This study reveals a high prevalence of MDR and XDR clinical isolates of A. baumannii at the study site over the last decade (34, 35). The high rates of MDR A. baumannii observed may be attributed to factors such as emergence of bacteria with different resistance mechanisms, increased likelihood of resistance dissemination in hospital environments, absence of a robust nosocomial infection surveillance system, and suboptimal infection control practices (36).
In the present study, biofilm production was observed in nearly all isolates of MDR A. baumannii, with 83.1% exhibiting strong biofilm production. This finding aligns with a study conducted in a tertiary care hospital in Nepal, where 99.6% of Acinetobacter isolates were identified as biofilm producers, and 89% of them were characterized as strong biofilm producers (37). Strong biofilm producers are significantly associated with recurrent infections and antimicrobial resistance (38, 39).
Our study revealed that among the 195 MDR A. baumannii isolates, twitching motility was observed in 42.0%, while surface-associated motility was observed in 66.2%. These findings are consistent with another study where 50.0% of isolates exhibited twitching motility, and 62.5% displayed surface-associated motility phenotypes (40). Approximately 4.1% and 61.0% of MDR A. baumannii isolates exhibited strong twitching motility and surface-associated motility, respectively, along with a robust capacity for strong biofilm production. Notably, this study marks the first from Nepal to establish a correlation between biofilm formation and the motility traits of MDR A. baumannii isolates. Our findings also revealed a significant difference in resistance to ampicillin-sulbactam and cefoperazone-sulbactam, specifically in relation to twitching motility among the isolates. Twitching motility was more commonly seen among the isolates from sputum (data not shown). We speculate that the greater degree of motility observed in sputum isolates may be attributed to the overexpression of type IV pili-related genes compared to isolates from other specimens. Motility, as a trait, relies on the presence of type IV pili, and it has been demonstrated in other bacteria that biofilm-forming cells often downregulate genes associated with motility (41).
Due to the escalating resistance of A. baumannii to various antibiotics, often attributed to biofilm formation, there is an urgent need to identify therapeutic strategies aimed at inhibiting biofilm formation and effectively treating established biofilms (42, 43). In our study, treatment with EDTA (125 mg/L) resulted in a notable 54.8% inhibition in biofilm formation, consistent with findings in other studies (44, 45). The ability of EDTA to chelate and potentiate bacterial cell walls, along with its capacity to destabilize biofilms by sequestering calcium, magnesium, zinc, and iron, positions it as a suitable agent for biofilm inhibition (46). Similarly, cinnamaldehyde demonstrated a promising inhibition of 77.3% in average biofilm formation, surpassing the findings by Mohamed et al. (17). In our study, Tween 80 exhibited a concentration-dependent reduction in biofilm formation, ranging from 61.8–76.8% as concentrations increased. Moreover, at a concentration of 100 mM, glutamic acid and glycine showed average reductions of 66.6% and 33.7%, respectively in biofilm formation. A study from Iraq also reported significant anti-biofilm activity of these compounds.(16) Additionally, inhibitory effects observed against Staphylococcus aureus, Pseudomonas aeruginosa, and Bacillus subtilis suggest that D-amino acids may serve as a general strategy for inhibiting biofilm formation in opportunistic pathogens (47–49). D-amino acids play a role in regulating bacterial cell wall remodeling during the stationary phase, contributing to biofilm dispersal in aging bacterial communities (50).
The use of anti-biofilm agents, capable of either inhibiting or eliminating biofilm without inducing resistance, is essential for a potential therapeutic approach to managing MDR biofilm-associated infections. As our study primarily focused on identifying alternative technologies for controlling A. baumannii biofilms, we turned to EOs derived from edible plants, which have been consumed by humanity since ancient times and are known for their diverse benefits. In our investigation, we employed EOs from ginger, garlic, turmeric, and Ageratina adenophora, along with ethanolic extracts of onion and chili, as anti-biofilm agents. Notably, to our knowledge, this study represents the first exploration in Nepal of bacterial biofilm inhibition using natural plant extracts.
The Gas Chromatography-Mass Spectrometry (GC-MS) analysis of the ginger EO revealed the presence of phenolic compounds (geraniol, citronellol), volatile sesquiterpenes (bisabolene), and monoterpenoids (curcumene, β-sesquiphellandrene), aligning with another study (51). In our study, the EO of ginger demonstrated an average 70.8% reduction in biofilm in A. baumannii isolates which is comparable to another study (52).
The EO of turmeric was found to contain secondary metabolites such as zingiberene, tumerone, ar-tumerone, curlone, and the phenolic compound curcumin, which is recognized as the most essential bioactive component among curcuminoids. Tumerone, in particular, exhibited anti-biofilm properties against bacterial isolates. Our study demonstrated an average 51.9% reduction in A. baumannii biofilm. Similarly, Ahamad et al. showed a reduction of A. baumannii biofilm from 2.0170 ± 0.14863 (mean ± SD) to 0.2470 ± 0.11314 by 200 mg/L turmeric extract. Additionally, Suwal et al. assessed the anti-biofilm activity of Curcuma longa (turmeric) rhizome extracts against, reporting inhibition ranging from 26.7–77.7% for Staphylococcus aureus and Pseudomonas aeruginosa (53).
The EO of garlic revealed major compounds such as allyl disulfide and allitridine, known for their biofilm inhibiting properties. In our study, we observed a substantial 68.6% inhibition of biofilm in A. baumannii at a concentration of 200 mg/L, aligning with findings by Somrani et al., who reported a 68.0% inhibition of biofilm when garlic EO was exposed to bacteria for one hour at its Minimum Inhibitory Concentration (MIC) (32). Garlic has also been recommended in different studies as an agent to prevent wound pathogen biofilm formation when formulated as garlic ointment (54). Additionally, it can be applied on catheters to prevent catheter-associated biofilm infections (55).
Furthermore, the GCMS analysis of the EO of Ageratina adenophora identified major chemical constituents such as α-phellandrene, camphene, bornyl acetate, p-cymene, γ-curcumene, germacrene, and α-bisabolol, consistent with previous reports (56–58) These compounds have demonstrated antibacterial activity against both Gram-positive and Gram-negative bacteria (56). In our study, 200 mg/L of Ageratina adenophora inhibited 67.6% of biofilm formation in A. baumannii. Chili pepper, another focus of our study, contained secondary metabolites such as dihydrocapsaicin and luteolin, which exhibit antimicrobial and anti-quorum sensing activities against bacterial pathogens (59, 60).
Another compound investigated in our study was the ethanolic extract of onion, which exhibited a notable 77.2% inhibition against A. baumannii biofilm. This efficacy aligns with findings demonstrating its effectiveness against Listeria monocytogenes biofilms as well (32). The anti-biofilm property of onion is attributed to its sulfur compounds. These compounds interact with the sulfhydryl (SH) groups of cellular proteins, forming mixed disulfides that have the potential to inflict damage upon microbial cells (61).
Numerous studies have elucidated the role of biofilm-related genes (csuE, ompA, bap, and blaPER−1) in A. baumannii in biofilm development and antibiotic resistance (62, 63). In our study, these genes were identified in 30 strong biofilm-producing isolates of MDR A. baumannii obtained from medical devices. The results revealed the most prevalent gene was csuE (89.9%), followed by ompA (53.3%), bap (46.7%), and blaPER−1 (26.6%). Similar result was reported by Thummeepak et al., who showed a prevalence of 48% and 30.2% for bap and blaPER−1 genes, respectively (39). The highest frequency of biofilm-related genes was observed in isolates from endotracheal aspirate samples. The csuE gene, a member of the usher-chaperone assembly system, plays a crucial role in mediating attachment and biofilm formation. The high prevalence of csuE in A. baumannii is consistent with findings from other studies (5, 62–64). OmpA, another biofilm-related gene in A. baumannii, is likely essential for attachment to human epithelial cells, biofilm development, and antimicrobial resistance.