Abstract: Staphylococcus aureus are organisms that have been detected as the major pathogens in hospital and community settings that are responsible for infections and their resistance to commonly used antibiotics has become worldwide concern. The indiscriminate use of antibiotics has led to the presence of drug resistant organisms. A total of 100 wound swabs were obtained from two tertiary hospitals in Port Harcourt, Nigeria and screened for S. aureus. This was done by streaking the swab sticks on sterile mannitol salt agar plates, positive growths were identified using conventional methods based on morphological and biochemical characteristics. Identified S. aureus was subjected to antibiotics susceptibility test using Kirby Bauer disc method. Plasmid profiling was also carried out on 30 randomly selected S. aureus using gel electrophoresis. Out of the 100 wound swabs examined, ninety-two were positive for S. aureus. Antibiotic susceptibility testing showed that S. aureus was 100% resistant to caftaxidim, ceftriazone, ofloxacin and ceptazdime. Staphylococcus aureus was sensitive to ofloxacin and gentamicin (88%), erythromycin (84%) and augumentin (82%). Plasmid profile result for S. aureus revealed single plasmid sizes of 2027, 2322 4361 and 23130 bp, respectively while double plasmid size was observed in one isolate. The determination of antibiotic sensitivity pattern and plasmid profiling of S. aureus isolated from wound will assist in the preliminary treatment of wound infections.
INTRODUCTION
Staphylococcus aureus is a facultative anaerobe, non-motile gram positive bacteria that occur singly in pairs (Akpaka et al., 2006). Staphylococcus species are known to be responsible for a wide number of diseases both in humans and animals. Staphylococcus aureus frequently colonizes the human skin, skin glands and mucous membrane of warm blooded animals. Staphylococcus aureus is present in about 25-30% of humans especially adults (Lowy, 1998; Akpaka et al., 2006; Haque et al., 2011; Den Heijer et al., 2013). Most times they can exist as normal flora of the skin, however if there is a break in the skin from a wound or surgery, or if there is a suppression of a person’s immune system, then the presence of S. aureus in the skin can cause an infection (Koneman et al., 1997). Staphylococcus aureus lives mostly on mucosal surfaces (Chiquet et al., 2007) and it is frequently involved in numerous hospital acquired infections (Chiquet et al., 2007). The mucoid strain observed in S. aureus is due to the capsule which enhances virulence (Alberich and Herbarth, 2001; Chiquet et al., 2007; Jensen and Lyon, 2009). Staphylococcus aureus is responsible for a broad spectrum of human and animal diseases, not just skin infections but severe diseases in humans and animals such as pneumonia and endocarditis among others (Dar et al., 2006). The genes that are responsible for antibiotic resistance in S. aureus are usually found on the plasmids or some other structures similar to plasmids (Sambrook et al., 1989). There has been an increase on drug resistance of S. aureus to most antibiotics in recent times, only few antibiotics are available for most of the staphylococci which are glycopeptides antibiotic such as vancomycin and teicoplanim (Chiquet et al., 2007; Dar et al., 2006). Infections by some species of staphylococci are difficult to treat because of frequency of multiple antibiotic resistant strains.
The genes for antibiotic resistance in S. aureus are located on the plasmid, these plasmids contain resistant genes against a number of antimicrobial agents (Simpson et al., 2007). Studies carried out to determine the role of plasmids on antibiotic resistance has been useful in determining the characteristics of plasmids in bacteria. Plasmids can be transferred from one close bacterium to another horizontally, while for bacterium that is distant from one another plasmids can be transferred phylogenetic (Dale and Park, 2004). These two modes of transfer of plasmids might be responsible for the spread of antibiotic resistance genes in the environment (Dale and Park, 2004). This gives rise to the suspicion that there is a possibility that these resistant genes can be transferred from one bacterium to another (Imade et al., 2010). Plasmid profiling and restriction endonuclease analysis of plasmid DNA are among the molecular techniques that are currently being used in differentiating strains of bacteria isolates. Plasmid profiles have been useful in the epidemiological surveillance of disease outbreaks and in tracing antibiotic resistance (Liu et al., 1995; Dutta et al., 2002; Tayfour et al., 2005).
The present study was carried out to determine the antibiotic resistant pattern and plasmid profile of S. aureus obtained from wound swabs.
MATERIALS AND METHODS
Source of specimen: A total of 100 wound swabs were obtained from patients attending two hospitals within Port Harcourtfrom from October 2013 to February 2014. Wound swabs were immediately transported to the University of Port Harcourt medical laboratory for isolation and characterization. After isolation and identification the isolates were taken to Nigerian Institute for Medical Research for plasmid profiling.
Isolation and identification of S. aureus: The swabs were aseptically cultured on mannitol salt (Oxoid) agar plates and incubated at 37°C for 24-48 h and examined for pure distinct colonies. The identification of the Staphylococcus isolates was done following standard microbiological technique which involves morphological, cultural characteristic, biochemical characteristics and coagulase test. The isolates were sub-cultured into nutrient agar (Oxoid) slant and incubated at 37°C for 24-48 h and later stored in the refrigerator for further use (Cowan et al., 2003; Cheesbrough, 2006; CLSI., 2011).
Antimicrobial susceptibility pattern of Staphylococcus isolates: Antibiotic susceptibility pattern was carried out on 92 isolates using Kirby Bauer disc diffusion method. From each slant culture of isolates, five representative colonies were touched with a sterile wire loop and suspended in sterile nutrient broth (Oxoid). Each isolate suspension was adjusted to 1×108 densities equal to the 0.5 McFarland standards before inoculation. From the nutrient broth 0.1 mL of the isolate was surface spread evenly on Muller-Hinton Agar (MHA, Oxoid)) plates. Sensitivity discs were picked with a pair of sterile forceps and placed on the plate which was incubated aerobically at 37°C for 24 h (CLSI., 2011). The antibiotics used were as follows: ceftazidime (CAZ) 30 μg, gentamicin (GEN)10 μg, cefuroxime (CRX) 30 μg, ceftriaxone (CTR) 30 μg, cloxacillin (CXC) 5 μg, ofloxacin (OFL) 5 μg, erythromycin (ERY) 30 μg, amoxicillin/clavulanate (AUG) 30 μg.
Plasmid profile of S. aureus isolates: Out of the 92 identified Staphylococcus isolates obtained, plasmid profiling was carried out on 30 randomly selected S. aureus isolates. Plasmid extraction was carried out based on the methods of Molina-Aja et al. (2002) with slight modifications. In brief, a single bacterial colony was picked up and grown in 5.0 mL of Muller Hilton broth in an eppendorf tube and centrifuged at 10,000 rpm for 2 min. The cell pellets obtained were re-suspended in 150 μL EDTA-Tris buffer and vortexes to mix. This was followed by the addition of 175 μL of 2% SDS and 175 μL of 0.4N NaOH. The tube was mixed vigorously, 250 μL of cold 5 M potassium acetate was added vigorously, the tube was centrifuged at 12,000 rpm for 5 min and the supernatant was transferred to a sterile 1.5 mL eppendorf tube and equal volume of cold isopropanol was added. After inverting gently, the mixture was immediately centrifuged at 12,000 rpm for 10 min and the DNA pellet was washed with 650 μL of cold (4°C) 70% ethanol by centrifuging at 12,000 rpm for 15 min. The supernatant was discarded and the pellet was dried for 30 min and re-suspended in 40 μL of sterile deionized water. Agarose gel was prepared and poured into electrophoresis tank and allowed to solidify. The sample 15 μL was mixed with 2 μL of the loading dye was loaded into wells. Gels were visualized and photographed using digital photo documentation system (Clinix, Japan) (Sambrook et al., 1989; Molina-Aja et al., 2002).
Calculation of molecular weight fragments: The fragment bands observed were directly compared with the molecular weight marker bands Hind III digest of Lambda phage from Roche Diagnostics Germany) which has eight distinct visually observed bands with molecular weight 1820, 21140, 23130, 9416, 6557, 4361, 2322 and 2027 bp.
RESULTS
Out of the 100 wound swabs collected, 92 of them were positive for S. aureus. Identified S. aureus were subjected to antibiotic sensitivity test using the disc diffusion method. Percentage of resistance and sensitivity of the isolates to antibiotics was shown in Table 1. Staphylococcus aureus was 100% resistant to ceftazidime followed by ceftriaxone (88.04%). Most of the isolates were highly susceptible to ofloxacin (95.65%) followed by gentamicin (88%), erythromycin (84%) and augumentin (82%) as shown in Fig. 1.
| Table 1: | Percentage of resistance and sensitive Staphylococcus aureus to antibiotics |
CAZ: Ceftazidime, CRX: Cefuroxime, GEN: Gentamicin, CTR: Ceftriaxone, ERY: Erythromycin, CXC: Cloxacillin, OFL: Ofloxacin, AUG: Amoxicillin/Clavulinate | |
| Fig. 1: | Percentage susceptibility pattern of Staphylococcus aureus to antibiotics |
| Fig. 2(a-c): | Plasmid profile of isolate in (a) Lane 1-12, (b) Lane 13-26 and (c) 27-30 |
Thirty isolates were randomly selected from the 92 S. aureus isolates for plasmid profiling. Results of plasmids are showed in Fig. 2a-c. The plasmid profile result for S. aureus revealed that S. aureus possessed mostly single plasmid, just a few had double plasmid size. In Fig. 2 isolates in lane 9 and 10 does not possess plasmid. The plasmid size for all the isolates in Fig. 2 is 23130 bp.
Figure 2b shows the plasmid profile of Staphylococcus aureus isolates in lane13-26. The isolate in lane15 does not possess plasmid while isolate 17 had four plasmid sizes of 2027, 2322, 4361and 23130 bp.
Figure 2c shows the plasmid profile of isolates in lane 27-30. Isolate 27 had double plasmid of 23130 and 9416 bp while isolate 28, 29 and 30 had plasmid size of 23130 bp.
DISCUSSION
The study was carried out to determine the antibiotic resistance and plasmid profile of S. aureus from wound swab. The antimicrobial susceptibility pattern was determined for all isolates. In this study, the frequency of S. aureus isolates was 92%. The frequency is higher than reports from previous studies from various clinical swabs (Akpaka et al., 2006; Rajaduraipandi et al., 2006; Al-Hamdani and Hamad, 2012). Their studies reported a frequency of S. aureus of 47.3% from various clinical swabs. All S. aureus isolates tested in this study were completely resistant to ceftazidime (100%) and ceftriaxone (88.04%) antibiotics. A similar result was also reported in a study where complete resistance of S. aureus to ceftazidime was observed while the isolates in our study were susceptible to erythromycin and gentamicin (Akpaka et al., 2006). Our study is also in agreement with previous study where result showed gentamicin and erythromycin was more effective in the management of S. aureus from surgical wound in that locality (Idighr et al., 2012). In the present study, S. aureus susceptibility to ofloxacin and gentamicin was 95.65%. This result is in agreement with reports from Nwankwo and Nasiru (2011) who reported 76.6% susceptibility to ofloxacin and 73.4% for gentamicin. This also compares favorably with results from other researchers. Previous studies have shown 100% resistance of S. aureus to AUG which is in contrast to the finding of this work. Similar work done carried out showed 6% resistance to gentamicin and resistance rate of 10.4% to ofloxacin (Rajaduraipandi et al., 2006). A study carried out by Stanley et al. (2013) on the antimicrobial susceptibility of S. aureus from isolated pregnant women showed that many were only moderately susceptible to tested antibiotics. In their study levofloxacin, ceftriaxone, clindamycin and erythromycin all appeared in decreasing order (Stanley et al., 2013). Their work was in agreement with the study carried out by Imade et al. (2010), where results obtained suggested ceftriaxone, ciprofloxacin, augumentin, ofloxacin and gentamicin as drugs of choice for S. aureus.
In most developing countries antimicrobial resistance is an aspect of great concern thus the need for further research. Plasmid profile studies are widely used for epidemiological studies and the role of plasmids in drug resistance. The wide spread of antibiotics resistance observed in isolates that possessed plasmids in the present study can be associated to the increased and abusive use of antibiotics (Liu et al., 1995; Ombui et al., 2000; Davis and Amabile-Cuevas, 2003). Most of the S. aureus in the present study showed similar antibiotic resistance pattern, while different plasmid sizes was observed in most of the isolates. This disparity can be due to R- plasmids of different sizes which are also responsible for the presence of multiple resistances. Staphylococcus aureus can acquire genetic elements from other bacteria. Large mobile genetic elements appear to encode both antibiotic resistant factors and proteins that are responsible for increase in virulence thus giving the organism the ability to adapt to the selective pressure of antibiotics (Alberich and Herbarth, 2001; Ojo et al., 2014). Plasmid profile analysis of isolates makes it possible to evaluate the dependence between antibiotic susceptibility and the presence of plasmids with respect to a particular isolate (Ojo et al., 2014). It can be said that resistance mediated by plasmid which has been observed in the various studies may be responsible for the differences observed in treatment by clinicians (Alberich and Herbarth, 2001; Ojo et al., 2014).
CONCLUSION
In this study, it was observed that S. aureus was a major pathogenic agent prevalent in wound. The high level of resistance could be associated with exposure of these drugs to isolates which may have enhanced development of resistance. There is a high level of antibiotic abuse in developing countries such as Nigeria arising from self-medication associated with inadequate dosage and failure to comply with treatment and availability of antibiotics to consumers across the counters with or without prescription.
ACKNOWLEDGMENTS
We acknowledge Dr. Stella Smith and staff of the Department of Molecular Microbiology, Nigeria Institute of Medical Research, Yaba where the plasmid analysis was carried out.