Bacterial isolates

According to the results, MIC90 values for ciprofloxacin and gentamicin were 256 and > 8000 µg/ml, respectively. For biocides, 71% (17/ 24) and 29% (7/24) of isolates showed reduced susceptibility to chlorhexidine digluconate (CHG) and benzalkonium chloride (BCC) respectively. Notably, other than efrA/B and emeA, no other efflux genes were identified in our isolates. Respectively, efrA/B and emeA were detected in 100% and 87.5% of isolates. Also, the presence of both genes was detected in 87.5% of isolates (Table 1).

Table 1 Distribution of efflux genes among enterococcal isolates according to reduced antimicrobial susceptibility patternZnONPs synthesis

The X-ray diffraction (XRD) analyzed by High Score Plus software (PANalytical B.V Almelo, The Netherlands, version 3.0e) verified the crystal planes of green synthesized ZnONPs (Fig. 1A). In this pattern, peaks appeared at 2θ positions of 31.9, 34.6, 36.4, 47.8, and 56.8°, are related to reflections from crystal planes of (010), (002), (011), (012) and (110), respectively. This pattern is compatible with that previously reported for ZnONPs [14]. Specific and clear peaks affirmed the high purity and crystalline nature of the arranged ZnONPs. In addition,transmission electron microscopy (TEM) examination appeared rectangular morphology of surface appearance of NPs in a range of 10 to 90 nm (Fig. 1B).

Fig. 1

A X-ray diffraction pattern of zinc oxide nanoparticles. B TEM image of the rectangular shaped zinc oxide nanoparticles

The MIC values of NPs, CCCP and Hoechst toxicity:

The MIC value of CCCP for all isolates was 62.5 µg/ml (except for 2 isolates: 31.25 µg/ml). In all isolates MIC values of ZnONPs and CuNPs, were 8 mg/mL while it was 2 mg/ml for AgNPs. Hoechst dyes at concentration of 2.5 µM showed no significant effect on the viability of bacterial isolates. Also, at sub-MIC concentration of CCCP (25 µg/mL) and NPs, no inhibitory effect on bacterial growth was detected.

Effect of CCCP on antibiotic resistance or biocide tolerance

To determine the role of efflux pumps in the antibiotic resistance and reduced susceptibility to biocides in 20 enterococcal isolates, we evaluated and compared the MICs of antimicrobial agents in the presence (25 μg/mL) and absence of CCCP. The results showed that in 70% (14/ 20) of gentamicin -resistant isolates, gentamicin MIC was decreased at least four folds in the presence of efflux pump inhibitor. Similarly, in 70% (14/ 20), 40% (8/ 20), and 30% (6/ 20) of isolates, antimicrobial susceptibility increased at least four folds to CIP, CHX and BCC in the presence of efflux pump inhibitor, respectively (Table 2). This confirms that efflux pump can be involved in extrusion of related antimicrobial agents. In following, accumulation assay was conducted.

Table 2 Effect of CCCP on fold reduction in MICs of antimicrobial agents in enterococcal isolatesDetermination of the most effective concentrations of NPs and CCCP for accumulation assay:

Monitoring the accumulation of Hoechst dyes in the presence of different concentrations of NPs and CCCP, was resulted to find concentrations of NPs and CCCP in which maximum level of fluorescence was reached. According to our findings, concentrations of 160 µg/ml for ZnONPs, and 40 µg/ml for Ag and CuNPs were determined for accumulation assay in enterococci. These concentrations for assessment in E. coli isolates were 40 µg/ml (for AgNPs) and 80 µg/ml (for ZnO and CuNPs). Also, the used concentrations of CCCP and Hoechst dyes were 25 µM and 2.5 µM, respectively.

Hoechst accumulation assay

Hoechst is a fluorescent probe which is widely used to indicate the efflux pumps activity in bacteria. In this study, both H33258 and H33342 were tested for accumulation assay, but in enterococci, the best result was derived from H33258 vs H33342 in E. coli strains (data not shown).

The effect of the efflux pump inhibitor (CCCP) and nanoparticles (ZnONP, AgNP and CuNP) on efflux pumps was evaluated by monitoring H33342 accumulation in E. coli strains. Heat-inactivated bacteria that served as a positive control accumulated maximal levels of H33342, rapidly (Fig. 2). None of the NPs could not significantly increase the accumulation of H33342 in E. coli isolates, compared to untreated ones (Fig. 2).

Fig. 2

Hoechst H33342 accumulation assay in E.coli isolate (#1063) in the presence and absence of CCCP and different nanoparticles (Ag, CU and ZnO). Heat-inactivated bacteria served as a positive control. The fluorescence intensity was recorded at excitation and emission wavelengths of 360 nm and 460 nm, respectively, over 80 min incubation period

Similarly, in enterococci, the presence of CuNPs did not increase the accumulation of H33258 compared to untreated strain. Although AgNPs were effective, the increase in fluorescence levels was lower than those in the presence of CCCP or ZnONPs. The steady-state level of H33258 was significantly higher in isolates treated with CCCP or ZnONP when compared with the untreated strain, suggesting an increased level of efflux (Fig. 3).

Fig. 3

Hoechst H33258 accumulation in enterococcal isolate (#B59) in the presence and absence of CCCP and different nanoparticles (Ag, CU and ZnO). The fluorescence intensity was recorded at excitation and emission wavelengths of 360 nm and 460 nm, respectively, over 120 min incubation period

The fold change in fluorescence in the steady-state phase in the presence and absence of CCCP and ZnONPs was calculated for each isolate. Addition of CCCP and ZnONPs caused a significant increase (P ≤ 0.05) in the level of H33258 accumulated at steady-state for most enterococcal isolates.

According to results, in 80% (16/20) of isolates, accumulation of H33258 increased significantly (P < 0.05) in the presence of ZnONPs. Of these, in 8 isolates, increased accumulation was also observed in the presence of CCCP and in eight remaining ones, only ZnONPs were able to increase dye accumulation, significantly. Conversely, in 10% of isolates (2/20), only the effect of CCCP was significant. The largest fold changes were1.44 & 1.63 for CCCP and ZnONP, respectively. Notably, in two isolates, no significant effect of CCCP or NPs was detected (Fig. 4).

Fig. 4

A, B, C Fold differences in levels of H33258 accumulated by enterococcal isolates at steady- state with or without efflux inhibitors (CCCP and ZnONPs). (*) Significant difference in dye accumulation in the presence of efflux inhibitors in comparison with the lack of them. P-value of ≤ 0.05 was considered significant. Each experiment was repeated three times

Effect of ZnONPs on antibiotic resistance or biocide tolerance

The susceptibility of 10 enterococcal isolates that were resistant or showed reduced susceptibility to CIP, GM and CHG was tested in the presence of ZnONPs. Results showed anti efflux activity (at least twofold reduction in MIC) in the presence of ZnONPs. The effect of ZnONPs on the MIC values of antimicrobial substances is presented in Table 3. According to results, ZnONPs caused 2 to 6 fold reduction in MIC values of antimicrobial agents.

Table 3 Effect of ZnONPs on MICs of antimicrobial agents in enterococcal isolates