1. Introduction

Staphylococcus aureus is one of the most important a frequent cause of nosocomial infections worldwide ^[1]. Emergence of Methicillin-resistant S. aureus strain (MRSA) in 1961 made staphylococcal infections as a major therapeutic challenge ^[2]. Methicillin Sensitive S. aureus (MSSA) and Methicllin Resistant S. aureus (MRSA). MRSA strains have been associated with a or hospital acquired infections world over and have also emerged as an important cause of community acquired infections ^[3]. MRSA infections are a problem across the whole health economy, and have been shown to be associated with a poorer outcome and higher mortality than similar infections caused by Methicllin-sensitive strains of S. aureus (MSSA).

Many of these isolates are becoming multidrug resistant and are susceptible only to glycopeptides antibiotics such as Vancomycin ^[4] Vancomycin-resistant Staphylococcus aureus (VRSA) infections, which are always methicillin-resistant, are a rare but serious public health concern ^[5]. In 1988, vancomycin-resistant enterococci (VRE) were first reported. These organisms quickly became endemic in hospital intensive care units. In vitro conjugative transfer of the vanA gene from enterococci to S.aureus was demonstrated in 1992. However, it was not until 1996, when the first case of vancomycin intermediate S.aureus (VISA; MIC, 8–16 µg/mL) was detected, that decreased susceptibility to vancomycin became a clinical reality. None of the VISA strains identified contained the vanA gene or any of the other vancomycin-resistant genes found in VRE. In vitro studies suggest that, with prolonged vancomycin exposure, VISA organisms produce a thickened cell wall matrix, limiting drug penetration ^{[6, 7]}. Recently, however, due to the use of Vancomycin for treatment of hospital-based infections, there has been growing resistance to Vancomycin by species such as Enterococcus ^[8]. The emergence of Vancomycin resistance in Enterococcus gave rise to the possibility of horizontal transmission of resistance elements to S. aureus. In the laboratory, Vancomycin resistance has been transferred from Enterococcus faecalis to S. aureus ^[9]. The objective of our study was to determine the prevalence of Vancomycin resistance Staphylococcus aureus (VRSA) among Methicillin resistant S. aureus (MRSA) in Khartoum State.

2. Methods

This cross sectional study was conducted during the period of March to October 2015. Swabs of wound, skin (axella, groin), sputum, ear, and throat, also urine, blood and body fluids and sputum specimens were collected. Patients in Intensive Care Unit (ICU) and wards, nasal swabs were collectred from different Khartoum hospitals after obtaining consent. Patients were aged between days to 84 years from both sexes. Mannitol Salt Agar, Müeller Hinton agar, Nutrient agar, Nutrient broth and DNAase Agar media were prepared in laboratory depending on the manufacturers' instruction. S. aureus were identified by fermentation of mannitol, colony morphology, Gram stain, Catalase test and sub-cultured on nutrient agar for coagulase test and deoxyribonuclease (DNase) test by conventional methods. All MRSA strains were determined by disk diffusion method using and confirmed by MIC (bioMerieux) method using the Oxacillin E-test then by Vancomucin E-test for VRSA strains according to National Committee for Clinical Laboratory Standards ^[10]. The following antibiotic disks were tested: Oxacillin, (OX 5μg), Vancomycin (VA 30μg), Ampicillin/Cavulinic acid (AMC 10μg), Amoxicillin (AMX 10μg), Tetracyclin (TE 30μg), Erythromycin (E 15μg), Gentamycin (GEN 10μg), Ciprofloxacin (CIP 5μg), Clindamycin (CD 2μg), Chloramphenicol (C 30μg) Ceftrixone (CTR 30μg), Trimethoprim-sulphanimide (COT 30μg, Nitrofurantoin (NIT 300μg) and Meropenem (MEM 10 μg). Potential anti-MRSA synergy was measured by fractional inhibitory concentration indices (FICI) This could be calculated as: The FIC of drug ‘A’, given by FICA = MICA combination / MICA alone while The FIC of drug ‘B’, given by FICB = MICB combination /MICB alone The FIC index of the combination in each tube is given by the sum of the FICs for each of the drugs present in the tube: FIC index = FICA + FICB ^[11].

Statistical analyses were conducted using SPSS (version 20; SPSS Inc., Chicago, IL) software. Descriptive analyses of percentages of categorical variables were reported using chi square x². p value of < 0.05 denoted a statistically significant difference in all statistical comparisons.

This study was approved by ethical clearance committee, Department of Microbiology- Tropical Medicine Research Institute., Khartoum, Sudan.

3. Results

Out of the 135 bacteria isolated from clinical specimens, 79(58.5%) were identified to be Staphylococcus species. Only 61 (97.2%) were Identified biochemically to be S. aureus, out of this 36(59%) Methicillin Sensitive S. aureus (MSSA) and 25(41%) Methicllin Resistant S. aureus (MRSA), Figure 1, Figure 3. MRSA were collected from patients in ICU units and wards from different Khartoum hospitals. Resistance to Methicillin was determined by two methods: Agar diffusion test to detect oxacillin (≤ 10 mm) was considered resistant (3.9) and E-test for determination of Oxacillin Minimum Inhibitory Concentration (MIC).

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Figure 1. Distribution of S. aureus isolates (MRSA/MSSA)

The resistance defined as an MIC of ≥4 µg/ml (10). In this study all MRSA isolates displayed an Oxacillin MIC of ≥256µg/ml. Resistance to vancomycin was determined by two methods: Agar diffusion test to detect vancomycin resistance (≤ 9 mm) was considered resistant (12) and E-test for determination of vancomycin MIC. The resistance defined as an MIC ≥16 µg/ml (5,10). In this study all VSSA isolates displayed vancomycin MIC of < 2µg/ml "except three intermediate resistant isolates MIC between 4-8 µg/ml" The percentage of the VISA was 12.0%, Table 1, Figure 4. We found a VISA strain in a patient who had underlying conditions including immunosuppressive therapy, long-time hospitalization, and serious disease. The majority of the MRSA isolates were resistant to multiple antimicrobial agents, in this study out of 25 MRSA strains isolated, exhibited high resistance to different classes of antibiotics including; Amoxicillin/Clavulinic acid and Ampicillin (100%), Ceftriaxone and Erythromycin (80%), Meropenem (60%), Ciprofloxacin, Clindamycin and Trimetoprim-Sulphanomide (56%), and Tetracycline (44%). Nitroforantoie exhibit no resistant pattern (100%) while 92% of MRSA isolates were susceptible to Chloramphenicol Table 2. No synergistic effects were detected from the above mentioned combined antibiotics according to FICI.

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Figure 2. prevalence of MRSA/MSSA strains obtained from different clinical specimens in Khartoum state

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Figure 3. E-test technique for MRSA/MSSA strains (a) MRSA strain with MIC > 256 µg/ml and (b) MSSA strain with MIC = 2µg/ml

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Figure 4. VISA strains by E-test method: (a) MIC =4 µg/ml, (b) MIC= 8 µg/ml

Table 1. Prevalence of VISA/VSSA obtained from clinical specimens according to E-test method (BíomMéríeux) of vancomycin

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Table 2. Antimicrobial resistance pattern of MRSA obtained from clinical specimens

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4. Discussion

In our study Oxacillin disk could not detect all MRSA isolates but Oxacillin MIC test had determined these MRSA isolates. Our result is similar to a study conducted in Iran ^[13]. It showed that the sensitivity of Cefoxitin disk and Oxacillin MIC test equal to PCR. Disc diffusion method of Vancomycin did not differentiate between VISA and VSSA strains. So the result is not considered unless the MIC of Vancomycin is used according to NCCLS. Besides those MRSA isolates, Coagulase Negative Staphylococcus (CoNS) was also isolated from clinical specimens in two patients. One of them was found to be methicillin and vancomycin resistant and the other was methicillin and vancomycin susceptible. Methicillin resistant coagulase negative isolate may be a result of the horizontal transfer of mecA gene between MRSA and coagulase negative staphylococcal species. In our study, all isolates ´except three intermediate resistant isolates in which MIC values between 4-8 μg/ml` were susceptible to vancomycin. They displayed MICs of < 2 μg/ml). The unprevalence of resistance to vancomycin may be related to the low usage of this antibiotic in the study area. Also our result substantiated the result of the study carried out in Khartoum ^[14]. Antibiotics resistance has become a serious public health concern with economic and social implications throughout the world, being community acquired infections like Streptococcal infections, pneumonia, typhoid fever or hospital acquired infections due to MRSA, VRE, VISA or extended spectrum β -lactamase (ESBL) enzyme producing Gram -ve bacteria.

These infections lead to higher rates of hospitalization, longer hospital stay, and increased cost of treatment and thus increased economic burden on the community ^[15]. MRSA strains were sensitive to Nitroforantoie, Chloramphenicol (100%), (92%) respectively while they were highly resistant to antibiotics including; amoxicillin /clavulinic acid and ampicillin (100%), ceftriaxone and erythromycin (80%), meropenem (60%), ciprofloxacin, clindamycin and trimetoprim- sulphanomide (56%), and tetracycline (44%). Our results go with the results of a study conducted in Sudan ^[4] that revealed a cross-resistance of MRSA strains. The rapid proliferation of antibiotic-resistant pathogens has spurred the use of drug combinations to maintain clinical efficacy and combat the evolution of resistance. Drug pairs can interact synergistically or antagonistically, yielding inhibitory effects larger or smaller than expected from the drugs’ individual potencies. The inhibitory effect of two drugs in combination can be larger or smaller than expected from their individual effects, corresponding to synergistic or antagonistic interactions between the drugs respectively. Synergistic interactions are usually thought of as advantageous since, for a given amount of drug. They inhibit the growth of drug-sensitive pathogens more effectively. However, in vitro studies have suggested that, for the same level of inhibition, more synergistic drug pairs may foster antibiotic resistance. Antagonistic drug combinations, on the other hand, are less effective at inhibiting drug-sensitive pathogens, but can reduce and even invert the selective advantage of single-drug resistant mutants, causing selection against resistance ^[16]. While this is so, we recommend more combinations of different antibiotics to solve this resistant problem.

5. Conclusion

In conclusion, S. aureus is highly prevalent in Khartoum hospitals, Sudan. MRSA is highly prevalent among populations of S. aureus isolated from different clinical specimens in different hospitals in Khartoum State, Sudan, with most of the MRSA isolates being from ICU patients.

The unprevalence of resistance to vancomycin may be related to the low usage of this antibiotic in the study area where it is used as a drug of choice for the treatment of MRSA infection.

Acknowledgments

The authors wish to acknowledge Professor Hassan Bashir EL-Amin for his helpful advice, Ms. Hadia in Zaitona hospital and Ms. Kamylia in ENT Hospital for their help in sample collection.

Competing Interests

The authors declare that they have no competing interests.

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