Biocides are basic compounds to control microorganism dissemination and ensuing infections and are frequently used as preservative, disinfectant and sterilizer against various microorganisms, in particular P. aeruginosa [17]. One of the most useful biocides against microbes, especially Gram-positive bacteria, is triclosan, which is widely used in toothpastes, soaps and other daily products [17]. Triclosan is an anionic and lipophilic compound, which its anti-bacterial function stems from inhibition of enoyl-acyl-carrier protein reductase (ENR), an enzyme involved in fatty acid synthesis [19, 21]. However, P. aeruginosa strains are inherently resistant to this biocide (MIC>2,000 μg/mL) [21]. ENR enzymes show diversity among different bacteria in terms of sequence and structure, and contain four isozymes including FabI (triclosan- sensitive ENR), FabL, FabV and FabK (triclosan-resistant ENRs) [19, 21]. The FabV isozyme is involved in swimming motility, energy metabolism, protein secretion and adherence, and is responsible for P. aeruginosa resistance to triclosan biocide [21]. Genes encoding FabI and FabV enzymes are found in most bacterial chromosomes such as P. aeruginosa [19, 21]. Bacterial resistance to triclosan is associated with mutation in the active site of fabI gene and the presence of fabV gene [17, 21]. In the current study, the prevalence fabI resistance gene among P. aeruginosa isolates was 0%. Unlike fabI gene, the frequency of fabV gene was high in the present study (84.2%). Zhu et al. showed that deletion of fabV gene confers extremely high susceptibility to triclosan (>2,000 folds) in P. aeruginosa isolates [19]. Similar result was reported by Huang et al. [21]. In this study, the MIC50 and MIC90 values of triclosan for fabV resistance gene- harboring P. aeruginosa strains was higher than fabV gene-negative strains (Table 4).
Chlorhexidine digluconate, an antiseptic, disinfectant and preservative, is a bactericidal biocide, which has higher antibacterial activity against Gram-positive compared with Gram-negative bacteria [22]. This biocide is used in oral health antiseptics, hand washes and other hygienic solutions. The antibacterial mechanism of chlorhexidine digluconate is via the bacterial cell membrane [17]. However, P. aeruginosa is intrinsically resistant to this biocide due to the presence of an outer membrane [22]. Adaptive resistance to chlorhexidine biocide is mediated by a membrane protein encoded by Acinetobacter chlorhexidine efflux gene (aceI). The AceI protein identified in Acinetobacter baumannii is involved in chlorhexidine efflux via an energy-dependent mechanism [23]. However, genes encoding this protein were not identified in P. aeruginosa strains in the current study (data not shown). The antiseptic resistance gene cepA, an efflux pump gene, is associated with chlorhexidine resistance in Gram-negative bacteria causing high chlorhexidine MICs [24, 25]. In our study, 62 (81.5%) cepA-positive strains were found, which is higher than those reported by Mendes et al. (44.5%) and Vijayakumar et al. (63.6%) [24, 25]. According to MIC results, chlorhexidine digluconate is more effective than other biocides against P. aeruginosa isolates (MIC range=4-64 μg/mL) (Table 2). In this study, the presence of cepA gene had variable effects on the MIC50 and MIC90 values of chlorhexidine (Table 4).
A major biocide resistance mechanism in Gram-negative bacteria including P. aeruginosa is the action of efflux pumps such as the small multidrug resistance family (SMR) [13, 18]. Biocide resistance genes qacEΔ1, qacE and qacG encode multidrug efflux pumps, which confer resistance to quaternary ammonium compounds like benzalkonium chloride [13, 18]. In our study, the qacEΔ1 gene was observed in 73.7% of clinical isolates of P. aeruginosa, while in studies conducted by Subedi et al., Roma˜o et al., Kücken et al., Helal et al. and Mahzounieh et al. the qacEΔ1 gene was detected in 46.1%, 48%, 10%, 48% and 91.5% of the isolates, respectively [13, 18, 26-28]. According to the reports of Subedi et al., Kücken et al., Helal et al. and Mahzounieh et al., 100%, 2.7%, 13.5% and 50% of P. aeruginosa strains, respectively, had the qacE gene [13, 26-28], while we detected this gene in 26.3% of isolates. The frequency of qacG gene in the present study was 11.8%, which is higher compared to the frequency reported by Subedi et al. (0%) [13]. The MIC50 and MIC90 values of benzalkonium chloride were significantly high for qacEΔ1-, qacE- and qacG-positive P. aeruginosa strains compared with the negative strains (Table 4).
Class I integron carries qacEΔ1 and antibiotic resistance genes in clinical isolates of P. aeruginosa [13]. Therefore, P. aeruginosa strains harboring class I integron are resistant to benzalkonium chloride and various antibiotics [26]. Comparison of our current and previous study (unpublished data) showed that the frequency of integron I-positive P. aeruginosa strains harboring qacEΔ1 gene was 32 out of 76 (42.1%). No significant association was observed between the presence of class I integron and biocide resistance genes (qacEΔ1, qacE, cepA and fabV), except for qacG gene (p = 0.00).
Formaldehyde is an organic electrophilic biocide, which its mechanism of action involves cross-linking of macromolecules (proteins, RNA and DNA) [17, 29]. Our results indicated that the MIC50 and MIC90 values of formaldehyde were high for biocide resistance genes-positive P. aeruginosa strains compared with the negative strains (Table 4).
A study by Chuanchuen et al. showed a cross-resistance between biocide and antibiotic resistance. They demonstrated a link between P. aeruginosa exposure to triclosan biocide and efflux-mediated resistance to ciprofloxacin [12]. In the present study, there was no significant association between biocide resistance genes and antibiotic resistance, except for levofloxacin and norfloxacin antibiotics and qacE and qacG genes. However, more studies are needed to substantiate the existence of biocide-antibiotic cross-resistance.