Variations in the Adaptive Response of <i>Burkholderia pseudomallei</i> to Cold Stress

The aim of the study was to analyze differential gene expression in Burkholderia pseudomallei strains with different survival rates under cold stress conditions.Materials and methods. Three strains of B. pseudomallei (sequence types ST 46, ST 70, and ST 85) were used as model strains. The RNA was isolated using the membrane columns method and stabilized through dehydration. The cDNA was sequenced on the Illumina MiSeq platform. Gene functions were classified using the KEGG PATHWAY database.Results and discussion. Based on the analysis of transcriptomes of B. pseudomallei strains after prolonged exposure to cold stress, the molecular mechanisms of B. pseudomallei adaptation to low temperatures have been described for the first time ever. It was shown that adaptation of B. pseudomallei to cold stress is associated with regulatory processes leading to a significant decrease in the total transcriptional activity. Two strategies of adaptation to low temperatures have been found: 1) modulation of regulatory processes leading to suppression of gene expression of the main metabolic pathways to the minimum level that ensures cell viability and activation of the minimum required set of stress response genes, and 2) less noticeable suppression of general metabolism in combination with activation of expression of an extended range of genes for cold and heat shock, general, osmotic, and universal stresses. Both mechanisms provide the causative agent of melioidosis with survival under conditions of prolonged cold stress at low positive temperatures. The first strategy showed greater efficiency at negative temperatures. The transition of B. pseudomallei to a viable but uncultivated state occurs in the long term (at least 2 years). While with the second strategy, this happens within 2 months. Assessment of the potential and molecular mechanisms of adaptation of this bacterium to cold stress is necessary to understand the degree of risk in case of a possible introduction of B. pseudomallei into regions with a temperate climate and to develop effective measures to ensure the biosafety of the environment

Melioidosis in aspects of epidemiology, clinic, and laboratory diagnostics

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

Design of Primers for Detection and Typing of β-Lactamase Genes from Pathogenic Species of <i>Burkholderia</i>

The set of oligonucleotide primers was designed to identify β-lactam-resitance determinats in isolates of pathogenic Burkholderia using PCR. Simultaneously identified was certain molecular class of detected β-lactamases. PCR with primers specific to metallo-β-lactamase (class B) and oxacillinase of D-ala carboxypeptidase family (class D of β-lactamase) gene sequences allowed to differentiate among pseudomallei group of Burkholderia species

Assessment of Resistance in <i>Burkholderia pseudomallei</i> to Benzalkonium Chloride and Antibiotics

The aim of the study was to investigate the features of resistance formation in Burkholderia pseudomallei to quaternary ammonium compounds, as well as to analyze its influence on the development of antibiotic resistance.Materials and methods. 10 strains of melioidosis causative agent with typical cultural and morphological properties have been studied. The selection of variants resistant to benzalkonium chloride was carried out by successive passages on a dense nutrient medium with the addition of a disinfectant in increasing concentrations. The determination of sensitivity to benzalkonium chloride was performed through serial dilutions in agar, to antibacterial drugs from the groups of sulfonamides, β-lactams and tetracyclines – using disk diffusion method. Statistical processing of the obtained results was conducted with the help of the Microsoft Excel 2019 software. Arithmetic mean values and errors of mean values were calculated. The significance of differences between the parameters was determined applying Student’s t-test.Results and discussion. All parental strains showed a similar degree of resistance to the disinfectant compound and most of the strains – susceptibility to the antibiotics tested. Cultivation of B. pseudomallei strains on a nutrient medium with the addition of benzalkonium chloride led to an increase in resistance to this disinfectant. In addition, an increase in the level of resistance to all studied antibiotics was found. Statistical processing of the data collected revealed a significant correlation between the change in sensitivity to benzalkonium chloride and the emergence of resistance to amoxicillin/ clavulanic acid and ceftazidime. It was found that the causative agent of melioidosis, with a natural high susceptibility to benzalkonium chloride, has a high potential for developing resistance to this disinfectant compound, which is of practical importance in the development of disinfection regimens using quarternary ammonium compounds. For the first time, a direct correlation between a decrease in the sensitivity to benzalkonium chloride in B. pseudomallei and emergence of resistance to amoxicillin/clavulonic acid and ceftazidime has been demonstrated

Current epidemiological situation on Particularly Dangerous Mycoses around the World and Forecast of Its Development

The literature review focuses on epidemiological aspects of the spread of particularly dangerous mycoses across the world (coccidioidomycosis, histoplasmosis, blastomycosis, paracoccidioidomycosis). Forecast of morbidity rates for the next few years is provided too. Out of all endemic mycoses, coccidioidomycosis, endemic for North America regions, poses the most dangerous threat. In case of the infection, complications are most likely to arise, including the dissemination of the process in immunocompromised persons. Histoplasmosis is also classed as particularly dangerous mycosis. It affects both humans and animals. It is endemic for North, Central and South America, as well as Asia and Australia. The most studied endemic areas of infection with blastomycosis are in the territory of North America, while paracoccidioidomycosis is endemic for Latin America countries. Analysis of academic publications on particularly dangerous mycoses over the last three years testifies to the increase in their morbidity rates around the world. This situation is associated, primarily, with the increment in the number of immunocompromised subjects. An important stage in the improvement of the agent diagnostics is introduction of advanced methods for early diagnostics of mycoses, in particular, molecular-genetic and genome sequencing tools. It could also allow for the detection of patients beyond the limits of endemic foci

Comparative Analysis of Potential Determinants of Resistance to Aminoglycosides in <i>Burkholderia pseudomallei</i> Strains with Different Level of Sensitivity to Gentamicin

The aim of the study was to identify and compare potential determinants of aminoglycoside resistance in gentamicin susceptible Burkholderia pseudomallei strains.Materials and methods. A bioinformatics analysis of whole genome shotgun sequences of three B. pseudomallei strains having different levels of sensitivity to gentamicin was carried out.Results and discussion. B. pseudomallei is intrinsically resistant to aminoglycosides. Such strains, as a rule, are not taken into account in the classical scheme of isolation and identification. At the same time, there were no significant differences in the clinical manifestations of melioidosis during infection with gentamicin-resistant and sensitive strains. In B. pseudomallei strains of different sequence types (ST70, ST948, and ST1566), point missense mutations were found in the genes of three efflux pumps of the RND family: AmrAB-OprA, BpeAB-OprB, BpeEF-OprC, and one with unknown functions, as well as in the gene aminoglycoside-6’-N-acetyltransferase AAC(6’)-III. All three strains had amino acid substitutions in the AmrA periplasmic linker: ARG160SER, Arg116Gln and Gly237Arg, Thr317Lys, respectively. In moderately sensitive strains (ST948 and ST1566), an identical Val222Met substitution was found in the repressor of the AmrAB-OprA operon, AmrR. It is likely that the intermediate level of sensitivity to gentamicin in the studied strains is mediated by the constitutive expression of the AmrAB-OprA operon, which partially compensates for the structural defects. It is also possible that a dinucleotide deletion in the AAC (6’)-III aminoglycoside-6’-N-acetyltransferase gene, as well as detected mutations in the homologues of the periplasmic linker (BPSL2234) of an uncharacterized efflux operon of the RND family, are involved in the loss of resistance to gentamicin

Infectious Diseases in the Americas Region that Are Relevant to the Global Surveillance and Control

The aim of the work was information-analytical assessment of the epidemiological situation on infectious diseases that are potentially or truly dangerous in terms of occurrence of emergencies of sanitary-epidemiological nature in the Region of Americas.Materials and methods. The study was based on the official reports of the WHO, the Pan American Health Organization, the Centers for Disease Control and Prevention, the national Ministries of Health, data from the ProMED information portal, the Global Network for the Epidemiology of Infectious Diseases, and published scientific papers.Results and discussion. By the model of the Americas, regional epidemiological features have been established, including the endemicity (enzooticity) of territories according to the most relevant nosological forms and the intensity of the epidemic process manifestations. It is shown that the main epidemiological risks in the countries of Central, South America and the Caribbean are associated with dengue, Zika, Chikungunya fevers characterized by a wide territorial dissemination and the ability to cause large-scale epidemic outbreaks, in the countries of North America – West Nile fever. Other infections of international concern include: cholera, that twice caused epidemics of imported origin during the seventh pandemic, which changed the structure of world morbidity; plague, manifested in an annual incidence, including with a complication by the pneumonic form, which determines an increased potential danger of anthropogenic spread; malaria, demonstrating an upward trend in morbidity and the number of intra-continental imported cases; yellow fever, characterized by the activation of natural foci and the expansion of the territories of potential pathogen transmission. The data obtained can serve as a basis for assessing the risks of infectious disease introduction from the American Region into safe territories, improving epidemiological forecasting and validity in making managerial decisions when conducting sanitary and anti-epidemic (preventive) measures

EMERGENCY PROPHYLAXIS OF EXPERIMENTAL MELIOIDOSIS USING SYNTHETIC IMMUNOMODULATORS AND HETEROLOGOUS VACCINES

Melioidosis is a particularly dangerous infection caused by Burkholderia pseudomallei , against which a vaccine has not yet been created. In this regard, the development of effective treatment regimens and emergency prevention of melioidosis is very relevant. To improve the effectiveness of emergency prophylaxis of melioidosis, synthetic peptides (bestim, imunofan) and thiopoietin preparation glutoxim were used when combined with the antibiotic doxycycline. In addition, in experiments on white mice, the ability of heterologous vaccines (plague and tularemia), used in the emergency prevention mode, to increase the resistance of animals to melioidosis infection was assessed. It was shown that the most effective was imunofan, which, when combined with doxycycline, increased by 20% the survival rate of 5LD50 Burkholderia pseudomallei infection and significantly increased the average life span of mice infected with 5-12 LD50 (p < 0.05). The efficiency of use for stimulation of non-specific resistance to melioidosis of a heterologous plague vaccine EV, once administered 1 day prior to infection, protected 90% of mice from 6 LD50 Burkholderia pseudomallei and 60% - with an increase in the infectious dose of the pathogen of melioidosis up to 15 LD50. The same level of protection from melioidosis was provided by a 3 day course of antibiotic therapy with doxycycline. It was concluded that the tularemia vaccine is not suitable for immunostimulation in melioidosis due to its high residual virulence and reactogenicity

Peculiarities of Burkholderia mallei and Burkholderia pseudomallei Identification Using Microbiological Analyzer Vitek 2 Compact 30

Automated Vitek 2 system, based on comparison of a biochemical profile of the studied bacterial cultures with the existing database, is widely used for B. pseudomallei and B. mallei identification in the laboratory practice.Objective of the study is to conduct extended phenotypic characterization of the strains of glanders and melioidosis causative agents, stored in the biobank of the Volgograd Research Anti-Plague Institute and analyze variations in their biochemical profiles, using Vitek 2 system.Materials and methods. Using Vitek 2 device, (bioMerieux, France) analyzed have been biochemical properties of 52 collection strains of B. pseudomallei and B. mallei grown on L-agar (Difco, USA) and trypticase-soy agar – TSA (HiMedia, India).Results and discussion. Most of the investigated strains (31 out of 40 B. pseudomallei and 8 of 12 B. mallei) have been identified with an acceptable probability for determining certain specie appurtenance, amounting to 90–99 %. The percentage of correct identification of B. pseudomallei and B. mallei is higher when strains are cultured on L-agar, than when on TSA. Due to the variability of the biochemical features, some strains have showed non-typical for its species results in certain tests (for B. pseudomallei strains – the absence of enzyme activity of β-N-acetyl-glucosaminidase, β-N-acetyl-galactosaminidase and ability to utilize D-cellobiose; for B. mallei strains – the absence of enzyme activity of L-proline-aryl-amidase and tyrosin-aryl-amidase, existence of glycin-aryl-amidase activity and ability to utilize sucrose, D-trehalose), which has led to their mal-identification. The probability of error diagnostics of microorganisms belonging to Burkholderia species necessitates up-dating of the database built into Vitek 2 analyzer as regards biochemical characteristics of the strains which have peculiar profiles