A small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones inhibits intracellular growth and persistence of Chlamydia trachomatis.

Chlamydia trachomatis is one of the most common sexually transmitted pathogens in the world and often causes chronic inflammatory diseases that are insensitive to antibiotics. The type 3 secretion system (T3SS) of pathogenic bacteria is a promising target for therapeutic intervention aimed at bacterial virulence and can be an attractive alternative for the treatment of chronic infections. Recently, we have shown that a small-molecule compound belonging to a class of 2,4-disubstituted 1,3,4-thiadiazine-5-ones produced through the chemical modification of the thiohydrazides of oxamic acids, designated CL-55, inhibited the intracellular growth of C. trachomatis in a T3SS-dependent manner. To assess the feasibility of CL-55 as a therapeutic agent, our aim was to determine which point(s) in the developmental cycle CL-55 affects. We found that CL-55 had no effect on the adhesion of elementary bodies (EBs) to host cells but significantly suppressed EB internalization. We further found that CL-55 inhibited the intracellular division of reticulate bodies (RBs). An ultrastructural analysis revealed loss of contact between the RBs and the inclusion membrane in the presence of CL-55. Finally, we found that our T3SS inhibitor prevented the persistence of Chlamydia in cell culture and its reversion to the infectious state. Our findings indicate that our T3SS inhibitor may be effective in the treatment of both productive and persistent infections.

The characteristics of ubiquitous and unique Leptospira strains from the collection of Russian centre for leptospirosis.

BACKGROUND AND AIM: Leptospira, the causal agent of leptospirosis, has been isolated from the environment, patients, and wide spectrum of animals in Russia. However, the genetic diversity of Leptospira in natural and anthropurgic foci was not clearly defined. METHODS: The recent MLST scheme was used for the analysis of seven pathogenic species. 454 pyrosequencing technology was the base of the whole genome sequencing (WGS). RESULTS: The most wide spread and prevalent Leptospira species in Russia were L. interrogans, L. kirschneri, and L. borgpetersenii. Five STs, common for Russian strains: 37, 17, 199, 110, and 146, were identified as having a longtime and ubiquitous distribution in various geographic areas. Unexpected properties were revealed for the environmental Leptospira strain Bairam-Ali. WGS of this strain genome suggested that it combined the features of the pathogenic and nonpathogenic strains and may be a reservoir of the natural resistance genes. Results of the comparative analysis of rrs and rpoB genes and MLST loci for different Leptospira species strains and phenotypic and serological properties of the strain Bairam-Ali suggested that it represented separate Leptospira species. CONCLUSIONS: Thus, the natural and anthropurgic foci supported ubiquitous Leptospira species and the pool of genes important for bacterial adaptivity to various conditions.

Detection of C. trachomatis in the serum of the patients with urogenital chlamydiosis.

Extragenital chlamydial complications may be associated with systemic spread of infection, but haematogenous route for C. trachomatis dissemination has not been clearly demonstrated. Here we report that serum specimens obtained from patients with chlamydiosis contain elementary bodies of C. trachomatis shown by culture and immunogold electron microscopy. We have found that 31 of the 52 patients had serum precipitates which were infective to McCoy cells. Immunostaining revealed very small inclusions resembling those reported during persistent C. trachomatis infection in vitro. DNA specimens from 49 (out of 52) patients with chlamydiosis gave positive PCR readings. The viability of the pathogen present in the sera was confirmed by chlamydial RNA detection in the cell monolayer inoculated by the serum precipitates. By using DNA isolation protocol from 1 mL of serum and quantitative TaqMan PCR, it was estimated that bacterial load in patients' sera was 2 × 10(2)-10(3) GE/mL. These findings for the first time demonstrated that C. trachomatis can be disseminated directly by the plasma, independently from blood cell, which may represent a new possible pathway of the chronic infection development. Therefore, new methodological approaches for detection of C. trachomatis in the serum of patients with complicated and chronic chlamydiosis could be important in the diagnosis of the infection regardless of its anatomical localization.

Isolation of Chlamydia pneumoniae from serum samples of the patients with acute coronary syndrome.

BACKGROUND: Limited body of evidence suggests that lipopolysaccharide of C. pneumoniae as well as C. pneumoniae-specific immune complexes can be detected and isolated from human serum. The aim of this study was to investigate the presence of viable elementary bodies of C.pneumoniae in serum samples of patients with acute coronary syndrome and healthy volunteers. MATERIAL AND METHODS: Serum specimens from 26 healthy volunteers and 56 patients with acute coronary syndrome were examined subsequently by serological (C.pneumoniae-specific IgA and IgG), PCR-based and bacteriological methods. Conventional, nested and TaqMan PCR were used to detect C.pneumoniae genetic markers (ompA and 16S rRNA) in DNA from serum specimens extracted with different methods. An alternative protocol which included culturing high-speed serum sediments in HL cells and further C.pneumoniae growth evaluation with immunofluorescence analysis and TaqMan PCR was established. Pellet fraction of PCR-positive serum specimens was also examined by immunoelectron microscopy. RESULTS: Best efficiency of final PCR product recovery from serum specimens has been shown with specific C. pneumoniae primers using phenol-chloroform DNA extraction protocol. TaqMan PCR analysis revealed that human serum of patients with acute coronary syndrome may contain genetic markers of C. pneumoniae with bacterial load range from 200 to 2000 copies/ml serum. However, reliability and reproducibility of TaqMan PCR were poor for serum specimens with low bacterial copy number (<200 /ml). Combination of bacteriological, immunofluorescence and PCR- based protocols applied for the evaluating HL cells infected with serum sediments revealed that 21.0 % of the patients with acute coronary syndrome have viable forms C.pneumoniae in serum. The detection rate of C.pneumoniae in healthy volunteers was much lower (7.7%). Immunological profile of the patients did not match accurately C.pneumoniae detection rate in serum specimens. Elementary bodies of C.pneumoniae with typical ultrastructural characteristics were also identified in serum sediments using immunoelectron microscopy. CONCLUSIONS: Viable forms C. pneumoniae with typical electron microscopic structure can be identified and isolated from serum specimens of the patients with acute coronary syndrome and some healthy volunteers. Increased detection rate of C. pneumoniae in serum among the patients with an acute coronary syndrome may contribute towards enhanced pro-inflammatory status in cardiovascular patients and development of secondary complications of atherosclerosis.

First detection of a viral agent causing disease in farmed sturgeon in Russia.

A virus was isolated from Siberian sturgeon Acipenser baeri and bester (beluga Huso huso x sterlet A. ruthenus hybrid) fingerlings in SSO-2, SSF-2 and WSSK-1 cell lines during an acute epizootic on a large fish farm producing fertilised sturgeon eggs and fry. Transmission electron microscopic examination of samples from both inoculated cell cultures and skin of affected fish revealed viral particles with a herpesvirus-like morphology. The etiological role of the Siberian sturgeon herpesvirus (SbSHV) was confirmed by fulfilment of Rivers' postulates. Experimental immersion of healthy Siberian sturgeon fingerlings in a suspension of SbSHV resulted in 100% mortality with signs of focal epidermal hyperplasia, skin necrosis and multiple skin haemorrhages. While infecting different organs and tissues, the virus showed clear integumentary tropism. Carp fry and rainbow trout fingerlings were neither susceptible to the virus nor did they transmit it to healthy Siberian sturgeon.

Application of Transmission Electron Microscopy for Analysis of Ultrastructural Organization of Circulating Immune Complexes in Patients with Typhoid Fever and Chronic Carriers of Salmonella typhi.

Transmission electron microscopy was used to study the ultrastructural organization of circulating immune complexes (CIC), isolated from patients with typhoid fever in the different periods of acute infectious process (febrile period, period of an early and late reconvalescence), relapse and from acute and chronic carriers of Salmonella typhi in the period of pathogen excreting. It has been shown that preparations of CIC from healthy donors consisted of amorphous mean electron density material, including a cell-like detritus. At acute and chronic infectious process there were bacterial cells in a structure of the CIC. Depending on the period of disease, bacteria had different ultrastructural organization in the CIC. In the febrile period, in the period of an early reconvalescence and in the period of formation of acute carriage of S. typhi, and also in relapse, bacteria had a typical structure, with reference to gram-negative microorganisms. In the period of formation of acute and chronic carriers of S. typhi, in a period of excretion of S. typhi, bacteria in the CIC had ultrastructural organization, relevant to the forms of bacteria with a defective cell wall. Immunocytochemistry research made with the purpose of visualization the O-antigen of S. typhi in bacteria has demonstrated positive immunolabeling on the O-antigen in the bacterial forms and forms with a defective cell wall, and on amorphous mean electron density material. The analysis of ultrastructural organization of the circulating immune complexes and immunolabeling on the O-antigen of S. typhi have allowed to conclude that S. typhi, both typical bacterial form, and the forms with a defective cell wall were the main structural component of circulating immune complexes in acute and chronic forms of infectious process.