Introduction
Staphylococcus aureus is a facultative anaerobic Gram-positive cocci bacterium. It is the most clinical important isolated human bacterial pathogen species cause to infection[1, 2] also It is an endogenous microorganism, colonizes approximately 30’50 % various part of healthy humans such as nasal cavity, skin, gastrointestinal, anus and vaginal vulvae . Frequently, cause a range of illnesses, from minor skin infections, such as pimples, impetigo, boils (furuncles), cellulitis,folliculitis,carbuncles, scalded skin syndrome, and abscesses, to life-threatening diseases such as osteomyelitis, meningitis, pneumonia, endocarditis, toxic shock syndrome (TSS), sepsis, bacterimia.[2-4] and S.aureus infections have become resistant to various antimicrobial agents including Beta-lactams such as penicillin, cephalosporin and carbapenems, and non Beta-lacatam antibiotics (Aminoglycoside, and Macrolides). Particularly Methicillin-resistant S. aureus (MRSA) have made these infections more difficult to treat and poses a serious therapeutic problem worldwide. mechanism of (MRSA) is the production of beta-lactamase which can protect the Microorganisms against all Beta-lactam by hydrolysis of its beta-lactam ring. Another mechanism is associated with penicillin-binding protein 2a (PBP2a), encoded by mecA. Another gene involved in penicillin resistance in staphylococci is blaZ which encodes beta-lactamase[5].
Colonized patients are the chief source of S. aureus in hospitals[6] and Several studies in worldwide have estimated that MRSA strains accounted for 84% of hospital-acquired S. aureus isolates and 45% of nonhospital acquired S. aureus isolates in Taiwan in 1998[7] while the incidence of MRSA were recorded from 25% in western part of India to 50 % in South India [8,9]. Therefore, the MRSA infections represent a burden for patients and healthcare systems because of their associated high morbidity, mortality and increased hospitalization costs. Norma et al reported 97.8% of MRSA isolates sensitive to Linezolid in Mexico [10] in contrast, Shahnaz et al from Iran reported 14.3% of MRSA isolates sensitive to linezolid [11]. While In 2003 Anupurba.S found 100% sensitivity of MRSA to Linezolid in Hyderabad-India.[12]
The intensive and extensive survey of literature reveals that there was a little research study on sensitivity of MRSA to linezolid reported in Bangalore- India, thus the main objective of the present work is to detect sensitivity of MRSA to linezolid among isolated clinical samples of patients which suspected S, aureus infection at Hospitals in Bangalore- India.
Material and methods
Samples Collection processes
A total fifty clinical swab samples (20 wound, 18 Nasopharyngeal and 12 High Vaginal swap ) (Table 1) were collected according to the standard collection techniques [13]from these patients attending in hospitals in Bangalore city, Karnataka state between February 2012 to May 2012 after ethical permission was obtained from these hospitals, Clinical swab samples were collected by using sterile cotton-tipped _swabs placed in Amies transport medium(Himedia,india), after the cape firmly screwed, Labeled and transport the medium to the lab within 24 hours in an ice-cooler but do not freeze according to the manufacture instruction.
Microbiological identification of Isolated S. aureus
The cotton-tipped swabs were received in Amies transport medium and were broken off into the Tryptic Soy Broth (TSB) (Himedia, India) for enrichment and vortex for 10 second to trap entire bacteria from cotton-tip to the broth then incubated at 37 for 48hours. After an incubation period using a new sterile cotton swab for inoculation of samples from TSB on the Mannitol Salt Agar plate(Himedia, India). The swabs were pressed to the side of the tube and rotated for removing excess media and inoculated on MSA plated by streaking technique to cover entire surface agar plate, then aerobically incubated for approximately 48 hours at 37 ”C and examine for fermentation of Mannitol [14].
Gram staining the colonies of fermented MSA agar was performed and examine Microscopically for cocci in grape like cluster shape, Gram positive colonies on MSA agar were inoculated by streak technique on Blood Agar (BA) (Himedia, India), TSA and incubated aerobically at 35 ”C for 48 hours. The following cultural characteristic such as smooth, convex, circular, yellow colonies and B-hemolysis on blood agar has been examined. Also further confirmed, the colonies from blood agar plates (BA) suspected Staphylococcus aureus were subjected to biochemical test to identify S.aureus such as Catalase test and Coagulase test, but Catalase test was performed from TSA because red blood test has catalase and thus yield a false positive reaction. S. aureus ATCC 25923 was known coagulase production used as control strain.[14,15].
Inoculums, preparation and Culturing Mueller-Hinton agar
In this research study sterile swab sticks were used to isolate three or four colonies of confirmed S.aureus from blood agar plates and suspended them in (5ml) of Tryptic Soy Broth, turbidity was adjusted to 0.5 Mc Farlands standards contain (1 x 108 cfu/ml). Within 15 min the sterile cotton swab was dipped into the inoculums and swabbing over the surface Mueller-Hinton agar plate (Himedia, India) so held the plates at room temperature for 15 minutes then applying the antimicrobial agent-impregnated discs.[16].
Antibiotic susceptibility testing
Kirby-Bauer disk diffusion method on Mueller-Hinton agar plate (Himedia, India) was used for the Antibiotic susceptibility test which was recommended by the Clinical and Laboratory Standards Institute [16]. The following eight antibiotic disks which were obtained from high media laboratories Pvt.Ltd, such as inhibitors of the cell wall synthesis [Ampicillin (AMP, 10”g), Amoxicillin (AMX, 10”g), Cefoxitin (Cef,30”g),]. Inhibitors of protein synthesis, [linezolid (Lin, 30”g) and Gentamicin (Gen,10”g)]. Inhibiting the DNA synthesis [Ciprofloxacin (Cip,10 ”g)] Chloramphenicol(Cl,30 ”g) and Cotrimoxazole (Cot,25 ”g)] were placed on the Mueller-Hinton agar 100-mm plate by using sterile forceps and were gently pressed down to ensure contacted. Within 15 minutes after the discs were applied, inverted the plates so incubated them aerobically at 37”C for 18-hrs. All the isolates of S. aureus were subjected to Cefoxitin disc to detect MRSA. After incubation the inhibition zone diameters were measured with a ruler and according to the recommendation CLSI guidelines, results were interpreted [17].
Result:
Based on Bergey’s Manual of Determinative Bacteriology, in this research study 30 out of 50 clinical swab samples were Gram positive cocci according to Gram stain test. 20 samples out of 30 were positive based on Catalase test for Staphylococcus and Micrococcus species. In addition to the above tests, 10 out of 20 samples were positive based on Mannitol Salt Agar fermentation for S.aureus colonies. It was found the color changed from red to yellow. In order to confirm the above results in another test has been carried out. It can be seen that the Coagulase test was positive for last 10 samples of Isolated S.aureus. From 50 clinical samples were analyzed, 10(20%) showed positive for S.aureus which were included high vaginal swab samples 3( 25%) , wound swab samples 4 (20%) and nasopharyngeal swab samples 3 (16.6%).
The result of an antibiotic susceptibility pattern of isolates S. aureus was presented in Table 2. It is clear from the table that S.aureus can be divided to the following groups: (i) resistance of S.aureus was recorded for the penicillin group as follows: Amoxicillin (90%), Ampicillin (90%) and Cefoxitin (90%). (ii) Fluoroquinones group: Ciprofloxacin (60%). (iii) Sulfonamides and Trimethoprim group: Cotrimoxazole (60%). (iv) Phenicol group Chloramphenicol (60%). Also High percentage (70%) of S.aureus resistance was recorded for Aminoglycoside group: Gentamicin as shown in figure 1. It is important to notice that all samples were sensitive (100%) to Oxazolidinones group: linezolid as can be seen in figure1.The maximum rate of resistance was recorded for five out of ten S.aureus samples which were (3,4,10,11,16) as illustrated in figure.2 The isolates were commonly resistant to seven out of eight classes of antimicrobial agents used in this study and the isolates were multi drug resistant S.aureus. ATCC 25923 Staphylococcus aureus was used as the quality control strain for disc diffusion.
Discussion
In this research study out of 50 clinical swab samples analyzed, 10 (20%) were positive for S. aureus, which was higher than the results reported by Obiazi et al.[3] who documented 20.8%.
The rate of isolated S.aureus vaginal swab samples (25%) , wound swab samples (20%) and nasopharyngeal swab samples (16%).In this study highest colonization of isolates S.aureus was obtained from high vaginal swab samples, which was about 25%, this result is similar to the result found by Nkwelang,et all[18] in Cameroon. This can be ascribed to the fact that female anatomy may exposes to contamination easily as a result of low personal hygiene, poor health education, which can be a major factor for colonizing bacteria and infection [19]
The resistance 90% of S.aureus was observed in this research study against Amoxicillin, Ampicillin and Cefoxitin higher than the results of Bolanle O,et all [2] was observed (73%), (88%) of the S.aureus resistance to Ampicillin and Amoxicillin respectively. Methicillin Resistant S. aureus detection is difficult because of its heterogeneous nature, but Cefoxitin disc was found for detection of Methicillin resistance S.aureus (MRSA) in isolated samples [20] and it has a sensitivity of 98.5% to detect MRSA [21]. In the present study, significantly higher number, 90% of S.aureus strains resistance to Cefoxitin. Thus, we can say 90% of isolated S.aureus has MRSA and the remaining strains (10%) were considered as Methicillin sensitivity S.aureus (MSSA) according to the CLSI criteria(17). The ability of the Staphylococcus aureus to resist of Beta lactam antibiotics is related to it is able to produce ”-lactamase that hydrolyses the rings of ”-lactam antibiotic. Consequently, Beta lactam antibiotics were losses its antimicrobial activity [2].
In this study S. aureus strains were shown 70%, 60% resistance to Gentamicin and Cotrimoxazole respectively. However, in a tertiary referral hospital in eastern Uttar Pradesh, India, researchers showed more than 80% resistance [12]. While, Rajaduraipandi K, reported 62%, 63.2% Resistance to and Gentamicin and Cotrimoxazole respectively in southern districts of Tamilnadu [23]. The results obtained for Ciprofloxacin and Chloramphenicol were 60% resistances higher than the results obtained by Onwubiko and Sadiq [24] which were about 38.1% resistance in Nigeria. Although Solmaz Dibah, et al, showed 68.4% resistance to Ciprofloxacin in Ardabil, Iran (25). While Rao and Prabhakar found (88.27%) resistance to Ciprofloxacin in Andhra Pradesh, India (26) higher than the results of the present work in India.
The drug resistances were related to misuse of these antibiotics without any prescription which is a common in developing countries (22).
The research studies mentioned above showed that the isolates S.aureus were often resistant to multiple antibiotics, but the most effective chemotherapeutic agents observed against Staphylococcus areas in this study was linezolid, which found 100% MRSA sensitive to it and this result is similar to the other result study recorded in Iran(27,28). However, another research study showed a lower percentage, such as Al-Zoubi et all, observed 96.6%, susceptible
of S. aureus to Linezolid in Northern area of Jordan [29]. Thus linezolid can be used as a drug of choice for treating MRSA infections.
Conclusion
In this research work it was appeared that MRSA recorded higher resistance rates. The results reveal that the treatment of infection caused by Staphylococcus aureus in Bangalore city with B-lactam groups and other drug groups would be unreliable except linezolid could be considered as the drug of choice for treatment of MRSA infection. But it was observed after prolong treatment buys linezolid; resistance may become a rise in the patients who used Linezolid for the first time. For that reason we recommend for monitoring antibiotic resistance pattern within and outside the hospital environments.
Acknowledgment
I would like to thank Dr.Prasad M, P, Director of Sangenomics research laboratory, valuable guidance Mr. Ganesh Rindhe, in the sangenomics research laboratory as external guide. This research was supported by the Ministry of Agricultural and Source water-Kurdistan region for financial support for this research project.a
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