Aminoglycoside and chlorhexidine resistance genes in Staphylococcus aureus isolated from surgical wound infections

Objective: Staphylococcus aureus especially the methicillin-resistant population (MRSA) is a major human pathogen resistant to many antimicrobial agents. This study aims to investigate the prevalence of the aminoglycoside-modifying enzymes (AMEs); genes aac(6’) aph(2’), aph(3’)-IIIa, Ant(4’)-Ia) and the biocide (quaternary ammonium compounds) resistance genes (qacA/qacB, qacC) in S. aureus isolated from surgical site infections. Methods: Swabs from 280 infected surgical sites were collected from different surgical words at Mansoura University Hospitals between January 2014 and December 2014). Sixty-six staphylococcal strains were isolated and included in this study. Verification of the presence of methicillin resistance gene (mecA), aminoglycoside resistance genes [(aac(6’)/aph (2’’), ant (4’)-1a and aph(3’)-IIIa)] and qac resistance genes (qacA/qacB, qacC) in S. aureus was carried out by PCR. Chlorhexidine MIC was also determined. Results: Out of the 66 S. aureus isolates included in this study, 17/66 isolates (25.8%) were phenotypically characterized as MRSA and mecA gene was detected in 19 S. aureus isolates (28.7 %) by PCR. Aminoglycoside resistant S. aureus accounted for 21/66 (31.8%) of the isolates. AME genes were detected in all aminoglycoside-resistant S. aureus; aac(6’)/aph (2’’) and was the most frequently detected 11/21(52.4%), followed by aph(3’)-IIIa 6/21 (28.6%) and the least frequent was ant (4’)-1a 4/21(19%). Aminoglycoside resistance in 9/21 (42.9%) of aminoglycoside resistant S. aureus isolates was found to be plasmid mediated as proved by plasmid curing experiment. A total of 14/21 aminoglycoside-resistant S. aureus isolates (66.7 %) carried the mecA gene. Nine of the 21 aminoglycoside-resistant S. aureus THE INTERNATIONAL ARABIC JOURNAL OF ANTIMICROBIAL AGENTS


Introduction
Staphylococcus aureus is a potentially serious opportunistic human pathogen that colonizes over 30% of individuals. MRSA is a major nosocomial pathogen that can easily circulate among patients and personnel in hospitals [1].
An important tool in controlling nosocomial MRSA infection is hand hygiene and decontamination of potentially contaminated rooms, utensils and colonized patients. Many antiseptic agents are in use for this purpose but overuse and sub-lethal concentrations of antiseptic agents have led to the emergence of MRSA with decreased antiseptic susceptibility [2].
Multidrug efflux pumps are membrane proteins that form channels to transport toxic compounds such as biocides and disinfectants out of the cell. Their substrates are diverse including quaternary ammonium compounds (Qac) for which they are named [3]. These qac genes, confer resistance to organic cations by means of a proton motive forcedependent multidrug efflux [4] Within Staphylococcus species, six different plasmid-encoded Qac efflux pumps were known: QacA and QacB are members of the Major Facilitator Superfamily (MFS) and are highly related and conserved in Staphylococcus species. Whereas, QacC, QacG, QacH, and QacJ belong to the Small Multidrug Resistance (SMR) family. Genes encoding these proteins are generally carried on plasmids [5,6]. Some of these plasmids (pST6, pSK4, and pSK41) contain antibiotic resistance genes encoding resistance to gentamicin, penicillin, kanamycin and tobramycin [7].
The qacA and qacB genes are closely related and confer resistance to a range of structurally disparate organic cations, including divalent cations like chlorhexidine. They differ only in the amino acid residue 323 that is (Asp) in QacA and (Ala) in QacB. Both are located on a variety of plasmids and qacA exists also on chromosomes [8].
Chlorhexidine gluconate is a water-soluble cationic bisbiguanide widely used as an antiseptic agent since 1954. It has been approved by the U.S. Food and Drug Administration for infection control in various applications, including general skin cleaning, central venous catheter site preparation, surgical hand and preoperative scrub as well as vascular catheter dressings [9]. Chlorhexidine destroys the isolates (42.9%) were positive for qacA/qacB genes and in all of them mecA gene co-existed.

Conclusion:
This study shows a high proportion of S. aureus isolated from infected surgical sites carried aminoglycoside and methicillinresistance genes together with quaternary ammonium compounds resistance genes.
Keywords: Staphylococcal Infections, Surgical Site Infections, Disinfectant Resistance, Drug Resistance. cell membrane and causes coagulation of intracellular contents of a variety of microorganisms, including Gram-positive and Gram-negative bacteria, lipophilic virus, protozoa, and fungi [10].
The purpose of the present study was to investigate the prevalence of the antiseptic-resistant genes qacA, qacB and qacC and aminoglycoside resistance genes in S. aureus clinical isolates from surgical site infections at Mansoura University Hospitals.

Sample collection, culture and identification
This study was approved by the ethical committee of the Faculty of Medicine, Mansoura University. Specimens from infected surgical sites were collected using sterile saline moistened cotton swabs and immediately transported to the laboratory in sterile screw-capped tubes containing 5 ml saline. Specimens were processed at the Microbiology Diagnostic and Infection Control Unit (MDICU) in the department of Medical Microbiology and Immunology, Mansoura faculty of medicine. Specimens were inoculated within 1-2 h onto mannitol salt agar and sub-cultured to Baird-Parker agar medium (Merck, Germany). Plates were incubated aerobically at 37°C for 48 hours. Colo-nies with typical black appearance surrounded by a clear zone were considered Staphylococci and when they grow in blood agar they were identified as S. aureus. Further identification of isolates included Gram-stain as well as catalase, and coagulase tests. S. aureus ATCC 12600 was used as positive control and S. epidermidis as a negative control [14].

Broth microdilution test
The minimum inhibitory concentrations (MICs) of chlorhexidine were determined by the reference broth microdilution procedure recommended by the CLSI using 96-well microtiter plates with serial 2-fold dilutions. Dilutions ranged from 32ug/ml to 0.25ug/ml. The MIC was defined as the lowest concentration of antimicrobial agent that produced no visible growth after 20-h incubation in Mueller-Hinton broth at 37°C [15].

Total DNA extraction
A single bacterial colony was grown overnight on L-broth with shaking at 37°C to stationary phase and 1.5 ml culture was harvested by centrifugation at 11600xg for 2 min in a microfuge. The manufacturer's recommendations (Sigma) using Thermo Scientific DNA extraction method were used. The extracted DNA was used for PCR amplification of the studied genes including the house-keeping rDNA genes.

Polymerase Chain Reaction (PCR)
PCR was performed in a 25μl reaction volume containing 1μl (50 ng) of extracted DNA, 1μl of each pair of primers 12.5μl of 2x Taq premix Mastermix (Sigma) and 9.5μl sterile double-distilled water. The specific PCR primers and the expected products' sizes are listed in (Table 1). The PCR program involved an initial denaturation step at 95°C for 5 min followed by 35 cycles of a denaturation step at 95°C for 30 seconds, a primer annealing step at 54-60°C for 30 seconds, an extension step at 72°C for 30 seconds, and a final step at 72°C for 10 min. In each reaction a positive and a negative control were included as appropriate. The negative control contained all the reagents without template DNA whereas the positive control was the staphylococcal 16S rDNA. All PCR products were analyzed by agarose gel electrophoresis using 1.5 % (w/v) agarose, performed at 100v in TAE buffer (0.5M Tris acetate, 5.7% acetic acid, 10mM EDTA pH 8.0) containing 0.5μg/ml ethidium bromide.

Plasmid curing
S. aureus isolates were sequentially passed in LB (approximately 100 cells into 100 ml) at 43°C with shaking for about 30 generations to eliminate the plasmid. Cured strains were diluted and plated on LA plates to obtain single colonies. Replica plating at 37°C was used to screen for loss of resistance [16]. Loss of the plasmid was confirmed by loss of unselected phenotypic traits (drug resistance) and by PCR of relevant genes [17]. All statistical analyses were performed using SPSS version 16.0 (SPAA Inc., Chicago, IL, USA), using Chi square and Independent sample T tests. p-value <0.05 is considered significant.

N (%) N (%) N (%) N (%) N (%)
The MIC for chlorhexidine ranged from 1 to 8 ug/ml while that for the standard S. aureus ATCC 12600 was 2 mg/ml. All aminoglycoside and methicillin sensitive S. aureus isolates did not carry any quaternary ammonium compound resistance genes and their MIC for chlorhexidine was <2ug/ml. Plasmid curing experiment eliminated AMEs genes and aminoglycoside resistance in 9/21 (42.9%) of aminoglycoside resistant S. aureus isolates.

Discussion
Aminoglycosides have an important role in the treatment of S. aureus associated surgical site infections in many countries including Egypt. There is a limited amount of data regarding the prevalence of aminoglycosides resistance genes (AMEs genes) among S. aureus clinical isolates in Egypt.
The results of this study shows that S. aureus isolated from infected surgical wounds were positive at a high rate for presence of aminoglycosides and methicillin-resistance genes (66.7%) in association with quaternary ammonium compounds resistance genes. Two thirds of the aminoglycoside resistant S. aureus tested in this study carried the mecA gene together with one of the AME genes. None of the S. aureus isolated in this work carried more than one AME gene. Other similar studies reported aac(6')/ aph (2'') as the most frequently detected AME gene (83%) among S. aureus isolates [24,25]. Some studies reported a prevalence of the three AME genes in 21% of isolates [26].
The co-existence of mecA gene and AME genes in a relevant number of tested S. aureus might suggest a genetic linkage as the mecA gene in MRSA is carried on the Staphylococcal Cassette Chromosome mec that encodes genes for other antibiotics including aminoglycosides [27]. Plasmid curing experiment eliminated AME genes and aminoglycoside resistance in (42.9%) of aminoglycoside resistance S. aureus isolates. This result confirmed the carriage of these genes on plasmids, and it is alarming since the horizontal gene transfer by plasmids is highly increasing the risk of drug resistant strain dissemination [28].
Quaternary ammonium compounds are commonly used disinfectants in hospitals to control hospital-associated infection but resistance to these compounds has lately increased [29]. Moreover, it is frequently linked to resistance to other antibiotics such as methicillin and aminoglycosides. However, inconsistent results were reported regarding the frequencies of antiseptic resistance genes including qacA/B and qacC genes in S. aureus in different countries and hospitals [2,30].
A study carried out by Noguchi et al. [2], reported an occurrence rate of qacA/B and qacC genes in Asian isolates to be 41.6% and 1.9%, respectively [2]. In our study qacA/B was more frequent than qacC and was detected in 47.4% of MRSA isolates, whereas qacC was positive only in 28.9% of those isolates. In agreement with this but with lower frequencies, Duran et al. [21] reported the isolation of qacA/B at a higher rate than qacC from MRSA with qacA/B (20.8%), while qacC gene existed at the frequency of 7.7% [29]. A Malaysian study showed much higher rate of detection for qacA/B gene among their MRSA isolates (83.3%), and only 1.9% of these strains were positive for qacC [31].
On the other hand, the study of Nakipoğlu et al., [32] reported an isolation rate of (36%) for qacC in MRSA isolates, while qacA/B was detected only in 4% of those isolates. Along with our results, a recent study reported the presence of antiseptic resistance genes in 68.4% of the aminoglycosides resistant MRSA isolated from surgical site infections, among of these 47.4% carried qacA/B and 28.9% were qacC positive [29].
In conclusion, the current work demonstrates that 66.7% of the aminoglycoside resistant S. aureus isolated from surgical site infections were harboring genes for antiseptic resistance and 42.8% and 23.8% were positive for qacA/B and qacC, respectively.
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