Synthesis of Silver Nanoparticles from Ganoderma Species and Their Activity against Multi Drug Resistant Pathogens.

The widespread and indiscriminate use of broad-spectrum antibiotics leads to microbial resistance, which causes major problems in the treatment of infectious diseases. However, advances in nanotechnology using mushrooms have opened up new domains for the synthesis and use of nanoparticles against multidrug-resistant pathogens. Mushooms have recently attracted attention and are exploited for food and medicinal purposes. The current study focuses on the molecular identification, characterization of biologically synthesized silver nanoparticles by X-ray diffraction (XRD) spectroscopy, Fourier Transform Infrared Spectroscopy (FTIR), UV-Vis spectroscopy and scanning electron microscopy (SEM) and antibacterial analysis of extract and silver nanoparticles (AgNPs) synthesis from Ganoderma resinaceum against multidrug resistant microbes. Accurate identification of mushrooms is key in utilizing them for the benefit of humans. However, morphological identification of mushrooms is time consuming, tedious and may be prone to error. Molecular techniques are quick and reliable tools that are useful in mushroom taxonomy. Blast results showed that G. resinaceum (GU451247) obtained from Pakistan was 97 % same to the recognized G. resinaceum (GU451247) obtained from China as well as G. resinaceum (GU451247) obtained from India. The antimicrobial potential of mushroom composite and AgNPs showed high efficacy against pathogenic Staphylococcus aureus (ZOI 23 mm) K. pneumonia (ZOI 20 mm), Pseudomonas aeruginosa (ZOI 24 mm) and E. fecalis and A. baumannii (ZOI 10 mm), and multidrug resistant (MDR) A. baumannii (ZOI 24 mm). XRD evaluation revealed the crystalline composition of synthesized NPs with diameter of 45 nm. UV-Vis spectroscopy obsorption peaked of 589 nm confirmed the presence of AgNPs. SEM results showed the cubic morphology of AgNPs. The FTIR analysis of NPs obtained from G. resinaceum containing C=O as well as (O=C-H) stretching revealed presence of hydrogen, carbonyl and amide groups. The synthesized extract and AgNPs showed promising minimum inhibitory concentration (MIC) at 2 mg concentration against the MDR strains. AgNPs are observed to be efficient as they need less quantities to prevent bacterial growth. In the view of challenges for developing antimicrobial NPs of variable shape and size by various other methods, tuning nanoparticles synthesized via mushrooms can be a wonderful approach to resolve existing hurdles.

TAC1, an unclassified bacteriophage of the family Myoviridae infecting Acinetobacter baumannii with a large burst size and a short latent period.

Bacteriophage TAC1 was isolated using a clinical isolate of Acinetobacter baumannii as the host. It showed stability over wide pH and temperature range and has exhibited in vitro antibacterial activity when applied at an MOI of 1. It demonstrated a broad intraspecies host range and infected 66% of the isolates tested. It has produced 454 virions from a single bacterium with a short latent period of 15 minutes. TAC1 has a linear dsDNA genome with a length of 101.77 kb and 37.5% GC content. The genome encodes 161 proteins and 13 putative tRNAs. Whole-genome sequence comparisons using BLASTn and phylogenetic analysis showed that TAC1 is related to unclassified bacteriophages of the family Myoviridae.

RLP, a bacteriophage of the family Podoviridae, rescues mice from bacteremia caused by multi-drug-resistant Pseudomonas aeruginosa.

Antimicrobial resistance is a serious threat to public health around the globe. According to the World Health Organization, there will be a return to the pre-penicillin era by 2050 if no new antimicrobials are discovered. It is therefore necessary to find new antimicrobials and alternatives. Pseudomonas aeruginosa exhibits resistance against many antibiotics and causes a variety of infections in immunocompromised individuals and especially in those with burn wounds and lung infections. Bacteriophage RLP against P. aeruginosa strain PA-1 was isolated from the Ravi River near Lahore. It showed marked stability at different pH values and temperatures, with the maximum storage stability at 4 °C. It demonstrated the ability to inhibit bacterial growth for up to 20 h, replicated in 25 min, and produced 154 virions per infected cell. RLP showed a broad host range, infecting 50% (19/38) of the multiple-drug-resistant (MDR) P. aeruginosa strains that were tested. The 43-kbp-long genome of RLP is a double-stranded DNA molecule that encodes 56 proteins in total: 34 with known functions, and 22 with no homolog in the gene databases. A cascade system of lytic machinery is also present in the form of four genes (R/z, R/z1, holin and endolysin). Therapeutic studies of RLP in bacteremic mice infected with P. aeruginosa strain PA-1 demonstrated a 92% survival rate in the treated group compared with 7.4% in the untreated group, and this result was statistically significant. Based on its physiological and genetic properties, ability to cause a reduction in bacterial growth in vitro and its in vivo therapeutic efficacy, RLP could be a good candidate for use in phage therapy.

A K-17 serotype specific Klebsiella phage JKP2 with biofilm reduction potential.

Klebsiella pneumoniae is an opportunistic pathogen responsible for nearly one-third of all Gram-negative infections. Increasing antibiotic resistance has pushed scientists to look for alternative therapeutics. Bacteriophages have emerged as one of the promising alternatives. In the current study, the Klebsiella phage JKP2 was isolated from a sewage sample and characterized against the K-17 serotype of K. pneumoniae. It produced bulls-eye-shaped clear plaques and has a latent period of 45 min with a burst size of 70 pfu/cell. It remained stable at tested pH (5 to 10) and temperatures (37 to 60 °C). Its optimum temperature for long-term storage is 4 °C and -80 °C. The JKP2 showed its infectivity against the K. pneumoniae K-17 serotype only. It controlled planktonic cells of K. pneumoniae 12 h post-incubation. At MOI-1, it efficiently eliminated 98% of 24 and 96% of 48-hour-old biofilm and 86% and 82% of mature biofilm of day 3 and 4, respectively. The JKP2 has an icosahedral capsid of 54 ± 0.5 nm with a short, non-contractile tail, measuring 12 ± 0.2 nm. It possesses a double-stranded DNA genome of 43.2 kbp with 54.1% GC content and encodes 54 proteins, including 29 with known functions and 25 with unknown functions. JKP2 was classified as Drulisvirus within the Autographiviridae family. It uses a T7-like direct terminal repeat strategy for genome packaging. JKP2 can be applied safely for therapeutic purposes as it does not encode an integrase or repressor genes, antibiotic resistance genes, bacterial virulence factors, and mycotoxins.

A single dose of a virulent bacteriophage vB PaeP-SaPL, rescues bacteremic mice infected with multi drug resistant Pseudomonas aeruginosa.

Multidrug resistant bacterial infections are difficult to treat and contribute to high morbidity and mortality. The phage vB PaeP-SaPL was isolated from a sewage drain (Lahore, Pakistan) against Pseudomonas aeruginosa PA-1 (NCBI Accession number MG763232). SaPL produced circular, transparent plaques, 4-5 mm in diameter and showed broad host range infecting 57 % of tested MDR P. aeruginosa clinical isolates (N = 38), while no infectivity was observed against any tested strains of other genera. SaPL inhibited PA-1 growth until 24 h post infection at MOI of 1. The SaPL showed stability at varying temperature and pH, with optimum stability at pH 7 and 45 °C. The latent period of SaPL was 20 min with burst size of 155 virions. The genome of SaPL was double stranded DNA of 45,796 bps having 63 CDS (13 for known proteins and 50 for hypothetical proteins) with a GC content of 52 %. The termini analysis revealed that SaPL genome ends are redundant and permuted. The packaging strategy used by SaPL was a headful (pac) strategy like P1 phage. Survivability of PA-1 challenged mice, treated with SaPL (100 %) was statistically significant (P < 0.05) than in untreated challenged mice (0%). Based on its efficacy in reducing bacterial growth, selective infectivity against majority of P. aeruginosa strains and its ability to increase survivability in PA-1 challenged mice, SaPL is proposed to be a potential candidate for bacteriophage therapy against difficult to treat MDR P. aeruginosa infections.

Characterization of a lytic EBP bacteriophage with large size genome against Enterobacter cloacae.

Enterobacter cloacae (E. cloacae) is an emerging nosocomial pathogen that had acquired antibiotic resistance against multiple classes of antibiotics. The current study was aimed to isolate and characterize lytic bacteriophage against E. cloacae. The bacteriophage EBP was isolated from a sewage water sample using E. cloacae as a host strain by double-layer agar technique. EBP was found stabile at a wide range of temperatures (25, 37, 60, and 80°C) and pH (5, 6, 7, 8, and 9) with antibacterial activity up to 24 h of infection. The latent period of EBP was 20 min with a burst size of 252 phages per cell. It showed a narrow host range and infected 12/21 (57%) isolates of E. cloacae tested. It has helical symmetry with a head size of 105 and 120 nm long tail with contractile sheath. The EBP has 179.1 kb long double-stranded DNA genome with 44.8% GC content. Majority of identified ORFs (187/281) were encoding putative proteins with unknown function. Necessary replication enzymes, structural proteins, and lytic enzymes were detected in the genome of EBP. Phylogenetic analysis revealed that EBP closely resembles with Coronobacter phage vB_CsaM_IeN, vB_CsaM_IeE, vB_CsaM_IeB, and Citrobacter phage Margaery. Based on electron microscopy and molecular characterization, EBP was classified as a Myoviridae phage.

Insight into Acinetobacter baumannii: pathogenesis, global resistance, mechanisms of resistance, treatment options, and alternative modalities.

Acinetobacter baumannii, once considered a low-category pathogen, has emerged as an obstinate infectious agent. The scientific community is paying more attention to this pathogen due to its stubbornness to last resort antimicrobials, including carbapenems, colistin, and tigecycline, its high prevalence of infections in the hospital setting, and significantly increased rate of community-acquired infections by this organism over the past decade. It has given the fear of pre-antibiotic era to the world. To further enhance our understanding about this pathogen, in this review, we discuss its taxonomy, pathogenesis, current treatment options, global resistance rates, mechanisms of its resistance against various groups of antimicrobials, and future therapeutics.

Assessment of biofilm removal capacity of a broad host range bacteriophage JHP against Pseudomonas aeruginosa.

Pseudomonas aeruginosa is an efficient biofilm-dwelling microbial pathogen, associated with nosocomial infections. These biofilm-associated infections are resistant to antibiotics and immune defenses, therefore pose major problem against their treatment. This scenario demands alternative therapeutic regimens, and bacteriophage therapy is one among potential strategies for clinical management of multiple drug resistance. In this investigation, the efficacy of a bacteriophage, JHP, is evaluated to eradicate P. aeruginosa biofilms. Growth kinetics of P. aeruginosa biofilm revealed that the highest cell density biofilm (1.5 × 1016 CFU/mL) was established within the polystyrene microtiter plate at 72 h post inoculation. Pseudomonas aeruginosa biofilms of different ages, treated with JHP (0.6 MOI) for different post-infection durations, reduced biomass from 2 to 4.5 logs (60-90%). JHP treatment before biofilm development reduced the bacterial load up to 9 logs (>95% bacterial load reduction) as compared with untreated control, which highlights its potential to prevent biofilm formation in indwelling medical devices. Combinations of JHP with other phages or antibiotics could be an efficient alternative for P. aeruginosa biofilm removal in clinical and industrial settings.