ANTISEPTICS RESISTANCE GENES ( QACA/B , SMR ) DETECTION AND EXPRESSION IN STAPHYLOCOCCUS AUREUS

This study was aimed to investigate the resistance of S. aureus to different antiseptics.This research indicate that the resistance of S.aureus to antiseptics is due to possessing either the smr gene or the qacA/B genes that associated with decreased susceptibility to antiseptics there for this study amid to determine the frequencies of S. aureus chloroxylenol resistant isolates and the presence of the previous genes in these isolates as well as the effect of chloroxylenol on the expression of these genes.189 clinical isolates isolated from skin infections identified as S. aureus in Baghdad by microscopical and biochemical tests. The chloroxylenol resistance S. aureus isolates was identified and chloroxylenol MIC was evaluated for these isolates. Antiseptic resistance genes ( qacA/B , smr ) were detected by PCR method and the results reveled that 21(84%) out of 25 isolates harbored qacA/B gene. While the smr gene was not demonstrated in any isolates. Furthermore, the chloroxylenol had no effect on qacA/B gene expression in these isolates.


INTRODUCTION
Staphylococcus aureus is a gram positive bacteria that grows in the form of clusters with 0.5 to 1.5μm in diameter portrayed by individual cocci since division of cell happens in more than one plane, these bacteria are non-motile, non-spore forming. (9). S. aureus are considered as a major human pathogen that causes a wide range of clinical infections. Disinfectants and antiseptics play a major role in control and the prevention of these infections. Dettol(chloroxylenol) are widely used for disinfection, antisepsis, and cleaning in Iraq. S. aureus colonizes both artificial and animate tissue in humans causing chronic persistent infections that are difficult to cure. It is a leading cause of wounds, skin and soft tissue infections, bacteremia, and infective endocarditis as well as osteoarticular, pleuropulmonary, and device-related infections. The annual incidence of S. aureus bacteremia in the United States is 38.2 to 45.7 per 100,000 person-years; elsewhere in the industrialized world, the incidence is approximately 10 to 30 per 100,000 personyears (19,26). Antibiotics have become the key factor for the rise of drug-resistant strains of groups of microorganisms Almost all synthetic or semi-synthetic antimicrobials presented with many side effects. To resolve such a problematic various plant-therapeutic and-antimicrobial compounds have the most attention. Especially when these substances have no or minimal side effects (14). Although strains of S. aureus have been emerged and developed resistance to some antibiotics, some strains still sensitive to more commonly known antibiotics.
Methicillin resistant staphylococcus aureus (MRSA) are resistant to antibiotic methicillin and others related antibiotics (1). Accordingly, one of the significant characteristics of S. aureus, its ability to secrete toxins which involving in severity diseases that including: exfoliative toxins, toxic shock syndrome toxin-1 and staphylococcal enterotoxins which implicated in staphylococcal scalded skin syndrome, toxic shock syndrome and staphylococcal food poisoning (SFP) ,respectively (28). Diarrhoea caused by different enteropathogens has been recognized as a major clinical problem for calves worldwide. Among these bacteria Salmonella typhyimurium, Clostridium perfringens and Staphylococcus aureus are believed to be the major microbial causes of diarrhea in calves (2). The emergence and rise of antibiotic and antiseptic resistance among staphylococci are a burden in health care facilities and communities (25) leading to increase mortality and morbidity in humans, highlighting the importance of infection control practices and the key role of biocides in healthcare. However, the widespread use of biocides in hospitals has led to concerns about the emergence of disinfectant-resistant bacteria (16). Large amounts of biocides are consumed within the different settings, including the medical environment where they are used for disinfection, antisepsis, and cleaning (25). One of these antiseptics commonly used in Iraq is chloroxylenol (Dettol), it's effect through the denaturation of protein and also acts on the cytoplasmic membrane of microorganisms (3). The plasmid-borne resistance genes qacA/B and smr encode multidrug efflux pumps that yield high-level resistance to quaternary ammonium compounds, chlorhexidine, chloroxylenol and other antiseptic compounds commonly used. The qacA/B genes are linked to higher minimal inhibitory concentrations (MICs) and antiseptic agent tolerance (10). Which was integrated into the cytoplasmic membrane via transmembrane segments and contained distinct subsets of amino acid residues involved in substrate recognition and binding (5). Research has shown that antiseptic exposure is linked to the carriage of qac and smr genes in Staphylococcus (11). This study aimed to determine the frequencies of S. aureus chloroxylenol resistance isolates prevalence and the presence of qacA/B and smr in chloroxylenol resistance S. aureus Iraqi isolate. as well as the effect of chloroxylenol on the expression of these genes. And the detection of the presence of qacA/B and smr in S.aureus was done by the nucleotidic sequences of the tpi amplicons ,were shown to be highly associated with the 16S rDNA gene sequences in a phylogenetic tree (8).

MATERIALS AND METHODS
Ethical statement : All participants agreed to provide the investigator with swab specimens.
Informed and written consent was obtained from all participants in the study approved by the College of Science Research Ethics Committee at University of Baghdad (reference number; CSEC/0920/0060).

Specimen collection
One hundred and eighty-nine different clinical specimens suspected as S. aureus were collected from patients attending hospitals, private clinics and from private clinical laboratories in Baghdad/Iraq. The collection of specimens (wounds, burn, and boils) were accomplished by rotating a sterile moistened swab with sterile normal saline, placed in brain-heart infusion broth (BHI) as a transport medium, and incubated overnight at 37°C.The specimens were re-cultured on blood agar, and mannitol salt agar. Isolates of S. aureus were identified in accordance to the morphological features on laboratory culture media and biochemical tests. Staphylococcus species was diagnosed by using Gram stain , colony shape, oxidase , catalase, growth on manitol salt agar, growth at 15 and 45 Co. according to their morphological, physiological and biochemical properties (12). Estimation of chloroxylenol minimum inhibitory concentration : The concentration of chloroxylenol in Dettol bottle used in this study was 4.85%. MICs of chloroxylenol were detected by a modified agar dilution method according to the recommendations of Clinical and Laboratory Standards Institute (CLSI)(CLSI, 2020). From a pure overnight culture, at least 3-4 colonies were picked and suspended entirely in about 5 ml normal saline using a vortex. Broth culture was visually compared with the McFarland 0.5 standard. The inoculum suspension (200μl) was spotted onto Muller Hinton agar surface. Subsequently, the inoculum spots were dried and plates were positioned upside down prior to incubation, then all plates were incubated overnight at 37°C. The MIC was read as the lowest concentration without visible growth (17). Detection of qaqA\B, smr , and tpi in S.aureus isolates DNA was extracted using ABIOpure TM Total DNA kit (ABIOpure, USA) from 25 S. aureus isolates resistant to chloroxylenol. Conventional PCR technique was carried out to amplify fragments of tpi, qacA\B, and smr using specific primer pairs (Table 1).The PCR reaction protocol listed in (Table 2) was carried out in 20μl as a final volume.PCR products were analyzed in1.5% agarose gels by electrophoresis (6). qaqA/B and smr is summarized in (| table 4). The results were normalized to the level of tpi expression. Tpi keephousing gene was used because it is more accurate in diagnosis of S.
aureus than the classical methods of S. aureus diagnosis, and it was also used to calculate the gene expression of the examined genes.  1). Identification of S.aureus by using tpi gene is more accurate than bacteriological and biochemical assays. 21(84%) out of 25 isolates harbored qacA/B gene (Figure 2). More likely, the rest four isolates employed other genes to resist the chloroxylenol. While the smr gene was not located in any isolate (Figure 3). The prevalence of qac and smr genes around the world is extremely diverse. In 2020 AlKhazraji found that qacA/B1 gene found in 2.5%, qacA/B2 in 5%, and smr gene was not detected in any S.aureus isolates collected from Baghdad (4). Saber in 2019 indicated that out of Sixty-five isolates of S. aureus from Baghdad were collected from different clinical sources, 14 of them were positive to qacA/B and only two were positive to smr gene (22). While Ignak in 2017 revealed the prevalence of qacA/B and smr genes in S.aureus isolates 10.3%, and 13.8%, respectively in Turkey(13).While, in Malaysia Antiseptic genes in S. aureus isolates (qacA/B and smr) were identified in 83.3% and 1.6%, respectively (23).In Iran, the frequency of qacA/B and smr genes among clinical isolates was 19.4% and 45.2%, respectively (21).

Figure 4. qac A/B gene expression in S. aureus isolates
Antiseptic resistance genes (qacA/B, smr, qacG, qacH,and qacJ), encoding multidrug efflux pumps which are carried by plasmids, were identified in Staphylococcus genus for the first time. Antiseptic resistance gene studies of qacA/B and smr mostly focused on S. aureus (27). Many studies have shown both the presence of plasmid-mediated antiseptic resistance genes such as qacA/B, smr, qacG, qacH, and qacJ and reduced susceptibility to antiseptic agents. Other researchers have reported that plasmids carry antiseptic resistance genes together with antibiotic resistance genes and contribute to the development of resistance in pathogens (13). Marked difference in the frequency and type of antiseptic resistance genes between countries or even between different hospitals in the same country, encourage us to determine the frequencies of qacA/B and smr genes in Iraqi clinical S. aureus isolates treated with chloroxylenol and to evaluate the expression of them. qacA/B gene was more frequent in S. aureus local isolate (84%) than smr gene and may be play a role in bacteria resistance to chloroxylenol. Further surveillance and research are necessary to better understand the effect and the role that the antiseptic resistance organisms play in the hospitals and in the community infections.

CONCLUSION
This study confirmed that the presence of qacA/B gene in some S. aureus in Iraqi isolates resistance to chloroxylenol, although chloroxylenol revealed no effect of on qacA/B gene expression in these isolates. However, the resistance to chloroxylenol may be achieved by another efflux pump mediated genes other than qacA/B or other factors. We found that the topical using of chloroxylenol as antiseptic for disinfection S. aureus and other nosocomial infection were effective on bacterial prevent or inhibition. but we could not consider chloroxylenol as an excellent antiseptic because it's could eradicate the bacterial infection only in moderate relative expression.