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 Orbital Segment of the International Space Station (ISS RS) is a unique closed living space characterized by constant temperature (around 22°C), increased humidity, space radiation and carbon dioxide levels higher than on the Earth, the presence of available organic substrates, microgravity, and permanent residence of people. All the above is a perfect environment for the development of microorganisms . Along with various fungal species, prokaryotes of different taxonomic groups also occur onboard the ISS RS . The fungi and bacteria onboard the ISS can disrupt the work of life-support systems, threaten the health of crew members and cause the corrosion of equipment; hence, much attention is focused on continuous monitoring of the composition of microbial communities in crewed spacecrafts .
The microbial contamination of the ISS RS is caused by crew rotation, delivery of equipment and consumable resources from the Earth, and exploitation of waste management systems. Most microorganisms discovered onboard the ISS RS are human-associated, and the microbiome of the ISS RS resembles that of some enclosed spaces on the Earth [4, 5] and can contain opportunistic microorganisms [2, 6, 7]. Some bacterial species can cause infections. Especially dangerous bacteria are spore-forming, in particular, representatives of the genus Bacillus, which can survive for a long time in an environment at low humidity and nutrient levels . Continuous monitoring of the microbiome in the ISS RS allows for assessing risk factors for crew health, integrity of ISS, and functioning of its systems. For example, the National Aeronautics and Space Administration of the United States (NASA) had determined the study of the ISS microbiome to be the main goal of both present and future orbital researches .
The goal of the present work was to study the resistance of bacterial strains of the genus Bacillus isolated from the samples taken in the ISS RS to a number of clinically significant antibiotics and to determine its possible mechanisms.
MATERIALS AND METHODS
The following bacteria of the genus Bacillus were used in this work: B. licheniformis 7-12, B. pumilis 8-12, and B. subtilis 14-12 isolated from the samples taken onboard the ISS RS in 2012–2014 and identified to a species by molecular methods  and the bacteria that we isolated and identified in the present work from the samples taken onboard the ISS RS in 2018. The comparative study was performed with three strains of bacterial species of the genus Bacillus from the collection of the Chair of Microbiology, Faculty of Biology, Moscow State University: B. licheniformis KM MSU 14, B. pumilus KM MSU 364, and B. subtilis KM MSU 25.
The bacteria isolated in 2018 were identified by analyzing the 16S pRNA genes . RNA was isolated from the cells of the strains grown in a liquid medium containing meat-peptone broth (MPB) with 1% glucose using a Genomic DNA Purification Kit KO 512 (Thermo Scientific, United States) according to the manufacturer’s instructions. The amplification of the 16S rRNA gene was performed using the following primers (Syntol, Russia): B63f (5'-CAG GCC TAA CAC ATG CAA GTC-3) and B1387r (5-GGG CGG WGT GTA CAA GGC-3'). The resultant DNA fragments were separated in agarose gel by electrophoresis (120 V). The agarose gel was prepared by adding agarose (1–1.2%) in 1× Tris-acetate (TAE) buffer, heating it up to 90–95°С and mixing until agarose is completely dissolved, followed by addition of 7.5 µL of ethidium bromide solution per every 150 mL of the solution, cooling to 45–50°С, and pouring into a special mold. After polymerization, the gel was placed into a Mini-SubCellGT horizontal electrophoresis chamber (BioRad, United States). The buffer (1 : 5) was added to the samples containing DNA fragments. Electrophoretic separation was performed in the 1× TAE buffer. Nucleic acid fragments were detected in UV light. DNA sequencing was performed with an ABIPRISM® BigDye™ Terminator reagent kit v. 3.1 (Thermo Scientific, United States), followed by the analysis of reaction products with an Applied Biosystems 3730 DNA Analyzer (Thermo Scientific, United States).
The minimum inhibitory concentration (MIC) of antibiotics was determined by serial twofold dilution method using a 96-well plate (Eppendorf, Germany). For this purpose, a 24-h bacterial culture was grown in MPB with 1% glucose to the optical density of 1.0 (OD 600 nm); the cell culture was then diluted by the MPB medium to the optical density of 0.1 (OD 600 nm) and added into the wells. After that the antibiotic was added into the wells so that its concentration in the first well was 4096 µg/mL followed by twofold dilution to the concentration 2 µg/mL. Nine β-lactam antibiotics and the aminocyclitol antibiotic spectinomycin were used in the work. Bacterial growth was assessed by adding resazurin (Sigma, United States) into the wells to a concentration of 50 μM .
To assess the efficiency of efflux systems, the nutrient medium and the respective antibiotic at a concentration from 4096 to 2 µg/mL were added to the 96-well plate as described previously, followed by the addition of a protonophore—carbonyl cyanide 3-chlorophenylhydrozone (CCCP) (Sigma, United States)—at the concentration of 2 µg/mL to each well. The wells with the antibiotic but without CCCР and the wells containing only the nutrient medium with the culture and CCCP were used as a control. The plates were incubated for 24 h at 37oC. The effect of СССP on the growth was assessed by adding resazurin at a concentration of 50 μM . All experiments were performed in triplicate. The efflux activity was determined by the ratio of reduction factor (RF) of the MIC of antibiotics in the cultures without CCCP to the MIC values in its presence. The absence of efflux was recorded at RF < 4; moderate activity was noted at RF within a range from 4 to 16; high activity was recorded at RF > 16 .
RESULTS AND DISCUSSION
Identification of cultures. The following bacteria were used in the work: the strains of the genus Bacillus isolated previously and identified to a species  as well as the new strains of spore-forming bacteria isolated from the samples taken onboard the ISS RS and identified in the present work. Based on the analysis of the 16S rRNA sequences, all four newly studied strains have been assigned to the genus Bacillus and designated as strains PWN2D, DLA64, LR2HG21, and HEP3B2. As the 16S rRNA gene sequences of the studied strains proved to be similar to those of several other species of the genus Bacillus, we failed to unambiguously identify particular species.
MIC determination. The resistance of bacterial strains of the genus Bacillus obtained in the ISS RS to a number of clinically significant antibiotics affecting Gram-positive bacteria has been studied. The reference cultures were some Bacillus strains obtained from the Culture Collection of the Chair of Microbiology, Moscow State University. We have analyzed the resistance of the studied strains to β-lactam antibiotics, such as penicillin, ampicillin, meropenem, cephalosporin derivatives of generation I (cefazolin), II (cefuroxime), III (ceftriaxone, cefoperazone, ceftazidime) and IV (cefepime), and the aminocyclitol antibiotic spectinomycin (Table 1). Due to high efficiency and low toxicity and availability of production technologies, β-lactam antibiotics are most widely used in modern medicine. Spectinomycin was the only non-β-lactam antibiotic of those used. It is often prescribed as a reserve group antibiotic to patients with contraindications to cephalosporins of the third and fourth generations.
The strain B. licheniformis 7-12 isolated from the ISS RS showed very high resistance to penicillin, ampicillin, and ceftriaxone: 1024 µg/mL (Table 1). This strain also demonstrated high resistance to all other tested antibiotics. The strain B. licheniformis KM MSU 14 from the Collection of the Chair of Microbiology (MSU) showed resistance only to penicillin, ampicillin, and spectinomycin with MIC values of 128, 256, and 256 µg/mL, respectively.
The strain B. pumilus 8-12 also showed very high resistance to all studied antibiotics, especially to ampicillin and ceftazidime: MIC 2048 µg/mL (Table 1). It was also quite resistant to cefoperazone and cefepime: MIC 1024 µg/mL. Cefuroxime (MIC 64 µg/mL) and ceftriaxone (MIC 128 µg/mL), which are cephalosporins of the second and third generations, as well as meropenem (MIC 16 µg/mL), proved to be the most effective inhibitors (Table 1). The strain B. pumilus KM MSU 364, similarly to B. pumilus 8-12, showed high resistance to ceftazidime and cefepime: MIC 1024 and 512 µg/mL, respectively; however, this strain showed lower resistance to all of the studied antibiotics compared to the strain 8-12, especially to penicillin, ampicillin, and cefoperazone (Table 1). The strain B. subtilis 14-12 showed considerable resistance only to four studied antibiotics: penicillin (MIC 2048 µg/mL), ampicillin (MIC 2048 µg/mL), cefuroxime (MIC 16 µg/mL), and spectinomycin (MIC 32 µg/mL) (Table 1). The strain B. subtilis showed low resistance to almost all generations of cephalosporin group antibiotics. The strain B. subtilis KM MSU 25 showed a resistance pattern similar to that of B. subtilis 14-12 (Table 1), with the exception of higher resistance to spectinomycin (MIC 2048 µg/mL).
Among the four bacterial strains of the genus Bacillus isolated and identified in the present work, the strain Bacillus sp. HEP3B2 showed the highest MIC values for the studied antibiotics (Table 1). The strain was resistant to all antibiotics: not only penicillin and ampicillin but also to cephalosporins of the first and third generations—cefazolin and ceftriaxone, as well as meropenem with IC 1024 µg/mL (Table 1). The strains Bacillus sp. PWN2D and Bacillus sp. DLA 64 showed the same MIC values for all of the studied antibiotics (Table 1). Both strains were resistant to penicillin, ampicillin, and spectinomycin (MIC 256 µg/mL). The strain Bacillus sp. LR2HG21 showed the maximum resistance to ampicillin (MIC 2048 µg/mL). The MIC values were 1024 µg/mL for cefuroxime and meropenem and 512 µg/mL for penicillin, spectinomycin, and ceftazidime from the group of cephalosporins (Table 1).
It should be noted that MIC was determined in the present work by the methods of serial dilutions in a microplate, and most experiments aimed at determining MIC values for bacteria of the genus Bacillus were performed by the method of gradient diffusion; hence, it would be incorrect to directly compare our results with the literature data, though some works indicate the resistance of strains of the genus Bacillus to penicillin and some other antibiotics .
Efflux detection. The main parameter that determines the efficiency of antimicrobial preparations against a microbial strain is MIC of an antibiotic, which can also be determined by the presence of active cell pumps extracting the antibiotic from a cell and functioning due to electrochemical potential across a membrane . The contribution of the efflux system to the stability of strains under study was assessed by measuring the MIC values for antibiotics before and after the exposure to the efflux pump inhibitor CCCP. It was shown that the addition of CCCP to the nutrient medium without antibiotics did not result in the inhibition of culture growth. The addition of CCCP to the culture inoculated in the nutrient medium in the presence of antibiotic leads to unhindered accumulation of this antibiotic in a cell and, consequently, to a decrease in the MIC of bacteria with active pumps.
All bacterial strains under study showed high resistance to penicillin and ampicillin (Table 1). The presence of efflux pumps active against these antibiotics was studied in B. licheniformis 7-12, B. licheniformis KM MSU 14, B. subtilis 14-12, Bacillus sp. R2HG21, and Bacillus sp. HEP3B2 (Table 2). It was shown that B. licheniformis 7-12 and B. licheniformis KM-MSU 14 had no efflux mechanism in the presence of penicillin or ampicillin. This fact indicates that these strains are apparently resistant to the antibiotics under study only due to the β-lactamase activity destroying the antibiotic. At the same time, the strains B. subtilis 14-12, Bacillus sp. R2HG21, and Bacillus sp. HEP3B2 showed a very high efflux activity in the presence of penicillin or ampicillin (Table 2).
The efflux in B. licheniformis 7-12 was studied, in addition to penicillin and ampicillin, also with ceftazidime as a cephalosporin group antibiotic of the third generation (the level of bacterial resistance to this antibiotic is relatively low (MIC 128 µg/mL), and it seems that the mechanism of this resistance has just started to take shape) and spectinomycin as an antibiotic, which is knowingly unsusceptible to β-lactamase and causes high resistance in B. licheniformis 7-12 with MIC 512 µg/mL. In the presence of ceftazidime and spectomycin, B. licheniformis 7-12 showed a moderate efflux activity.
For detecting the presence of efflux pumps in B. pumilis 8-12, the cephalosporins ceftriaxone and cefepime of the third and fourth generation were selected with MIC values of 128 and 1024 µg/mL, respectively (Table 2). The MIC values of these antibiotics in the presence of CCCP indicate a moderate activity of efflux pumps (Table 2).
The presence of efflux pumps in the strains Bacillus sp. LR2HG21 and Bacillus sp. HEP3B2 was studied with the use of cefepime and spectinomycin; in case of the strains PWN2D and DLA64, only spectinomycin was used. Cefepime was taken as cephalosporin of the fourth generation being of the highest interest due to its novelty, and spectinomycin was used as an antibiotic, which is knowingly not destroyed by β-lactamase. Bacillus sp. LR2HG21 and Bacillus sp. HEP3B2 showed a moderate and high activity of efflux pumps, respectively, against both cefepime and spectinomycin (Table 2). Two Bacillus strains, PWN2D and DLA64, showed the high activity of efflux pumps against spectinomycin (Table 2).
The study of the ISS microbiome for many years has shown that bacterial species of the genus Staphylococcus are detected most frequently and bacteria of the genus Bacillus are ranked second [3, 13]. The predominance of Staphylococcaceae can be explained by the anthropogenic factor [4, 5], while the high frequency of occurrence of representatives of the genus Bacillus is associated with their extremely high resistance to drying and deficiency of nutrients . Bacteria of the genus Bacillus are universal in occurrence, and it already includes more than 200 species. In the ISS, however, the species diversity of the studied Bacillus representatives is no more than 20 species, with the predominance of B. licheniformis, B. pumilus, and B. subtilis [2, 3, 13]. Historically, B. anthracis was recognized as the only pathogenic species of the genus; however, some strains of B. cereus, B. megaterium, B. thuringiensis, B. licheniformis, B. pumilus, B. subtilis, and B. alvei were described as opportunistic and even pathogenic .
The strains B. licheniformis 7-12 and B. pumilus 8-12 showed much higher resistance to penicillin and ampicillin than the strains B. licheniformis KM MSU 14 and B. pumilus KM MSU 364 taken for comparative analysis from the collection of the Chair of Microbiology, Moscow State University. Many Bacillus strains isolated in the ISS RS showed high resistance to cephalosporins of generation I–IV as well as to meropenem (Table 1). At the same time, all strains taken from the collection of Moscow State University (B. licheniformis KM MSU 14, B. pumilus KM MSU 364, B. subtilis KM MSU 25) and some other “space” strains of Bacillus (Bacillus sp. PWN2D and Bacillus sp. DLA64) showed a very low resistance to cephalosporins of generation I–IV and to meropenem (Table 1). The low resistance of collection cultures can be explained by their long-term storage, so that they had no time to form resistance, while the causes and mechanisms of antibiotic resistance in “space” strains can be different.
The first potential source of antibiotic-resistant Bacillus strains in space can be, first of all, the mutations caused by preflight sterilization of equipment with UV light, hydrogen peroxide, or other disinfectants as well as the mutations caused by specific cosmic conditions: cosmic radiation and microgravity. It is not improbable that resistant Bacillus strains are brought to the ISS by astronauts since these bacteria are part of the microbiota of human skin and intestines . In any case, the clinically significant resistance of the strains found in the ISS to antimicrobial agents can spread via horizontal gene transfer: for instance, by plasmids [13, 16]. For example, most of the 40 Bacillus strains isolated from the samples taken in the Antarctic and ISS were shown to have one or two plasmids, some of them being linked with the replicons of the element of virulence of B. anthracis pXO1 and pXO2. In addition, it was established that six of 25 tested strains acquired foreign DNA through conjugation [13, 16]. As has been mentioned before, Staphylococcus is the predominant bacterial genus in the ISS. It is also known that staphylococci were the first microorganisms that became resistant to β-lactam antibiotics, which, in turn, significantly reduced the efficiency of traditional therapy . It is possible that the studied bacilli could acquire the β-lactamase resistance from staphylococci, which led to an increase in their antibiotic resistance.
Finally, the studied Bacillus strains could acquire one of the mechanisms of protection from antibiotics, such as modification of antibiotic target, its inactivation, active removal from a microbial cell (efflux), and disruption of the permeability of external cell structures. All these mechanisms are capable of functioning both independently and as a complex. We have investigated the possibility of efflux in our strains. This mechanism emerged in the course of evolution as the protection of microorganisms from substances inhibiting their metabolism.
The findings indicate the absence of active efflux systems in B. licheniformis 7-12 and B. licheniformis KM MSU 14 showing high MIC values to penicillin and ampicillin (Table 2). The high resistance of both strains to these antibiotics is apparently due to their inactivation by β-lactamases. At the same time, the strains B. subtilis 14-12, Bacillus sp. R2HG21, and Bacillus sp. HEP3B2 isolated from the ISS samples showed the very high activity of efflux in the presence of penicillin and ampicillin. Thus, the mechanism of resistance to penicillin or ampicillin can vary in different strains of Bacillus.
In the presence of ceftazidime, ceftriaxone, cefepime, and spectinomycin, B. licheniformis 7-12, B. pumilus 8-12, and Bacillus sp. LR2HG21 demonstrated a moderate activity of efflux pumps (Table 2), suggesting the presence in these strains of resistance mechanisms, such as β-lactamase activity, in addition to the efflux systems. The high activity of efflux pumps in the strain HEP3B2 against cefepime and spectinomycin and in the strains PWN2D and DLA64 against spectinomycin indicates that the main mechanism of resistance to the studied antibiotics in these strains is the efflux (Table 2).
Efflux pumps can be found in almost all bacterial species, and the genes encoding this class of proteins can be localized on chromosomes or plasmids and transmitted by horizontal transfer [18, 19]. Efflux pumps can be divided into two groups: primary and secondary transporters. The family of ABC transporters belongs to the first group. For functioning, these transporters use the energy of ATP hydrolysis. Secondary transporters include the MSF, SMR, RND, and MATE families and function due to the electrochemical potential across a membrane [17, 20]. One of the most common ways to suppress efflux pumps, which are secondary transporters, is using CCCP. This uncoupler of oxidative phosphorylation disturbs the proton gradient of the membranes, which is necessary for the activity of efflux pumps [12, 21, 22].
The activity of efflux pumps can vary due to the emergence of different mutations, which can increase the efficiency of antibiotic removal from the cell in some cases . For gram-positive bacteria, the clinically significant efflux pumps determining their resistance to a broad range of antibiotics are transporters of the MFS family . In gram-positive bacteria, the efflux pump structure has been best studied in B. subtilis. They were shown to have transporters from the MFS and SMR families [18, 21, 23, 24]. Based on the literature data and the effect of CCCP on the studied strains (Table 2), it can be supposed that the discovered systems of active efflux of antibiotics in Bacillus strains belong to the group of secondary transporters of the MFS and SMR families described for the genus Bacillus [12, 18, 21, 23, 24].
As of yet, no serious infections or disease outbreaks in the ISS have been reported . However, the discovered bacterial strains with high resistance to some antibiotics require further screening of the ISS microbiota in order to prevent the potential health risk of some of these microorganisms for people with a weakened immune system, e.g., astronauts as a result of work under the extreme conditions of long-term space flight.
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The research was self-funded.
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The authors declare that they have no conflict of interests. This article does not contain any studies involving animals or human participants performed by any of the authors.
Yenikeyev ORCID http://orcid.org/0000-0002-8467-9051
Tatarinova ORCID http://orcid.org/0000-0002-9883-5780
Zakharchuck ORCID http://orcid.org/0000-0003-3783-3279
Translated by E. V. Makeeva
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Yenikeyev, R.R., Tatarinova, N.Y. & Zakharchuk, L.M. Mechanisms of Resistance to Clinically Significant Antibiotics of Bacterial Strains of the Genus Bacillus Isolated from Samples from the International Space Station. Moscow Univ. Biol.Sci. Bull. 75, 224–230 (2020). https://doi.org/10.3103/S0096392520040045
- Russian Segment of the International Space Station (ISS RS)
- bacteria of the genus Bacillus
- antibiotic resistance
- minimum inhibitory concentration
- efflux pumps
- closed habitat