Isolation and Antibiogram of Staphylococcus aureus from Patients Admitted at Specialist Hospital Okitipupa, Ondo State, Nigeria

Aims: To isolate and determine the antimicrobial resistance and susceptibilities of Staphylococcus aureus from patients hospitalized at State Specialist Hospital, Okitipupa, Ondo State, Nigeria. Study Design: Prospective cross sectional. Place and Duration of Study: Department of Biological Sciences, Ondo State University of Science and Technology, Okitipupa, between March and April 2015. Methodology: Forty three (43) samples were obtained from nasal cavity, skin, wound, or umbilical cord of 20 patients aged 4 days to 80 years. Isolates were identified by cultural characteristics on Mannitol Salt Agar, Nutrient agar and biochemical tests. Antibiotic susceptibility testing was performed by the disk diffusion method (Kirby-Bauer). Multiple antibiotic resistance (MAR) index for each S. aureus was determined. Results: Seventeen S. aureus isolates were identified. Nine S. aureus strains recovered from 18 samples from male patients was higher than 8 strains recovered from 25 samples from females. 21-30 age-group yielded the highest number of S. aureus isolates (7 strains), followed by 31-40 Original Research Article Eucharia; BMRJ, 14(6): 1-8, 2016; Article no.BMRJ.25360 2 age group (5 strains). Eleven (64.7%) strains showed multiple resistances ranging from 55.6% to 88.8%, four (23.5%) strains showed multiple resistances ranging from 33.3% to 44.4%, one (5.9%) strain (N1) showed 100% resistance, while one (5.9%) strain (O1) showed 100% sensitivity. 70.6% of the isolates had a MAR index above 0.5 indicating that they probably originated from an environment where antibiotics are frequently used or abused. The highest resistance was offered to the β-lactam antibiotics, including oxacillin (88.2%), cloxacillin (82.4%) and amoxicillin/clavulanic acid (76.5%); and the cephalosporins: cefuroxime (70.6%) and ceftazidime (64.7). Resistance to ceftriaxone (cefuxitin) was the lowest (23.5%). Resistance to erythromycin was high (70.6%), but gentamicin and ofloxacin were offered relatively low resistance (41.2%) respectively. Conclusion: The study revealed multiple antibiotic resistances in S. aureus isolated from hospitalized patients. The preponderance of multiple antibiotic S. aureus in the hospital environment continues to present challenges to the health sector. The high rates of resistance to the β-lactams – oxacillin, cloxacillin, amoxicillin-clavulanic acid and cephalosporins highlight the decreasing importance of β-lactams in the therapy of S. aureus infections.


INTRODUCTION
Staphylococcus aureus is a Gram-positive, facultative anaerobic, unencapsulated cocci which occur in clusters. The bacterium occurs as a commensal on the human anterior nares and on the skin (and less commonly in other locations) of the human body [1,2]. It is a normal inhabitant of the healthy lower reproductive tract of women [3,4]. S. aureus colonizes approximately 20% to 30% of the human population [5], and is reported to be found in 25% to 27% of health workers [6]. The presence of S. aureus does not always indicate infection. S. aureus can survive from hours to weeks, or even months, on dry environmental surfaces, depending on strain [7].
Though the bacterium occurs as normal flora, it can infect tissues when the skin or mucosal barriers have been breached. This can lead to many different types of infections ranging from furuncles and carbuncles to life-threatening diseases such as pneumonia, meningitis, osteomyelitis, endocarditis, toxic shock syndrome (TSS), bacteremia, and sepsis [8,9]. S. aureus is still one of the five most common causes of nosocomial infections and is often the cause of postsurgical wound infections [10]. Each year, some 500,000 patients in United States' hospitals contract a staphylococcal infection [11]. The control of infections caused by S. aureus relies heavily on intensive use of antibiotic drugs. However, there is increasing evidence of drug resistance by this microorganism [12].
The treatment of choice for S. aureus infection is penicillin, a βlactam. Penicillin is bactericidal by inhibiting the formation of peptidoglycan crosslinkages that provide the rigidity and strength in a bacterial cell wall. In most countries, but penicillin resistance is extremely common. This is because many members of the genus Staphylococcus produce penicillinase (a βlactamase), which inactivate penicillins, making them naturally resistant to these antibiotics [13]. Following the emergence of penicillin resistance, penicillinaseresistant β-lactam antibiotics such as oxacillin or flucloxacillin were developed and used as firstline therapy. However resistance to penicillinaseresistant β-lactams soon emerged in many bacterial strains and has become a global health problem [14].
Staphylococcus aureus continues to offer challenges to medical science in the area of resistance to chemotherapeutic agents leading to treatment failures using common antibiotics [15]. Adejuwon et al. [16] reported the presence of S. aureus in pus from acne which was sensitive to gentamicin, tetracycline, amoxicillin, amoxicillin/clavulanic acid, chloramphenicol and sulphamethoxazole but resistant to ampicillin, erythromycin, cloxacillin, cotrimoxazole, streptomycin and penicillin.
Attempts to control diseases caused by S. aureus through the use of antibiotics have resulted in increased prevalence of resistant strains of the organism [17,18]. Therefore, in order to effectively treat infections caused by S. aureus, culture and antibiotic sensitivity tests must first be performed. Once culture and sensitivity results confirm the type of bacterial infection and sensitivity pattern, treatment may be modified [19].
An antibiogram is the result of an in vitro sensitivity test of an isolated bacterial strain to different antibiotics. The antibiogram shows the profile of the antimicrobial resistance and susceptibility of a particular microorganism. The correlation of in vitro to in vivo is often high enough for the test to be clinically useful. In the hospital, antibiograms are commonly used to help guide empirical antimicrobial treatment and are an important component of detecting and monitoring trends in antimicrobial resistance [20].
Strategies for monitoring antibiotic susceptibility patterns should include periodic isolation and testing of isolates for antibiotic susceptibility in all localities, using a state-owned hospital as a sampling center for a cross sectional study. Results of cross sectional studies usually provide a good overview of situations in the entire community.
The aims of the present study were to isolate and determine the profile of antimicrobial resistance and susceptibilities of S aureus isolated from patients hospitalized at State Specialist Hospital, Okitipupa, Ondo State, Western Nigeria. No previous study of this nature has been carried out in Okitipupa L.G.A in Ondo South senatorial district. This study seeks to provide data on the resistance pattern for S. aureus isolated from the hospital in Okitipupa.

Samples Collection, Handling, Culture and Identification
Samples were collected from State Specialist Hospital, Okitipupa from March to April 2015. Sterile cotton swabs moistened in sterile distilled water were used to collect samples from the skin, nostrils, wound exudates or umbilical cord excision sites of hospitalized patients. The swabs sticks were inserted into their tubes and transported immediately into the Microbiology laboratory for analysis. Prior to sample collection, permission was obtained from the Hospital Authorities through a written application. The patients also had to give their consent before samples were collected from them.
All swab samples were directly inoculated onto Nutrient agar and Mannitol salt agar (MSA) plates. The plates were incubated at 37°C for 24 h, and then examined for colony characteristics.
Antimicrobial susceptibility tests were performed for all isolates according to the criteria of the Clinical and Laboratory Standards Institute (CLSI) [24]. Bacterial suspensions were prepared, adjusted to the 0.5 McFarland Standards, and inoculated onto Mueller-Hinton agar (Oxoid) by surface swabbing. Using sterile forceps, the antibiotic-containing discs were placed aseptically on the inoculated plates and left on the table for 1 hour for proper diffusion to occur. The plates were iincubated in an inverted position, at 35°C for 16-18 hours and thereafter examined for clear zones of inhibition. Inhibition zone diameters (IZD) around each antibiotic disk (if any), were measured using a transparent ruler, and recorded in millimeters (mm). A standardized table was used to determine if the bacterium was "Resistant", "Intermediate" or "Sensitive".

Determination of Multiple Antibiotic-Resistance (MAR) Index
Multiple antibiotic-resistance (MAR) index of each S. aureus isolate was calculated using the formula: MAR Index = a/b, where a is number of antibiotics the isolate is resistant to; and b is number of antibiotics tested.

Isolation of S. aureus
A total of 43 samples were collected from skin, nares, wound or unbilical cord scar of twenty patients and analyzed. Seventeen (17)

Percentage of resistances, intermediates and sensitivities of S. aureus isolates
The seventeen (17) S. aureus isolates showed varying degrees of susceptibilities and resistances to the antibiotics. The percentage resistances, intermediates and sensitivities of each S. aureus isolate to the antibiotics are represented by a stacked column chart (Fig. 1).
The chart in Fig. 1 clearly depicts the degrees of resistances, intermediates and sensitivities shown by each S. aureus isolate to the antibiotics. Strain N1 showed resistance to all the antibiotics used (100% resistance), and strain O1 showed sensitivity to all the antibiotics used (100% sensitivity). Eleven (64.7%) isolates showed multiple resistances ranging from 55.6% to 88.8%. Four (23.5%) isolates showed multiple resistances ranging from 33.3% to 44.4%.

Multiple Antibiotic-Resistance (MAR) indices S. aureus isolates
Multiple antibiotic-resistance (MAR) index was calculated using the formula: MAR Index = a/b, where a is number of antibiotics the isolate is resistant to; and b is number of antibiotics tested. MAR Indices of the isolates are presented in Table 1. MAR index shows the level of multiple antibioticresistance attained by a particular bacterial strain. From data presented in Table 1, MAR Indices of the isolates varied from 0.00 (not resistant to any antibiotic tested) to 1 (resistant to all antibiotics tested). 70.6% of the isolates had a high MAR index above 0.5.

Resistance profile of S. aureus isolates
The number and percentage of S. aureus isolates resistant to each antibiotic used is presented on Table 2.

Fig. 1. Percentage resistances (R), intermediates (I) and sensitivities (S) shown by S. aureus isolates (A1 -V2) to the antibiotics Axis-Y: percentage of susceptibility. Axis-X: isolate number
The Figure shows the Table 2 shows how each antibiotic fared with the isolates. For instance, to ceftazidime (CAZ) 64.7% of all the isolates were resistant.
Results from the present study revealed multiple antibiotic resistance (MAR) in S. aureus strains isolated from Specialist Hospital, Okitipupa.
70.6% of the isolates had a MAR index above 0.5 indicating that they probably originated from an environment where antibiotics are frequently used or abused. Multiple resistance in S. aureus seems to be a longstanding problem yet to be solved, since a similar finding was reported from Zaria, Nigeria in 2003 [25].
The resistance profile showed a high rate of resistance to β-lactam antibiotics including oxacillin (88.2%), cloxacillin (82.4%), amoxicillin/clavulanic acid (76.5%), and the cephalosporins: cefuroxime (70.6%) and ceftazidime (64.7). However, resistance to ceftriaxone (cefuxitin) was the lowest (23.5%). Resistance to β-lactam antibiotics in this study may be due to hyper-production of β-lactamase enzymes which inactivate this class of antibiotics [13,14]. Resistance to erythromycin was high (70.6%), but gentamicin and ofloxacin were offered relatively low resistance (41.2%). Findings in this study agree with previous workers who reported high rates of antibiotic resistance in S. aureus [15,16,26]. However, the findings differ from reports from another part of Nigeria where phenotypic resistance to oxacillin was 40.3%, cefuxitin 46.5%, gentamicin 11.5% and erythromycin 21.2% [27]. treatment regimes administered to hospital patients. In hospitals, inappropriate use of antimicrobial agents tends to create a selective pressure that promotes the emergence of resistant strains and predisposes patients to colonization with such organisms. The carriage of S. aureus on the bodies, especially hands of 25%-27% of health workers reported by previous workers [6] is an important factor in transferring resistant microbes to patients hospitalized in the wards.
The dominance of antibiotic resistant pathogens in the hospital environment is a phenomenon that must be approached in a more proactive manner. More emphasis should be placed on proper and regular hand washing by health workers, sterilization/disinfection of instruments, materials and surfaces, as well as maintenance of sanitary conditions in the wards. All these will greatly reduce the occurrence and transfer of resistant microbes in the hospitals. Regular washing of bed clothes with soaps and disinfectants, and use of sterile hand gloves when handling patients will ensure that antibiotic resistant microbes do not survive and are not transferred from patient to patient.
Research efforts to develop alternative treatments for resistant bacterial infections should attract more funds from the government and stakeholders on the private sector. Special attention should be focused on medicinal plant extracts which have been used as antimicrobials for centuries with no development of resistance. Vaccine development is another area that should not be undermined in the search for solution for antimicrobial resistance in bacterial pathogens. More research effort is needed in the area of DNA vaccines, which is reported to be effective, with no adverse effects in experimental animals [28,29].

CONCLUSIONS
High MAR indices above 0.5 shown by 70.6% of S. aureus strains isolated from Specialist Hospital, Okitipupa, reveals the challenges in the area of empirical therapy for diseases caused by S. aureus. The high rates of resistance to the βlactam antibiotics including oxacillin (88.2%), cloxacillin (82.4%), amoxicillin/clavulanic acid (76.5%), and the cephalosporins: cefuroxime (70.6%) and ceftazidime (64.7) highlights the decreasing importance of these drugs in antimicrobial therapy for S. aureus infections.
Antibiotic resistance is highest where a majority of the population is poor and ignorant of appropriate use of antibiotics as is the case with the study area in this investigation. The authorities in the health sector should mount public enlightenment programs to educate the masses on the dangers of abusing antimicrobials, as well as the appropriate ways to use them. Development of and strict adherence to sound antibiotic policies in the health sector will help slow the emergence of drug resistance.