МИКРОБИОЛОГИЧЕСКАЯ ЭФФЕКТИВНОСТЬ КОМБИНАЦИЙ АНТИБИОТИКОВ В ОТНОШЕНИИ ИНВАЗИВНЫХ ШТАММОВ KLEBSIELLA PNEUMONIAE

Введение. Комбинированная антибиотикотерапия – основной способ лечения инфекций, вызванных антибиотикорезистентными бактериями. Цель исследования – выявить комбинации антибиотиков с синергидной активностью в отношении инвазивных экстремально-антибиотикорезистентных штаммов K.pneumoniae, продуцирующих карбапенемазы. Материал и методы. Для 12 инвазивных штаммов K.pneumoniae, продуцирующих карбапенемазы KPC, OXA-48 и NDM, определены минимальные подавляющие концентрации антибиотиков. Определение чувствительности к комбинациям антибиотиков выполнено модифицированным диско-диффузионным методом. Результаты. Отмечен эффект потенцирования колистина азтреонамом (58,3% штаммов), азитромицином (33,3%), кларитромицином (41,7%), рифампицином (33,3%), доксициклином (50,0%). Показана высокая микробиологическая эффективность цефтазидима/авибактама в отношении всех продуцентов карбапенемаз KPC и OXA-48. Для продуцентов MBL NDM выявлен синергидный эффект комбинации цефтазидима/авибактама и азтреонама с восстановлением чувствительности. Выводы. Полученные результаты открывают перспективы для комбинированной терапии инфекций кровотока, вызванных XDR-штаммами K.pneumoniae

The Role of the st313-td Gene in Virulence of Salmonella Typhimurium ST313

Multidrug-resistant Salmonella enterica serovar Typhimurium ST313 has emerged in sub-Saharan Africa causing severe infections in humans. Therefore, it has been speculated that this specific sequence type, ST313, carries factors associated with increased pathogenicity. We assessed the role in virulence of a gene with a yet unknown function, st313-td, detected in ST313 through comparative genomics. Additionally, the structure of the genomic island ST313-GI, harbouring the gene was determined. The gene st313-td was cloned into wild type S. Typhimurium 4/74 (4/74-C) as well as knocked out in S. Typhimurium ST313 02-03/002 (Δst313-td) followed by complementation (02-03/002-C). Δst313-td was less virulent in mice following i.p. challenge than the wild type and this phenotype could be partly complemented in trans, indicating that st313-td plays a role during systemic infection. The gene st313-td was shown not to affect invasion of cultured epithelial cells, while the absence of the gene significantly affects uptake and intracellular survival within macrophages. The gene st313-td was proven to be strongly associated to invasiveness, harboured by 92.5% of S. Typhimurium blood isolates (n = 82) and 100% of S. Dublin strains (n = 50) analysed. On the contrary, S. Typhimurium isolates of animal and food origin (n = 82) did not carry st313-td. Six human, non-blood isolates of S. Typhimurium from Belarus, China and Nepal harboured the gene and belonged to sequence types ST398 and ST19. Our data showed a global presence of the st313-td gene and in other sequence types than ST313. The gene st313-td was shown to be expressed during logarithmic phase of growth in 14 selected Salmonella strains carrying the gene. This study reveals that st313-td plays a role in S. Typhimurium ST313 pathogenesis and adds another chapter to understanding of the virulence of S. Typhimurium and in particular of the emerging sequence type ST313

Антибактериальное покрытие имплантатов на основе полиметилметакрилатного костного цемента in vitro и in vivo

Nowadays, the infection recurrence rate in osteomyelitis is still high. New hardware not only allowing one to stabilize bone fragments, but also having antibacterial activity seems to be an extremely useful and promising task. The goal of the study was to assess the effectiveness of use of an antibacterial coating based on polymethylmethacrylate cement in experiment and in infected nonunions of long tubular bones of the lower limbs. Bone cement-based coatings impregnated with antibiotics were formed on titanium plates. A plate rinse was carried out; antibiotic concentrations in the rinsed solutions were estimated by a serial broth microdilution method. The antibacterial activity of control and rinsed samples against the antibiotic-sensitive and multiple-antibiotic-resistant Staphylococcus aureus and Pseudomonas aeruginosa strains was estimated by a bilayer agar method. Clinical part. The study included 70 patients divided into 2 groups: osteosynthesis with antibacterial-coated interlocking nail (40 patients, main group) and osteosynthesis with an external fixation device (30 patients, control group) comparable in age, sex and disease duration. The effectiveness of the antibacterial coating was analyzed by the duration of systemic use of antibiotics and infection recurrence rate. The concentrations of meropenem and phosphomycin in the rinsed solutions obtained at one-fold and two-fold treatments were sufficient to suppress the growth of control strains. Vancomycin-containing samples possessed sufficient antibacterial activity against both methicillin-sensitive (MSSA) and methicillin-resistant (MRSA) S. aureus strains, double rinse of the samples eliminated their bactericidal properties. The duration of systemic antibacterial therapy in the main group was statistically significantly lower than in the control group (U = 77.5, p < 0.001), and in the main group the infection recurrence rate was lower than 32.5 % vs. 86.7 % (χ2 = 20.39, p < 0.001). The PMMA-based coating impregnated with phosphomycin, meropenem or vancomycin possesses sufficient and long-lasting antibacterial activity, mainly against antibiotic-sensitive strains. An adequate use of such a coating in clinical practice allows one to obtain the desired result of treatment.Частота рецидивов после костных инфекций в настоящее время высока. Создание конструкции, которая не только позволяет стабилизировать костные отломки, но и обладает антибактериальной активностью, представляется чрезвычайно актуальной и перспективной задачей. Цель исследования – оценить эффективность применения антибактериального покрытия на основе полиметилметакрилатного цемента в эксперименте и при инфицированных несращениях длинных трубчатых костей нижних конечностей. На титановых пластинах сформированы покрытия из костного цемента, импрегнированного антибиотиками. Выполнена отмывка пластин методом последовательных микроразведений в бульоне, оценена концентрация антибиотиков в отмывочных растворах. Антибактериальная активность образцов в отношении чувствительных и множественно-резистентных штаммов S. aureus и P. aeruginosa оценена двуслойным агаровым методом. В исследование включены 70 пациентов, разделенных на 2 группы: остеосинтез стержнем с блокированием и антибактериальным покрытием (40 пациентов, основная группа) и остеосинтез аппаратом внешней фиксации (30 пациентов, контрольная группа), сопоставимые по возрасту, полу и продолжительности заболевания. Эффективность антибактериального покрытия анализировали по продолжительности системного применения антибактериальных препаратов и наличию рецидивов инфекции. Концентрации меропенема и фосфомицина в отмывочных растворах, полученных при однократной и двукратной обработке образцов, были достаточными для подавления роста контрольных штаммов. Ванкомицин-содержащие образцы обладали достаточной антибактериальной активностью в отношении как метициллинчувствительного (MSSA), так и метициллинрезистентного (MRSA) штаммов S. aureus, двукратная отмывка образцов устраняла их бактерицидные свойства. Продолжительность системной антибактериальной терапии в основной группе была статистически значимо меньше, чем в контрольной (U = 77,5, p < 0,001), также в основной группе наблюдалось меньше рецидивов инфекции 32,5 % vs. 86,7 % (χ2 = 20,39; p < 0,001). Покрытия из костного цемента, импрегнированного фосфомицином, меропенемом или ванкомицином, обладают достаточной и длительной антибактериальной активностью, проявляющейся главным образом в отношении антибиотикочувствительных штаммов. Адекватное применение такого покрытия в клинической практике позволяет получить желаемый результат лечения

МЕХАНИЗМ ФОРМИРОВАНИЯ ПОЛИЛАКТИДНЫХ ПОКРЫТИЙ ИЗ АКТИВНОЙ ГАЗОВОЙ ФАЗЫ

The mechanism of deposition of a poly-L-lactide film from the active gas phase has been shown to be associated with destructing a macromolecule into large fragments rather than with depolymerizing a poly-L-lactide film to a monomer followed by its polycondensation on substrate. The transformation of the initial polymer powder into a thin film under the great energetic effect is accompanied by decreasing the molecule mass, while the D-isomer content increases from 4 to 12 %. In so doing, not only the phase state of the polymer changes from semi-crystalline to amorphous one, but the poly-L-lactide relaxation state is transformed from glass to rubber one; these changes could promote an accelerated release of different biocide additives from the film.Методами спектроскопии ЯМР 1 H и 13С и поляриметрии установлено, что при формировании тонких пленок поли-L-лактида путем осаждения из активной газовой фазы происходит деструкция исходного полимера до крупных фрагментов с сохранением полимерного состояния вещества. Одновременно конфигурационные превращения макромолекулы приводят к увеличению содержания в ней D-звеньев с 4 до 12 мол. %, в результате чего исходный аморфно-кристаллический полимер превращается в аморфный. Переход поли-L-лактида в тонкопленочное состояние сопровождается изменением не только фазового, но и релаксационного состояния полимера, что является дополнительной причиной ускорения высвобождения из пленки биоцидных добавок

THE ANTIBACTERIAL ACTIVITY OF OFFICINAL HERBS IN REGARD TO EXTENSIVELY ANTIBIOTIC-RESISTANT GRAM-NEGATIVE RODS

Objective: to estimate the antibacterial activity of officinal herbs available in Belarus in regard to extensively antibiotic-resistant Gram-negative bacteria. Material and methods . Minimal inhibitory concentrations and minimal bactericidal concentrations of water infusions from officinal medicinal herbs in regards to antibiotic-resistant and antibiotic-sensitive strains of Gram-negative rods have been determined. Results. 4 out of the 17 herbs revealed the expressed antibacterial activity in regards to Gram-negative non-fermenting rods. The herbs did not reveal any antibacterial activity of water infusions in regard to K. pneumoniae . Conclusion. The revealed data on the antibacterial activity make it possible to recommend medicinal herbs for additional local application along with current systemic antibiotic therapy.</jats:p

Activity of Cefiderocol and Other New Antibiotics Against Extensively Drug-Resistant &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; Strains

Background. The spread of extensive drug-resistance among gram-negative bacteria calls for the search for antimicrobics with new mechanisms of actions.The aim was to assess susceptibility of extensively drug-resistant K. pneumoniae strains to cefiderocol and other new inhibitor-protected β-lactams, and to determine genetic mechanisms of antibiotic resistance.Methods. This study included 30 extensively drug-resistant K. pneumoniae strains collected in 2016–2021 from 4 regions of Belarus. Carbapenemase genes were detected by real-time PCR. Minimum inhibitory concentrations (MICs) for cefiderocol and other new antibiotics were assessed by microdilution method using the Sensititre system. Whole genome sequencing was performed for 2 resistant and 3 cefiderocol-susceptible strains. Genome assemblies and annotation were performed using UGENE v. 37.0 software. Nucleotide sequences were translated using CLC  Sequence Viewer v. 8.0 (QIAGEN) package. The PROVEAN software was used to assess amino asides substitutions and their influence on the functional activity of proteins.Results. KPC carbapenemase-producers were 4 strains, OXA-48 — 17, KPC+OXA-48 — 1, NDM — 7, OXA-48 + NDM — 1. All KPC-producers were susceptible to imipenem/relebactam and meropenem/vaborbactam. Resistance to ceftazidime-avibactam was noted in all NDM  producers and OXA-48+NDM  co-producer. The study has identified 9 cefiderocol-resistant strains. These were NDM and OXA-48-producers isolated from hospitalized patients with COVID-19 infection from 3 regions of Belarus. Resistant strains had functionally significant nonsynonymous  substitutions in the genes of TonB-dependent receptors for catecholate siderophores FepA (F472V, P64S) and Fiu (T92S).Conclusion. The study has shown high efficacy of new inhibitor-protected carbapenems and cephalosporins against certain types of carbapenemase-producers. Strains with mutational resistance to cefiderocol, an antibiotic not previously used in Belarus, have been identified.</jats:p

Susceptibility to antibiotic combinations of &lt;i&gt;Klebsiella pneumoniae&lt;/i&gt; and &lt;i&gt;Acinetobacter baumannii&lt;/i&gt; strains isolated from COVID-19 patients

Objective. To assess the susceptibility of K.pneumoniae and A.baumanii strains isolated from hospitalized COVID-19 patients to antibiotics and their combinations.Materials and methods. The minimum inhibitory concentrations (MICs) of meropenem and colistin were determined for 47 A.baumannii and 51K.pneumoniaestrains isolated from the hospitalized COVID-19 patients by the broth microdilution method. The susceptibility to 11 antibiotic combinations was assessed using the method of multiple combination bactericidal testing.Results. Colistin resistance was detected in 31.9 % of A.baumannii strains (MIC50 — 0.5 mg/l, MIC90 — 16 mg/l) and in 80.4 % of K.pneumoniaestrains (MIC50 — 16 mg/l, MIC90 — 256 mg/l). It has been shown that double antibiotic combinations with the inclusion of colistin exhibit bactericidal or bacteriostatic activity against 76.6–87.2 % of A.baumannii strains. Combinations with the addition of meropenem, colistin and macrolides exhibited bactericidal activity against 78.4–80.4 % of K.pneumoniae strains. Combinations of two carbapenems were not active, the combination of meropenem-colistin had a bactericidal effect only in 13.7 % of K.pneumoniae strains.Conclusion. Widespread colistin resistance was found in carbapenem-resistant K.pneumoniae and A.baumannii strains isolated from the hospitalized COVID-19 patients. The combinations of antibiotics that have a synergistic antibacterial effect in their pharmacokinetic/pharmacodynamic concentrations have been determined.</jats:p

Experience of using modern genomic technologies to study microorganisms and their communities

Objective. The aim of this work was to review the main results of genomic studies of microorganisms and their communities performed on the base of the Research Laboratory of Gomel State Medical University.Materials and methods. Genomic, transcriptomic and metagenomic analysis of the microorganisms of the stomach and respiratory tract.Results. The capabilities of modern next-generation sequencing platforms have been analyzed, and the authors` own results of the use of genomic, transcriptomic and metagenomic analysis of the microbiota in patients with various gastric and respiratory pathologies have been described.Conclusion. The analysis of the obtained data has revealed peculiarities of the structure of the microbial communities of the stomach of the patients infected with H. pylori with different gastric pathology: the proportion participation of H. pylori in the metagenome of the samples with different forms of gastric cancer did not exceed 25 %, of gastritis — 6 %, of peptic ulcer — 1 %. At the same time, the minimal amount of H. pylori in all the cases could reach 0.1 %. A signifcant degree of CagA and CagY loci variability of H. pylori was detected. Streptoccocus genus bacteria dominated (36 %) in the bacterial microbiome of a patient diagnosed with the coronavirus disease, and in the viral microbiome, SARS-CoV-2 constituted 59 % of the total number of viruses in the material. The analysis of 13 strains of Klebsiella pneumoniae with multiple and extreme resistance to antibiotics has found that the studied strains belong to fve MLST-types, three of which are classifed as high epidemic risk groups.</jats:p

Prevalence of carbapenemase-producing &lt;i&gt;Klebsiella pneumonia&lt;/i&gt; in Gomel Region of Belarus and their sensitivity to antibiotics, antibiotic combinations, and decontaminants

Here, we characterized in public health organizations prevalence of carbapenemase-producing Klebsiella pneumoniae, sensitivity to antimicrobial agents (AMAs), combined antimicrobial agents, and decontaminants. For this, there were selected 58 clinical isolates of K. pneumoniae resistant to carbapenems and/or polymyxins and examined within the microbiological monitoring program. Genes encoding KPC, OXA-48, VIM, IMP, NDM carbapenemases were detected by real-time multiplex PCR. Sensitivity to antimicrobial agents was determined by an automated method on a microbiological VITEK-2 Compact analyzer (bioMérieux, France) and by serial broth microdilution method. Sensitivity to 11 dual antimicrobial agent combinations was determined by a modified method of multiple combination bactericidal antibiotic testing. As a part of combinations, AMAs at pharmacokinetic/pharmacodynamics (PK/PD) threshold concentrations (meropenem — 8 μg/ml, amikacin — 16 μg/ml, levofloxacin — 1 μg/ml, tigecycline — 0.5 μg/ml, phosphomycin — 32 μg/ml, colistin — 2 μg/ml) were tested. Susceptibility to 7 combined decontaminants of different composition was determined by the suspension method. Carbapenemase genes were detected in 22 K. pneumoniae clinical isolates, of which 19 isolates contained a blaOXA-48 gene and 3 isolates — gene blaNDM. Carbapenemase producing K. pneumoniae were identified in 10 Gomel public health organizations and five regional centers of the Gomel region. The majority of such strains were isolated from patients in ICU (63.6%) and surgical departments (27.3%). Tigecycline (100% of the sensitive isolates, МIC50 — 1 μg/ml, MIC90 — 1 μg/ml) and colistin (86.4% of the sensitive isolates, МIC50 — 0.5 μg/ml, MIC90 — 4 μg/ml) exhibited the highest activity against carbapenemase-producing K. pneumoniae, whereas aminopenicillins, cephalosporins, aztreonam, aminoglycosides, fluoroquinolones, chloramphenicol (no sensitive isolates) had exhibited the lowest efficacy. Bactericidal activity of all antibiotic combinations containing colistin was shown against 86.4–95.5% of K. pneumoniae isolates. At least 3 distinct combinations of antimicrobial agents with bactericidal activity were efficient against 21 K. pneumoniae isolates (95.5%). Only 1 bactericidal combination (meropenem–amikacin) was unveiled for one isolate (producer of NDM MBL with MIC of colistin 32 μg/ml). Geksadekon, duacid, oksidez, hlorocid and diajsid exerted a bactericidal effect at 1/4 work dose against all isolates. Duacid, oksidez, hlorocid and diajsid showed bactericidal effect at 1/16 work dose against 95.5–100% isolates. Thus, several decontaminant groups (oxidizing agents, chlorine-containing preparations) were characterized by bactericidal activity against multidrug-resistant and extremely drug-resistant of K. pneumoniae even at 4–16 times lower than recommended concentration.</jats:p

Formation &lt;i&gt;in vitro&lt;/i&gt; of colistin resistance in carbapenem-resistant Gram-negative bacteria and its biological cost

Introduction. The spread of resistance to carbapenems among gram-negative bacteria have led to an increase in the consumption of polymyxins and the emergence of certain strains resistant to them. Polymyxin resistance is mainly associated with mutations in chromosomal genes. The development of mutational resistance to antibiotics can lead to a decrease in the viability of bacteria; which is manifested by an increase in the duration of the cell cycle; a decrease in virulence and competitive fitness.The purpose of the study was to assess in vitro the intensity of the formation of colistin resistance in carbapenemresistant clinical isolates of gram-negative bacteria; the stability of the formed emerged resistance and its biological cost.Materials and methods. For 46 strains of Klebsiella pneumoniae; 77 strains of Pseudomonas aeruginosa and 42 strains of Acinetobacter baumannii; real time polymerase chain reaction (PCR) was used to detect the genes of carbapenemases; the minimum inhibitory concentrations (MIC) of meropenem and colistin were determined by broth microdilution method. The selection of resistant subpopulations on Muller–Hinton agar with the addition of 16 mg/l colistin was carried out. For colistin-resistant mutants and their isogenic sensitive strains; the kinetic parameters of growth in broth culture were determined. Incubation and result recording were performed on an Infinite M200 microplate reader for 18.5 hours at 35°C with measurement of light scatter in the wells every 15 minutes.Results. The production of carbapenemases MBL VIM in P. aeruginosa; MBL NDM; KPC and OXA-48 in K. pneumoniae; OXA-23 and OXA-40 in A. baumannii was observed. All strains were sensitive to colistin (MIC varied from 0.062 to 2 mg/l). The colony growth on a selective medium with16 mg/l colistin was observed for 97.8% of K. pneumoniae strains; 16.9% of P. aeruginosa strains; and 61.9% of A. baumannii strains. The mutational nature of colistin resistance was confirmed for 21.7% of K. pneumoniae strains. For colistin-resistant mutants of K. pneumoniae; a significant increase in the duration of the lag phase (Tlag) was observed: 225.6 ± 7.037 min in the wild-type susceptible strains and 245.5 ± 8.726 in resistant mutants; p = 0.037. The indicators of the doubling time of the number of microbial cells in the exponential growth phase (Tdoubling) and the area under the bacterial growth curve did not differ significantly.Conclusion. A high frequency of formation of colistin resistance in vitro in carbapenemase-producing strains of K. pneumoniae was observed. The absence of significant changes in the kinetics of microbial growth in resistant strains makes it possible to predict the further spread of mutational resistance to colistin; as well as its preservation in microbial populations of K. pneumoniae even in the case of limiting the use of this antibiotic.</jats:p