from the female genital tract. A symbiosis between Mycoplasma species and T. vaginalis has been described. The aim of this study was to conduct molecular-based analyses of vaginal specimens, thus assessing the prevalence of non-STI Mycoplasma infections. In total, 582 samples from female patients and an additional 20 T. vaginalis isolates were analyzed by PCR using Mycoplasma specific 16S rRNA primers, and the obtained PCR products were sequenced. Mycoplasma species were detected in 28.2% of the collected vaginal samples. Mycoplasma hominis was found in 21.5% of the specimens, Ureaplasma species were found in 7.5% of the samples. The molecular data of the newly described species, CandidatusMycoplasma girerdii, were obtained for the first time in Austria, in a sample also positive for T. vaginalis. Analyses of the cultivated T. vaginalis strains confirmed the presence of M. hominis in two out of 20 samples. A comparably high prevalence of genital mycoplasmas was revealed through advanced diagnostic assays, with M. hominis and U. parvum being the most prevalent species. The previously described symbiotic relationship between M. hominis and T. vaginalis was confirmed.

Prevalence of selected sexually transmitted infectious agents in a cohort of asymptomatic soldiers in Austria.

BACKGROUND: According to the World Health Organization (WHO), more than one million sexually transmitted infections (STIs) are acquired every day worldwide. Although STIs may be asymptomatic in many cases, they can cause severe symptoms and can also lead to adverse pregnancy outcomes and both male and female infertility. Asymptomatic carriers seem to play an important role in terms of the distribution of STIs; however, studies revealing the prevalence of STIs in asymptomatic individuals are rare. METHODS: In the current study, 654 leftovers of standard urine samples from healthy, asymptomatic Austrian soldiers were investigated for the prevalence of Trichomonas vaginalis, Chlamydia trachomatis, and genital mycoplasmas (Mycoplasma hominis, Mycoplasma genitalium, Ureaplasma urealyticum, Ureaplasma parvum, and Candidatus Mycoplasma girerdii) by specific PCRs. RESULTS: We detected T. vaginalis, M. hominis, U. urealyticum, U. parvum, and C. trachomatis in the investigated samples with prevalence of 7.6%, 4%, 2.4%, 5.4%, and 3.2%, respectively; neither M. genitalium nor Ca. Mycoplasma girerdii was found in our sample collection. CONCLUSIONS: Our study introduces data on STIs of a mainly male cohort, which are scarce because most of the available information on sexually transmitted infectious agents arises from fertility clinics (mainly women) or symptomatic patients.

Characterisation of Trichomonas vaginalis Isolates Collected from Patients in Vienna between 2019 and 2021.

Trichomonas vaginalis (TV) is the causative agent of trichomoniasis, the most common nonviral sexually transmitted disease. TV can carry symbionts such as Trichomonas vaginalis virus (TVV) or Mycoplasma hominis. Four distinct strains of TV are known: TVV1, TVV2, TVV3, and TVV4. The aim of the current study was to characterise TV isolates from Austrian patients for the presence of symbionts, and to determine their effect on metronidazole susceptibility and cytotoxicity against HeLa cells. We collected 82 TV isolates and detected presence of TVV (TVV1, TVV2, or TVV3) in 29 of them (35%); no TVV4 was detected. M. hominis was detected in vaginal/urethral swabs by culture in 37% of the TV-positive patients; M. hominis DNA was found in 28% of the TV isolates by PCR. In 15% of the patients, M. hominis was detected in the clinical samples as well as within the respective TV isolates. In 22% of the patients, M. hominis was detected by culture only. In 11 patients, M. hominis was detected only within the respective cultured TV isolates (13%), while the swab samples were negative for M. hominis. Our results provide a first insight into the distribution of symbionts in TV isolates from Austrian patients. We did not observe significant effects of the symbionts on metronidazole susceptibility, cytotoxicity, or severity of symptoms.

Chlamydia trachomatis serovars in urogenital and ocular samples collected 2014-2017 from Austrian patients.

Infection of humans with Chlamydia trachomatis, a bacterial pathogen with a unique intracellular replication cycle, may cause a variety of clinical manifestations. These are linked to various serovars of the pathogen; trachoma to serovars A-C, oculogenital infections to serovars D-K, and lymphogranuloma venereum to serovars L1-L3. Nineteen serovars are known as human pathogens. The aim of the study was to determine the serovars of 401 C. trachomatis DNA positive extracts from original clinical specimens of patients in Austria including cervical and urethral swabs, urine, genital secretions and conjunctival swabs - collected from 2014 to 2017. Sequence analysis of the omp1 gene, encoding major outer-membrane protein was performed on each sample. In 50.1% of samples serovar E was identified and serovars F, D/Da and G/Ga were found in 16.2%, 9.7% and 9.0%, respectively. Remaining serovars were J (6.0%), K (4.7%), H (2.7%), B/Ba (1.0%), and I/Ia (0.5%). In 19 patients follow up samples could be tested. The majority of C. trachomatis serovars were associated with urogenital tract infections (D-K), however, one of them - serovar B/Ba - is linked to both, ocular and genital tract infection.

Candida albicans Hgt1p, a multifunctional evasion molecule: complement inhibitor, CR3 analogue, and human immunodeficiency virus-binding molecule.

BACKGROUND: The complement system is tightly controlled by several regulators. Two of these, factor H (FH) and C4b-binding protein (C4BP), can be acquired by pathogens conveying resistance to complement attack. The aim of the study was to characterize the FH binding molecule of Candida albicans, a potentially life-threatening yeast. METHODS: The gene coding for this molecule was identified by probing an expression library and homozygous deletion mutants of the respective gene were constructed. Binding and functional assays were undertaken to compare wild-type and knockout strains. RESULTS: The high-affinity glucose transporter 1 (CaHgt1p) was identified as an FH-binding molecule. Homozygous hgt1Δ/Δ deletion mutants, but not the restored strain in which HGT1 was reintegrated, showed a decreased binding of FH and even of C4BP, demonstrating its function as an FH- and C4BP-binding protein. This led to an enhanced terminal complement complex deposition after incubation with human serum; CaHgt1p thus functions as complement inhibitor. hgt1Δ/Δ mutants failed to form rosettes with complement-coated sheep erythrocytes, and show reduced binding to HIV-gp160, implying that a complement receptor 3 (CR3) moiety, known as fungal HIV binding molecule is lacking. CONCLUSIONS: CaHgt1p is a multifunctional evasion molecule, as complement inhibitor, CR3 analogue and HIV receptor.

Conventional dendritic cells mount a type I IFN response against Candida spp. requiring novel phagosomal TLR7-mediated IFN-ß signaling.

Human fungal pathogens such as the dimorphic Candida albicans or the yeast-like Candida glabrata can cause systemic candidiasis of high mortality in immunocompromised individuals. Innate immune cells such as dendritic cells and macrophages establish the first line of defense against microbial pathogens and largely determine the outcome of infections. Among other cytokines, they produce type I IFNs (IFNs-I), which are important modulators of the host immune response. Whereas an IFN-I response is a hallmark immune response to bacteria and viruses, a function in fungal pathogenesis has remained unknown. In this study, we demonstrate a novel mechanism mediating a strong IFN-ß response in mouse conventional dendritic cells challenged by Candida spp., subsequently orchestrating IFN-α/ß receptor 1-dependent intracellular STAT1 activation and IFN regulatory factor (IRF) 7 expression. Interestingly, the initial IFN-ß release bypasses the TLR 4 and TLR2, the TLR adaptor Toll/IL-1R domain-containing adapter-inducing IFN-ß and the ß-glucan/phagocytic receptors dectin-1 and CD11b. Notably, Candida-induced IFN-ß release is strongly impaired by Src and Syk family kinase inhibitors and strictly requires completion of phagocytosis as well as phagosomal maturation. Strikingly, TLR7, MyD88, and IRF1 are essential for IFN-ß signaling. Furthermore, in a mouse model of disseminated candidiasis we show that IFN-I signaling promotes persistence of C. glabrata in the host. Our data uncover for the first time a pivotal role for endosomal TLR7 signaling in fungal pathogen recognition and highlight the importance of IFNs-I in modulating the host immune response to C. glabrata.