Synergistic and Additive Effects of Menadione in Combination with Antibiotics on Multidrug-Resistant Staphylococcus aureus: Insights from Structure-Function Analysis of Naphthoquinones.

Antimicrobial resistance (AMR) interferes with the effective treatment of infections and increases the risk of microbial spread and infection-related illness and death. The synergistic activities of combinations of antimicrobial compounds offer satisfactory approaches to some extent. Structurally diverse naphthoquinones (NQs) including menadione (-CH3 group at C2) exhibit substantial antimicrobial activities against multidrug-resistant (MDR) pathogens. We explored the combinations of menadione with antibiotic ciprofloxacin or ampicillin against Staphylococcus aureus and its biofilms. We found an additive (0.590 %) were also observed. However, preformed biofilms were not affected. Dent formation was also evident in S. aureus treated with the test compounds. The structure-function relationship (SFR) of NQs was used to determine and predict their activity pattern against pathogens. Analysis of 10 structurally distinct NQs revealed that the compounds with -Cl, -Br, -CH3 , or -OH groups displayed the lowest MICs (32-256 µg/mL). Furthermore, 1,4-NQs possessing a halogen or -CH3 moiety showed elevated ROS activity, whereas molecules with an -OH group affected cell integrity. Improved activity of antimicrobial combinations and SFR approaches are significant in antimicrobial therapies.

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.

Antibacterial and Antibiofilm Potency of Menadione Against Multidrug-Resistant S. aureus.

Menadione is an analogue of 1,4-naphthoquinone (1,4-NQ) that possesses enormous pharmaceutical potential. The minimum inhibitory concentration (MIC) of menadione was determined against eighteen pathogens of the ESKAPE category, including thirteen multidrug-resistant (MDR) and five standard strains. From a total of eighteen pathogens, five strains of S. aureus (four: MDR and one: Standard strain) were considered further for detailed studies. This study included the determination of minimum bactericidal concentration (MBC), time-kill assay, scanning electron microscopic technique (SEM), and detection of reactive oxygen species (ROS). Additionally, the effect of menadione on biofilms of three strains of S. aureus was performed through crystal violet assay, SEM, and confocal laser scanning microscopy (CLSM). Menadione exerted substantial antibacterial activity against S. aureus (S8, S9, NCIM 5021) at a lower MIC (64 µg/mL). Whereas, the MIC of 256 µg/mL was displayed against J2 and J4 (MDR and biofilm-forming strains). The time-killing effect of menadione against S. aureus strains was observed after 9 h at MBCs of 64 µg/mL (NCIM 5021), 128 µg/mL (S8, S9), and 512 µg/mL (J2, J4). Enhanced levels of ROS in all five S. aureus were observed in presence of menadione (MICs and MBCs). The relation of enhanced ROS due to menadione activity invigorated us to explore its effect on S. aureus biofilms. We report menadione-mediated inhibition (> 90%) of biofilm formation (at respective MICs) and effect on preformed biofilms (> 85%) at 1024 µg/mL. Menadione possessing antibacterial and antibiofilm potentials are imperative in the era of multidrug resistance developed by bacterial pathogens.

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.