Vol 11, No 3 (2021)

Cover Page

Full Issue

REVIEW ARTICLES

Melioidosis in aspects of epidemiology, clinic, and laboratory diagnostics

Zakharova I.B., Toporkov A.V., Viktorov D.V.

Abstract

Melioidosis is a life-threatening infection caused by Burkholderia pseudomallei, an environmental Gram-negative bacterium, inhabitant of moist soils in the tropics and subtropics. There is no licensed vaccine against melioidosis. The main routes of B. pseudomallei infection are percutaneous inoculation, inhalation, or ingestion. Individual cases of vertical, sexual, zoonotic, and nosocomial transmission of melioidosis are described. Risk factors for infection are contact with soil or water (especially during the rainy season). The age over 45, type 2 diabetes, alcoholism, liver disease, chronic lung disease, chronic renal disease, and thalassemia, as well as long-term use of steroids and immunosuppressive therapy, are the main susceptibility factors for melioidosis. Among the affected adult residents of endemic regions, 80% had one or more predisposing factors, among children — about 20%. No significant influence of concomitant diseases on the development of melioidosis in travelers was found. Less than 50% of patients had predisposing factors. The incubation period of melioidosis ranges within 1—21 days; on average, 9 days, in case of sizeable infectious dose, it can be less than one day. There is no post-infectious immunity, and reinfection can occur with a different B. pseudomallei strain after successful treatment. B. pseudomallei is a facultative intracellular pathogen that can invade and multiply inside a wide range of cells, including phagocytic. The acute form of melioidosis is characterized by pneumonia, multiple abscesses, bacteremia, and systemic sepsis. Chronic, subacute, and latent forms are also possible. Antimicrobial therapy is divided into the initial intensive phase and the subsequent eradication phase. B. pseudomallei is resistant to penicillins, first- and second-generation cephalosporins, aminoglycosides, macrolides, chloramphenicol, fluoroquinolones, tetracyclines, trimethoprim, and in some cases to co-trimoxazole, and rarely to ceftazidime. Early diagnosis and appropriate management are crucial in reducing severe complications leading to high mortality, and in preventing disease recurrences. However, there is no pathognomonic melioidosis-specific feature, and the disease is not well known to physicians and microbiologists. The results of serological tests for detection of specific antibodies are ambiguous. The bacterial load of the tested sample limits the detection of antigens. Among the accelerated methods for identifying the causative agent of melioidosis, PCR has the highest sensitivity and specificity. Automated identification using microbiological analyzers generally shows good results, but about 15% of isolates are misidentified. Time-of-flight mass spectrometry with matrix-assisted laser desorption ionization is potentially useful for rapid identification of B. pseudomallei. However, existing databases require optimization by adding the reference spectra for B. pseudomallei.

Russian Journal of Infection and Immunity. 2021;11(3):409-422
pages 409-422 views

Pathogenic characteristics of intracellular infection in chlamydiosis

Markina A.N., Kapustina T.A., Parilova O.V., Belova E.V.

Abstract

This review analyzes scientific data and systematized information regarding microbiological aspects and pathogenesis of chlamydia infection. Chlamydia are obligate intracellular microorganisms that are sensitive to any human cells where energy parasitism is possible: various types of epithelium, fibrocytes, histiocytes, glial and muscle cells, squamous epithelium of the meninges and eyeball, neurons, monocytes, macrophages. Structure, chemical composition and enzymatic activity of Chlamydia closely resemble those in Gram-negative bacteria, as they retain their morphological identity throughout the life cycle, possess cell wall, ribosomes, DNA, RNA, biochemical elements of glycolysis, tissue respiration, peptose production being sensitive to some broad-spectrum antibiotics, and are able to vegetative form division. In recent decades, the pathogenesis and clinical symptoms of chlamydial lesions in the urogenital tract, nervous, cardiovascular, musculoskeletal and bronchopulmonary systems have been actively studied, and new approaches and treatment schemes for chlamydia-infected patients are still developing. Over many decades, Chlamydia infection has been an important and urgent problem, not only due to its high prevalence, but also because of high rate of complications negatively affecting populational health and related demographic parameters. Chlamydia causes multiple diseases resulting in chronization of inflammatory process in all human organs and systems, and affects reproduction of population. Mandatory statistical recording of chlamydiosis cases introduced as early as in 1994 in the Russian Federation does not reflect the actual incidence rate. A rise in number of inflammatory diseases of the upper respiratory tract caused by Chlamydia undoubtedly increases its etiological importance, but data regarding etiopathogenetic role of Chlamydia in ENT pathology are ambiguous and contradictory. Available publications describe a large range of variation in rate of detected Chlamydia due to poor awareness of doctors about the microbiological properties of chlamydia, pathogenetic and clinical features of clinical signs of this infection, and lack of common and clear understanding on the algorithms for identifying and treating chlamydiosis. Since the majority of chlamydia-associated nosological forms are asymptomatic, knowing pathogenetic features of related will allow to prevent the spread of the infection and reduce subsequent socio-economic consequences.

Russian Journal of Infection and Immunity. 2021;11(3):423-432
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Cytokines and neuro-specific proteins in viral encephalitis and convulsive syndrome in children. II. Convulsive syndrome

Alekseeva L.A., Zheleznikova G.F., Gorelik E.Y., Sckripchenko N.V., Zhirkov A.A.

Abstract

In this Section we provide new data on the pathogenetic factors in pediatric convulsive syndrome, including a prominent role of viral infection in developing seizures and epilepsy (EPL) in children, as evidenced by clinical and experimental studies. Various forms of convulsive syndrome associated with viral infection include febrile convulsions and febrile epileptic status, encephalitis-related acute symptomatic seizures, and postencephalitic epilepsy. The human herpesvirus-6 isolated in temporal lobe epilepsy is a frequent causative agent of febrile seizures and febrile epileptic status. Febrile seizures and, especially, febrile epileptic status are associated with further developing epilepsy. Of special note is the febrile infection-related epileptic syndrome (FIRES) more often affecting school-aged children and characterized by extremely severe course and unfavorable outcome. Convulsive syndrome is associated with systemic inflammation and overproduced pro-inflammatory cytokines that increase permeability of the blood-brain barrier and functional activity of brain-resident cells, which are involved in eliciting seizures and maintaining epileptogenesis. Taking into consideration the key role of inflammation underlying convulsive syndrome, in recent decades cytokines and chemokines have been widely studied as possible prognostic criteria for epileptogenesis. Neuron-specific proteins are examined as markers of brain cell damage in various inflammatory diseases of the central nervous system. The first Section of the review presented current understanding on systemic and local cytokine/chemokine response in viral encephalitis. Here we present clinical trials published within the last 5—7 years assessing cytokines/chemokines and neuron-specific proteins in children with various forms of convulsive syndrome, including epilepsy. Association between biomarker level and disease clinical parameters as well as potential for their use to diagnose and predict its further course are discussed.

Russian Journal of Infection and Immunity. 2021;11(3):433-446
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Safety of dengue vaccine (CYD-TDV) in Asia: a systematic review

Wan Ismail W.R., Abdul Majid M.S., Li H.Y., Madrim M.F., Abdul Rahim S.S., Jeffree M.S., Azhar Z.I., Ghazi H.F., Hassan M.R.

Abstract

The use of the vaccine in Asia is still very much limited and remained controversial due to its safety, which has yet been properly assessed and evaluated. Hence, the objective of this review is to assess the safety of the CYD-TDV dengue vaccine of the efficacy trials conducted in Asia. A total of 309 related articles were generated from the electronic database search using relevant keywords and only four of the articles were selected for the final review process. The seroprevalence at baseline of the participants involved in the studies was between 50 percent and 80 percent. In terms of the safety of the CYD-TDV vaccine, injection site reaction (pain, swelling, erythema) recorded a relative risk (RR) at 95% CI of (0.46—1.76) and systemic reactions (fever, headache, myalgia) also with RR at 95% CI of (0.89—1.81) were detected among the participants. Among the four studies reviewed, three studies reported some severe adverse effect experienced by the participants with RR at 95% CI of (0.92—2.11). In terms of the immunogenicity, high GMT values were reported for DENV-2 at 67.8 (95%CI of 64.8-70.8), DENV-3 at 73.1 (95% CI of 69.9-76.3) and DENV-4 at 65 (95%CI of 62-67.9) where even though lower values were reported it is consistent with other published studies on the immunogenicity of the CYD-TDV against the DENV serotypes. This review showed that the CYD-TDV can be considered for use in Asia, but with several conditions and following current safety recommendations.

Russian Journal of Infection and Immunity. 2021;11(3):447-453
pages 447-453 views

ORIGINAL ARTICLES

Features of peripheral blood B-cell subset phenotype are associated with clinical outcome of widespread purulent peritonitis

Belenjuk V.D., Savchenko A.A., Borisov A.G., Kudryavtsev I.V.

Abstract

The aim of the study was to investigate the phenotypic features of peripheral blood B-lymphocytes in patients with widespread purulent peritonitis (WPP) during postoperative treatment depending on the disease outcome. 52 patients with acute surgical diseases and injuries of the abdominal organs complicated by WPP were examined. Blood sampling was performed before surgery (preoperative period), as well as on day 7, 14 and 21 of the postoperative period. All patients with WPP were divided into two groups depending on the disease outcome during postoperative period: patients with favorable outcome (n = 34), patients with unfavorable outcome (n = 18). 68 healthy subjects were included into control group. Immunophenotyping of blood B-lymphocytes was assessed by flow cytometry and direct immunofluorescence of whole peripheral blood stained with monoclonal antibodies. It was found that patients with WPP in preoperative period contained lowered B1-cell subset compared to control group that was paralleled with low absolute B-lymphocyte count. Moreover, a higher number of activated (based on upregulated CD23 expression) peripheral blood B1-lymphocytes was observed in the patients with unfavorable than in patients with favorable WPP outcome. In addition, dynamics of changes in frequency of B-lymphocytes during postoperative period (day 7—21) varied profoundly depending on the disease outcome. In particular, patients with favorable WPP outcome during postoperative period were found to contain decreased percentage of the most B-cell subsets (including activated cells) examined, whereas patients with unfavorable disease outcome had virtually unaltered B-cell composition in the postoperative period. Moreover, count of total B-cells, naive B-cells and B2-cells negative or positive for CD23 expression was higher in patients with unfavorable outcome than in patients with favorable WPP outcome throughout entire postoperative period. Percentage of some other B-cell subsets in patients with unfavorable than with favorable outcome was also higher only at certain stages of postoperative treatment. It is assumed that such features in peripheral blood B-cell subset composition were closely linked to the disease outcome so that patients with unfavorable WPP outcome turned out to have disturbed B-cell maturation and migration in developing immune response that might be due to lowered total sensitivity of host body to postoperative antibiotic therapy.

Russian Journal of Infection and Immunity. 2021;11(3):454-462
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Induction of cross-reactive antibodies in mice immunized with conserved influenza A virus neuraminidase-derived linear B-cell epitopes

Sychev I.A., Kopeikin P.M., Tsvetkova E.V., Cheredova K.V., Milman B.L., Shamova O.V., Isakova-Sivak I.N., Desheva Y.A.

Abstract

Introduction. Influenza is a socially significant infection that causes profound damage to populational health and national economy annually. Preventive vaccination is the most effective means to manage influenza and its complications. Diverse influenza vaccines exist, but their common drawback is the narrow specificity, the need for annual renewal of the strain composition, not always satisfactory immunogenicity and effectiveness. In this regard, close attention is paid to developing universal influenza vaccines aimed to induce cross-reactive immune-related cues against most conserved parts of viral proteins. Antibodies against neuraminidase (NA) are able to provide heterosubtypic protection, which is important due to potential threat posed by influenza viruses differed in hemagglutinin and neuraminidase sequence in comparison to currently circulating viruses. The present study is aimed to search for new and analysis of earlier predicted NA linear B-cell epitopes conserved among all influenza A virus subtypes. Results. Eight conserved linear B-cell epitopes were identified around the active site of neuraminidase, three of which (MNPNQKIITIGS, ILRTQESEC, and DNWKGSNRP) were synthesized de novo, conjugated with bovine serum albumin to be next used for mouse immunization. IgG antibodies were detected by ELISA in the sera of immunized mice. Antibodies were found to specifically bind to various influenza A viruses containing NA subtypes N1, N2, N3, and N9. Immunization with NA peptides did not protect mice from substantial body weight loss after infection with lethal H1N1 influenza virus. However, all immunized mice survived during the observation period, whereas in the control group the survival rate was as low as 28.6%. Analyzing lung viral load in the mice infected with the H1N1 virus revealed no differences in virus titers on either day 4 or 8 post-infection. Nevertheless, the protective effect lacked after the mice were challenged with lethal H7N9 influenza virus: Mortality rate, body weight loss, and lung virus titers were comparable in immunized and control mice. Conclusions. The data obtained evidenced about serum cross-reactive anti-NA antibodies induced by immunization with NA peptides, as well as protective efficacy against infection caused by H1N1 virus, but not H7N9 virus. Such results hold promise and indicate that NA linear B-cell epitopes can be used for designing epitope-directed influenza vaccines, but a deeper and more comprehensive study on the specificity of conserved NA epitopes, as well as optimization of immunization schemes for achieving higher protective efficacy are required.

Russian Journal of Infection and Immunity. 2021;11(3):463-472
pages 463-472 views

Saposin D acting on macrophage bacteriostatic function in experimental tuberculosis infection

Shepelkova G.S., Evstifeev V.V., Ergeshov A.E., Yeremeev V.V.

Abstract

The protection against tuberculosis infection is largely determined by the ability of host tissue macrophages to limit the growth and spread of mycobacteria. Able to multiply within the host macrophages, mycobacteria have developed a number of protective mechanisms preventing phagosome-lysosome fusion, thereby evading damaging effects of lysosomal enzymes. Saposins are small, acidic, thermostable, non-enzymatic glycoproteins that participate as co-fac-tors in degradation of short oligosaccharide head group glycosphingolipids. Saposins A, B, C and D are formed in acidic endosomes due to cleavage of initial prosaposin molecule. The effect of saposins on human immune response is mediated by their involvement in presenting mycobacterial antigens on CD1 molecules. Preliminary studies with electron microscopy allowed to uncover saposin D-bound damaging effect on Mycobacterium tuberculosis in acidic environment. These data allowed us to suggest that saposin D is an important protective component fighting against TB infection. The aim of the study was to explore how saposin D deficiency might affect formation of anti-tuberculosis immune response and ability of macrophages to inhibit M. tuberculosis growth. Materials and methods. Interstitial pulmonary macrophages and peritoneal macrophages were isolated from wild type C57BL/6 strain and saposin D deficient C57BL/6-SapD-/- mouse strains. Results. It was found that as compared to macrophages from mice, macrophages from wild type strain significantly better controlled mycobacteria growth in vitro. To study an opportunity of compensating for deficient saposin D in peritoneal macrophages from C57BL/6-SapD-/- mice, a saposin D gene-bearing lentiviral vector was created. Transfection of SAPD-deficient peritoneal macrophages with expression vector compensated for saposin D deficiency in such cells and restored bactericidal function. The mechanisms of action for current anti-TB drugs are mediated by various metabolic pathways in mycobacteria (inhibited biosynthesis of fatty acids, arabinogalactan, peptidoglycan and protein; inhibition of DNA-dependent processes, proton pumps and cytochrome P450-dependent monooxygenases). Conclusion. It was shown that saposin D deficiency affects activation of macrophage bactericidal function in vitro. Our study data may be a prerequisite for biologically substantiated potential of using a vector construct bearing natural human protein gene such as saposin D, as a new anti-tuberculosis drug.

Russian Journal of Infection and Immunity. 2021;11(3):473-480
pages 473-480 views

Genetic adhesion profiles and adhesive variability of uropathogenic Escherichia coli strains

Kuznetsova M.V., Gizatullina J.S.

Abstract

Our study was aimed at investigating prevalence of adhesion genetic determinants among uropathogenic E. coli strains and assessing their correlation with level of specific and non-specific adhesion. E. coli bacterial cultures (n = 33) isolated from patients with urinary tract infection were examined. A phylogenetic group of strains was detected by using Clermont quadriplex-PCR method. Detection of fimbrial and afimbrial adhesin genes was carried out with end-point PCR. Level of erythrocyte-specific, non-specific hydrophobic and hydrophilic adhesion as well as biofilm formation were estimated by using standard methods. Adhesin genes were detected with the following frequencies: fimH — 75.76%, flu — 66.67%, iha — 39.40%, papC — 33.33%. Each of the genes sfaDE, upaG, afa/draBC and yqi was found with frequency 18.18%, whereas eaeA was not detected. Seven strains (21.21%) carried solely fimbrial adhesin genes, three strains (9.09%) — afimbrial adhesin genes, and twenty-one strains (63.64%) had genes of both adhesin types. Twenty-three individual adhesion genotypes were found among thirty-three UPEC strains. A combination of at least four genes were detected in 45.45% strains, among which 60% belonged to phylogroup B2. Odds ratio for adhesin gene prevalence in B2 group was calculated. It was shown that in B2 group yqi and sfaDE genes were detected by 14-fold more frequently (OR = 14.286) than in other phylogroups, and flu gene was observed at 10-fold higher rate (OR = 10.000). No correlation between such genes and level of adhesion to erythrocytes was found, whereas fimH+, papC+ and upaG+ strains had higher level of non-specific hydrophilic adhesion. It was shown that fimbrial adhesins accounted for bacterial adhesion and biofilm formation stronger than afimbrial ones. Thicker biofilm tended to form on latex catheter surface for strains with positive genetic profile for adhesin gene carriers.

Russian Journal of Infection and Immunity. 2021;11(3):481-490
pages 481-490 views

Molecular diagnostics for verification of pleural tuberculosis in Morocco

Chaoui I., Taoudi S., Oudghiri A., Benamor J., Bourkadi J., El Mzibri M.

Abstract

Pleural tuberculosis (pTB) is a very common form of extrapulmonary tuberculosis (TB). pTB diagnostics represents a major burning challenge worldwide due to the limitations of available conventional diagnostic tools. These latter include microscopic examination of the pleural fluid for acid-fast bacilli, mycobacterial culture of pleural fluid in solid or liquid media, sputum or pleural tissue, and histopathological examination of pleural tissue; these tests have recognized limitations for clinical use. Hence, to overcome these limitations, attention has been devoted to new nucleic acid amplification (NAA) diagnostic tests such as the polymerase chain reaction (PCR) and real-time PCR (RT-PCR), owing to their accuracy, rapidity, high sensitivity and specificity. Within this context, this prospective study was conducted to evaluate the performance of molecular diagnosis methods for differentiation between tuberculosis and non-tuberculosis pleural effusions. Fifty patients with pleural effusion were enrolled in this prospective study in Rabat, Morocco. The efficacy of conventional polymerase chain reaction (PCR) in the diagnostics of tuberculous pleurisy by targeting IS6110 and mycobacterial internal transcribed spacer (MYITS) was evaluated compared to histopathologic examination and culture data. Our results showed that IS6110 PCR could “rule in” pTB, the sensitivity and specificity being 41.6% and 85.7%, respectively. Therefore, the findings confirmed that molecular tests exert a relatively high specificity in EPTB but lower sensitivity, thus a positive test is considered as a pTB case whereas negative one cannot exclude the disease. Although the study was limited by a small sample size, it adds to the body of evidence of usefulness of molecular testing as adjuncts to histopathologic examination for accurate diagnosis of pTB, to treat timely and to avoid the emergence and spread of drug resistant pTB. However, further efforts should be made to increase the sensitivity of NAA methods and to identify the best molecular targets to be useful in clinical practice.

Russian Journal of Infection and Immunity. 2021;11(3):491-496
pages 491-496 views

Molecular and genetic analysis of Mycobacterium tuberculosis population in the Vologda Region with low tuberculosis incidence

Vyazovaya А.A., Lebedeva I.A., Ushakova N.B., Pavlov V.V., Gerasimova A.A., Solovieva N.S., Zhuravlev V.Y., Narvskaya O.V.

Abstract

The Vologda Region is characterized by a relatively calm epidemic situation for tuberculosis in Russia: the incidence rate in 2010—2018 is decreased from 45.2 to 15.8 per 100 thousand of the population (44.4 in Russia). However, the proportion of patients with multiple drug resistance (MDR) of the pathogen increased from 12.1% in 2016 to 23.7% in 2018. The aim of the study was to characterize the genetic structure of the M. tuberculosis population and identify the main genotypes associated with the primary multidrug resistance of the pathogen in the Vologda Region. A total of 82 strains of M. tuberculosis isolated in 2018 from newly diagnosed tuberculosis patients were studied. Drug susceptibility testing was performed using the standard method of absolute concentration and BACTEC MGIT 960 kit. M. tuberculosis strains were assigned to the Beijing genotype and its main subtypes based on the analysis of specific markers. The Beijing strains were subtyped by the MIRU-VNTR method (24 standard loci), calculating the Hunter-Gaston Discriminatory Index (HGDI). Other strains of the non-Beijing group were spoligotyped. The majority of the strains were of the Beijing genotype (62.2%; 51 of 82). The most numerous cluster was Central Asian/Russian (41.5%; 34 of 82 strains). The shares of the Central Asia Outbreak (CAO) subtype and cluster B0/W148 amounted to 8.5% and 7.3%, respectively. The non-Beijing strains belonged to the genetic families T (11%; 9 of 82), LAM (11%), Haarlem (6.1%), and Ural (4.9%). Among 82 M. tuberculosis isolates, 33 (40.2%) MDR strains were identified, counting 27 of the Beijing genotype, including those of the Central Asian/Russian — 18 (66.7%), B0/W148 and CAO — 4 each (14.8%) clusters. MIRU-VNTR typing of 51 Beijing strains revealed 22 profiles (HGDI = 0.852); the largest clusters were 94-32 (35.3%) and 95-32 (15.7%), which included strains Central Asian/Russian and CAO. Four strains of genotype B0/W148 belonged to cluster 100-32. The loci QUB26 (HGDI = 0.493) and MIRU26 (HGDI = 0.388) had the highest polymorphism. For the first time, a molecular genetic study carried out in the Vologda region revealed the heterogeneity of the M. tuberculosis population with strains of the Beijing genotype dominated. At the same time, the share of the associated with MDR, epidemiologically and clinically significant cluster Beijing B0/W148, well defined in Russia and abroad, was only 7.3%, which is significantly less than in other regions of the Northwestern Federal District of the Russian Federation (~19%). Concurrent, representatives of the Central Asian/Russian cluster of the Beijing genotype prevailed in the structure of genotypes and among MDR M. tuberculosis strains.

Russian Journal of Infection and Immunity. 2021;11(3):497-505
pages 497-505 views

Molecular genetic characteristics of the carbapenem resistant Klebsiella pneumoniae KP254 strain as a representative of the highly virulent strain evolutionary branch

Alekseeva A.E., Brusnigina N.F., Gordinskaya N.A.

Abstract

Here we provide molecular and genetic characteristics of the Klebsiella pneumoniae KP254 clinical strain belonging to clonal group 23 based on the genome-wide sequencing data. It is known that representatives of such clonal group exert highly virulent properties and cause community-acquired infections. Phenotypically, K. pneumoniae KP254 strain is characterized by multidrug resistance, including carbapenems. The determinants of antibiotic resistance (blaSHV-1, oqxAB, fosA) and pathogenicity encoding fimbriae 1, 3 types and the siderophore yersineobactin synthesis were found in the chromosome structure. However, there was uncovered the lack of conjugative element ICEKp1, the pathogenicity island KPHPI208, and the allantoin regulon genes which are often found in highly virulent strains. Analyzing nucleotide sequences in silico allowed to reveal the replicons of incompatibility group plasmids for FII, FIAHI1/FIIK, Col440I, ColpVC, FIBK, FIIpCRY. Combining contigs relative to reference sequences by using the BLASTN service allowed to identify two putative antibiotic resistance plasmids IncFII and IncFIIpCRY as well as one virulence plasmid IncFIBK. The determinants of the aerobactin siderophore, the RmpA2 mucoid phenotype regulator as well as heavy metal resistance genes constitute the virulence plasmid structure. The virulence plasmid nucleotide sequence coverage comprised 93% relative to the virulence plasmid pK2044 with 99.38% identity level; the genomic regions responsible for the salmochelin and RmpA protein synthesis were deleted. The set of antibiotic resistance determinants identified in the mobilome structure includes the genes for beta-lactamase LAP-2 (IncFIIpCRY plasmid) — a TEM-1 analogue, as well as extended-spectrum beta-lactamase CTX-M-55 (IncFII plasmid), both of which are rarely recorded in the Russian Federation. Additionally, widespread genes blaOXA-1, aac(3’)-IIa, ΔcatB4, aac (6’)-Ib-cr, tet(A), qnrSI, sul2, catA2 were also found in the plasmid DNA. The carbapenemase genes are absent in the resistome structure, whereas the examined strain exerts carbapenem resistance. The analysis of the ompK35 and ompK36 porin gene translated sequences revealed mutational changes which resulted in emerged stop codon within the ompK35 gene, whereas OmpK36 amino acid sequence contains a large number of substitutions, insertions, and deletions. The changes identified serve as one of the factors determining the carbapenem resistance. A synergistic effect may be accounted for by activity of the efflux pumps found in the structure of the K. pneumoniae KP254 genome, particularly AcrAB-TolC and KpnEF. Thus, the strain examined by us preserves the most significant signs specific to the highly virulent evolutionary branch Klebsiella strains, and at the same time, acquires the multidrug resistance genetic determinants.

Russian Journal of Infection and Immunity. 2021;11(3):506-516
pages 506-516 views

Comparing humoral immune response in adult measles patients and measles vaccinated subjects

Toptygina A.P., Andreev Y.Y., Smerdova M.A., Navruzova L.N., Maleev V.V.

Abstract

 

Introduction. The implementation of the WHO Measles Elimination Program has yielded serious results, but in recent years an increase in the incidence rate of this infection has been observed. In particular, according to the WHO, in 2019 vs. 2018 measles morbidity was elevated by 3-fold worldwide. While investigating measles outbreaks among patients, apart from unvaccinated subjects, a substantial group of adults vaccinated in childhood was distinguished. The aim of this work was to examine the characteristics of humoral measles immunity in adult measles patients as well as subjects after measles vaccination. Materials and methods. 50 adult measles patients aged 20 to 55 years were examined. In all patients, the diagnosis was confirmed clinically and by laboratory assays by detecting measles IgM antibodies. The second group consisted of 50 conditionally healthy seronegative age-matched adults, vaccinated with the live measles vaccine (Microgen, Russia). Peripheral blood samples were collected from the cubital vein in total volume of 4 ml on 6±1 day after the onset of rash in patients as well as 6 weeks after vaccination. Specific measles antibodies and their avidity were determined by ELISA using the commercial Avidity: Anti-Measles Viruses ELISA/IgG kit (Euroimmun, Germany). Results. It was shown that people aged 20—35 years more likely suffered from measles than elderly. And it was in this age group that healthy measles seronegative individuals were more abundant. Among vaccinees, 44% responded to vaccination with the primary type of immune response, and 56% responded with the secondary type, while among measles patients, 34% and 66% responded with the primary and secondary type, respectively, as follows from the spectrum of specific IgG subclasses and the antibody avidity assay. The secondary type of immune response indicates that these subjects were apparently vaccinated against measles in childhood, but lost with time long-lived plasma cells producing protective antibodies. While comparing the parameters of specific humoral immunity in groups with acute measles infection (day 6 from the onset of rash) and early convalescents (3 weeks after the onset of rash), it was shown that the level of specific IgG increased threefold in early convalescents (p < 0.01) compared with those at acute phase. The level of specific IgA, on the contrary, decreased from 73.44 (69—75.3) Me/ml to 48.64 (45—56.4) Me/ml, but remained very high. At the same time, the spectrum of specific IgG subclasses shifted from primary immune response (high IgG3 and low IgG1) to secondary response (low IgG3 and high IgG1), which is typical for the response of emerging memory B cells.

Russian Journal of Infection and Immunity. 2021;11(3):517-522
pages 517-522 views

Measles humoral immunity in health-care workers

Krieger E.A., Samodova O.V.

Abstract

Healthcare-workers are at risk of contact with measles patients and disease transmission. Measles-infected employees of healthcare facilities may contribute to the nosocomial measles spread and serve as a source of infection for most susceptible cohorts such as pregnant women, neonates, and immunocompromised patients. In order to study the humoral immunity against measles in healthcare workers and reveal factors associated with seronegative status, we performed a cross-sectional study by enrolling 847 healthcare workers of the Arkhangelsk Regional Clinical Hospital. Anti-measles virus serum immunoglobulin G antibodies were quantified by using VectorMeasles-IgG ELISA kit (Vector-Best, Russia). According to the manufacturer's recommendations specific IgG anti-measles titer cut-off value higher than 0.18 IU/ml, equal to 0.12—0.17 IU/ml, or lower than 0.12 IU/ml was considered as positive (protective), equivocal, or negative, respectively. Assessing an impact of employee's gender, age, affiliation (department), current position was carried out by using binary logistic regression analysis while analyzing seronegative status of healthcare workers. Study participants dominated by females (92.1%). The median age was 48 (39; 57) years. The employees of somatic departments prevailed (26.7%). It was found that 93.7% of medical workers had concentration of anti-measles antibodies exceeding magnitude of protective titer (above 0.18 IU/ml), 4.4% and 1.9% were measles seronegative and equivocal, respectively. The level of antibodies against measles was associated with age of healthcare workers, but not with gender. All employees older than 60 years were measles seropositive, whereas those younger than 35 or within range 35 to 60 years of age had protective antibody titer in 77% and 95.5% of cases, respectively. The proportion of subjects with seronegative results did not depend on employee's position (doctor, nurse, cleaning worker), but varied a lot between different hospital departments. According to the data of logistic regression, the odds to have a seronegative test result among employees from somatic and remaining departments were 4.4-fold higher. Importantly, the rate of seronegative results was by 10-fold lower in employees born between 1968 and 1984 than those found in subjects born after 1985. On the other hand, subjects older than 60 years of age were seronegative at 50-fold lower rate than those who were under 35. The median concentration of measles immunoglobulin G among vaccinated healthcare workers vs. subjects recovered after natural infection was significantly lower reaching 0.56 IU/ml and 4.2 IU/ml (p < 0.001), respectively. Five-year monitoring showed that titer of measles IgG antibodies decreased by 1.2-1.9-fold (average — 1.5). Thus, a cohort of healthcare workers from multidisciplinary healthcare facility demonstrated that the proportion measles-susceptible subjects was 6.3%. Importantly, age of examined subjects mainly affected seronegative status. Taking into consideration age-related lowered serum antibody level in vaccinated healthcare workers, seroprevalence studies and subsequent revaccination of seronegative people should be performed at least once every 5 years to prevent measles spread in healthcare facilities.

Russian Journal of Infection and Immunity. 2021;11(3):523-529
pages 523-529 views

Features of IgG-antibodies production to individual Cytomegalovirus proteins in various eye diseases (age-related macular degeneration and central serous chorioretinopathy)

Neroev V.V., Krichevskaya G.I., Alatortseva G.I., Ryabina M.V., Sarygina A.P., Nesterenko L.N., Dotsenko V.V., Luhverchik L.N.

Abstract

Age-related macular degeneration (AMD) and central serous chorioretinopathy (CSC) are diseases targeting the posterior segment of the eye that often lead to lowered visual functions. Pathogenesis of such disorders largely remains unclear. Among the risk factors of developing chronic inflammation, various microorganisms are considered, particularly Cytomegalovirus (CMV ). The study was aimed at analyzing the humoral response to individual viral proteins during chronic and reactivated CMV infection in AMD and CSC patients. Materials and methods. 104 CMV-seropositive patients were enrolled in the study including 75 AMD and 29 CSC subjects. IgM- and IgG-antibodies specific to CMV late viral antigens as well as IgG antibodies against the main non-structural immediate early (IE) antigen were evaluated by ELISA. IgG antibodies to individual CMV phosphoproteins such as the main non-structural immediate early protein (IE), the DNA-binding phosphoprotein pp52, and the tegument phosphoproteins (pp150, pp65, and pp28) were assessed by using Line-Immunoassay: recombinant antigens containing immunodominant protein fragments derived from viral antigens (p52, p150, p65, p28) were used. Positive (bands 2+) and strongly positive (bands 3+) data were only used for analysis. Results. It was shown that in both groups patients with chronic CMV infection had comparable rate of detected antibodies specific to individual antigens. The level of seropositivity to CMV р150 and р65 was significantly higher than that to CMV р52 and р28 (p < 0.05). Patients with AMD compared to patients with CSС had significantly higher moderate positive response (2+) to all the antigens examined. Upon reactivation of chronic CMV infection in AMD patients, the level of seropositivity to all antigens was increased, the number of cases with an intensely positive response to individual antigens was elevated, but patients with moderate positive response still prevailed. However, reactivation of chronic CMV infection was observed only in 6 CSС patients, allowing to perform no comparative analysis between these two groups. Conclusion. The main difference between CMV-chronically infected patients with AMD and CSC was not found at the level of seropositivity to individual CMV recombinant antigens, but rather in magnitude of antibody production so that AMD patients in comparison to CSC patients displayed moderate antibody production (bands 2+). A marked difference was related to the level of antibodies against CMV p150: AMD patients showed moderate antibody response (bands 2+), whereas CSC subjects dominated with strong positive response (bands 3+) (p < 0.05). It seems that moderate antibody production to recombinant CMV antigens examined in AMD patients occurs due to a weak expression of such viral antigens during chronic infection, resulting in long-term maintenance of antigenic stimulation leading to prolonged inflammation.

Russian Journal of Infection and Immunity. 2021;11(3):530-538
pages 530-538 views

Immunoglobulins and predicted mortality in clinical course of concomitant HIV and TB infection

Mal’tseva N.V., Victorova I.B., Kazantseva O.M., Arkhipova S.V., Khanin A.L.

Abstract

A search for prognostic markers of HIV and tuberculosis coinfection (HIV/TB), especially in case of Mycobacterium tuberculosis multidrug resistance (MDR MBT) associated with low rates of TB eradication, is of relevance in connection with the problem of choosing adequate anti-TB therapy which is able to decrease mortality. 113 HIV/TB patients aged 24 to 58 years were examined: 70 males and 43 females hospitalized at the Novokuznetsk TB Clinic during the 2017—2019 period. MDR MBT (concomitant resistance to Isoniazid and Rifampicin) was found in 50 patients (12 patients with MDR MBT had additional resistance to Fluoroquinolones) aged 24 to 54 years — 31 males and 19 females. The control group consisted of 49 healthy individuals aged 27 to 72 years (26 females and 23 males) lacking focal and systemic infections with moderately pronounced age-related changes. In plasma samples, concentration of total (non-specific) immunoglobulins of classes E, M, G, A (including secretory immunoglobulin A, sIgA) were measured by using enzyme-linked immunosorbent assay. Data statistical processing was performed by using licensed software packages InStatII, Microsoft Excel, IBM SPSS Statistics 22. An extended range of individual variability in count of peripheral blood CD4 lymphocytes was revealed both among non-survivor and survivor patients with HIV/TB examined, being a drawback of using such parameter as lethality predictor. It was found that the serum level of total IgE, IgM, IgG, IgA and sIgA in patients with HIV/ TB was higher than that one in control group, whereas in non-survivor vs. survivor patients the concentration of IgE and sIgA was elevated. The coefficient of disease outcome prediction (CP) for patients with HIV/TB and MDR MBT was calculated being equal to the ratio of the multiplication of serum concentration of IgE, IgM, IgA and secretory IgA to CD4 lymphocyte count (CP = IgE x IgM x IgA x sIgA/CD4). CP higher than 200 was detected in 77% non-survivor and 6% of survivor patients. The relative risk of death with CP > 200 was very high (OR = 56.7, p < 0.0001) being 8.5 times higher than that one upon CD4 < 200 (OR = 6.7, p = 0.0237). A positive correlation between CP and lethal outcome was more valuable than that of CD4. The data presented allow us to propose CP for clinical use as an effective prognostic criterion for HIV/TB with MDR MBT.

 

Russian Journal of Infection and Immunity. 2021;11(3):539-548
pages 539-548 views

An impact of the combined drug containing antimicrobial, antiprotozoal, antifungal and glucocorticoid agents on local immunity in women with bacterial vaginosis and non-specific vaginitis

Bokach O.M., Murasheva M.S., Selkova M.S., Storozheva K.V., Chepanov S.V., Selkov S.A.

Abstract

Inflammatory diseases of the vagina related to imbalanced microflora composition represent one of the most common gynecological diseases primarily dealt with by obstetrician/gynecologists. Such diseases markedly worsen female patient quality of life as well as affect female reproductive system resulting both in altered fertility and adverse pregnancy outcomes. By now, multiple therapeutic and preventive protocols have been proposed to manage such diseases, but insufficient therapeutic efficacy and frequent relapses require further investigations. The aim of the study was to evaluate an effect of the drug Elgyna (Wertex, Russia) consisting of antibacterial, antifungal and glucocorticoid agents on local immunity in females with bacterial vaginosis and nonspecific vaginitis. For this, there were examined and treated 24 females enrolled to the study, who underwent gynecological examination (speculum and bimanual vaginal examination), cervical epithelium microscopy, and calculating karyopicnotic index. Special attention was paid to quantifying level of cytokines IL-6, IL-10, TNFα in vaginal smears before and after therapy with the drug Elgyna. It was found that by the end of the therapy course vaginally administered Elgyna resulted in significantly ameliorated clinical symptoms and normalized microscopic parameters of vaginal discharge. Moreover, this drug consisting of antibacterial, antifungal and glucocorticoid agents did not suppress vaginal epithelium proliferation, but lowered production of pro-inflammatory cytokine TNFα in vaginal smears and shifted toward anti-inflammatory cytokine production. This evidence pathogenetically justifies administration of the drug Elgyna in females with bacterial vaginosis and nonspecific vaginitis as an anti-inflammatory medicine. Efficacy and safety of the two-step therapeutic approach consisting of antibacterial therapy followed by vaginal microbiota recovery for treating inflammatory diseases of vaginal mucosa were confirmed in multiple randomized clinical trials. We envision that such therapeutic concept might be added with a third component implying correction of local vaginal immunity aimed at normalizing the balance between pro- and anti-inflammatory cytokines.

Russian Journal of Infection and Immunity. 2021;11(3):549-555
pages 549-555 views

Splenic morphometric characteristics in infectious mononucleosis (ultrasonic study)

Perepelitsa S.A., Perminova L.A., Stepanyan I.A., Zakhar E.V.

Abstract

The spleen is a secondary immune and the largest organ of the reticuloendothelial system, actively involved during infectious mononucleosis. Clinically, assessing a degree of organ involvement in the pathological process seems unlikely. As a rule, only palpation and percussion are used to determine the size of the spleen, which represents a late and subjective sign of potential splenomegaly. Ultrasound examination provides ample opportunities to estimate the spleen size during infectious mononucleosis. Our study was aimed at identifying morphometric and Doppler ultrasound changes in the spleen in patients with infectious mononucleosis. Materials and methods. There were enrolled 24 patients with infectious mononucleosis to be compared with 30 healthy medical institute students in control group. All participants underwent splenic ultrasound examination. Spleen echotexture and contour clarity together with measuring linear dimensions: length, width, and thickness were analyzed. Splenic artery and vein velocity parameters were measured at the gates of the spleen. Elastographic window for fibroelastometry was positioned within the window for standard grayscale examination. Measurements were performed at 5 or more points of the spleen parenchyma at least 4—5 mm away from the capsular zone and the zone of large vessels. Based on the morphometric measurements obtained, the mass, spleen mass coefficient (SMC), as well as the spleen mass/body height ratio and spleen mass/body surface area ratio were calculated. Results. We found that patients with infectious mononucleosis had the length, thickness and weight of the spleen significantly greater than those observed in control group. However, the mass of the spleen varies widely and justified to calculate the CMS as an objective criterion to assess the size of the spleen. It was revealed that infectious mononucleosis may proceed in one of the three variants of the splenic response: decreased size, CMR < 1.5; normal size, CMR ranged from 1.5 to 4; splenomegaly, CMR > 4. Splenomegaly is associated with increased organ stiffness, peak blood flow velocity and pressure in v. lienalis, as well as periportal lymphadenopathy. Identified changes are characteristics of young patients. Conclusion. Ultrasound examination of the spleen is of high diagnostic value for patients with infectious mononucleosis allowing highly accurate assessment state of the immune organs in the acute disease period. At the preclinical stage, gradation of splenomegaly is possible depending on its intensity, assessed hemodynamics and organ stiffness.

Russian Journal of Infection and Immunity. 2021;11(3):556-564
pages 556-564 views

SHORT COMMUNICATIONS

Polymorphism of pro-inflammatory cytokine genes in acute intestinal infections

Epifantseva N.V., Vitkovsky Y.A., Emelyanova A.N.

Abstract

Acute intestinal infections are widespread and hold the second place among infectious diseases, giving way solely to respiratory diseases. In this regard, much attention has been paid to examining acute intestinal infections, including immunopathogenetic mechanisms. And since proinflammatory and antiinflammatory cytokines play an important role in development of inflammatory reactions affecting disease severity and outcome, it becomes reasonable to study polymorphism of genes governing production of related molecules. Thus, the aim of our study was to examine the polymorphism in the IL-1β Т31С and IL-2 T330G genes; such mutations were characterized by nucleotide replacement affecting the gene promoters, which influenced production rate and level of the relevant cytokines. There were enrolled 108 patients with acute intestinal infections comprising main group as well as 94 apparently healthy subjects in the control group. Genomic DNA was isolated from the whole blood leukocytes by using a DNA-express-blood reagent, followed by conducting amplification reaction with two pairs of allele-specific primers. The polymorphism in the IL-1β, IL-2 genes was determined by PCR with primers purchased from Litech LLC (St. Petersburg). Data processing was carried out by using the statistical Statistica 6 suite software. While assessing the carriage rate of IL-1β T31C gene polymorphic markers by using the multiplicative inheritance model in both groups, the prevalence of the normal T allele and, respectively of the —31TT and —31TC genotypes with OR = 1.83 and an interval of 1.04—3.22 (χ2 = 6.35, p = 0.04, df = 2) was found, which allowed us to identify a relationship between the carriage of IL-1β в gene heterozygous variant and potentially elevated risk of AII. Regarding the IL-2 T330G gene, it was found that pathological G alleles was more markedly abundant in patients with acute intestinal infections compared to control group. Analyzing diverse IL-2 T330G carriage rate in patients with acute intestinal infections revealed that carriers of the TG heterozygous variant predominated — 56.48% (χ2 = 17.75, F = 0.000031), whereas pathological genotype GG was found in 13.89% (χ2 = 12.31, F = 0.000663, p < 0.05), with high probability of the relationship between carriage of these genotypes and a risk of disease development (OR — 3.63 [1.97—6.68] and OR — 6.91 [2.12—22.59]). Hence, the carriage of polymorphic variants of the IL-1β T31C and IL-2 T330G genes was associated with elevated risk of developing AII in case of infection with pathogenic microorganisms.

Russian Journal of Infection and Immunity. 2021;11(3):565-569
pages 565-569 views

Metabolic activity of peripheral blood neutrophils in patients with Opisthorchis felineus invasion and skin syndrome

Tsukanov V.V., Gorchilova E.G., Kolenchukova O.A., Gvozdev I.I., Savchenko A.A., Vasyutin A.V., Tonkikh J.L., Rzhavicheva O.S., Borisov A.G.

Abstract

The aim was to examine functional activity of peripheral blood neutrophilic granulocytes in patients with Opisthorchis felineus invasion and skin syndrome. Materials and methods. A total of 92 patients with chronic opisthorchiasis were examined, of which 38 patients (20 males and 18 females, average age 35.7±3.9 years) and 54 patients (28 males and 26 females, average age 36.5±4.1 years) with/without skin syndrome, as well as 32 apparently healthy subjects (17 males and 15 females, average age 41.5 years). Detection of adult parasite eggs or bodies in the duodenal contents and/or feces was used as the major diagnostic method of opisthorchiasis used in all 92 opisthorchiasis patients. Liver fibrosis was assessed by elastometry method according to the METAVIR scale in all 92 opisthorchiasis patients. Functional activity of peripheral blood neutrophilic granulocytes was examined in all 92 opisthorchiasis patients and 32 healthy individuals from the control group by chemiluminescent analysis measuring intensity of reactive oxygen species production in spontaneous and zymosan-induced reactions in lucigenin and luminol-dependent processes. Results. Opisthorchiasis patients with vs. without skin syndrome suffered at higher rate from pain in the right hypochondrium, dyspepsia, astheno-vegetative syndrome, skin itching, blood eosinophilia, hyperbilirubinemia, cholestatic syndrome, cholecystitis, hepatomegaly, and had elevated serum immunoglobulin E level. Liver fibrosis F3—F4 according to METAVIR was determined in 18.4% and 11.1% of opisthorchiasis patients with vs. without skin syndrome (OR = 1.78; CI 0.57—5.57; p = 0.05). Both luminol- and lucigenin-dependent processes in opisthorchiasis patients with vs. without skin syndrome revealed significantly decreased functional activity of neutrophilic granulocytes which was evidenced by significantly decreased maximum intensity of reactive oxygen species production and the area under the chemiluminescence curve both in spontaneous and zymosan-induced reaction. Conclusion. The results suggest that the presence of skin syndrome in patients with opisthorchiasis is accompanied by more prominent clinical and laboratory manifestations, decreased functional activity of peripheral blood neutrophils, thereby allowing to consider it as a marker of intense pathology.

Russian Journal of Infection and Immunity. 2021;11(3):570-576
pages 570-576 views

Immunity to measles virus in diverse population groups

Liubimova A.V., Zlokazov M.D., Ivanova L.A., Arzamastseva L.Y., Ryzhman N.N., Kolosovskaya E.N., Gumilevskaya O.P.

Abstract

The aim of the study was to assess the measles immunity level among employees of medical organizations of various specialties, young subjects, and neonates. Materials and Methods. The measles immunity status was assessed for all the employees of St. Petersburg State Budgetary Healthcare Institution Maternity Hospital No. 10 (386 subjects), civilian medical staff of the clinics of the Military Medical Academy named after S.M. Kirov (Military Medical Academy) (1399 subjects), cadets of the Military Medical Academy (304 subjects) and 34 neonates. Laboratory testing was performed by using ELISA with test kits “VektoKor-IgG” (RF). Results. Medical workers displayed high total level of protection against measles virus serologically confirmed in 87.5% of the maternity hospital staff and 81.6% of the employees at the Military Medical Academy clinics. No differences in the immune status between the varying categories of medical personnel from the departments of various specialties were revealed. Lower population immunity was observed in the 20-40-year age group. Among the employees of the maternity hospital, the proportion of measles virus seropositive subjects aged 20—40 years was 75.9% (95%, CI 67.2—82.9%), 40—59 years — 91.5% (95%, CI 86.7—94.7%), 60 year and older subjects were all seropositive (95%, CI 94.6—100%). Dual vaccination did not guarantee emergence of effective measles immunity. The majority of seronegative subjects were dually vaccinated, hence, even in case of previous dual measles vaccination, it is necessary to perform a serological examination prior to recruitment to medical facilities. Females comprised more than 90% of medical workers. Therefore, women of fertile age previously dually vaccinated, but immunologically seronegative, are at risk of measles and intrauterine fetal infection, as well as the risk of its development is increased in vaccination age children. It was confirmed by demonstrating that as few as 70.6±15.3% of neonates were immune to the measles virus. The proportion of seronegative infants (29.4±15.3%) was comparable to that one in seronegative subjects aged 20—40 years (24.1±8.1%) working at the maternity hospital. Among the younger age group (aged 18—23 years) such as cadets of the Military Medical Academy, a significantly higher proportion of seronegative individuals was found — 48.3±5.6%, thereby increasing probability for measles outbreak therein. Conclusions. In order to eradicate measles, it is necessary to develop a screening program for detecting antibodies against measles among young and middle-aged subjects.

Russian Journal of Infection and Immunity. 2021;11(3):577-584
pages 577-584 views

Assessing Serratia spp. pathogenic potential from cryogenic habitats

Goncharov A.E., Solomenny A.P., Panin A.L., Grigoriev S.E., Cheprasov M.Y., Ahremenko Y.A., Kolodzieva V.V., Goncharov N.E., Kraeva L.A.

Abstract

The genus Serratia are opportunistic bacteria widely spread in natural environment. At the same time, this bacterial genus consists of the species associated with outbreaks of nosocomial infections. Serratia species are found in extreme habitats, but pathogenic potential of polyextremophilic strains in this genus remains unexplored. The aim of this study was to compare the genomes of two Serratia strains isolated in polar regions, primarily examining genetic factors of virulence and adaptation to cryogenic environment. During the 56th Russian Antarctic Expedition the Serratia liquefaciens 72 strain was isolated from a guano sample of the Adelie Penguin (Pygoscelis adeliae) colony on Tokarev Island (Haswell Archipelago, East Antarctica). The Serratia fonticola 5l strain was isolated from the frozen carcass of moose (Alces alces) fossils found on the Buor-Khaya Peninsula near the Laptev Sea coast (Yakutia Region, Russia). The whole-genome sequencing of such strains allowed to reveal genetic structures evidencing about their successful adaptation to low temperatures. Thus, it was found that both genomes contain genes encoding the main cold shock proteins, phylogenetically close to the corresponding genes in the hypobarotolerant Serratia liquefaciens strain ATCC 27592. Furthermore, both strains bear a cluster of tc-fABCD genes determining the bacterial adhesion to epithelial tissues, and the genes for RTX toxins — adhesins, crucial factors of biofilm formation in pathogenic Gram-negative bacteria. Experimental studies confirmed the ability of Serratia liquefaciens 72 and Serratia fonticola 5l to actively form biofilms in a wide temperature range (from 6°C to 37°C). The results obtained indicate that the examined genus Serratia strains isolated in Arctica and Antarctica exert overall similar adaptation strategies to polar climate, including the ability to produce pili, show active adhesion, and biofilm formation under low temperatures. Genetic adaptive factors may also act as pathogenicity factors allowing extremotolerant Serratia strains to exert traits of opportunistic and nosocomial pathogens and spread via chilled food-borne transmission. The wide use of food technologies, such as cooling and vacuum sealing, can potentially create a new ecological niche favourable for selection of psychrotolerant and hypobarotolerant pathogens. The data obtained allow to raise a question about necessity of further studies to monitor genetic diversity among psychrophilic hypobarotolerant microbial populations possessing pathogenic and epidemic potential.

Russian Journal of Infection and Immunity. 2021;11(3):585-590
pages 585-590 views

METHODS

Method for phenotypic identification of Acinetobacter nosocomialis bacteria

Sivolodskii E.P., Zueva E.V.

Abstract

The study's purpose was the developing of the method for Acinetobacter nosocomialis bacteria identification by the totality of phenotypic characteristics of the Acinetobacter baumannii (Ab) group based on the urease activity trait.

The research's objects were clinical strains of the Ab group, isolated in 2018—2019 in the bacteriological laboratory of Kirov Military Medical Academy (St. Petersburg), of which were A. baumannii (n = 85), A. nosocomialis (n = 12), A. pittii (n = 10). In addition, 9 strains of A. nosocomialis were isolated from water samples from the Neva River and 2 strains of A. calcoaceticus from the cyanobacterial mats. The belonging of the strains to Ab group was determined by the combination of the metabolic and physiological characteristics of the taxonomic tests for this group. The species identification of the studied strains was determined by the matrix-associated laser desorption/ionization time of flight mass spectrometry (MALDI-TOF MS). The urease activity of bacteria was determined using the microvolume method. We used a medium with urea in the following composition (g/l): Na2HPO4 — 1.1; KH2PO4 — 1.1; NaCI — 5.0; 40% alkaline solution phenol red — 5 ml; urea — 10—15; distilled water — 1 l. The ingredients, without urea, were dissolved in distilled water, dispensed to vials, sterilized for 20 minutes at 121°C, urea was added in the calculated amount. The medium with urea was added in 0.1 ml to wells of the plate, then inoculated the daily agar culture of the studied and control strains by full loop (2 mm in diameter) and incubated under aerobic conditions at 37°C. The reaction results for a rapid urease activity were determined after 3 hours by a change in the initial color of the medium. The reactions were accounted after 7—24 hours to detect activity urease. It was found that 100% of A. nosocomialis strains and 18.82% of A. baumannii strains had urease activity. At the same time, high activity urease was found only in one strain of A. baumannii (1.17%) and in all strains of A. nosocomialis. Therefore, the presence of high urease activity is of taxonomic importance for the species A. nosocomialis as the marker of distinguishing this species from other bacteria species of the Ab group. The sensitivity and specificity of the identifying A. nosocomialis strains by suggested approach compared to the studied strains identification by MALDI-TOF MS were 100 and 98.83% (x2 = 103.2; p < 0.0001), respectively.

Russian Journal of Infection and Immunity. 2021;11(3):591-596
pages 591-596 views

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