Evaluation of TD test for analysis of persistence or tolerance in clinical isolates of Staphylococcus aureus.

Besides natural and acquired mechanisms of resistance, bacteria can cope with presence of antibiotics by using complex mechanisms such as persistence or tolerance. The main purpose of this study was to evaluate the suitability of newly developed Tolerance Disk Test (TDtest) (Gefen et al., 2017) to detect persistent or tolerant bacterial cells in clinical isolates of Staphylococcus aureus. The principle of the test is to resuscitate the subpopulation of persistent or tolerant bacterial cells following a disk diffusion test by glucose. Results of the TDtest were evaluated using time killing experiments for three pairs of consecutive S. aureus isolates from lower respiratory airway samples of three cystic fibrosis patients with chronic staphylococcal infections. TDtest enabled semi-quantitative detection of persistent or tolerant bacterial populations in all analyzed isolates for oxacillin, vancomycin, and ciprofloxacin to which isolates studied were susceptible. Therefore, TDtest is a promising method for rapidly determining persistence/tolerance in clinical isolates of S. aureus.

[The phenomenon of bacterial persistence to antibiotics].

Bacterial persistence in clinical microbiology is a phenomenon where the bacterial subpopulation of any bacterial strain, without having been exposed to an antibiotic, is already persistent to it. In clinical bacterial strains, persistence is not tested at all and the role of this phenomenon in the treatment of bacterial infections has not yet been evaluated. Therefore, the aim of the article is to highlight the significance of this probably global phenomenon in the treatment of bacterial infections with antibiotics. Also described are the mechanisms of its origin and some manner that could potentially reduce the frequency of these antibiotic-resistant bacterial cells in the bacterial population.

Environmental Stress Affects the Formation of Staphylococcus aureus Persisters Tolerant to Antibiotics.

The ability to form persisters has been observed in many microorganisms, including Staphylococcus aureus, mainly in the context of chronic infections and the pathogenicity of these microbes. In our research, we have demonstrated that salt or oxidative stress could play a role in the formation of S. aureus persisters outside the host's intracellular interface. We pre-exposed planktonic growing bacterial culture to an oxidative or salt stress and monitored the dynamics of persister formation after ciprofloxacin and gentamicin treatment. In parallel, using the quantitative PCR (qPCR) approach, we determined the expression level of the stress sigma factor SigB. The pre-exposure of bacteria to salt stress caused a 1-2.5 order of magnitude increase in persister formation in the bacterial population after antibiotic exposure, depending on the type and concentration of the antibiotic used. In contrast, oxidative stress only minimally influenced the formation of persisters, without correlation to the antibiotic type and concentration. We have demonstrated that both stress and antibiotic exposure increase the expression of sigB in bacterial populations from very early on. And that the expression level of sigB differs with the type of antibiotic and stress, but no correlation was observed between persister formation and sigB expression. The method used could be helpful in testing the ability that strains can have, to form persisters.

NAD(P)H-hydrate dehydratase- a metabolic repair enzyme and its role in Bacillus subtilis stress adaptation.

BACKGROUND: One of the strategies for survival stress conditions in bacteria is a regulatory adaptive system called general stress response (GSR), which is dependent on the SigB transcription factor in Bacillus sp. The GSR is one of the largest regulon in Bacillus sp., including about 100 genes; however, most of the genes that show changes in expression during various stresses have not yet been characterized or assigned a biochemical function for the encoded proteins. Previously, we characterized the Bacillus subtilis168 osmosensitive mutant, defective in the yxkO gene (encoding a putative ribokinase), which was recently assigned in vitro as an ADP/ATP-dependent NAD(P)H-hydrate dehydratase and was demonstrated to belong to the SigB operon. METHODS AND RESULTS: We show the impact of YxkO on the activity of SigB-dependent Pctc promoter and adaptation to osmotic and ethanol stress and potassium limitation respectively. Using a 2DE approach, we compare the proteomes of WT and mutant strains grown under conditions of osmotic and ethanol stress. Both stresses led to changes in the protein level of enzymes that are involved in motility (flagellin), citrate cycle (isocitrate dehydrogenase, malate dehydrogenase), glycolysis (phosphoglycerate kinase), and decomposition of Amadori products (fructosamine-6-phosphate deglycase). Glutamine synthetase revealed a different pattern after osmotic stress. The patterns of enzymes for branched amino acid metabolism and cell wall synthesis (L-alanine dehydrogenase, aspartate-semialdehyde dehydrogenase, ketol-acid reductoisomerase) were altered after ethanol stress. CONCLUSION: We performed the first characterization of a Bacillus subtilis168 knock-out mutant in the yxkO gene that encodes a metabolite repair enzyme. We show that such enzymes could play a significant role in the survival of stressed cells.

Draft Genome Sequence of Stenotrophomonas maltophilia Strain 5BA-I-2, a Soil Isolate and a Member of a Phylogenetically Basal Lineage.

Stenotrophomonas maltophilia is an omnipresent environmental bacterium emerging as an opportunistic human pathogen and exhibiting multidrug resistance. Here, we report the draft genome sequence of S. maltophilia strain 5BA-I-2, a soil isolate and a member of a phylogenetically basal lineage.

Evolution of REP diversity: a comparative study.

BACKGROUND: Repetitive extragenic palindromic elements (REPs) constitute a group of bacterial genomic repeats known for their high abundance and several roles in host cells´ physiology. We analyzed the phylogenetic distribution of particular REP classes in genomic sequences of sixty-three bacterial strains belonging to the Pseudomonas fluorescens species complex and ten strains of Stenotrophomonas sp., in order to assess intraspecific REP diversity and to gain insight into long-term REP evolution. RESULTS: Based on proximity to RAYT (REP-associated tyrosine transposase) genes, twenty-two and thirteen unique REP classes were determined in fluorescent pseudomonads and stenotrophomonads, respectively. In stenotrophomonads, REP elements were typically found in tens or a few hundred copies per genome. REPs of fluorescent pseudomonads were generally more numerous, occurring in hundreds or even over a thousand perfect copies of particular REP class per genome. REP sequences showed highly heterogeneous distribution. The abundances of REP classes roughly followed host strains´ phylogeny, differing markedly among individual clades. High abundances of particular REP classes appeared to depend on the presence of the cognate RAYT gene, and deviations from this state could be attributed to recent or ancient mutations of rayt-flanking REPs, or RAYT loss. RAYTs of both studied bacterial groups are monophyletic, and their cognate REPs show species-specific characteristics, suggesting shared evolutionary history of REPs, RAYTs and their hosts. CONCLUSIONS: The results of our large-scale analysis show that REP elements constitute intriguingly dynamic components of genomes of fluorescent pseudomonads and stenotrophomonads, and indicate that REP diversification and proliferation are ongoing processes. High numbers of REPs have apparently been retained during the entire evolutionary time since the establishment of these two bacterial lineages, probably because of their beneficial effect on host long-term fitness. REP elements in these bacteria represent a suitable platform to study the interplay between repeated elements, their mobilizers and host bacterial cells.

Identification and characterization of repetitive extragenic palindromes (REP)-associated tyrosine transposases: implications for REP evolution and dynamics in bacterial genomes.

BACKGROUND: Bacterial repetitive extragenic palindromes (REPs) compose a distinct group of genomic repeats. They usually occur in high abundance (>100 copies/genome) and are often arranged in composite repetitive structures - bacterial interspersed mosaic elements (BIMEs). In BIMEs, regularly spaced REPs are present in alternating orientations. BIMEs and REPs have been shown to serve as binding sites for several proteins and suggested to play role in chromosome organization and transcription termination. Their origins are, at present, unknown. RESULTS: In this report, we describe a novel class of putative transposases related to IS200/IS605 transposase family and we demonstrate that they are obligately associated with bacterial REPs. Open reading frames coding for these REP-associated tyrosine transposases (RAYTs) are always flanked by two REPs in inverted orientation and thus constitute a unit reminiscent of typical transposable elements. Besides conserved residues involved in catalysis of DNA cleavage, RAYTs carry characteristic structural motifs that are absent in typical IS200/IS605 transposases. DNA sequences flanking rayt genes are in one third of examined cases arranged in modular BIMEs. RAYTs and their flanking REPs apparently coevolve with each other. The rayt genes themselves are subject to rapid evolution, substantially exceeding the substitution rate of neighboring genes. Strong correlation was found between the presence of a particular rayt in a genome and the abundance of its cognate REPs. CONCLUSIONS: In light of our findings, we propose that RAYTs are responsible for establishment of REPs and BIMEs in bacterial genomes, as well as for their exceptional dynamics and species-specifity. Conversely, we suggest that BIMEs are in fact a special type of nonautonomous transposable elements, mobilizable by RAYTs.