Summary

Prolonged overuse and misuse of antibiotics, ineffective infection control strategies, and lack of new drug development have led to the occurrence of a phenomenon known as antibiotic resistance. Recently, Acinetobacter baumannii (A. baumannii) emerged as an ESKAPE pathogen of utmost significance in nosocomial settings where they cause various infections with mortality rates as high as 80%. Formation of biofilm and virulence factors are under the control of quorum sensing (QS). Therefore, disruption of QS seems a tempting approach to decrease virulence and biofilm biosynthesis which in turn make them more susceptible to human defense system and antibiotics without imposing evolutionary pressure. In this regard, nanoparticles in particular silver-nanoparticles are reported by numerous studies to be impressive quorum-quenchers. However, studies concerned with QS inhibition in A. baumanni are limited. The scarcity of quorum sensing inhibitor (QSI) for this critical pathogen in parallel with the increasing prevalence of multi-drug resistant (MDR) A. baumannii strains is of a mounting concern. The present study was conducted to investigate the antibiofilm and anti-QS effect of biogenic nano-silvers synthesized by Pseudomonas aeruginosa (P. aeruginosa) against drug-resistant A. baumannii clinical strains. For this purpose, 26 A. baumannii isolates were collected from several laboratories of bacteriology located in Erbil from November 2021 to February 2022 along with an ATCC strain of A. baumannii which was employed as control during the research. Several biochemical tests were used to identify the recovered isolates and Vitek-II system was operated for validation of their identification.

The susceptibility of the clinical bacterial isolates to a panel of antimicrobials was investigated. The results of the Kirby-Bauer and static biofilm assay showed that the isolates exhibited high resistance to the tested antibiotic agents where they were 100% resistant to ceftazidime and cefepime. Moreover, biggest share (66%) of the isolates were extensively drug-resistant (XDR) while 30% and 4% were MDR and non-MDR respectively. As for biofilm formation capacity, the isolates exhibited varying degrees of biofilm production where 11%, 48% and 41% were labeled as strong, moderate and weak biofilm creators.

For extracellular synthesis of silver nanoparticles (AgNPs) an ATCC strain of P. aeruginosa was employed via using silver-nitrate solution as substrate. Alteration in the color of the reaction-mixture to deep brown was indicated as a preliminary sign for presence of AgNPs which was later confirmed by UV-vis (Ultraviolet-visible) spectrophotometer. FTIR (Fourier-transform infrared-spectroscopy) was operated to analyze the possible functional groups that could be accountable for the bio-reduction/capping of AgNPs. SEM (Scanning electron microscopy), EDX (Energy-dispersive X-ray) spectroscopy and XRD (X-ray diffraction) analysis were used to further characterize the harvested AgNPs as crystalline, polydispersed spherical nanocrystals with hardly any agglomerations in the size range of 45-50nm. Average crystallite size equivalent to 28.32nm was calculated from the XRD data. Elemental make-up of the bacteriogenic nanocrystals was studied via EDX which manifested a sharp peak at 3Kev and had higher silver distribution (84.15%) indicating biosynthesis and purity of our nanoparticles. Anti-Acinetobacter effect of the harvested AgNPs was first screened via agar-well diffusion assay and results showed 16.3mm mean inhibition zones in the tested isolates. Following this, broth-microdilution technique was considered to allocate minimum inhibitory concentration (MIC) and corresponding sub-MIC values of the harvested AgNPs.

 Antibiofilm potency of the AgNPs at sub-MIC value was tested via flat-bottom microplates and results showed significant reduction in the biofilm formation capacity compared to untreated strains. Moreover, real-time PCR was operated to study the impact of the harvested AgNPs on level of expression for each of the candidate genes (abaI, abaR, OmpA, Bap) in the selected isolates in the presence and absence of AgNPs. Obtained results indicated that the treated isolates exhibited pronounced reduction in the level of QS, OmpA and Bap genes expression which are responsible for the development of resistance and biofilm in A. baumannii.  Henceforth, with the remarkable antibacterial, antibiofilm and anti-QS results obtained in our study, we suggest employing bacteriogenic AgNPs as a possible quorum-quenching (QQ) agent against drug-resistant bacteria such as A. baumannii which might provide an avenue to overcome drug resistance crisis in the post-antibiotic era. To our knowledge, the current study represents the first report regarding antibacterial, antibiofilm and anti-QS effect of green AgNPs synthesized via P. aeruginosa against drug-resistant A. baumannii.