Antibacterial and Anti-Biofilm Efficacy of Endolysin LysAB1245 against a Panel of Important Pathogens.

Infections caused by antibiotic-resistant bacteria pose a significant global challenge. This study explores the antibacterial effects of a bacteriophage-derived endolysin, LysAB1245, against important pathogens, including Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Staphylococcus aureus. We determined the minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) for all tested isolates. A time-kill study was conducted to evaluate the reduction in bacterial survival following treatment with LysAB1245. Additionally, the effects of LysAB1245 on P. aeruginosa K1455 and methicillin-resistant S. aureus (MRSA) NPRC 001R-formed biofilms were investigated. The MIC and MBC of LysAB1245 against all the tested isolates ranged from 4.68 to 9.36 µg/mL and 4.68 to 18.72 µg/mL, respectively. The time-kill study demonstrated more than a 4 log CFU/mL (99.99%) reduction in bacterial survival within 6 h of LysAB1245 treatment at 2MIC. LysAB1245 (1/8-1/2MIC) treatment significantly reduced biofilms formed by P. aeruginosa and MRSA in a concentration-dependent manner. Furthermore, scanning electron and confocal laser scanning microscopy confirmed the potential inhibition effects on 3-day established biofilms formed on abiotic surfaces upon treatment with LysAB1245 at 2MIC. The findings indicate that endolysin LysAB1245 could be employed as a new alternative therapeutic antibacterial and anti-biofilm agent for combating biofilm-related infections.

Characterization of a novel bacteriophage endolysin (LysAB1245) with extended lytic activity against distinct capsular types associated with Acinetobacter baumannii resistance.

Capsular polysaccharides are considered as major virulence factors associated with the ability of multidrug-resistant (MDR) Acinetobacter baumannii to cause severe infections. In this study, LysAB1245, a novel bacteriophage-encoded endolysin consisting of a lysozyme-like domain from phage T1245 was successfully expressed, purified, and evaluated for its antibacterial activity against distinct capsular types associated with A. baumannii resistance. The results revealed a broad spectrum activity of LysAB1245 against all clinical MDR A. baumannii isolates belonging to capsular type (KL) 2, 3, 6, 10, 47, 49, and 52 and A. baumannii ATCC 19606. At 2 h following the treatment with 1.7 unit/reaction of LysAB1245, more than 3 log reduction in the numbers of bacterial survival was observed. In addition, LysAB1245 displayed rapid bactericidal activity within 30 min (nearly 3 log CFU/mL of bacterial reduction). Thermostability assay indicated that LysAB1245 was stable over a broad range of temperature from 4 to 70°C, while pH sensitivity assay demonstrated a wide range of pH from 4.5 to 10.5. Furthermore, both minimal inhibitory concentration (MIC) and minimal bactericidal concentration (MBC) of LysAB1245 against all MDR A. baumannii isolates and A. baumannii ATCC 19606 were 4.21 µg/mL (0.1 unit/reaction). Conclusively, these results suggest that LysAB1245 possesses potential application for the treatment of nosocomial MDR A. baumannii infections.

Isolation and Characterisation of Bacteriophage Selective for Key Acinetobacter baumannii Capsule Chemotypes.

Nineteen bacteriophages against five main capsular types of multidrug-resistant Acinetobacter baumannii were isolated from tertiary care hospital sewage. Eight representative phages from each capsular type were characterized and tested for their biological properties. The biological features revealed that phages T1245, T444, and T515 had a large burst size of more than 420 pfu/mL, together with a short latent period lasting less than 6 min, and were readily adsorbed to a bacterial host within 10 min. Moreover, these phages demonstrated host specificity and stability over a broad range of temperatures (-20 to 60 °C) and pH (5.0-9.0). A whole-genome analysis of six lytic and two temperate phages revealed high genomic similarity with double-stranded DNA between 40 and 50 kb and G + C content of 38-39%. The protein compositions disclosed the absence of toxin-coding genes. The phylogenic results, together with morphological micrographs, confirmed that three selected phages (T1245, T444, and T515) belong to the Podoviridae family within the order Caudovirales. The biological data and bioinformatics analysis indicated that these novel A. baumannii phages possess important enzymes, including depolymerase and endolysin, which could be further developed as promising alternative antibacterial agents to control A. baumannii infections.

Interaction of lytic phage T1245 with antibiotics for enhancement of antibacterial and anti-biofilm efficacy against multidrug-resistant Acinetobacter baumannii.

Biofilms associated with multidrug-resistant (MDR) Acinetobacter baumannii on medical devices remain a big clinical problem. Antibiotic susceptibility tests were performed with eight commonly employed antibiotics against clinical isolates. The effects of antibiotics in combination with well-characterized lytic phage T1245 were studied to assess their antibacterial and anti-biofilm efficacy. Ceftazidime, colistin, imipenem, and meropenem significantly reduced bacterial density up to approximately 80% when combined with phage T1245, compared with control. Phage T1245 in combination with ceftazidime, colistin, and meropenem at subinhibitory concentrations demonstrated significant reduction in biomass and bacterial viability of 3-day established biofilms, compared with antibiotic alone. In addition, electron microscopy further confirmed the disruption of biofilm structure and cell morphology upon treatment with phage T1245 and antibiotics, including ceftazidime, colistin, and meropenem. Combined treatment of phage T1245 with these antibiotics could be employed for the management of A. baumannii infections and eradication of the bacterial biofilms.

Genomic and Phenotypic Analyses of Acinetobacter baumannii Isolates From Three Tertiary Care Hospitals in Thailand.

Antibiotic resistant strains of Acinetobacter baumannii are responsible for a large and increasing burden of nosocomial infections in Thailand and other countries of Southeast Asia. New approaches to their control and treatment are urgently needed and an attractive strategy is to remove the bacterial polysaccharide capsule, and thus the protection from the host's immune system. To examine phylogenetic relationships, distribution of capsule chemotypes, acquired antibiotic resistance determinants, susceptibility to complement and other traits associated with systemic infection, we sequenced 191 isolates from three tertiary referral hospitals in Thailand and used phenotypic assays to characterize key aspects of infectivity. Several distinct lineages were circulating in three hospitals and the majority belonged to global clonal group 2 (GC2). Very high levels of resistance to carbapenems and other front-line antibiotics were found, as were a number of widespread plasmid replicons. A high diversity of capsule genotypes was encountered, with only three of these (KL6, KL10, and KL47) showing more than 10% frequency. Almost 90% of GC2 isolates belonged to the most common capsule genotypes and were fully resistant to the bactericidal action of human serum complement, most likely protected by their polysaccharide capsule, which represents a key determinant of virulence for systemic infection. Our study further highlights the importance to develop therapeutic strategies to remove the polysaccharide capsule from extensively drug-resistant A. baumanii during the course of systemic infection.

Eucalyptus citriodora leaf extract-mediated biosynthesis of silver nanoparticles: broad antimicrobial spectrum and mechanisms of action against hospital-acquired pathogens.

Pathogen resistance to conventional antibiotics has become a serious clinical and public health problem, making the development of an alternative mean a very urgent issue. Recently, biosynthesis of silver nanoparticles (AgNPs) was successfully accomplished in the presence of Eucalyptus citriodora leaf extract as a reducing agent. In this study, the antimicrobial mechanisms of AgNPs against important hospital-acquired pathogens, including Gram-positive, Gram-negative bacteria, and fungi were further assessed. The results indicated that AgNPs could enhance a broad antimicrobial spectrum against drug-resistant organisms, with a range of minimum inhibitory concentration from 0.02 to 0.36 µg/mL. Time-kill assay showed that AgNPs produced bactericidal effects on the microorganisms. AgNPs could significantly reduce biofilm production in pathogens without affecting growth of the pathogens (p < 0.05). AgNPs inhibited cell viability and biofilm formation in a dose-dependent manner. Cell membrane damage in microorganisms resulting from effects of AgNPs was observed. A significant increase in per cent uptake of crystal violet was observed in all isolates treated with AgNPs when compared with the control (p < 0.05). Upon treatment with AgNPs, the surface charge of the reference strains and clinical isolates of pathogens moved towards neutral. The alteration of surface potential after exposure to AgNPs could contribute to membrane disruption and cell viability. Scanning electron microscopy further confirmed morphological cell changes and disrupted the cell membrane. Increasing resistance to AgNPs was not induced by stepwise isolation of the bacteria after 45 passages on Luria-Bertani agar supplemented with AgNPs. Furthermore, AgNPs was not toxic to red blood cells.