Effect of ZnO nanoparticles on methicillin, vancomycin, linezolid resistance and biofilm formation in Staphylococcus aureus isolates

Background Multidrug resistant (MDR) and biofilm producing Staphylococcus aureus strains are usually associated with serious infections. This study aimed to evaluate the antibacterial and antibiofilm-formation effects of zinc oxide nanoparticles (ZnO-NPs) against staphylococcus aureus (S. aureus) isolates. Methods A total of 116 S. aureus isolates were recovered from 250 burn wound samples. The antimicrobial/antibiofilm effects of ZnO-NPs against methicillin, vancomycin and linezolid resistant S. aureus (MRSA, VRSA and LRSA) isolates were examined using phenotypic and genotypic methods. The minimum inhibitory concentration (MIC) of ZnO-NPs was determined by microdilution method. The effects of sub-MIC concentrations of ZnO-NPs on biofilm formation and drug resistance in S. aureus were determined by the microtiter plate method. The change in the expression levels of the biofilm encoding genes and resistance genes in S. aureus isolates after treatment with ZnO-NPs was assessed by real time reverse transcriptase PCR (rt-PCR). Results MICs of ZnO-NPs in S. aureus isolates were (128–2048 µg/ml). The sub-MIC of ZnO-NPs significantly reduced biofilm formation rate (the highest inhibition rate was 76.47% at 1024 µg/ml) and the expression levels of biofilm genes (ica A, ica D and fnb A) with P < 0.001. Moreover, Sub-MIC of ZnO-NPs significantly reduced the rates of MRSA from 81.9 (95 isolates) to 13.30% (15 isolates), VRSA from 33.60 (39 isolates) to 0% and LARSA from 29.30 (34) to 0% as well as the expression levels of resistance genes (mec A, van A and cfr) with P value < 0.001. Conclusion ZnO-NPs can be used as antibiofilm and potent antimicrobial against MRSA, VRSA and LRSA isolates. Supplementary Information The online version contains supplementary material available at 10.1186/s12941-021-00459-2.

Page 2 of 11 Abdelraheem et al. Ann Clin Microbiol Antimicrob (2021) 20:54 aureus isolates (LRSA) was reported in USA shortly after its use [8]. Development of Linezolid resistance in these early studies was due to mutation in the 23S rRNA gene, but it seemed to be an uncommon finding [9,10]. However, a different mechanism of Linezolid resistance has reported: the acquisition of plasmid-mediated ribosomal methyltransferase cfr gene, which also mediates chloramphenicol resistance [11,12]. In addition to development of multi-drug resistance among S. aureus strains, biofilm formation is reported as an important cause of treatment failure and recurrent infections [13]. Biofilm formation by S. aureus is encoded by (ica) ADBC genes which mediate synthesis of polysaccharide intracellular adhesin (PIA) [14]. Therefore, detection of one or more of these genes can determine the ability of S. aureus strains to produce biofilm [15]. Biofilm protects the organism from antimicrobials and also from killing by the host immune system [13]. With the emergence, spread, and persistence of resistance to different antimicrobials, the development of novel and effective alternatives to the traditional antibiotics has become an urgent need. The progressions in nanotechnology hold a promising future of nanomaterials as antimicrobial agents. Nanomaterials have a broader microbicidal spectrum than traditional antibiotics [16]. ZnO nanoparticles (ZnO-NPs) have been identified as one of the most promising metallic nanomaterials. In recent years, there is an increasing interest in ZnO-NPs as effective antibacterial agents due to their safety and stability for human cells [17,18]. The current study aimed to use phenotypic and molecular methods to assess the efficacy of ZnO-NPs against MRSA, VRSA, LRSA and biofilm formation among S. aureus isolated from burn wounds. PCR products were analyzed by gel electrophoresis, to exclude any unspecific products.

Biofilm formation testing among S. aureus isolates
The isolated organisms were tested for their ability to form biofilm as previously described [28]. Each isolate was inoculated into trypticase-soy broth and incubated overnight. After adjusting the turbidity of bacterial suspensions to the turbidity of 0.5 McFarland, 100 μl of each isolate was inoculated into sterile 96 well microtiter plate except last column that used as negative control. The inoculated plate was incubated for 24 h. The contents of wells were gently decanted and washed by saline. The wells were stained by 150 μl of crystal violet (0.2%) for 15 min at room temperature. The stain was gently discarded and wells were washed by water. The plate was dried at room temperature and the crystal violet in stained cells was solubilized with 95% ethanol. The optical density (OD) of each well was measured at 620 nm by ELISA reader. The average OD values were calculated for all tested isolates and negative controls. The isolates were divided into four categories non biofilm, weak, moderate and strong biofilm producer as previously described [28].

Molecular identification of biofilm formation among S. aureus isolates
Gene expression of biofilm encoding genes (ica A, ica B, ica D and fnb A) were tested using quantitative realtime reverse transcriptase-polymerase chain reaction (rt-PCR). 16 s RNA gene was used as a reference gene. One step Sybr green kits (SensiFAST SYBR Lo-ROX Kit, Meridian Life science, UK) were used according to manufacturer's instructions. Primers used were listed in Table 1 Phenotypic identification of MRSA, VRSA and LRSA after application of ZnO-NPs Sub-inhibitory concentration of ZnO-NPs (1/2 MIC) of each sample was measured and added to wells that inoculated by bacterial broth adjusted to the turbidity of 0.5 McFarland standard then, the plates were incubated at 37 °C for 24 h. Micro-dilution tests for oxacillin, vancomycin and linezolid were repeated with the same previous steps to all ZnO-NPs treated isolates and MICs were determined according to CLSI 2019 guidelines [20].

Biofilm formation testing among S. aureus isolates after ZnO-NPs application
Biofilm-forming isolates were inoculated in trypticasesoy broth and incubated 24 h at 37 °C. About 100 μl of each isolate' suspension was inoculated into sterile 96 well microtiter plate and mixed with 100 μl of 1/2 MIC of ZnO-NPs. The microplate was incubated at 37 °C until the biofilm formation. The results were interpreted by ELISA plate reader at 620 nm using the same steps that used before.

Effect of ZnO-NPs on genes expression
Gene expression of biofilm encoding genes (ica A, ica B, ica D and fnb A) and antimicrobial resistance genes (mec A for methicillin, van A for vancomycin and cfr for Linezolid) were tested using quantitative real-time reverse transcriptase-polymerase chain reaction (rt-PCR) after treating of S. aureus isolates with sub-inhibitory concentration of ZnO-NPs (1/2 MIC) of each sample and incubating the plates at 37 °C for 24 h, bacterial RNA was extracted again using the same method that used before. Gene expression of biofilm encoding genes and antimicrobial resistance genes were tested again using the same method that used before. PCR products were analyzed by gel electrophoresis, to exclude any unspecific products. The relative expression of target genes was calculated using the equation; RQ = 2 −ΔΔCt as described previously [29]. Table 1 The Primers sequence of the tested genes

Statistical analysis
All data collected in this study were stored in a computer database. Statistical analysis was done on SPSS package version 23.0 (SPSS Inc., Chicago, IL, USA). Chi-squared tests were performed for categorical data, while Mann Whitney U test and Z test were performed for comparison of continuous data. Roc curve analysis was used to detect specificity and sensitivity of the used methods.

Isolation of S. aureus
Out of 250 wound samples, 116 (46.40%) isolates were identified as S. aureus.

Effect of ZnO-NPs on genes expression
Using real time RT-PCR; ica A gene and fnb A gene expressed in 116 isolates (100%) of S. aureus isolates and ica D expressed in 96 isolates (82.7%). However ica B gene was not detected in the study isolates. Regarding antimicrobial resistance genes, the rate of mec A expression among S. aureus isolates was (94 isolates; 81%), van A was (22 isolates; 18.96%) and cfr gene was (29 isolates; 25%). Antimicrobial resistance genes were not detected in sensitive or intermediate resistant isolates. Analyzing expression levels of biofilm encoding genes (ica A, ica B, ica D and fnb A) among S. aureus isolates compared to their expression levels after treating of the isolates with ZnO-NPs using Mann-Whitney U-test revealed that; there was statistically significant decrease of the expression levels of ica A, ica D and fnb A genes after treating of the S. aureus isolates with ZnO-NPs (P < 0.001, for each) (Fig. 4a-c; Additional file 1: Table S1). As regards to antimicrobial resistance genes (mec A, van A and cfr); the expression levels of the three genes significantly decreased after treating of S. aureus isolates with ZnO-NPs (P < 0.001, for each) as shown in (Fig. 5a-c). The receiver operating characteristic curve (ROC) analysis using the expression levels of the studied genes was used to assess the accuracy of these results and revealed that; there were highly significant decreases in the expression levels of the studied genes after treating isolates with sub-MICs of ZnO-NPs with high sensitivity and specificity as shown in (Table 3).\

Discussion
The multidrug resistant strains of S. aureus are increasing, making the treatment more difficult. The prevalence of MRSA among S. aureus isolates is the highest in Egypt compared to other African countries [30]. The prevalence of MRSA in Egypt is ranging from 24 to 82% [31], which is comparable with the prevalence reported in the current study (95/116, 81.90%). Our prevalence is comparable also with other reports from developing countries; (80%) [32], 75% [33] and 76% [34]. The most effective drugs against MRSA are vancomycin and linezolid [35], however, isolates with reduced susceptibility to vancomycin are increasing [36]. The global prevalence of VRSA and VISA before 2010 was 1.2% and after 2010 were 2.40% and 4.3% respectively and the prevalence of VRSA in Egypt was 5.50% [37]. However, the current study has reported a high prevalence of VRSA and VISA; 33.62% (39/116) and 5% (6/116) respectively, that agrees with a similar report from Egypt [33] where the author reported that 20.68% of the isolates were VISA, and 20.68% were VRSA. In Egypt, yet a few researches studied the resistance against linezolid among staphylococcal isolates using phenotypic methods only, where the prevalence of LRSA ranged from 5 to 15.4% [38,39]. The prevalence of LRSA in the current study was (34/116, 29.31%), that is higher than the previous Egyptian reports. Harcharan singh et al. also reported a high percentage of LRSA in Rajasthan (20.3%) [40]. On the other context, the current finding is higher than the global rates reported in the LEADER or ZAAPS studies [41,42]. This could be due to the availability of linezolid in the Egyptian market, its use as an empiric treatment in our locality and absence of guidelines that control its use. One of the main reasons of antibiotic resistance is the rebellious nature of biofilms produced by these pathogens. In this study, 85/116 (73%) of isolates were identified as biofilm producers, that is compatible with other studies investigated biofilm production by S. aureus isolated from wound samples [43,44]. Our study focused on the development of promising alternative agents for treatment of these serious infections such as ZnO-NPs. Interestingly, ZnO-NPs were identified by several reports as non-toxic to human cells [45]. ZnO-NPs should penetrate into bacterial cells to express the antibacterial activity [46]. Therefore, the broth dilution assay can be considered as accurate and confirmative method for identification of antibacterial activity of ZnO-NPs [47]. By using the broth dilution method, our study showed that, MICs of ZnO-NPs among S. aureus isolates were ranging from 128 to 2048 µg/ml. Other studies have also reported that bactericidal effect of ZnO-NPs is concentration-dependent [48][49][50].  [56,57]. Several previous studies investigated the prevalence of mec A, van A and cfr genes among S. aureus isolates [34,58,59]. However, information about the expression levels of these genes is very little. Therefore, the current study assessed the expression levels of these genes by investigating RNA of S. aureus isolates before and after application of ZnO-NPs by quantitative rt-PCR. Mec A gene was expressed in 100% of oxacillin resistant isolates and van A gene was expressed in (22/39; 50%) of the VRSA isolates. Cfr gene expression was detected in (29/34; 85.30%) of LRSA isolates, this high rate could be explained by horizontal spread of cfr gene among different species [60]. Cfr-mediated resistance was also identified in 100% of LRSA isolates in previous studies [12].

Conclusion
In this study, promising activities of ZnO-NPs as an antibacterial agent against MRSA, VRSA and LRSA as well as anti-biofilm activity were reported. The study demonstrated that the ZnO-NPs are able to reduce the expression levels of the ica A, ica D and fnb A genes (the main genes associated with biofilm formation in S. aureus) and also reduce the expression levels of the mec A, van A and cfr genes (the main genes associated with resistance to methicillin, vancomycin and linezolid in S. aureus).
Finally, we recommend the use of ZnO-NPs for resistant infections. However, further researches must be done to evaluate the safety of ZnO-NPs use in vitro and in vivo.
Additional file 1: Table S1. Mean relative quantity (RQ) of gene expression before and after application of ZnO NPs.