Characterization of eDNA from the Clinical Strain Acinetobacter baumannii AIIMS 7 and Its Role in Biofilm Formation

Release of extracellular DNA (eDNA) was observed during in vitro growth of a clinical strain of Acinetobacter baumannii. Membrane vesicles (MV) of varying diameter (20–200 nm) containing DNA were found to be released by transmission electron microscopy (TEM) and atomic force microscopy (AFM). An assessment of the characteristics of the eDNA with respect to size, digestion pattern by DNase I/restriction enzymes, and PCR-sequencing, indicates a high similarity with genomic DNA. Role of eDNA in static biofilm formed on polystyrene surface was evaluated by biofilm augmentation assay using eDNA available in different preparations, for example, whole cell lysate, cell-free supernatant, MV suspension, and purified eDNA. Biofilm augmentation was seen up to 224.64%, whereas biofilm inhibition was 59.41% after DNase I treatment: confirming that eDNA facilitates biofilm formation in A. baumannii. This is the first paper elucidating the characteristics and role of eDNA in A. baumannii biofilm, which may provide new insights into its pathogenesis

An MFS Transporter-Like ORF from MDR Acinetobacter baumannii AIIMS 7 Is Associated with Adherence and Biofilm Formation on Biotic/Abiotic Surface

A major facilitator superfamily (MFS) transporter-like open reading frame (ORF) of 453 bp was identified in a pathogenic strain Acinetobacter baumannii AIIMS 7, and its association with adherence and biofilm formation was investigated. Reverse transcription PCR (RT-PCR) showed differential expression in surface-attached biofilm cells than nonadherent cells. In vitro translation showed synthesis of a ~17 kDa protein, further confirmed by cloning and heterologous expression in E. coli DH5α. Up to 2.1-, 3.1-, and 4.1- fold biofilm augmentation was observed on abiotic (polystyrene) and biotic (S. cerevisiae/HeLa) surface, respectively. Scanning electron microscopy (SEM) and gfp-tagged fluorescence microscopy revealed increased adherence to abiotic (glass) and biotic (S. cerevisiae) surface. Extracellular DNA(eDNA) was found significantly during active growth; due to probable involvement of the protein in DNA export, strong sequence homology with MFS transporter proteins, and presence of transmembrane helices. In summary, our findings show that the putative MFS transporter-like ORF (pmt) is associated with adherence, biofilm formation, and probable eDNA release in A. baumannii AIIMS 7

Emerging Strategies to Combat ESKAPE Pathogens in the Era of Antimicrobial Resistance: A Review

The acronym ESKAPE includes six nosocomial pathogens that exhibit multidrug resistance and virulence: Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp. Persistent use of antibiotics has provoked the emergence of multidrug resistant (MDR) and extensively drug resistant (XDR) bacteria, which render even the most effective drugs ineffective. Extended spectrum β-lactamase (ESBL) and carbapenemase producing Gram negative bacteria have emerged as an important therapeutic challenge. Development of novel therapeutics to treat drug resistant infections, especially those caused by ESKAPE pathogens is the need of the hour. Alternative therapies such as use of antibiotics in combination or with adjuvants, bacteriophages, antimicrobial peptides, nanoparticles, and photodynamic light therapy are widely reported. Many reviews published till date describe these therapies with respect to the various agents used, their dosage details and mechanism of action against MDR pathogens but very few have focused specifically on ESKAPE. The objective of this review is to describe the alternative therapies reported to treat ESKAPE infections, their advantages and limitations, potential application in vivo, and status in clinical trials. The review further highlights the importance of a combinatorial approach, wherein two or more therapies are used in combination in order to overcome their individual limitations, additional studies on which are warranted, before translating them into clinical practice. These advances could possibly give an alternate solution or extend the lifetime of current antimicrobials

Adiantum philippense

Development of an ecofriendly, reliable, and rapid process for synthesis of nanoparticles using biological system is an important bulge in nanotechnology. Antioxidant potential and medicinal value of Adiantum philippense L. fascinated us to utilize it for biosynthesis of gold and silver nanoparticles (AuNPs and AgNPs). The current paper reports utility of aqueous extract of A. philippense L. fronds for the green synthesis of AuNPs and AgNPs. Effect of various parameters on synthesis of nanoparticles was monitored by UV-Vis spectrometry. Optimum conditions for AuNPs synthesis were 1 : 1 proportion of original extract at pH 11 and 5 mM tetrachloroauric acid, whereas optimum conditions for AgNPs synthesis were 1 : 1 proportion of original extract at pH 12 and 9 mM silver nitrate. Characterization of nanoparticles was done by TEM, SAED, XRD, EDS, FTIR, and DLS analyses. The results revealed that AuNPs and AgNPs were anisotropic. Monocrystalline AuNPs and polycrystalline AgNPs measured 10 to 18 nm in size. EDS and XRD analyses confirmed the presence of elemental gold and silver. FTIR analysis revealed a possible binding of extract to AuNPs through –NH2 group and to AgNPs through C=C group. These nanoparticles stabilized by a biological capping agent could further be utilized for biomedical applications

Antibiofilm and antipersister activity of acetic acid against extensively drug resistant Pseudomonas aeruginosa PAW1.

Pseudomonas aeruginosa is an ESKAPE pathogen associated with difficult-to-treat burn wound and surgical-site infections. This study aimed to characterise an extensively drug resistant (XDR) P. aeruginosa isolate (designated PAW1) and to investigate the antibiofilm and antipersister effect of acetic acid on PAW1. PAW1 was identified using biotypic (VITEK) and genotypic (16S rDNA) analysis. Minimum inhibitory concentration (MIC) and disc susceptibility testing showed high level resistance against all antibiotics from classes including beta lactams, cephems, carbapenems and fluoroquinolones. It was therefore identified as extensively drug resistant (XDR), showing resistance to all antibiotics except for, aminoglycoside (gentamicin and netilmicin) and lipopeptides (polymyxin B). Time kill assays showed antibiotic tolerant, persister cell formation in presence of 100X MICs of gentamicin and polymyxin B. Other virulence traits such as ability to produce lipase, protease, haemolysin, and siderophores and to form biofilms were additional factors which may contribute to its pathogenicity. PAW1 showed promising susceptibility against acetic acid with MIC and minimum biofilm inhibitory concentration of 0.156% (v/v). Percent viability of PAW1 was dependent on dose and treatment time of acetic acid. 0.625% acetic acid treatment of 5 minutes was effective in killing >90% planktonic cells showing lesser toxicity to L929 cells (IC50 = 0.625%). Biofilm disruption caused due to acetic acid was also dose dependent, showing 40.57% disruption after treatment with 0.625% acetic acid for 5 minutes. FESEM imaging and live dead staining of planktonic and biofilm forms of PAW1 confirmed that acetic acid treatment caused 19.04% of cell shrinkage and disruption of extracellular matrix resulting in killing of cells. Antipersister activity of acetic acid was demonstrated by showing complete killing of PAW1 at 4X MIC. Overall, this study characterised an XDR isolate P. aeruginosa showing resistance and tolerance to various antibiotics. Antipersister and antibiofilm effect of acetic acid demonstrates the importance of forgotten topical agents as an effective strategy to treat XDR pathogens

Adiantum philippense L. frond assisted rapid green synthesis of gold and silver nanoparticles

Development of an ecofriendly, reliable, and rapid process for synthesis of nanoparticles using biological system is an important bulge in nanotechnology. Antioxidant potential and medicinal value of Adiantum philippense L. fascinated us to utilize it for biosynthesis of gold and silver nanoparticles (AuNPs and AgNPs). The current paper reports utility of aqueous extract of A. philippense L. fronds for the green synthesis of AuNPs and AgNPs. Effect of various parameters on synthesis of nanoparticles was monitored by UV-Vis spectrometry. Optimum conditions for AuNPs synthesis were 1 : 1 proportion of original extract at pH 11 and 5 mM tetrachloroauric acid, whereas optimum conditions for AgNPs synthesis were 1 : 1 proportion of original extract at pH 12 and 9 mM silver nitrate. Characterization of nanoparticles was done by TEM, SAED, XRD, EDS, FTIR, and DLS analyses. The results revealed that AuNPs and AgNPs were anisotropic. Monocrystalline AuNPs and polycrystalline AgNPs measured 10 to 18 nm in size. EDS and XRD analyses confirmed the presence of elemental gold and silver. FTIR analysis revealed a possible binding of extract to AuNPs through –NH2 group and to AgNPs through C=C group. These nanoparticles stabilized by a biological capping agent could further be utilized for biomedical applications