Background. To date, there is little data on the clinical characteristics of Bacillus bacteria found in clean rooms and aseptic facilities. The aim of the study was to identify and determine the resistance of Bacillus strains isolated from the International Space Station and medical laboratory to clinically significant antibiotics. Methods. Isolates were identified using 16S rRNA gene analysis, MALDI-TOF, and whole-genome sequencing. Antibiotic sensitivity was assessed using the disk diffusion method. Results . Seven Bacillus strains out of 13 showed resistance to imipenem, and each of B. cereus LR2HG21, HSA01, HSA03, and HSA12 showed resistance to imipenem, ciprofloxacin, levofloxacin, and norfloxacin. Whole-genome sequencing of B. cereus LR2HG21, HSA01, HSA03, HSA12 and B. safensis SE192, resistant to imipenem and meropenem, showed that resistance to them is provided by the TEM-116 gene. In addition to TEM-116, the resistance of B. cereus LR2HG21 to imipenem and meropenem, and B. cereus HSA01 and HSA03 to imipenem, is provided by the BcI and/or BcII genes. Resistance to erythromycin in B. subtilis SE15 and B. subtilis SE171 is encoded by the mphK gene. Conclusion. Resistance to a particular antibiotic in different Bacillus strains can be achieved by one or more mechanisms simultaneously.

Pure cultures of 19 strains of spore-forming bacteria were obtained from the equipment surfaces of the Russian segment of the International Space Station. The study of morphological, cultural, and physiological-biochemical properties of these bacteria allowed the authors to attribute all strains to the genus Bacillus. As a result of using MALDI-TOF methods and genome-wide sequencing, it was found that six of 19 bacillus strains belong to the species B. paralicheniformis, four to B. pumilus, four to B. subtilis, two to B. cereus, and one to B. amyloliquefaciens. In accordance with the requirements and norms of EUCAST 2023, the resistance of bacillus strains obtained from the Russian segment of the International Space Station to such antibiotics as imipenem, meropenem, ciprofloxacin, levofloxacin, norfloxacin, vancomycin, erythromycin, clindamycin, and linezolid was studied. Resistance to erythromycin was found in 11 strains of Bacillus, and five strains showed resistance to clindamycin. Only one strain showed resistance to imipenem, levofloxacin, and norfloxacin, respectively. Analysis of the complete genome of bacterial strains in which resistance to erythromycin and (or) clindamycin was found made it possible to establish that resistance to these antibiotics in B. paralicheniformis strains SE71, SE131, SE181, SE182, and SE183 provides the ermD antibiotic resistance gene. In B. cereus SE43, resistance to erythromycin encodes the mphL gene.

Isolates of bacterial strains dominating on the surface of medical equipment used for blood sampling have been studied. Pure cultures of these bacteria have been identified as Bacillus cereus HSA01, Bacillus cereus HSA12, Bacillus cereus HSA03, Bacillus subtilis HSA06, and Bacillus amyloliquefaciens HSA09, and their resistance to a number of β-lactam antibiotics and spectinomycin has been determined. All strains are shown to be resistant to penicillin and ampicillin with the minimum inhibitory concentration (MIC) varying from 256 to 2048 μg/mL as well as to cephalosporin antibiotics with the MIC values varying from 2 to 2048 μg/mL. Resistance to spectinomycin administered to patients with allergic reactions to penicillin and cephalosporins is within the MIC range of 16–256 μg/mL. The ampicillin and cefuroxime resistance of B. cereus HSA01 is provided by the work of efflux pumps, while ceftazidime resistance is determined by the action of metal-β-lactamases (MBL); penicillin resistance is provided by the functioning of both mentioned systems. A high ampicillin and cefuroxime resistance of B. cereus HSA12 is provided by MBL and efflux activities, respectively, whereas ceftazidime resistance is determined by both MBL and efflux pumps. In the case of B. cereus HSA03, the observed resistance to penicillin, ampicillin, cefepime, and ceftazidime is explained by the efflux activity, resistance to cefazolin and ceftazidime is provided by MBL, while ampicillin and ceftazidime resistance is provided by both MBL and efflux. The penicillin and ampicillin resistance of B. subtilis HSA06 is provided by the MBL activity only. In B. amyloliquefaciens HSA09, resistance to ampicillin is provided by MBL and the action of efflux pumps, while penicillin resistance is provided only by the efflux activity. Thus, resistance to penicillin, ampicillin, and cephalosporin derivatives in the studied Bacillus group is provided, depending on the strain and the specific antibiotic, by metal-β-lactamase and/or efflux pumps representing secondary transporters.

The Russian Segment of the International Space Station, as a closed habitat, is a favorable environment for the development of microorganisms. There are bacteria and fungi of various systematic groups, some of which can lead to infections. Thus, certain species of spore-forming bacteria of the genus Bacillus are dangerous. Seven strains of bacteria of this genus isolated from samples obtained at the station showed resistance to β-lactam antibiotics, such as penicillin, ampicillin, meropenem, a number of cephalosporin derivatives I (cefazolin), II (cefuroxime), III (ceftriaxone, cefoperazone, ceftazidime), IV (cefepime) generations, and the aminocyclitol antibiotic spectinomycin. It has been found that all these strains are resistant to penicillin and ampicillin with a minimum inhibitory concentration (MIC) from 16 to 2048 μg/mL as well as to cephalosporin antibiotics and meropenem with a MIC value from 2 to 2048 μg/mL. Bacterial resistance to spectinomycin used in patients with an allergy to β-lactams (penicillins and cephalosporins) is in the MIC range from 32 to 2048 μg/mL. The absence of active efflux pumps in B. licheniformis 7-12 with high MIC values for penicillin and ampicillin suggested that this strain has a β-lactamase defense mechanism against these antibiotics. Three more strains resistant to penicillin and ampicillin— B. subtilis 14-12, Bacillus sp. R2HG21, Bacillus sp. HEP3B2—have another defense mechanism: the active transport of antibiotic from the cell, which is mediated by the presence of efflux pumps functioning due to the electrochemical potential of the cell membrane. In six strains of the studied bacilli, it has been shown that the resistance to cephalosporin derivatives of the third to fourth generations—ceftriaxone, ceftazidime, cefepime and the aminocyclitol antibiotic spectinomycin—is also apparently provided by the systems of active outflow of xenobiotics belonging to the group of secondary transporters.

The Russian segment of the International Space Station, as a closed habitat, is a favorable environment for the development of microorganisms. There are bacteria and fungi of various systematic groups, some of which can lead to infections. Thus, certain species of sporeforming bacteria of the genus Bacillus are dangerous In seven strains of bacteria of this genus, isolated from samples obtained at the station, resistence to such β-lactam antibiotics as penicillin, ampicillin, meropenem, a number of cephalosporin derivatives I (cefazolin), II (cefuroxime), III (ceftriaxone, cefoperazone, ceftazidime), IV (cefepime) generations, as well as the aminocyclitol antibiotic spectinomycin. It was found that all these strains are resistant to penicillin and ampicillin with a minimum inhibitory concentration (MIC) from 16 to 2048 µg/ml, as well as to cephalosporin antibiotics and meropenem with a MIC value from 2 to 2048 µg/ml. Bacterial resistance to spectinomycin used in patients with allergy to β-lactams penicillins and cephalosporins is in the MIC range from 32 to 2048 µg/ml. The absence of active efflux pumps in B. licheniformis 7-12 with high MIC values for penicillin and ampicillin suggested that this strain has a β-lactamase defense mechanism against these antibiotics. In three more strains resistant to penicillin and ampicillin – B. subtilis 14-12, Bacillus sp. R2HG21, Bacillus sp. HEP3B2 functions another defense mechanism – active transport of the antibiotic from the cell, mediated by the presence of efflux pumps, functioning due to the electrochemical potential of the cell membrane. It has been shown that, in six strains of the studied bacilli, resistance to cephalosporin derivatives of the 3rd-4th generations of ceftriaxone, ceftazidime, cefepime and the aminocyclitol antibiotic spectinomycin is also apparently provided by systems of active outflow of xenobiotics belonging to the group of secondary transporters.

From swabs of surfaces of equipment and air samples of the Russian segment of the International Space Station, nine strains of spore-forming bacteria of the genus Bacillus belonging to the species B. pumilus, B. licheniformis, B. subtilis, B. megaterium, and B. amyloliquefaciens were isolated. The last species of bacilli on the equipment of RS ISS was detected for the first time. For these species of bacilli, there are known strains that can be opportunistic to humans, and their metabolites can cause biodegradation of equipment and materials. B. pumilus found on ISS belongs to the group of bacteria that exhibits a particularly high resistance to adverse environmental conditions, such as dehydration, ultraviolet and gamma radiation, and chemical disinfection.

For cultures of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus N1 and SSO, S. thermosulfidooxidans subsp. asporogenes 41, and the thermotolerant strain S. thermotolerans Kr1 grown under forced aeration and in a high medium layer without aeration, growth characteristics, substrate consumption, and exometabolite formation were compared. Sulfobacilli grown under oxygen limitation exhibited greater generation time, longer growth period, cell yield decreased by from 40 to 85% (depending on the strain), suppressed cell respiration ( demonstrated for S. sibiricus N1 ), accumulation of exometabolites (acetate and propionate) in the medium, and emergence of resting forms. For strains N1, SSO, and Kr1, oscillations of Fe(II) and Fe(III) content in the medium were revealed. For S. sibiricus N1 and S. thermotolerans Kr1, grown under hypoxia (0.07% O-2 in the gas phase), coupling of substrate oxidation with Fe(III) reduction was revealed, as well as utilization of Fe(III) as an electron acceptor alternative to oxygen. The role of labile energy and constructive metabolism for survival of sulfobacilli under diverse conditions is discussed.

Проведено сравнение активности ферментов углеродного метаболизма умеренно термофильных хемолитотрофных бактерий Sulfobacillus sibiricus, штаммов N1 и SSO, и S. thermosulfidooxidans subsp. asporogenes 41, выращенных без принудительной аэрации, в высоком слое среды, с активностью ферментов тех же культур, выращенных при интенсивной аэрации. Снижение доступа воздуха к растущим клеткам S. sibiricus N1 приводило к переключению метаболизма глюкозы с пентозофосфатного на путь Энтнера–Дудорова при сохранении пути Эмбдена–Мейергофа–Парнаса. У штамма SSО S. sibiricus и штамма 41 S. thermosulfidooxidans subsp. asporogenes, независимо от кислородного режима культивирования, расщепление глюкозы осуществлялось по путям Энтнера–Дудорова и пентозофосфатному соответственно, а также по пути Эмбдена–Мейергофа–Парнаса. Продолжительный рост S. sibiricus, N1 и SSO, в высоком слое среды, без принудительной аэрации приводил к репрессии синтеза большинства ферментов ЦТК, особенно дегидрогеназ, и ряда карбоксилаз, в том числе РБФ-карбоксилазы. Обсуждаются особенности углеродного метаболизма у различных штамов сульфобацилл при росте в условиях дефицита кислорода.

A comparative study of the activities of the enzymes of carbon metabolism from the cells of moderately thermophilic chemolithotrophic bacteria Sulfobacillus sibiricus (strains N1 and SSO) and Sulfobacillus thermosulfidooxidans subsp. asporogenes (strain 41) was carried out grown in a high layer of medium without forced aeration and cells grown with intense aeration. Limited air access to the growing S. sibiricus N1 cells resulted in switching from the pentose phosphate pathway of glucose metabolism to the Entner-Doudoroff pathway while the Embden-Meyerhof-Parnas pathway persisted. Irrespective of the level of the aeration, in the cells of S. sibiricus SSO and S. thermosulfidooxidans subsp. asporogenes 41, degradation of the glucose occurred via the Entner-Doudoroff and pentose phosphate metabolic pathways, respectively, as well as via the Embden-Meyerhof-Parnas pathway. Prolonged growth of S. sibiricus, strains N1 and SSO, in a high layer of the medium without forced aeration led to the repression of synthesis of most of the tricarboxylic acid cycle (TCA cycle) enzymes, in particular dehydrogenases, as well as of some carboxylases including RuBisCO. The traits of carbon metabolism in various strains of Sulfobacillus under conditions of oxygen deficiency are discussed.

Дано подробное описание фенотипических свойств вида Sulfobacillus thermotolerans Kr1Т в зависимости от условий культивирования. Высокие скорости роста 0.22–0.30 ч-1 и окислительной активности штамма показаны в оптимальных миксотрофных условиях при температуре 40°С на среде с неорганическими (Fe(II), S0, пиритно-арсенопиритным концентратом) и органическими (глюкозой и/или дрожжевым экстрактом) субстратами. В клетках, выросших в оптимальных условиях на среде с железом, идентифицированы гемы A, B и, вероятно, С, указывающие на наличие соответствующих цитохромов. Впервые в клетках выявлены специфические внутрицитоплазматические протяженные структуры в виде тяжей цилиндрической формы. В клетках сульфобациллы, растущих лито-, органо- и миксотрофно при 40°С, обнаружены ферменты трех основных путей ассимиляции углеводов и отдельных реакций ЦТК. Активность ферментов была максимальной в условиях миксотрофного роста. В области предельно высоких, 55°С, и низких, 12–14°С, температур скорость роста уменьшалась соответственно в 2 и 10 раз, не обнаруживалась активность одного из ключевых ферментов окислительного пентозофосфатного пути, 6-фосфоглюконатдегидрогеназы, и наблюдалось снижение активности почти всех ферментов метаболизма глюкозы и ЦТК. Скорость фиксации 14-углекислоты клетками в авто-, миксо- и гетеротрофных условиях составляла 31.8, 23.3 и 10.3 нмоль/(ч мг белка) соответственно. Показаны активности РБФК-карбоксилазы (максимальная – при литотрофном росте) и карбоксилаз гетеротрофной фиксации углекислоты. Обсуждаются физиолого-биохимические особенности термотолерантной сульфобациллы в сравнении с умеренно термофильными.

The phenotypic characteristics of the species Sulfobacillus thermotolerans Kr1 T , as dependent on the cultivation conditions, are described in detail. High growth rates (0.22–0.30 h −1 ) and high oxidative activity were recorded under optimum mixotrophic conditions at 40 °C on medium with inorganic (Fe(II), S 0 , or pyrite-arsenopyrite concentrate) and organic (glucose and/or yeast extract) substrates. In cells grown under optimum conditions on medium with iron, hemes a , b , and, most probably, c were present, indicating the presence of the corresponding cytochromes. Peculiar extended structures in the form of cylindrical cords, never observed previously, were revealed; a mucous matrix, likely of polysaccharide nature, occurred around the cells. In the cells of sulfobacilli grown litho-, organo-, and mixotrophically at 40 °C, the enzymes of the three main pathways of carbon utilization and some enzymes of the TCA cycle were revealed. The enzyme activity was maximum under mixotrophic growth conditions. The growth rate in the regions of limiting temperatures (55 °C and 12–14 °C) decreased two-and tenfold, respectively; no activity of 6-phosphogluconate dehydrogenase, one of the key enzymes of the oxidative pentose phosphate pathway, could be revealed; and a decrease in the activity of almost all enzymes of glucose metabolism and of the TCA cycle was observed. The rate of 14 CO 2 fixation by cells under auto-, mixo-, and heterotrophic conditions constituted 31.8, 23.3, and 10.3 nmol/(h mg protein), respectively. The activities of RuBP carboxylase (it peaked during lithotrophic growth) and of carboxylases of heterotrophic carbon dioxide fixation were recorded. The physiological and biochemical peculiarities of the thermotolerant bacillus are compared versus moderately thermophilic sulfobacilli.