The presence of Trichomonas vaginalis in urogenital samples can affect the sensitivity of Mycoplasma hominis identification techniques, leading to an underestimation of bacterial infections.

Trichomonas vaginalis and Mycoplasma hominis, two microorganisms causing infections of the urogenital tract, are closely associated in that they establish an endosymbiosis relationship, the only case among human pathogens. As a result, the presence of one microorganism may be considered a sign that the other is present as well. Identification of the two pathogens in clinical samples is based on cultivation techniques on specific media, even though in recent years, new sensitive and rapid molecular techniques have become. Here, we demonstrate that the concomitant presence of T.vaginalis in urogenital swabs may lead to a delay in the identification of M.hominis, and thus to an underestimation of bacterial infections when cultural techniques are used.

Patterns of antibiotic resistance of Mycoplasma hominis endosymbiont of Trichomonas vaginalis and the influence of bacterial intracellular location on drug susceptibility.

OBJECTIVES: Mycoplasma hominis, an opportunistic pathogen of the human lower urogenital tract, can survive and replicate within the protozoan Trichomonas vaginalis, establishing an endosymbiotic relationship. The intracellular location may provide a means for the bacteria to evade the immune system and protection from antimicrobial activities. Our aim was to investigate the influence of the endosymbiotic association of M. hominis with trichomonad cells on bacterial antibiotic susceptibility. METHODS: We evaluated antibiotic resistance patterns in a group of M. hominis isolated from T. vaginalis clinical specimens as well as in M. hominis isolated from patients without trichomoniasis. Using an experimental model system, we compared the minimum inhibitory concentration (MIC) and lethal concentration (MLC) of tetracycline on M. hominis endosymbionts of T. vaginalis and extracellular bacteria. RESULTS: The incidence rate of M. hominis strains resistant to C14 and C15 macrolide antibiotics was higher in intracellular strains associated with T. vaginalis compared with extracellular bacteria isolated from women not affected by trichomoniasis. However, sensitivity to tetracycline and quinolones was similar in both groups. In vitro experiments demonstrated that M. hominis strains, when isolated as endosymbionts from T. vaginalis, exhibited reduced sensitivity to tetracycline when cultured extracellularly for at least eight weeks. CONCLUSION: The intracellular localization of bacteria within trichomonad cells may affect antibiotic susceptibility.

Benzimidazole-2-Phenyl-Carboxamides as Dual-Target Inhibitors of BVDV Entry and Replication.

Bovine viral diarrhea virus (BVDV), also known as Pestivirus A, causes severe infection mostly in cattle, but also in pigs, sheep and goats, causing huge economical losses on agricultural farms every year. The infections are actually controlled by isolation of persistently infected animals and vaccination, but no antivirals are currently available to control the spread of BVDV on farms. BVDV binds the host cell using envelope protein E2, which has only recently been targeted in the research of a potent and efficient antiviral. In contrast, RdRp has been successfully inhibited by several classes of compounds in the last few decades. As a part of an enduring antiviral research agenda, we designed a new series of derivatives that emerged from an isosteric substitution of the main scaffold in previously reported anti-BVDV compounds. Here, the new compounds were characterized and tested, where several turned out to be potent and selectively active against BVDV. The mechanism of action was thoroughly studied using a time-of-drug-addition assay and the results were validated using docking simulations.

Evaluation of Recombinant Kpkt Cytotoxicity on HaCaT Cells: Further Steps towards the Biotechnological Exploitation Yeast Killer Toxins.

The soil yeast Tetrapisispora phaffii secretes a killer toxin, named Kpkt, that shows ß-glucanase activity and is lethal to wine spoilage yeasts belonging to Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. When expressed in Komagataella phaffii, recombinant Kpkt displays a wider spectrum of action as compared to its native counterpart, being active on a vast array of wine yeasts and food-related bacteria. Here, to gather information on recombinant Kpkt cytotoxicity, lyophilized preparations of this toxin (LrKpkt) were obtained and tested on immortalized human keratinocyte HaCaT cells, a model for the stratified squamous epithelium of the oral cavity and esophagus. LrKpkt proved harmless to HaCaT cells at concentrations up to 36 AU/mL, which are largely above those required to kill food-related yeasts and bacteria in vitro (0.25-2 AU/mL). At higher concentrations, it showed a dose dependent effect that was comparable to that of the negative control and therefore could be ascribed to compounds, other than the toxin, occurring in the lyophilized preparations. Considering the dearth of studies regarding the effects of yeast killer toxins on human cell lines, these results represent a first mandatory step towards the evaluation the possible risks associated to human intake. Moreover, in accordance with that observed on Ceratitis capitata and Musca domestica, they support the lack of toxicity of this toxin on non-target eukaryotic models and corroborate the possible exploitation of killer toxins as natural antimicrobials in the food and beverages industries.

Production of a lyophilized ready-to-use yeast killer toxin with possible applications in the wine and food industries.

Kpkt is a yeast killer toxin, naturally produced by Tetrapisispora phaffii, with possible applications in winemaking due to its antimicrobial activity on wine-related yeasts including Kloeckera/Hanseniaspora, Saccharomycodes and Zygosaccharomyces. Here, Kpkt coding gene was expressed in Komagataella phaffii (formerly Pichia pastoris) and the bioreactor production of the recombinant toxin (rKpkt) was obtained. Moreover, to produce a ready-to-use preparation of rKpkt, the cell-free supernatant of the K. phaffii recombinant killer clone was 80-fold concentrated and lyophilized. The resulting preparation could be easily solubilized in sterile distilled water and maintained its killer activity for up to six months at 4 °C. When applied to grape must, it exerted an extensive killer activity on wild wine-related yeasts while proving compatible with the fermentative activity of actively growing Saccharomyces cerevisiae starter strains. Moreover, it displayed a strong microbicidal effect on a variety of bacterial species including lactic acid bacteria and food-borne pathogens. On the contrary it showed no lethal effect on filamentous fungi and on Ceratitis capitata and Musca domestica, two insect species that may serve as non-mammalian model for biomedical research. Based on these results, bioreactor production and lyophilization represent an interesting option for the exploitation of this killer toxin that, due to its spectrum of action, may find application in the control of microbial contaminations in the wine and food industries.

Yeast killer toxins: from ecological significance to application.

Killer toxins are proteins that are often glycosylated and bind to specific receptors on the surface of their target microorganism, which is then killed through a target-specific mode of action. The killer phenotype is widespread among yeast and about 100 yeast killer species have been described to date. The spectrum of action of the killer toxins they produce targets spoilage and pathogenic microorganisms. Thus, they have potential as natural antimicrobials in food and for biological control of plant pathogens, as well as therapeutic agents against animal and human infections. In spite of this wide range of possible applications, their exploitation on the industrial level is still in its infancy. Here, we initially briefly report on the biodiversity of killer toxins and the ecological significance of their production. Their actual and possible applications in the agro-food industry are discussed, together with recent advances in their heterologous production and the manipulation for development of peptide-based therapeutic agents.