Expression and characterization of novel chimeric endolysin CHAPk-SH3bk against biofilm-forming methicillin-resistant Staphylococcus aureus.

The continuous evolution of antibiotic resistance in methicillin-resistant Staphylococcus aureus (MRSA) due to the misuse of antibiotics lays out the need for the development of new antimicrobials with higher activity and lower resistance. In this study, we have expressed novel chimeric endolysin CHAPk-SH3bk derived from LysK to investigate its antibacterial activity against planktonic and biofilm-forming MRSA. The molecular docking and MD simulation results identified critical amino acids (ASP47, ASP56, ARG71, and Gly74) of CHAPk domain responsible for its catalytic activity. Chimeric endolysin CHAPk-SH3bk showed an effective binding to peptidoglycan fragment using 14 hydrogen bonds. The in-vitro antibacterial assays displayed higher activity of CHAPk against planktonic MRSA with 2-log10 reduction in 2 h. Both CHAPk and CHAPk-SH3bk displayed bactericidal activity against MRSA with ∼4log10 and ∼3.5log10 reduction in 24 h. Biofilm reduction activity displayed CHAPk-SH3bk reduced 33 % and 60 % of hospital-associated ATCC®BAA-44™ and bovine origin SA1 respectively. The CHAPk treatment reduced 47 % of the preformed biofilm formed by bovine-origin MRSA SA1. This study indicates an effective reduction of preformed MRSA biofilms of human and animal origin using novel chimeric construct CHAPk-SH3bk. Stating that the combination and shuffling of different domains of phage endolysin potentially increase its bacteriolytic effectiveness against MRSA.

A systematic review on antimicrobial activities of green synthesised Selaginella silver nanoparticles.

BACKGROUND: Metallic nanoparticles from different natural sources exhibit superior therapeutic options as compared to the conventional methods. Selaginella species have attracted special attention of researchers worldwide due to the presence of bioactive molecules such as flavonoids, biflavonoids, triterpenes, steroids, saponins, tannins and other secondary metabolites that exhibit antimicrobial, antiplasmodial, anticancer and anti-inflammatory activities. Environment friendly green synthesised silver nanoparticles from Selaginella species provide viable, safe and efficient treatment against different fungal pathogens. OBJECTIVE: This systematic review aims to summarise the literature pertaining to superior antifungal ability of green synthesised silver nanoparticles using plant extracts of Selaginella spp. in comparison to both aqueous and ethanolic raw plant extracts by electronically collecting articles from databases. METHODS: The recommendations of the Preferred Reporting Items for Systematic Reviews and Meta-Analysis were taken into consideration while preparing this review. The titles and abstracts of the collected data were stored in Endnote20 based on the inclusion and exclusion criteria. The search strategy included literature from established sources like PubMed, Google Scholar and Retrieval System Online using subject descriptors. RESULTS: The search yielded 60 articles with unique hits. After removal of duplications, 46 articles were identified, 40 were assessed and only seven articles were chosen and included in this review based on our eligibility criteria. CONCLUSION: The physicochemical and preliminary phytochemical investigations of Selaginella suggest higher drug potency of nanoparticles synthesised from plant extract against different diseases as compared to aqueous and ethanolic plant extracts. The study holds great promise as the synthesis of nanoparticles involves low energy consumption, minimal technology and least toxic effects.

Novel aadA5 and dfrA17 variants of class 1 integron in multidrug-resistant Escherichia coli causing bovine mastitis.

Mobile genetic elements (MGEs) are associated with the emergence of multidrug resistance in extended-spectrum ß-lactamase (ESBL)-producing Enterobacteriaceae. This study explores the role of class 1 integrons and IS26 elements in breaching taxonomic barriers. A total of 110 E. coli bacteria were isolated from 300 clinical mastitis milk samples. The 98% E. coli isolates were extended-spectrum beta-lactamase- producers. About 83% of these isolates carried co-resistance for fluoroquinolones. The co-existence of (extended-spectrum beta-lactamase + quinolone resistance determining region mutations) and (extended-spectrum beta-lactamase + plasmid-mediated quinolone resistance genes) was found in 76% and 44% of isolates, respectively. The MGEs were detected in 88% of isolates with IS26 in 82% and class 1 integrase in 40% of isolates. The types of class 1 integron gene cassettes detected includes dfrA7, (dfrA17 + aadA5), and (dfrA1 + aadA1). We discovered 2 and 4 novel variants of the dfrA17 and aadA5 genes, respectively. We report a variant of aadA5 with mutation E235G in the Indian subcontinent earlier reported only in a human clinical isolate from Belgium. About 19 isolates carried IS26 linked to integrase gene intI1 with an internal deletion of 265 bp in the 5`CS of integrase gene intI1, earlier reported only in E. coli ST131 isolates from human clinical, wastewater samples. This study suggests intercontinental dissemination of antibiotic resistant genes (ARGs) across different microbiomes via mobile genetic elements. KEY POINTS: • The role of mobile genetic elements in the emergence of multidrug-resistant E. coli in bovine mastitis. • Novel variants of the aadA5 (aminoglycoside adenyl transferase) and dfrA17 (dihydrofolate reductase) genes were identified in pathogenic E. coli isolated from bovine mastitis in class 1 integron gene cassette. • Sequence analysis of mobile genetic components revealed the physical connection between IS26 and intI1 genes with an internal deletion in 5'CS of class 1 integrase.

Global epidemiology of CTX-M-type ß-lactam resistance in human and animal.

CTX-M ESBL are widely found in animal and human infections. For better understanding of CTX-M variations and epidemiology, a total of 2210 CTX-M sequences were retrieved from NCBI as at 20 December 2020. The maximum incidences of CTX-M were reported in China (n = 508), USA (n = 354) and Japan (n = 180). Single amino acid substitution in the domain region of CTX-M ESBL lead to survival benefits to the bacteria. A total of 31 different variations were found of which D240G was the most common followed by A77V and V103I substitutions. The variations in CTX-M enzymes were explained continent-wise revealing the maximum variation reported in America followed by Asia and Europe of which D240G substitution was the most prevalent. India contained only three variations (E166A, P167S D240G) found in New Delhi, Karnataka, West Bengal and Tamil Nadu. The P167 and D240 were under strong positive selection with dN/dS calculation.

An efficient method for generating a germ cell depleted animal model for studies related to spermatogonial stem cell transplantation.

BACKGROUND: Spermatogonial stem cell (SSC) transplantation (SSCT) has become important for conservation of endangered species, transgenesis and for rejuvenating testes which have lost germ cells (Gc) due to gonadotoxic chemotherapy or radiotherapy during the prepubertal phase of life. Creating a germ cell-depleted animal model for transplantation of normal or gene-transfected SSC is a prerequisite for such experimental studies. Traditionally used intraperitoneal injections of busulfan to achieve this are associated with painful hematopoietic toxicity and affects the wellbeing of the animals. Use of testicular busulfan has been reported recently to avoid this but with a very low success rate of SSCT. Therefore, it is necessary to establish a more efficient method to achieve higher SSCT without any suffering or mortality of the animals. METHODS: A solution of busulfan, ranging from 25 µg/20 µl to 100 µg/20 µl in 50 % DMSO was used for this study. Each testis received two diagonally opposite injections of 10 µl each. Only DMSO was used as control. Germ cell depletion was checked every 15 days. GFP-expressing SSC from transgenic donor mice C57BL/6-Tg (UBC-GFP) 30Scha/J were transplanted into busulfan-treated testis. Two months after SSCT, mice were analyzed for presence of colonies of donor-derived SSC and their ability to generate offspring. RESULTS: A dose of 75 µg of busulfan resulted in reduction of testis size and depletion of the majority of Gc of testis in all mice within 15 days post injection without causing mortality or a cytotoxic effect in other organs. Two months after SSCT, colonies of donor-derived Gc-expressing GFP were observed in recipient testes. When cohabitated with females, donor-derived offspring were obtained. By our method, 71 % of transplanted males sired transgenic progeny as opposed to 5.5 % by previously described procedures. About 56 % of progeny born were transgenic by our method as opposed to 1.2 % by the previously reported methods. CONCLUSIONS: We have established an efficient method of generating a germ cell-depleted animal model by using a lower dose of busulfan, injected through two diagonally opposite sites in the testis, which allows efficient colonization of transplanted SSC resulting in a remarkably higher proportion of donor-derived offspring generation.

ß-Endorphin inhibits phagocytic activity of lizard splenic phagocytes through µ receptor-coupled adenylate cyclase-protein kinase A signaling pathway.

The receptor-coupled intracellular signaling mechanism of endogenous opioid peptide ß-endorphin (ß-end) is explored for the first time in ectothermic vertebrates using wall lizard as a model. ß-End inhibited the percentage phagocytosis and phagocytic index of lizard splenic phagocytes in a dose-dependent manner. The inhibitory effect of ß-end on phagocytosis was completely antagonized by non-selective opioid receptor antagonist naltrexone and also by selective µ-receptor antagonist CTAP. However, selective antagonists for other opioid receptors like NTI for δ-receptor and NorBNI for κ-receptor did not alter the effect of ß-end on phagocytosis. This suggests that ß-end mediated its inhibitory effect on phagocytic activity of splenic phagocytes exclusively through µ opioid receptors. The µ opioid receptor-coupled downstream signaling cascade was subsequently explored using inhibitors of adenylate cyclase (SQ 22536) and protein kinase A (H-89). Both SQ 22536 and H-89 abolished the inhibitory effect of ß-end on phagocytosis in a concentration-related manner. Implication of cAMP as second messenger was corroborated by cAMP assay where an increase in intracellular cAMP level was observed in response to ß-end treatment. It can be concluded that ß-end downregulated the phagocytic activity of lizard splenic phagocytes through µ opioid receptor-coupled adenylate cyclase-cAMP-protein kinase A pathway.