Alkyl Sulfatase of Cholera Vibrios

The aim of the work was to study the structure of the alkyl sulfatase (asu) gene in Vibrio cholerae strains of various serogroups, as well as to compare nucleotide and amino acid sequences of alkyl sulfatases using various methods of bioinformatic analysis.Materials and methods. 483 strains of V. cholerae O1, O139 and nonO1/nonO139 serogroups were employed in the work. The search for the gene, its recurrence, and localization was carried out applying the Blast software. The nucleotide and corresponding amino acid sequences of the gene, as well as its structure, were studied using bioinformatic analysis. Sequencing was performed on the MiSeq (Illumina) platform. The enzymatic activity was detected using a medium, confirming the presence/absence of the gene by PCR in vitro and in silico.Results and discussion. Bioinformatic analysis of the nucleotide and corresponding amino acid sequences of the asu gene has been carried out and its structure investigated. Four functional domains have been identified. In the beta-lactamase domain, a conservative amino acid sequence -HAHADH- has been found in all strains of cholera vibrios, which is part of the Zn2+ binding motif. It has been established that the alkyl sulfatase of cholera vibrios belongs to the family of Zn2+-dependent β-lactamases. Blast analysis has revealed the similarity of nucleotide and amino acid sequences of alkyl sulfatases in representatives of V. cholerae O1 and O139 serogroups (ctxAB+tcpA+) and representatives of the genera Aeromonas and Pseudomonas, which is in the line with the data of 3D modeling of the amino acid sequence structures of the alkyl sulfatase enzyme in these microorganisms. The bioinformatic analysis of nucleotide and amino acid sequences of alkyl sulfatases in cholera vibrios has showed the conservativeness of these sequences in toxigenic strains and the presence of a number of single mutations in the asu gene in atoxigenic ones. The presence or absence of the asu gene has been established by PCR in vitro and in silico and confirmed by the results obtained using the Blast program. It is demonstrated that the presence/absence of the asu gene correlates with the ability/inability of O139 strains to hydrolyze SDS on the medium. These results can be used in studying mechanisms of cholera vibrios adaptation, persistence and pathogenicity

Features of post-vaccination humoral immune response in the persons who underwent COVID-19 of various severity

The studies on humoral immune response in the individuals who have undergone COVID-19 and vaccinated with anti-COVID vaccines allows us to assess the  development  of  “hybrid”  immunity,  which contributes to understanding the mechanisms of its formation from the effector phase to the step of immunological memory. We assessed the relative and absolute contents of B cell populations and subpopulations, development of humoral immunity in the patients who suffered with COVID-19 of varying severity being thereafter vaccinated with “KoviVak” and “Sputnik V”. The study involved volunteers (age 47.3±14.5 years) who beared COVID-19 asymptomatically (n = 32), at moderate severity (n = 21), or had severe form of the disease (n = 12), then being vaccinated with “KoviVak” and “Sputnik V” 6-9 months after their recovery. The groups of vaccinated persons consisted of those who beared severe disease being vaccinated with “KoviVak” (n = 6) or “Sputnik V” (n = 6); moderate cases, vaccinated with “KoviVak” (n = 10) and “Sputnik V” (n = 11); asymptomatic cases vaccinated with “KoviVak” (n = 10) and “Sputnik V” (n = 22). We have determined relative and absolute numbers of B lymphocytes (CD45+CD19+), B1 lymphocytes (CD45+CD5+CD19-CD27-), B2 lymphocytes (CD45+CD19+CD5-CD27-), total population of memory B cells (CD45+CD19+CD5-CD27+), non-switched (CD45+CD19+IgD+CD27+), and switched (CD45+CD19+IgD-CD27+) memory  B  cells;  mature  naive   B   lymphocytes   (CD45+CD19+CD27-IgD+),   plasmoblasts   (CD45+CD19+ CD38+++IgD-CD27+),   as  well as presence of IgG to S(RBD)-SARS-CoV-2 protein.We have found that the humoral immunity among survivors of COVID-19 of varying severity is expressed for up to nine months. The largest number of volunteers who raised antibodies to SARS-CoV-2 S-protein was registered in the group of seriously ill patients. As soon as 1 month after “Sputnik V” vaccination and until the end of the observation, all the examined subjects in this group became seropositive. 4-5 months after injection of this vaccine, specific immunoglobulins were present in all patients who had asymptomatic or average-severity infection. All volunteers who received “KoviVak” had antibodies to the COVID-19 viral S protein from the beginning to the end of the study. Vaccination, especially with “KoviVak”, contributed to the highest increase, both in relative and absolute numbers of memory B lymphocytes in asymptomatic patients. Less pronounced changes in the content of B lymphocytes in COVID-19 patients who had severe and moderate clinical course may be associated with higher levels of these cells prior to injection of the vaccines. A positive correlation was found between the number of memory B cells and presence of immunoglobulins to the S protein SARS-CoV-2 in all examined patients

Retrospective Molecular-Epidemiological Analysis of Cholera Epidemic in the Republic of Dagestan in 1994

Objective of the study is the retrospective evaluation of epidemic cholera manifestations in the Republic of Dagestan in 1994, taking into account VNTR-typing and studies of genome peculiarities in the isolated V. cholerae O1, Biovar El Tor. Materials and methods. Utilized have been the data on the infection rates in different periods of the epidemic. The strains have been investigated using VNTR-analysis and whole genome sequencing. Results and conclusions. Heterogeneity of the agent population has been established on the basis of the circulating VNTR-genotypes. The whole-genome sequencing has revealed the presence of B-subunit allele ctxB1 in CTXφ prophage of El Tor V. cholerae O1 strains. The strains isolated in the early and the following stages of epidemic development have been found to be closely related to strains from India, Bangladesh, and Nepal. Some strains possessed a cluster of multiple antibiotic resistance genes, SXTET: floR, strA, strB, sul2 – of “Indian” type – SXT-ICE-Ind, as well as ICE mobile elements, containing determinants of resistance to antibiotics of tetracycline group – tetA. Phylogenetic relatedness of the Dagestan strains to the strains from Asia indicates to the origin of the strains, as well as to the independent cholera importations that took place at different stages of the epidemic. The role of genetically altered V. cholerae O1 El Tor variants with identified peculiarities on molecular-genetic level, as a system-forming factor in the emergence and development of epidemic in the Dagestan Republic, was directly linked to the social conditions

Results of the Triennial Monitoring over the Ship’s Ballast Water at the Ports of the Rostov-Region

Demonstrated are the results of microbiological monitoring over the ship’s ballast water at the sea ports of the Rostov region. Analyzed for the presence of Vibrio cholerae are the samples of the ship’s ballast water collected within the period of May-September 2010-2012, a total of 179 samples collected from 127 ships. Among 30 V. cholerae non O1/ non O139 strains isolated in 2010-2011 only 13 have been identified serologically. Application of MALDI-TOF mass spectrometry in 2012 has enhanced informational capacity of the investigations. It has expanded the range and increased the frequency of bacteriological findings. The detection of vibrios implies possibility of V. cholerae importation into the aquatic area of Azov Sea

Organizational and Functional Reassignment of a Scientific Plague Control Institution of the Rospotrebnadzor under Conditions of Coronavirus Infection Pandemic

The aim of the work was to summarize the experience of the Rostov-on-Don Research Anti-Plague Institute in the organizational and functional response aimed at ensuring the implementation of a complex of on-going and scheduled anti-epidemic (preventive) measures at different territorial levels in the context of COVID-19 pandemic. Stage-wise reassignment of scientific and operational subdivisions of the facility was carried out in a timely manner in a flexible mode for a prompt response to the challenges that arose from community transmission of the new coronavirus infection in the territory of the Russian Federation. It allowed an effective distribution of the forces and assets without damage for specialized functioning in the main areas of scientific and practical activities. Realized was an effective interdepartmental interaction with territorial scientific and practical institutions of the Rospotrebnadzor and specialized medical organizations. Practical, methodological and advisory assistance was provided to personnel of specialized medical institutions within the framework of conversing in-patient facilities into hospitals for the treatment of patients with COVID-19. Priority research to study the level of the humoral and cellular immune response to SARS-CoV-2 among the population of the Rostov Region, the etiological spectrum of the causative agents of community-acquired pneumonia associated with COVID-19, full genome sequencing of SARS-CoV-2 isolates was carried out. Specialists of the task forces reinforced human resources of individual laboratory facilities in Moscow city, the Republic of Crimea and the Rostov Region. Complex objective of effective functioning has been implemented: in an operational mode – participation in anti-epidemic (preventive) measures in the face of changes in the dynamics and intensity of the epidemic manifestations of new coronavirus infection; in scheduled mode – the implementation of activities in the main areas of the scientific work of the organization

Comparative Analysis of the Pathogen Structure in Patients with Community-Acquired and Nosocomial Pneumonia in Medical Organizations of the Rostov, Tyumen Regions and Khabarovsk Territory at the Current Stage of a New Coronavirus Infection Pandemic

The aim of the study was to conduct a comparative analysis of the spectrum and antibiotic resistance of secondary pneumonia pathogens isolated in the territories of the Rostov, Tyumen Regions and Khabarovsk Territory against the background of a new coronavirus infection pandemic.Materials and methods. We investigated sputum samples from coronavirus-positive and coronavirus-negative patients with community-acquired pneumonia from medical organizations using bacteriological method, PCR mass spectrometry.Results and discussion. The study of the etiological structure of secondary pneumonia agents isolated from patients in medical organizations of the Southern, Ural and Far Eastern Federal Districts has revealed that the dominant cultures in SARS‑CoV‑2 “+” and SARS‑CoV‑2 “–” patients were yeast and yeast-like fungi. It has been found that under diversity of isolated fungi, Candida albicans species prevailed. The bacterial microflora is represented by a variety of gram-positive and gram-negative bacteria, of which Staphylococcus aureus and Klebsiella pneumoniaе were most often present in sputum. It has also been established that even before hospitalization of patients, community-acquired pneumonia could be caused by microorganisms of the ESKAPE group (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniaе, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp.), which are usually considered as polyantibiotic-resistant pathogens of nosocomial infections. Moreover, in coronavirus-positive patients with secondary community-acquired pneumonia, those pathogens were isolated 2–3 times more frequently than in coronavirus-negative ones. Assessment of sensitivity/resistance of isolated strains to antibacterial drugs has revealed a general trend: the majority of the strains, regardless of the type, were characterized by a narrow spectrum of sensitivity, having 3 or more markers of antibiotic resistance. This confirms the necessity and expediency of microbiological support of the patient during the entire infectious process. The most adequate drugs of choice, providing activity against 60–70 % of strains of the Enterobacteriaceae family, are amikacin and cefoperazone/sulbactam

Cholera: Analysis and Assessment of Epidemiological Situation around the World and in Russia (2013–2022). Forecast for 2023

The aim of the review was to analyze and assess the epidemiological situation on cholera in the world and Russia in 2013–2022 and to make a forecast for 2023. Over the period of 2013–2022, 500 administrative territories affected by cholera were identified in various regions of 71 countries of Asia, Africa and America (the Caribbean region) with formation of 69 endemic foci in 16, 41 and 12 countries, respectively. In 2022, 1 209 301 cases of cholera were registered in 36 countries of the world. The intensity of epidemic process in Asian countries (Syria, Lebanon) increased. Unfavorable epidemiological situation on the African continent persisted. Epidemics and large outbreaks, which began in 2021–2022 due to emergency situations (ES) of social and natural character, continued. The prediction of the stability of the epidemiological situation on cholera in the territories of constituent entities of the Russian Federation (RF), given for 2022, has been confirmed. In 2022, 43 non-toxigenic strains of Vibrio cholerae О1 were isolated from surface water bodies, 8 V. cholerae nonO1/nonO139 strains – from humans. Similarity of those strains with genetically closely related ones isolated in the course of monitoring in previous years in Russia, Donetsk People’s Republic (DPR) and Zaporozhye Region was demonstrated. In 2023, the risks of importation of the infection into RF are retained. It is associated with the intensification of epidemic processes in Asian, African and Caribbean region countries. The extended border with Ukraine, to where importation of cholera from endemic countries is possible, contributes to increased degree of threat. Bioterrorism is also not excluded. The consequences of a possible cholera epidemic complication in DPR, Lugansk People’s Republic, Zaporozhye and Kherson Regions are exacerbated by social emergencies resulting in disruption of infrastructure, interruptions in water supply, etc. In the absence of implementation of the risks of importation of this infection on the territory of RF, a stable epidemiological situation on cholera will be retained. Detection of non-toxigenic V. cholerae O1 strains (including probability of clonal complexes formation), as well as strains of non-O1/non-O139 serogroups, which can be etiological factors of sporadic cases or outbreaks of diarrheal diseases, in surface water bodies is predicted

Cholera: Trends in the Development of the Epidemic Process in 2021, Forecast for 2022

The aim of the work was to summarize the results of cholera monitoring in 2021, to assess current trends in the development of the epidemic process, and to predict the epidemiological situation in the Russian Federation for 2022. It was established that within the period of 2012–2021, 4117264 cases of cholera with the spread of infection across 83 countries on all continents were registered in the world and there was a downward trend in the incidence in Asia and Africa. The dynamics of monthly morbidity in 2021 was associated with emergencies as factors of epidemiological risk. Epidemics and outbreaks of cholera were documented against the background of COVID-19 pandemic and laid a double burden on healthcare systems. At the same time, based on the overview of the results of cholera monitoring in the constituent entities of the Russian Federation, it was shown that the forecast of epidemic well-being given for 2021 was fully justified. It has been determined that the increase in the number of non-toxigenic strains of Vibrio cholerae O1 (67) isolated from water bodies compared to 2020 (25) is mainly due to the appurtenance of a number of isolates to clonal complexes. The study of phylogenetic relation has demonstrated that the detection of strains with genotypes which were previously identified in the isolates evidences the persistence potential. The identification of strains with new genotypes, which were earlier established in the strains circulating in other territories, pointed at the possibility of the occasional importations. The forecast of the epidemiological situation on cholera in Russia for 2022 is associated with the continuous existence of risks of introduction. If these epidemiological risks are not realized, a favorable epidemiological situation is predicted regarding this infection in the country. It is expected that the detection of epidemiologically insignificant strains of V. cholerae O1 in environmental water bodies, along with their clones and/or clonal complexes, will remain, including strains that may be an etiological factor in sporadic cases or outbreaks of disease

THE USE OF MASS SPECTROMETRIC ANALYSIS FOR THE DETECTION OF BACTERIAL TOXINS

Toxins - molecular weight compounds produced by microorganisms, animals, plants and possessing antigene properties. Recently due to the perceived threat of terrorist actions identification of a number of bacterial toxins is especially important. A new approach in the identification of toxins associated with the development of mass spectrometry and can be successfully used for analysis of most environmental toxins. The method of MALDI-MS allows the detection of toxins such as Shiga-toxin Escherichia coli, delta-toxin of Staphylococcus aureus, Bacillus cereus particular, botulinum neurotoxin, cholera toxin. Analytical and diagnostic characteristics of the method, the simplicity and speed studies indicate the long term implementation of a method in the practice of laboratory diagnostics in determining toxinproducing of the studied microorganisms