Diversity of resistant determinants, virulence factors, and mobile genetic elements in Acinetobacter baumannii from India: A comprehensive in silico genome analysis.

Introduction: The frequency of infections associated with multidrug resistant A. baumannii has risen substantially in India. The use of next-generation sequencing (NGS) techniques combined with comparative genomics has great potential for tracking, monitoring, and ultimately controlling the spread of this troublesome pathogen. Here, we investigated the whole genome sequences of 47 A. baumannii from India. Methods: In brief, A. baumannii genomes were analyzed for the presence of antibiotic resistance genes (ARGs), virulence factors genes (VFGs), and mobile genetic elements (MGEs) using various in silico tools. The AbaR-type resistance islands (AbaRIs) were detected by examining the genetic environment of the chromosomal comM gene. Multilocus sequence types were determined using the Pasteur scheme. The eBURST and whole genome SNPs-based phylogenetic analysis were performed to analyze genetic diversity between A. baumannii genomes. Results and discussion: A larger number of A. baumannii isolates belonging to the ST2 genotype was observed. The SNPs-based phylogenetic analysis showed a diversity between compared genomes. The predicted resistome showed the presence of intrinsic and acquired ARGs. The presence of plasmids, insertion sequences, and resistance islands carrying putative ARGs conferring resistance to antibiotics, quaternary ammonium compounds, and heavy metals was predicted in 43 (91%) genomes. The presence of putative VFGs related to adherence, biofilm formation and iron uptake was observed in the study. Overall, the comprehensive genome analysis in this study provides an essential insight into the resistome, virulome and mobilome of A. baumannii isolates from India.

Characterization of Novel Klebsiella Phage PG14 and Its Antibiofilm Efficacy.

The increasing frequency of infections caused by multidrug-resistant Klebsiella pneumoniae demands the development of unconventional therapies. Here, we isolated, characterized, and sequenced a Klebsiella phage PG14 that infects and lyses carbapenem-resistant K. pneumoniae G14. Phage PG14 showed morphology similar to the phages belonging to the family Siphoviridae. The adsorption curve of phage PG14 showed more than 90% adsorption of phages on a host within 12 min. A latent period of 20 min and a burst size of 47 was observed in the one step growth curve. Phage PG14 is stable at a temperature below 30°C and in the pH range of 6 to 8. The PG14 genome showed no putative genes associated with virulence and antibiotic resistance. Additionally, it has shown lysis against 6 out of 13 isolates tested, suggesting the suitability of this phage for therapeutic applications. Phage PG14 showed more than a 7-log cycle reduction in K. pneumoniae planktonic cells after 24 h of treatment at a multiplicity of infection (MOI) of 10. The phage PG14 showed a significant inhibition and disruption of biofilm produced by K. pneumoniae G14. The promising results of this study nominate phage PG14 as a potential candidate for phage therapy. IMPORTANCE Klebsiella pneumoniae is one of the members of the ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter species) group of pathogens and is responsible for nosocomial infections. The global increase of carbapenem-resistant K. pneumoniae has developed a substantial clinical threat because of the dearth of therapeutic choices available. K. pneumoniae is one of the commonly found bacteria responsible for biofilm-related infections. Due to the inherent tolerance of biofilms to antibiotics, there is a growing need to develop alternative strategies to control biofilm-associated infections. This study characterized a novel bacteriophage PG14, which can inhibit and disrupt the K. pneumoniae biofilm. The genome of phage PG14 does not show any putative genes related to antimicrobial resistance or virulence, making it a potential candidate for phage therapy. This study displays the possibility of treating infections caused by multidrug-resistant (MDR) isolates of K. pneumoniae using phage PG14 alone or combined with other therapeutic agents.

Corrigendum: Diversity of resistant determinants, virulence factors, and mobile genetic elements in Acinetobacter baumannii from India: A comprehensive in silico genome analysis.

[This corrects the article DOI: 10.3389/fcimb.2022.997897.].

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.

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 ß-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.