General Pharmacology, Toxicology and Pharmaceutics

Trichomoniasis, the most common non-viral sexually transmitted infection worldwide, is caused by the protozoon Trichomonas vaginalis. The 5- nitroimidazole drugs, of which metronidazole is the most prescribed, are the only effective drugs to treat trichomoniasis. Resistance against metronidazole is increasingly reported among T. vaginalis isolates. T. vaginalis can establish an endosymbiosis with two Mycoplasma species, Mycoplasma hominis and Candidatus Mycoplasma girerdii, whose presence has been demonstrated to influence several aspects of the protozoan pathobiology. The role of M. hominis in T. vaginalis resistance to metronidazole is controversial, while the influence of Ca. M. girerdii has never been investigated. In this work, we investigate the possible correlation between the presence of Ca. M. girerdii and/or M. hominis and the in vitro drug susceptibility in a large group of T. vaginalis isolated in Italy and in Vietnam. We also evaluated, via RNA-seq analysis, the expression of protozoan genes involved in metronidazole resistance in a set of syngenic T. vaginalis strains, differing only for the presence/absence of the two Mycoplasmas. Our results show that the presence of M. hominis significantly increases the sensitivity to metronidazole in T. vaginalis and affects gene expression. On the contrary, the symbiosis with Candidatus Mycoplasma girerdii seems to have no effect on metronidazole resistance in T. vaginalis.

Background: ‘Candidatus Liberibacter asiaticus’ (CLas) is a major causal agent of citrus greening disease. The disease primarily involves an asymptomatic, often latent infection of CLas. However, there is no effective technique to distinguish latent-infected trees from healthy ones. This study describes the development of a new detection method for latent CLas infection using cuttings. Methods: Root tissues regenerated from cuttings using symptomatic and asymptomatic citrus trees were prepared for real-time a polymerase chain reaction (PCR) test which was used to investigate latent CLas. When some of the regenerated roots were negative for CLas in the first real-time PCR assay, a subsequent cultivation in soils was performed using the CLas-negative cuttings. CLas development during cultivation was evaluated by a second real-time PCR assay using soil-grown roots from seedlings. Results: Previously, CLas had not been detected from leaves of the latent-infected trees in our greenhouse by real-time PCR. In this study, however, CLas was detected at a moderate frequency from the root tissues of cuttings derived from the latent-infected trees, by the same PCR test. For cuttings with regenerated roots that tested negative for CLas by real-time PCR, CLas was frequently detected from roots grown in nursery soil with autoclaving, after cultivation for a month or more. Conclusions: Latent infection with CLas was detectable by real-time PCR using root tissues regenerated by cuttings and roots grown in nursery soil with autoclaving. These results suggest that the new method of investigation would provide great opportunities for early detection of CLas in asymptomatic citrus trees from field surveys, and would accelerate the eradication practice of citrus greening.

Peptidoglycan (PG) is essential for bacterial survival and maintaining cell shape. The rod-shaped model bacterium Escherichia coli has a set of seven endopeptidases that remodel the PG during cell growth. The gamma proteobacterium Candidatus Thiosymbion oneisti is also rod-shaped and attaches to the cuticle of its nematode host by one pole. It widens and divides by longitudinal fission using the canonical proteins MreB and FtsZ. The PG layer of Ca. T. oneisti has an unusually high peptide cross-linkage of 67% but relatively short glycan chains with an average length of 12 disaccharides. Curiously, it has only two predicted endopeptidases, MepA and PBP4. Cellular localization of symbiont PBP4 by fluorescently labeled antibodies reveals its polar localization and its accumulation at the constriction sites, suggesting that PBP4 is involved in PG biosynthesis during septum formation. Isolated symbiont PBP4 protein shows a different selectivity for β-lactams compared to its homologue from E. coli. Bocillin-FL binding by PBP4 is activated by some β-lactams, suggesting the presence of an allosteric binding site. Overall, our data point to a role of PBP4 in PG cleavage during the longitudinal cell division and to a PG that might have been adapted to the symbiotic lifestyle.

Citrus huanglongbing (HLB) is a devastating disease for the citrus industry. The previous studies demonstrated that oxytetracycline and penicillin are effective antibiotics against Candidatus Liberibacter asiaticus (CLas). However, since CLas is uncultured, the mechanisms of action of antibiotics against CLas are still unclear. It was recently reported that the endophytic microbial communities are associated with the progression of citrus HLB after oxytetracycline and penicillin treatment. Therefore, we hypothesize that penicillin has greater antibacterial activity against CLas than oxytetracycline, which may be associated with the alteration of the structure and function of endophytic microbial communities in HLB-affected citrus in response to these antibiotics. To test this hypothesis, the microbiome of HLB-affected citrus leaves treated with these two antibiotics was analyzed using a metagenomic method. Our results indicate that the microbial structure and function in HLB-affected citrus were altered by these two antibiotics. The relative abundance of beneficial bacterial species, including Streptomyces avermitilis and Bradyrhizobium, was higher in penicillin-treated plants compared to those treated with oxytetracycline, and the relative abundance of the bacterial species (such as Propionibacterium acnes and Synechocystis sp PCC 6803) associated with CLas survival was lower for penicillin-treated plants compared to oxytetracycline-treated plants. These results indicate that penicillin has greater antibacterial activity against CLas. Based on the metagenomic analysis, this study elucidated the mechanism for the observed increase in antibacterial activity of penicillin against CLas. The data presented here are not only invaluable for developing eco-friendly and effective biocontrol strategies to combat citrus HLB, but also provide a method for revealing mechanism of antimicrobial against uncultured bacteria in host.