Molecular distribution of biocide resistance genes and susceptibility to biocides among vancomycin resistant Staphylococcus aureus (VRSA) isolates from intensive care unit (ICU) of cardiac hospital- A first report from Pakistan.

Background: The study was conducted with the aim to investigate the VRSA isolates in terms of their susceptibility to routinely used biocides influenced by the co-occurrence of biocide resistant gene (BRGs) and efflux pumps genes. Methodology: Frequently touched surfaces within intensive care unit (ICU) of cardiac hospital were classified into three primary sites i.e., structure, machines and miscellaneous. Over a period of six months (January 2021 to July 2021) twenty three swabs samples were collected from these sites. Subsequently, these samples underwent both phenotypic and molecular methods for VRSA isolation and identification. Susceptibility and efficacy testing of biocides (benzalkonium chloride (BAC), cetrimide (CET) and chlorhexidine gluconate (CHG)) were evaluated using microdilution broth and suspension method. Furthermore, specific primers were used for singleplex PCR targeting BRGs (cepA, qacA, and qacE) and efflux pump (norA, norB, norC, sepA, mepA and mdeA) associated genes. Results: We found that 72.2 % S. aureus demonstrate the presence of vanA or vanB genes with no significant difference among three sites (p > 0.05). cepA is the most dominant BRGs followed by qacA and qacE from structure site as compared to other sites (p < 0.05). BAC showed reduced biocide susceptibility and MIC50. There was no significant difference between presence or absence of BRGs and high MIC values of VRSA isolates from all three sites. However, efflux pump genes (EFPGs) particularly norA and norA + sepA had a significant association with BRGs and reduced biocide. Conclusion: BAC is the most effective disinfectant against VRSA. Proper and controlled use of BAC is required to overcome the VRSA contamination. We recommend continuous monitoring of the BRGs prevalence for better prevention of microorganism dissemination and infection control in hospitals.

Genomic and Phenotypic Insight into the Probiotic Potential of Lactic Acid Bacterial spp. Associated with the Human Gut Mucosa.

Commensal microbiome-based health support is gaining respect in the medical community and new human gut-associated Lactic Acid Bacteria (LAB) strains must be evaluated for their probiotic potential. Here we characterized the phenotype and genomes of human ileocecal mucosa-associated LAB strains using metagenomic sequencing and in vitro testing. The strains characterized belonged to the genus Enterococcus (Enterococcus lactis NPL1366, NPL1371, and Enterococcus mundtii NPL1379) and Lactobacillus (Lactobacillus paragasseri, NPL1369, NPL1370, and Lactiplantibacillus plantarum NPL1378). Genome annotation suggested bacterial adaptation to both human physiological and industrial manufacturing-related stressors. Genes for histidine kinases in enterococci and Na + /K + antiporters and F0F1 ATP synthases in Lactobacillus strains may support their tolerance to acid seen in vitro. The bile salt hydrolase (BSH) gene in Lp. plantarum and L. paragasseri may help explain their reported bile salt deconjugation and cholesterol-lowering behavior. Thioredoxin is the principal antioxidant system, and several oxidases and general stress-related proteins are found in lactobacilli, most notably in L. plantarum NPL1378. Multiple adhesion and biofilm-related genes were predicted in the LAB genomes. Adhesion and biofilm-related genes figured prominently in the genomes of enterococcal strains, especially E. lactis, corresponding to its biofilm formation capacity in vitro. Bacteriocin and secondary metabolite biosynthetic gene clusters in the sequenced genomes of E. lactis NPL1366 and Lp. plantarum NPL1378 may explain their in vitro pathogenic antagonism. Moreover, folate producing Lp. plantarum strain holds potential to be used in therapeutics or biofortification of food. All the strains were deemed safe through in vitro and in silico analysis. This basic genetic and phenotypic information supports their contention as probiotic adjuncts to conventional medical therapy.

Metataxonomic analysis of microbiota from Pakistani dromedary camelids milk and characterization of a newly isolated Lactobacillus fermentum strain with probiotic and bio-yogurt starter traits.

This study was undertaken to investigate the starter and probiotic potential of lactic acid bacteria isolated from dromedarian camel's milk using both culture-dependent and -independent approaches and metataxonomic analysis. Strains of lactic acid bacteria recovered were examined in vitro for tolerance to gastric acidity, bile, and lysozyme. Bile salt hydrolysis, serum cholesterol-lowering, oxalate degradation, proteolytic activity, exopolysaccharide production, and cell surface characteristics necessary for colonizing intestinal mucosa were also evaluated. A single strain of the species, Lactobacillus fermentum named NPL280, was selected through multivariate analysis as it harbored potential probiotic advantages and fulfilled safety criteria. The strain assimilated cholesterol, degraded oxalate, produced exopolysaccharides, and proved to be a proficient alternate yogurt starter with good viability in stored bio-yogurt. A sensorial analysis of the prepared bio-yogurt was also found to be exemplary. We conclude that the indigenous L. fermentum strain NPL280 has the desired traits of a starter and adjunct probiotic culture for dairy products.

Mining indigenous honeybee gut microbiota for Lactobacillus with probiotic potential.

The present study was done to explore the diversity of lactic acid bacteria (LAB) associated with the gastrointestinal tract (GIT) of honeybee species endemic to northeastern Pakistan. Healthy worker bees belonging to Apis mellifera, A. dorsata, A. cerana and A. florea were collected from hives and the surroundings of a major apiary in the region. The 16S rRNA amplicon sequencing revealed a microbial community in A. florea that was distinct from the others in having an abundance of Lactobacillus and Bifidobacteria. However, this was not reflected in the culturable bacteria obtained from these species. The isolates were characterized for safety parameters, and 20 LAB strains deemed safe were evaluated for resistance to human GIT stresses like acid and bile, adhesion and adhesiveness, and anti-pathogenicity. The five most robust strains, Enterococcus saigonensis NPL780a, Lactobacillus rapi NPL782a, Lactobacillus kunkeei NPL783a, and NPL784, and Lactobacillus paracasei NPL783b, were identified through normalized Pearson (n) principal components analysis (PCA). These strains were checked for inhibition of human pathogens, antibiotic resistance, osmotic tolerance, metabolic and enzymatic functions, and carbohydrate utilization, along with antioxidative and cholesterol-removing potential. The findings suggest at least three strains (NPL 783a, 784 and 782a) as candidates for further in vitro and in vivo investigations of their potential health benefits and application as novel probiotic adjuncts.

Cholate-stimulated biofilm formation by Lactococcus lactis cells.

Bile acid resistance by Lactococcus lactis depends on the ABC-type multidrug transporter LmrCD. Upon deletion of the lmrCD genes, cells can reacquire bile acid resistance upon prolonged exposure to cholate, yielding the ΔlmrCD(r) strain. The resistance mechanism in this strain is non-transporter based. Instead, cells show a high tendency to flocculate, suggesting cell surface alterations. Contact angle measurements demonstrate that the ΔlmrCD(r) cells are equipped with an increased cell surface hydrophilicity compared to those of the parental and wild-type strains, while the surface hydrophilicity is reduced in the presence of cholate. ΔlmrCD(r) cells are poor in biofilm formation on a hydrophobic polystyrene surface, but in the presence of subinhibitory concentrations of cholate, biofilm formation is strongly stimulated. Biofilm cells show an enhanced extracellular polymeric substance production and are highly resistant to bile acids. These data suggest that non-transporter-based cholate resistance in L. lactis is due to alterations in the cell surface that stimulate cells to form resistant biofilms.

The ABC-type multidrug resistance transporter LmrCD is responsible for an extrusion-based mechanism of bile acid resistance in Lactococcus lactis.

Upon prolonged exposure to cholate and other toxic compounds, Lactococcus lactis develops a multidrug resistance phenotype that has been attributed to an elevated expression of the heterodimeric ABC-type multidrug transporter LmrCD. To investigate the molecular basis of bile acid resistance in L. lactis and to evaluate the contribution of efflux-based mechanisms in this process, the drug-sensitive L. lactis NZ9000 DeltalmrCD strain was challenged with cholate. A resistant strain was obtained that, compared to the parental strain, showed (i) significantly improved resistance toward several bile acids but not to drugs, (ii) morphological changes, and (iii) an altered susceptibility to antimicrobial peptides. Transcriptome and transport analyses suggest that the acquired resistance is unrelated to elevated transport activity but, instead, results from a multitude of stress responses, changes to the cell envelope, and metabolic changes. In contrast, wild-type cells induce the expression of lmrCD upon exposure to cholate, whereupon the cholate is actively extruded from the cells. Together, these data suggest a central role for an efflux-based mechanism in bile acid resistance and implicate LmrCD as the main system responsible in L. lactis.