Detection of Biocides Resistance Genes among Clinical Isolates of Both Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus Aureus

BACKGROUND: Biocides compounds are widely used in infection control programs, including hand washing and skin decolonization before invasive procedures. However, its overuse leads to the emergence of Staphylococcus aureus with decreased biocides susceptibility, which became a problem in hospitals in different parts of the world. AIM OF THE WORK: The present work aims to study the susceptibility of methicillin-resistant isolates of staphylococci aureus, obtained from different clinical samples to commonly used biocides, determines the prevalence of the biocide resistance genes, qacA/B, and qacC, among these isolates. METHODS: Biocide’s resistance genes [qacA/B, qacC] in Both Methicillin-Resistant and Methicillin-Sensitive Strains of Staphylococcus aureus were detected by PCR method. The minimum inhibitory concentrations [MICs] of Biocide agents were determined by broth dilution method, whereas antibiotic susceptibility was analyzed by disk diffusion and MICs methods according to Clinical and Laboratory Standards Institute [CLSI] criteria. RESULTS: The frequency of Biocides resistance genes [qacA/B, qacC] was found to be high in our clinical staphylococci isolates. The qacA/B gene was detected in [63.3%] of MRSA isolates compared with only [23.3%] of MSSA isolates. Simultaneously, the qacC gene was detected in [63.3%] of MRSA isolates compared to only [13.3 %] of MSSA isolates., with the significant difference between both groups [P<0.05]. We also found an association between the presence of antiseptic resistance genes qacA/B, and increased MIC values to both savlon, hypochlorite, and Phenol [P<0.05]. We also showed that an association between the presence of antiseptic resistance genes qacC and the MICs to both savlon, hypochlorite, and Phenol [P<0.05]. CONCLUSION: Our study detects biocide resistance genes [qacA/B, qacC] in Staphylococcus aureus isolates in Egypt; more studies are needed to correct the biocide uses and to support infection control programs to avoid the development of new resistance mechanisms.


Methicillin-resistant Staphylococcus aureus
is an important nosocomial pathogen infecting individual in hospitals worldwide [1] . Many studies detect the important role of staphylococci aureus in disease in immunecompromised patients and the increased prevalence of multidrug-resistant strain, the emergence and rise of antibiotic resistance among staphylococci aureus is a burden in health care facilities and communities [2] Biocides are chemical agents applied to inactivate or destroy microorganisms in different sites. Large amounts of biocides are used within different sites, including the medical environment used for disinfection, antisepsis, and cleaning. A wide variety of biocidal agents are commonly used in hospitals and healthcare facilities to decontaminate surfaces, disinfect the hands of health care personnel, and treat hospital-acquired infections [3] .
The worldwide increase in antimicrobial resistance in bacterial, leading to increased mortality and morbidity in humans, highlights the importance of infection control practices and the key role of healthcare biocides. However, the widespread use of biocides in hospitals has led to the emergence of disinfectant resistant bacteria. Excessive use may also result in the appearance of cross-resistance between widely used biocides and antibiotics [4] .
Epidemiological data on antiseptic susceptibility and the Distribution of resistance genes are useful for nosocomial infection control. Multidrug efflux systems are possibly the most described resistance mechanism regarding acquired resistance to biocides. Antiseptic resistance genes, qacA/B, qacC have been identified in Staphylococcus species [5] .
The qacA/B, qacC are found mainly in staphylococci aureus clinical isolates and could be responsible for reduced susceptibility to certain antiseptic agents. The incidence rate of MRSA in our hospital has increased to more than 80% of clinical isolates, and the prevalence of methicillin-resistant coagulase-negative staphylococci is also rising [6] .

AIM OF THE WORK
The present work aims to study the susceptibility of methicillin-resistant isolates of staphylococci Aureus, obtained from different clinical samples, commonly used biocides [antiseptic and disinfectant agents] to determine the prevalence of the biocide resistance genes, qacA/B, and qacC among these isolates.

1-Data collection
Special questionnaires were prepared, including sex, age, health status, work type, medical history of the patients. Other data were also recorded for each HCWs. The samples were obtained from admitted patient's samples, Healthcare workers [HCWs] and environmental samples. Then, each sample was undergone to the following:

I-Isolation and identification of staphylococci aureus by the following:
[A] Direct Smear: Gram-stained smears: Typical staphylococci appear as gram-positive cocci in clusters.
[B] Culture and confirming the result: • by inoculating them on blood agar and mannitol salt agar plates, given rise to typical colonies after 18-24 hours at 37°C in aerobic incubation [7] .

II-Detection of Methicillin Resistance:
A] Determination of the susceptibility to Oxacillin and vancomycin antibiotics by the disc diffusion and minimal inhibitory concentration [MIC] methods: according to the recommendations of the [CLSI,2010, CLSI 2020] [8] .
B] Detection of penicillin-binding protein 2a by the SlidexR MRSA detection: MRSA isolates were tested for the presence of [PBP2a] [product of the mecA gene] by the Slidex ® MRSA agglutination test [9]. [MSSA] isolates to Biocides by the Broth Dilution Method. The procedure was done by the serial two-fold broth dilution method as described by Mazzola et al. [10] . Biocides tested in the present study were selected from the chemical agents used to disinfect patient care items and instruments in the hospital. They were glutaraldehyde or cidex [ [11] .   NS=non-significant; HS = highly significant  [12] . One hundred and six staphylococci aureus were isolated from patients and Healthcare workers. The incidence of staphylococci aureus in this study was 42.4%. which was lower than our results [58.3%]. Other study conducted at Al-Zahraa University Hospital included admitted 300 female patients to identify the causative organism[s] responsible for surgical site infection following Cesarean section. The most common organism isolated was staphylococci aureus 31/300 [10.3%] [13] , which was lower than our results [58.3%].

VI-Statistical analysis:
In our study, the staphylococci aureus isolated strains were 350 out of 600 [58.3%]. This result compared to those obtained by [14] with staphylococci aureus percentages of 31.27%. The high rates of staphylococci aureus contamination in our study are likely to reflect both poor hand hygiene practices and compromised hygienic measures and increased duration of hospital stay.  [15] . There is a difference in the incidence of both staphylococci aureus [ [16] . The rate of MRSA was 5.98 % [7/117] in patients. which was lower than our results [13.2%.], the rate of MRSA was 36% [9/25] in HCWs which was higher than our results [7%.]. Two MRSA strains were isolated from the 12 environmental samples compared to no environmental MRSA in our results. In another trial was undertaken between August 2011 and June 2012, at Al-Kadhamia teaching Hospital and Al-Numan hospital, Baghdad, Iraq [11] . One hundred and six staphylococci aureus were isolated from patients and Healthcare workers. The incidence of staphylococci aureus in this study was 42.4%. which was lower than our results [58.3%]. The rate of MRSA in patients was 97 % which was higher than our results [13.2%.], the rate of MRSA in HCWs was 82%% which was higher than our results [7%.], which confirm the higher incidence rate of MRSA in both patients and HCWs in this study thank our study.
In another study was conducted at MNH in Dar es Salaam, Tanzania, between May and June 2018, on environmental samples, the results of this study showed that staphylococci aureus incidence was [20%] from environmental samples, which was lower than our results [33%], these results including 19.5% for MRSA, compared with our results [0%] for MRSA [17].
Another study was conducted at Al Zahraa University Hospital on 300 females admitted patients to identify the most causative organism[s] responsible for surgical site infection after Cesarean section. The most common organism isolated was staphylococci aureus [10.3%] [13] , which was lower than our results [58.3%]. Other study conducted at Al-Azhar University Hospital, Damietta branch. The results obtained in Egyptian study by Alaa Eldeen et al. [18] revealed MRSA nasal carriage rate of [2.3%] among medical residents in Gynecology department which is lower than our study results about MRSA isolates in [HCWs] [7%]. Our study result is higher than the study result conducted by Siegel et al. [19] , who reported an MRSA colonization rate of 2.1% in the nasal cavity and axilla of HCWs.
The high rates of MRSA contamination in [HCWs] are likely to be a source of infection and transmission of pathogens within the hospital and remains a hazard for hospitalized patients. Also, cross-transmission of microorganisms from patients and the environment may have an important role.
The high MRSA contamination rates reflect both poor hand hygiene practices and compromised hygienic measures. Increased duration of stay in hospitals. The difference in MRSA rates in different studies might be due to the difference in locations and periods of the studies, the difference in hygienic conditions maintained in different hospitals, the hospital's healthcare facilities, and the implementation of the program for infection control, and rational use of antibiotics.  [20] , who also found that MRSA isolates had increased MIC to povidone-iodine [betadine] as compared to the MSSA isolates. By contrast, Bamber and Neal [21] , reported no differences between the sensitivity of MRSA and MSSA to betadine. Also, a highly significant difference was observed between both [MRSA and MSSA] regarding the MICs to Savlon [P<0.05]. This finding goes in accordance with the previous finding of Irizarry et al. [22] , who also found the increased value of MICs of MRSA strains for some biocides, including cetrimide and chlorhexidine. Also, Wootton et al. [23] observed that the MICs for QACs for MRSA were 1.5-3-fold higher than those for MSSA. However, Al-Masaudi et al. [24] found that there was no important difference between MRSA and MSSA regarding their susceptibility to savlon.
Unlike the results by Al-Masaudi et al. [24] who found that both MRSA and MSSA are equally sensitive to phenols, a highly significant difference in the present study, between the MRSA and MSSA isolates regarding the MICs to Phenol [P0.05] was observed.
Regarding hypochlorite sensitivity in the present work, the MRSA isolates had significantly higher MICs than the MSSA isolates [P0.05]. The MRSA isolates MIC value exceeded the user-defined concentrations [MIC= 0.825]. This finding supports the finding of Mitchell et al. [25] , who reported an increased MIC for MRSA than MSSA isolates to hypochlorite.
In our study, no significant difference was reported between both MRSA and MSSA isolates regarding the MICs of the glutaraldehyde and glutaraldehyde concentration as low as 0.5% effectively inhibited all isolates. This finding is in accordance with the findings of Reynaldo et al. [26] who reported that MRSA isolates are sensitive to 2% glutaraldehyde as MSSA. Again, Kheiralla et al. [27] noted, the highest concentration of glutaraldehyde [2%] was the most concentration effective against the MRSA isolates. Also, the authors noted that the 0.5% concentration was more effective on the MRSA isolates than the concentration of 1%.
The studied MRSA isolates were sensitive to the most used disinfectants/ antiseptics at the user's defined concentrations, and there was an important difference between the MICs of MRSA isolates as compared to the MSSA isolates. This is important as it may mean that certain isolates have an ability to survive at lower concentrations of biocides and that the use of biocides may act as a selective pressure to allow these isolates to predominate.
As regards the presence of biocides resistance genes [qacA/B and qacC] in both MRSA and MSSA isolates. In the current work, the qacA/B gene was present in 43.3% of the studied staphylococci aureus isolates. There was a significant difference between the MRSA isolates and the MSSA isolates regarding the presence of the qacA/B gene since the gene was presented in 63.3% of MRSA isolates and in 23.3% of MSSA isolates [P<0.05]. This goes in agreement with the study of Mayer et al. [28] , who detected the qacA/B genes in 42% of their studied staphylococci aureus strains were isolated between 1997 and 1999 from 24 different European university hospitals. The authors noticed significantly more MRSA than MSSA isolates possessed the qacA/B genes [63% versus 12%, respectively]. Also, Taheri et al. [29] detected the qacA/B gene in 76.46% of their studied staphylococci aureus strains isolates. The authors noticed that significantly more MRSA than MSSA isolates possessed the qacA/B genes [63.3% versus 25.9%, respectively].
Also, Noguchi et al. [30] detected the qacA/B gene in 41.6% of MRSA strains isolated from different Asian countries between 1998 and 1999. Later, Sekiguchi et al. [31] reported a higher detection rate of the qacA/B gene [52.3%] among their studied MRSA isolates from a Tokyo hospital. Furthermore, Noguchi et al. [30] detected the [qacA/B] gene in 45.9% of MRSA isolates cultured from patients. A much higher rate was reported by Miyazaki et al. [32] , who found the [qacA/B] gene in 80% of MRSA strains isolated in Brazil. Also, Conceicao et al. [33] detected the qacA/B gene among 40.5% of MRSA strains isolates. Also, Wang et al. [34] detected the [qacA/B] gene among 61.1% of MRSA strains isolated in China.
On the other side, Vali et al. [35] detected the [qacA/B] gene in only 10% of their studied isolated MRSA from hospitals in Edinburgh. Also, Ignak et al. [11] detected the [qacA/B] gene among 10.3% of MRSA isolates.
In the present work, the qacC gene was presented in 38.3% of the studied staphylococci aureus isolates. The gene was presented in 63.3% of the MRSA strains isolates and in 13.3% of the MSSA isolates [P<0.05]. These results are much higher than those reported by Alam et al. [36] and Noguchi et al. [29] , who reported the qacC gene in 3.3% and 2.5% only of their studied MRSA isolates, respectively. Also, our results are much higher than those reported by Conceicao et al. [33], and Taheri et al. [29] , who detected the qacC gene in 3.7%, and 6.7% only of their studied MRSA isolates, respectively. However, Vali et al. [35] noted that the qacC gene was more spread [44.2%] than the [qacA/B] gene [10%] among their studied MRSA strains isolated from hospitals in Edinburgh. Also, Ignak et al. [11] noted that the qacC gene was more spread [13.8%] than the [qacA/B] gene [10.3%] among their studied MRSA strains isolated from hospitals. The high spreading of these genes is likely to be due to selective pressure by the various disinfection agents used in hospitals. Moreover, it is noted that more MRSA than MSSA isolates was positive for the [qacA/B] gene. This may be due to [the location of the qacA/B genes on multi-resistance plasmids like the pSK qacA] and the βlactamase/heavy metal resistance families], such as pSK23. These plasmids harbor a few determinants responsible for resistance to a range of antimicrobial agents. This finding plays an important role in qacA/B genes in biocide resistance.
In the present trial, a highly significant difference was found between group 1 [qacA/B +ve] and group 3 [qacA/B and qacC -ve] regarding the MICs to savlon, hypochlorite, and Phenol. Yet, no significant difference was observed between both groups regarding the susceptibility to betadine [PVP-I2] and glutaraldehyde. Behr et al. [37] also observed a significant difference between these isolates that carry qacA/B and those that do not carry qacA/B regarding MICs of QACs and chlorohexidine.
In our study, a highly significant difference was found between group 2 [qacC +ve] and group 3 [qacA/B -ve and qacC -ve] regarding the MICs to savlon, hypochlorite, and Phenol. Yet, no significant difference was detected between both groups regarding to the susceptibility to betadine [PVP-I2] and glutaraldehyde. Similarly, Mycock et al. [35] reported the increased MICs for isolates carrying the qacC gene of biocides, including chlorhexidine, cetrimide, benzalkonium chloride, and hypochlorite. Also, Sekiguchi et al. [31] reported a significant difference between the isolated strains that carry the qacC gene and those that do not carry the qacC gene regarding the MICs of QACs.
In the current study, a highly significant difference was found between group 4 [qacA/B +ve, qacC +ve] and group 3 [qacA/B-ve and qacC -ve] regarding the MICs to betadine [PVP-I2], savlon, hypochlorite and Phenol. In agreement with Al-Masaudi et al. [24] who reported increased MICs against plasmid-carrying MRSA strains for biocides including chlorhexidine, cetrimide, benzalkonium chloride, hypochlorite, and betadine. In addition, a significance difference was found between group1 [qacA/B +ve] and group 2 [qacC +ve] regarding the MICs to savlon with the qaCA/B +ve group being more resistant than the qacC +ve group to savlon. No significant difference was detected between both groups regarding their susceptibility to betadine [PVP-I2], glutaraldehyde, hypochlorite and phenol. This finding goes in accordance with Russell et al. [39] who reported that the qacA/B gene specifies resistance to chlorhexidine and QACs whereas the qacC gene carry resistance to some QACs as previously reported by Leelaporn et al. [40] From the current study we concluded that although the studied MRSA isolates strains were susceptible to most of the commonly used disinfectants/antiseptics at the user's defined concentrations, there was a significant difference between the MICs of MRSA isolates strains as compared to the MSSA isolates. This is of importance as it may mean that certain isolates will have an ability to survive at lower concentrations of biocides, the use of biocides act as a selective pressure to allow these isolates to predominate. Thus, we recommend eradicating MRSA colonization in both patients and HCWs to prevent its spread to the community. Further studies on larger number of isolates to confirm our findings. Other studies on other multi-drug resistant organisms particularly gram-negative organisms are also recommended.

Financial and Non-financial Relationships and Activities of Interest
None