Survey on Phenotypic Resistance in Enterococcus Faecalis: Comparison of Expression of Biofilm Related Genes in Persister and Non Persister Cells

Background Currently, phenotypic resistance is a serious therapeutic challenge, and a definitive remedy has not been discovered yet. Biofilm formation and persister cells are two well-studied phenotypic resistance, leading to the recalcitrance and relapse of different chronic infections. It appears that the presence of persister cells in biofilm is the main factor in the relapse of infections and treatment failure. Thus, we aimed to evaluate the expression of biofilm-associated genes in persister and non-persister E. faecalis isolates. Methods Ninety-five clinical E. faecalis isolates were investigated using microtiter broth dilution (MBD) and microtiter plate (MTP) assay to determine the vancomycin-sensitive isolates and biofilm formation, respectively. To this end, 91 vancomycin-sensitive E. faecalis isolates (biofilm producers) were screened by PCR to determine the presence of biofilm-related genes (gelE, esp, and agg). The vancomycin-tolerant isolates were determined by MTP assay. Bacterial persister assay was performed using an enzymatic lysis assay. Finally, the expression of biofilm-related genes was evaluated in persisters and non-persister isolates of E. faecalis by real-time PCR assay. Results E. faecalis isolates indicated a high (95.8%) sensitivity to vancomycin. PCR assay identified gelE, esp, and agg genes in 91 (100%), 72(79.12), and 74(81.32) of isolates, respectively. All E. faecalis biofilm producers were tolerant to vancomycin, and minimum bactericidal concentration for biofilms (MBCB) was > 2500 µg/ml. Based on enzymatic lysis assay, among 91 isolates, only 3 persister were isolated. The increased expression level of biofilm-related genes was observed in persister than non-persister E. faecalis isolates. Conclusion The expression of biofilm-related genes is higher in persister than non-persister isolates of E. faecalis.

Optimisation and Benchmarking of Targeted Amplicon Sequencing for Mycobiome Analysis of Respiratory Specimens

(1) Background: Firm consensus has yet to be established in relation to taxonomic classification and primer choice in targeted amplicon sequencing of the mycobiome. While the nuclear ribosomal internal transcribed spacer (ITS) region are recognized as the formal fungal taxonomic barcode, appraisal of different ITS sub-regions and the influence of DNA extraction methods have not been comprehensively undertaken using human respiratory specimens. (2) Methods: We performed ITS analysis of respiratory (sputum) samples by assessing (a) the effect of alternate DNA extraction techniques and (b) an evaluation of four different ITS primer pairs (ITS1F and ITS2; ITS1-30F and ITS1-217R; gITS7ngs and ITS4ng; and Fseq and Rseq) on the mycobiome profiles generated for mock fungal communities and their respective clinical (airway) specimens. (3) Results: Primer pairs varied in their resulting ITS mycobiome profiles, suggesting that particular pairs may be more relevant for analysis of respiratory samples compared to others. Assessment of DNA extraction methods highlighted lower final DNA concentrations achieved by mechanical disruption compared to enzymatic lysis. However, despite lower yields, DNA liberated by mechanical lysis more readily yielded ITS bands with highest success in combination with the Fseq and Rseq primers. (4) Conclusion: Choice of extraction method, primers used, and sequencing approach are all important considerations in sequencing the mycobiome and should be tailored to sample type. A standardization of approach to mycobiome studies using respiratory specimens will permit more reliable comparisons between studies and improve our understanding of the role of fungi in the human airway.

DETECTION OF POTENTIALLY PATHOGENIC BACTERIA IN THE BRACKISH RIVERS FLOWING INTO THE ELTON LAKE BY HIGH-THROUGHPUT SEQUENCING

Aim. To indicate potentially pathogenic bacteria in plankton of the brackish rivers flowing into the Elton Lake by high-throughput sequencing of 16S ssuRNA gene. Materials and methods. The water samples from brackish rivers Lantsug and Chernavka, flowing into the Elton Lake, were taken up in a volume of 50 ml, filtered through membrane filters (pore diameter - 0.22 pm). Total DNAwas obtained by phenol-chloroform extraction with preliminary homogenization and enzymatic lysis. DNA libraries for sequencing were created by protocol Illumina with primers to a variable V3-V4 region of 16S ssuRNA gene. Sequencing was performed on a platform MiSeq («Illumina», США). Results.There were found the phylotypes of potentially pathogenic bacteria of Proteobacteria phylum from the families Enterobacteriaceae, Pseudomonadaceae, Campylobacteraceae, Vibrionaceae, Aeromonadaceae, Moraxellaceae, Legionellaceae, Alcaligenaceae, Campylobacteraceae, and also of Firmicutes, Bacteroidetes, Actinobacteria phyla in the plankton samples of the brackish rivers. Probable source of bacterial contamination is large and small cattle. Conclusion. These data demonstrate that the continental brackish waters, along with freshwater and marine habitats perform a reservoir function to potentially pathogenic microorganisms. High-throughput sequencing can be used to screen the presence of pathogens in water.

C-phycocyanin from Arthrospira maxima LJGR1: production, extraction and protection

C-phycocyanin (C-PC) overproduced by Arthrospira maxima LJGR1 under high urea and salt concentrations was extracted by different methods and evaluated as an antioxidant activity. A. maxima was grown in flat plate photobioreactors, using modified Zarrouk medium with 624 mg L-1 urea and 15 g L-1 NaCl to produce 11 mg C-PC L-1d-1. Agitation, sonication at 35 and 45°C and enzymatic lysis of the cell wall were tried. The C-PC yields, purity and inhibition of the oxidant radicals (DPPH & ABTS) obtained by the best extraction methods were 215 mg C-PC g-1, 0.74, 92% and 29%; when agitation and 221, 0.75, 87% and 27% using sonication at 45°C. Chia mucilage and amaranth flour coatings best preserved C-PC antioxidant activity from temperature variations and up to six days shelf life at 20°C. An activation energy of 160 kJ mol-1 has been obtained with chia mucilage, and the least color degradation rate (r∆E*) of 4.2 d-1 with amaranth flour.

Critical cell wall hole size for lysis in Gram-positive bacteria

Gram-positive bacteria can transport molecules necessary for their survival through holes in their cell wall. The holes in cell walls need to be large enough to let critical nutrients pass through. However, the cell wall must also function to prevent the bacteria's membrane from protruding through a large hole into the environment and lysing the cell. As such, we hypothesize that there exists a range of cell wall hole sizes that allow for molecule transport but prevent membrane protrusion. Here, we develop and analyse a biophysical theory of the response of a Gram-positive cell's membrane to the formation of a hole in the cell wall. We predict a critical hole size in the range of 15–24 nm beyond which lysis occurs. To test our theory, we measured hole sizes in Streptococcus pyogenes cells undergoing enzymatic lysis via transmission electron microscopy. The measured hole sizes are in strong agreement with our theoretical prediction. Together, the theory and experiments provide a means to quantify the mechanisms of death of Gram-positive cells via enzymatically mediated lysis and provides insights into the range of cell wall hole sizes compatible with bacterial homeostasis.