Phenotypic and metabolic adaptations of Rhodococcus cerastii strain IEGM 1243 to separate and combined effects of diclofenac and ibuprofen

IntroductionThe increasing use of non-steroidal anti-inflammatory drugs (NSAIDs) has raised concerns regarding their environmental impact. To address this, understanding the effects of NSAIDs on bacteria is crucial for bioremediation efforts in pharmaceutical-contaminated environments. The primary challenge in breaking down persistent compounds lies not in the biochemical pathways but in capacity of bacteria to surmount stressors.MethodsIn this study, we examined the biodegradative activity, morphological and physiological changes, and ultrastructural adaptations of Rhodococcus cerastii strain IEGM 1243 when exposed to ibuprofen, diclofenac, and their mixture.Results and DiscussionOur findings revealed that R. cerastii IEGM 1243 exhibited moderate biodegradative activity towards the tested NSAIDs. Cellular respiration assay showed higher metabolic activity in the presence of NSAIDs, indicating their influence on bacterial metabolism. Furthermore, catalase activity in R. cerastii IEGM 1243 exposed to NSAIDs showed an initial decrease followed by fluctuations, with the most significant changes observed in the presence of DCF and the NSAID mixture, likely influenced by bacterial growth phases, active NSAID degradation, and the formation of multicellular aggregates, suggesting potential intercellular synergy and task distribution within the bacterial community. Morphometric analysis demonstrated alterations in size, shape, and surface roughness of cells exposed to NSAIDs, with a decrease in surface area and volume, and an increase in surface area-to-volume ratio (SA/V). Moreover, for the first time, transmission electron microscopy confirmed the presence of lipid inclusions, polyphosphates, and intracellular membrane-like structures in the ibuprofen-treated cells.ConclusionThese results provide valuable insights into the adaptive responses of R. cerastii IEGM 1243 to NSAIDs, shedding light on the possible interaction between bacteria and pharmaceutical compounds in the environment

Biotransformation of Oleanolic Acid Using <i>Rhodococcus rhodochrous</i> IEGM 757

Using the bioresources of the Regional Specialised Collection of Alkanotrophic Microorganisms (acronym IEGM, Perm, Russia; WFCC # 285), R. rhodochrous IEGM 757 was selected, which catalyzed the C5, C22, and C23 functionalization of pentacyclic triterpenoid oleanolic acid (OA, 3β-hydroxyolean-12-en-28-oic acid, 1.0 g/L) to form a new 5α,22α-dihydroxy derivative of gypsogenic acid (3β,5α,22α-trihydroxyolean-12-ene-23,28-dioic acid) for 5 days. In silico analysis showed that, compared to the native triterpenoid, the OA metabolite may be more soluble in water and less ecotoxic, act as an apoptosis agonist and insulin promoter, and have chemopreventive and analgesic effects. Phase-contrast, fluorescent, scanning, and transmission electron microscopy and X-ray spectroscopy demonstrated the high resistance of R. rhodochrous IEGM 757 to OA. This creates opportunities for further research and development of a method for the production of the OA metabolite. New-generation sequencing of the R. rhodochrous IEGM 757 whole genome, annotation and bioinformatics analysis of the obtained sequences, and real-time PCR were applied. As a result, 24 genes encoding CYP450 enzymes were found, which are highly likely to be involved in the process of OA oxidation

Distinct Effects of Moxifloxacin and Bedaquiline on Growing and ‘Non-Culturable’ <i>Mycobacterium abscessus</i>

Mycobacterium abscessus has recently emerged as the cause of an increasing number of human infections worldwide. Unfortunately, it is highly resistant to existing drugs, and new specific agents to combat M. abscessus have not yet been found. The discovery of antibiotics that are effective not only against replicating but also against dormant and often recalcitrant cells is a daunting challenge. In this study, we developed a model of non-replicating M. abscessus, which represents a valuable screening tool for antibacterial agents. Thus, we demonstrated that, under a deficiency of potassium ions in the growth media and prolonged incubation, M. abscessus entered a ‘non-culturable’ state with a significant loss of colony-forming ability, but it retained viability, as confirmed using the most-probable-number (MPN) assay. The ‘non-culturable’ mycobacteria possessed decelerated cellular metabolism and noticeable differences in cell morphology from actively growing mycobacteria. ‘Non-culturable’ cells were used in a comprehensive screening of the efficacy of antibiotics, along with actively growing cells. Both CFU and MPN tests confirmed the prominent bactericidal effect of moxifloxacin on actively growing and ‘non-culturable’ M. abscessus, as proven by less than 0.01% of cells surviving after antibiotic treatment and prolonged storage. Bedaquiline exhibited a comparable bactericidal effect only on metabolically inactive non-culturable cells aged for 44 days. There were reductions ranging from 1000 to 10,000-fold in CFU and MPN, but it was not so efficient with respect to active cells, resulting in a bacteriostatic effect. The demonstrated specificity of bedaquiline in relation to inert non-replicating M. abscessus offers a new and unexpected result. Based on the findings of this research, moxifloxacin and bedaquiline can be regarded as potential treatments for infections caused by M. abscessus. In addition, a key outcome is the proposal to include the combination of viability assays for comprehensive testing of drug candidates. Relying on CFU-based assays alone resulted in overestimates of antibacterial efficacy, as demonstrated in our experiments

Syntrophic Growth of <i>Biomaibacter acetigenes</i> Strain SP2 on Lactate and Glycerol

A moderately thermophilic Gram-positive chemo-organotrophic bacterium, strain SP2, was isolated by serial dilutions with crotonate and yeast extract as substrates from a butyrate-degrading methanogenic enrichment obtained from thermophilically digested sludge of the Kuryanoskaya wastewater treatment plant (Moscow, Russia). Cells of strain SP2 are spore-forming rods, sometimes occurring in short chains. The bacterium is an obligate anaerobe that grows at temperatures from 20 to 70 °C (55–60 °C optimum) within a pH range of 3.5–8 (7.5 optimum) and with NaCl concentrations of up to 2.5%. The strain utilized yeast extract and simple sugars as carbon and energy sources. Thiosulfate was used as an electron acceptor when grown on sucrose, resulting in the formation of hydrogen sulfide and the accumulation of elemental sulfur globules inside the cells. Strain SP2 is phylogenetically related to Biomaibacter acetigenes strain SK-G1T as revealed by comparison with the 16S rRNA gene (99.9% identity) and genome (ANI 99%, dDDH 90%) of both strains. It is interesting that strain SP2 was capable of syntrophic conversion of glycerol and lactate when co-cultivated with hydrogenotrophic methanogen, which was not previously shown for the SK-G1T type of strain. The isolation and in-depth study of new facultatively syntrophic microorganisms is important for wastewater treatment ecotechnologies due to their ability to switch to an alternative source of carbon and energy and therefore greater resistance to changing environmental conditions in bioreactors