Characterization of an endo-beta-1,4 glucanase gene from paper-degrading and denim bio-stoning cellulase producing Aspergillus isolates.

Cellulases are used in textile, pulp and paper, brewery and wine, sugars, and ethanol industries. Four fungal isolates obtained from organic municipal solid wastes (OMSW) were selected based on their cellulolytic activity on carboxymethyl cellulose (CMC) agar medium. Based on the internal transcribed spacer (ITS) sequence of the ribosomal DNA, the four cellulolytic isolates were identified as Aspergillus fumigatus AKAL1, Aspergillus oryzae AKAL4, Aspergillus flavus AKAL8, and Aspergillus flavus AKAL9. After 9 days of fermentation at 30°C and pH 6.5 under 110 rpm agitation, these isolates produced the maximum amount of cellulase. The cellulase showed optimum activity at temperature 35-40°C and pH 6.0-7.0 and was stable for 1 h at 25-45°C and pH 5.0-7.0. The Mg2+ and Zn2+ significantly increased but Hg2+ , K+ , and Ca2+ severely repressed the cellulase activity. Degradation of filter papers and bio-stoning of denim was successfully done with the crude cellulase. An endo-ß-1,4-glucanase was isolated and characterized from Aspergillus isolates. Genome-wide analysis revealed that the genomes of A. oryzae, A. fumigatus, and A. flavus, the pertinent species of the fungal isolates, had 23, 25, and 22 cellulase genes, respectively. Phylogenetic analysis revealed that the cellulases in these fungal species were divided into three major groups, and the isolated endo-ß-1,4-glucanase clustered to Group II. Ten different motifs are present in cellulases of the three species. Results herein provide a valuable resource for understanding cellulase genes in Aspergillus species and potential application of cellulase in textile and fermentable sugars production industries.

Empowering antimicrobial photodynamic therapy of Staphylococcus aureus infections with potassium iodide.

Infections caused by the Gram-positive bacterium Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), impose a great burden on global healthcare systems. Thus, there is an urgent need for alternative approaches to fight staphylococcal infections, such as targeted antimicrobial photodynamic therapy (aPDT). We recently reported that targeted aPDT with the S. aureus-specific immunoconjugate 1D9-700DX can be effectively applied to eradicate MRSA. Nonetheless, the efficacy of aPDT in the human body may be diminished by powerful antioxidant activities. In particular, we observed that the efficacy of aPDT with 1D9-700DX towards MRSA was reduced in human plasma. Here we show that this antagonistic effect can be attributed to human serum albumin, which represents the largest pool of free thiols in plasma for trapping reactive oxygen species. Importantly, we also show that our targeted aPDT approach with 1D9-700DX can be empowered by the non-toxic inorganic salt potassium iodide (KI), which reacts with the singlet oxygen produced upon aPDT, resulting in the formation of free iodine. The targeted iodine formation allows full eradication of MRSA (more than 6-log reduction) without negatively affecting other non-targeted bacterial species or human cells. Altogether, we show that the addition of KI allows a drastic reduction of both the amount of the immunoconjugate 1D9-700DX and the irradiation time needed for effective elimination of MRSA by aPDT in the presence of human serum albumin.

Extracellular metabolites of endophytic fungi from Azadirachta indica inhibit multidrug-resistant bacteria and phytopathogens.

Aim: To evaluate antimicrobial activity of extracellular metabolites (EMs) of endophytic fungal isolates (EFIs) from Azadirachta indica. Materials & methods: EFIs were identified by internal transcribed spacer (ITS) sequencing. Antimicrobial activity, and minimum inhibitor concentration (MIC) and minimum bactericidal concentration (MBC) were determined using agar diffusion and microdilution method, respectively. Results: Seventeen EFIs were isolated from different organs of A. indica. Eight of them were identified based on ITS sequencing. The EMs of EFIs inhibited the growth of six multidrug-resistant (MDR) bacterial superbugs and three phytopathogenic fungi. The MDR bacterial superbugs are resistant to six commercial antibiotics of different generations but susceptible to EMs of EFIs. The MIC (0.125-1.0 µg/µl), MBC (0.5-4.0 µg/µl) and minimum fungicidal concentration (1.0-4.0 µg/µl) of the EMs from EFIs are lower enough. Conclusion: The EMs of the EFIs have promising antimicrobial activity against MDR bacteria and phytopathogenic fungi.

Fighting Staphylococcus aureus infections with light and photoimmunoconjugates.

Infections caused by multidrug-resistant Staphylococcus aureus, especially methicillin-resistant S. aureus (MRSA), are responsible for high mortality and morbidity worldwide. Resistant lineages were previously confined to hospitals but are now also causing infections among healthy individuals in the community. It is therefore imperative to explore therapeutic avenues that are less prone to raise drug resistance compared with today's antibiotics. An opportunity to achieve this ambitious goal could be provided by targeted antimicrobial photodynamic therapy (aPDT), which relies on the combination of a bacteria-specific targeting agent and light-induced generation of ROS by an appropriate photosensitizer. Here, we conjugated the near-infrared photosensitizer IRDye700DX to a fully human mAb, specific for the invariantly expressed staphylococcal antigen immunodominant staphylococcal antigen A (IsaA). The resulting immunoconjugate 1D9-700DX was characterized biochemically and in preclinical infection models. As demonstrated in vitro, in vivo, and in a human postmortem orthopedic implant infection model, targeted aPDT with 1D9-700DX is highly effective. Importantly, combined with the nontoxic aPDT-enhancing agent potassium iodide, 1D9-700DX overcomes the antioxidant properties of human plasma and fully eradicates high titers of MRSA. We show that the developed immunoconjugate 1D9-700DX targets MRSA and kills it upon illumination with red light, without causing collateral damage to human cells.

Identification of AcrAB-TolC Efflux Pump Genes and Detection of Mutation in Efflux Repressor AcrR from Omeprazole Responsive Multidrug-Resistant Escherichia coli Isolates Causing Urinary Tract Infections.

Antimicrobial resistance poses a threat in the treatment of infectious diseases in Bangladesh as well as in the world. Multidrug-resistant (MDR) Enterobacteriaceae, the most common cause of one such infectious disease, urinary tract infection (UTI), has contributed to the escalating problem of selecting empiric antibiotics against UTIs. The aim of this study was to investigate the presence of the efflux pump in MDR Escherichia coli isolates from UTI in the North-East region of Bangladesh, to isolate and characterize the AcrAB-TolC efflux pump genes of these locally isolated strains and to do mutation analysis of the efflux pump repressor AcrR gene to understand the AcrAB-TolC efflux pump mechanism. In the presence of omeprazole, an efflux pump inhibitor, every MDR E. coli isolate showed increased susceptibility to at least 1 of the 7 antibiotics investigated, indicating that efflux pump might be involved in their antibiotic resistance. Omeprazole decreased the minimum inhibitory concentration of every antibiotics being investigated by 2- to 8-fold. DNA and the deduced amino acid sequences of the polymerase chain reaction (PCR) products analyzed by bioinformatics tools revealed that the chromosomal AcrAB-TolC and AcrR genes were present in all MDR and antibiotic-susceptible E. coli isolates. However, the deduced amino acid sequences of the amplification refractory mutation system (ARMS) PCR product of the AcrR gene revealed that the substitution of arginine to cysteine at position 45 of AcrR was observed only in the MDR E. coli whose antibiotic susceptibility increased in the presence of omeprazole. Data reported herein support the notion that the increased antibiotic susceptibility of the MDR E. coli isolates in the presence of omeprazole might be due to efflux pump(s) inhibition and the AcrAB-TolC efflux pump might be a contributor to antibiotic resistance when the mutation of arginine to cysteine occurs at position 45 of AcrR.