| Abstract|| |
Background : Staphylococcus aureus is one of the most commonly isolated organisms in nosocomial infections. While the prevalence of methicillin--resistant Staphylococcus aureus (MRSA) continues to increase worldwide, there is a concern about an increase in vancomycin minimum inhibitory concentrations (MIC,s) among S. aureus strains. An attempt was, therefore, made to study vancomycin susceptibility in 156 MRSA isolates from various clinical samples during the period of February 2008 to January 2009. Materials and Methods: A total of 156 isolates of S. aureus were collected from various clinical specimens. MIC of vancomycin was detected by agar dilution method. Results : Out of 156 MRSA isolates all were susceptible to vancomycin by disc diffusion method. By agar dilution method, 138 isolates were susceptible to vancomycin (VISA MIC 0.5-2 μgm/ml) and 18 isolates showed intermediate susceptibility to vancomycin (VISA MIC 4-8 μgm/ml). No vancomycin resistant S. aureus (VRSA) with MIC ≥ 16 μgm/ml was detected. Conclusion : The present study reveals the emergence of MRSA with reduced susceptibility to vancomycin and indicates the magnitude of antibiotic resistance in and around Davangere.
Keywords: Methicillin-resistant Staphylococcus aureus, minimum inhibitory concentration, vancomycin intermediate Staphylococcus aureus / vancomycin resistant Staphylococcus aureus
|How to cite this article:|
Sharma P, Vishwanath G. Study of vancomycin susceptibility in methicillin-resistant Staphylococcus aureus isolated from clinical samples. Ann Trop Med Public Health 2012;5:178-80
|How to cite this URL:|
Sharma P, Vishwanath G. Study of vancomycin susceptibility in methicillin-resistant Staphylococcus aureus isolated from clinical samples. Ann Trop Med Public Health [serial online] 2012 [cited 2018 Nov 16];5:178-80. Available from: http://www.atmph.org/text.asp?2012/5/3/178/98609
| Introduction|| |
Staphylococcus aureus is commonly associated with hospital and community acquired infections.  Infection caused by S. aureus used to respond to beta-lactam antibiotics. Vancomycin is the drug of choice for therapy of infections due to MRSA, but increase in vancomycin use has led to the emergence of two types of glycopeptides resistant S. aureus. The first one, a vancomycin intermediate resistant S. aureus (VISA), is due to a thickened and poorly cross-linked cell wall. The second type, vancomycin-resistant S. aureus (VRSA), is due to acquisition from Enterococcus spp. of the vanA operon, resulting in high-level resistance.  In 1996, the first case of S. aureus with reduced susceptibility to vancomycin (MIC 8 μgm/ml) was reported in Japan.  In June 2002, first clinical isolate of VRSA with minimum inhibitory concentration (MIC) of ≥ 32 μgm/ml was identified in the United States. 
| Materials and Methods|| |
A total of 156 isolates from various clinical samples like pus, blood, urine, sputum, and body fluids were collected from patients attending outpatient department and patients admitted in the hospital. All clinical samples except urine were inoculated into blood agar plate (Hi-Media, India) and MacConkey agar plate (Hi-Media, India), whereas the urine samples were inoculated only on cystine lactose electrolyte deficient (CLED) agar plates (Hi-Media, India). Identification of S.aureus was done by tube coagulase test, catalase test, and mannitol fermentation test according to Clinical and Laboratory Standards Institute (CLSI) guidelines.  Methicillin-resistant Staphylococcus aureus (MRSA) was detected by Kirby Bauer's disk diffusion method using Muller Hinton agar (Hi-Media, India) with 4% Nacl and 1 μgm oxacillin disk (Hi-Media, India). Vancomycin resistance was also detected by Kirby-Bauer's disk diffusion method using 30 μgm disk, according to CLSI guidelines.  Sensitivity was also tested for other antibiotics like pencillin (10U), gentamycin (10 μgm), amikacin (30 μgm), ciprofloxacin (5 μgm), azithromycin (15 μgm) ofloxacin (5 μgm), cotrimoxazole (1.25/23.75 μgm), and cefazolin (30 μgm). S. aureus ATCC 29213 was used as reference strain.
MIC of vancomycin was detected by agar dilution method as described elsewhere.  Briefly, plates of Muller Hinton agar were prepared with vancomycin with varying concentration from 0.5-128 μgm/ml. By direct colony suspension method, 0.5McFarland equivalent inoculum was prepared in normal saline from 18-24 hrs agar plate culture. The suspension was further diluted to achieve concentration of 10 5 CFU/ml. Samples were inoculated into plates with various concentrations, and then incubated at 35 0 C for 24hrs. The MIC was checked by examining the plates for lowest concentration of vancomycin that inhibited visible growth. S. aureus ATCC 29216 and Enterococcus faecalis ATCC 29212 were used as vancomycin susceptible control and vancomycin resistant control, respectively.
According to CLSI guidelines, VISA are those isolates with MIC between 4 μgm/ml - 8 μgm/ml, While heterogeneous strains (hVISA) appear to be susceptible to vancomycin, but contain a subpopulation of cells with reduced susceptibility to vancomycin (MIC ≥ 4 μgm/ml) and VRSA with MIC of ≥ 16 μgm/ml. 
| Results|| |
Out of the 156 MRSA isolates, 120 were from pus samples, 26 from blood samples, 6 from sputum samples, and 4 from urine samples were isolated. Of these, 54 were from diabetic ulcer, 38 from post-operative wound infection, 19 from Pyogenic abscess, 16 from non-healing ulcers, 14 from fracture sites, and the remaining 15 were from cases like septicaemia, cellulites, osteomyelitis, and respiratory tract infection. All the MRSA isolates were uniformly sensitive to vancomycin by disc diffusion method. By agar dilution method, MIC of 138 isolates was between 0.5-2 μgm/ml (VSSA), but 18 isolates showed MIC of 4-8 μgm/ml (VISA). No VRSA and hVISA were detected [Figure 1]. The detection of reduced susceptibility to vancomycin by routine disc diffusion method is unreliable and vancomycin non-susceptibility is most probably being under reported. Most of the VISA isolates were susceptible only to amikacin, cotrimoxazole, ofloxacin, and resistant to other antibiotics. [Table 1] shows VISA isolates and their MIC.
| Discussion|| |
Patients mostly at the risk of developing VISA and VRSA are those with previous exposure to vancomycin. Reduced susceptibility to vancomycin is seen in the elderly and in those with chronic leg or decubitus ulcers mainly containing vancomycin resistant enterococci (VRE), which were probably donor organisms of the vanA gene to S. aureus. The current vancomycin resistant staphylococci in hospital as well as in community are alarming situations to the clinicians. The development of antibiotic resistance in developing countries seems to be very much related to the irrational antibiotic usage due to its easy availability at the drug store without prescription, injudicious use in hospitals and uncontrolled use in agriculture, animal husbandry and fisheries. 
In 2003, Assadullah et al. has reported some strains of vancomycin intermediate staphylococci in India.  Song et al. have reported some strains of heterogeneous vancomycin resistant staphylococci (hVRSA) in India and neighbouring countries in 2004.  Tiwari et al. had reported two strains of VRSA in the northern parts of India, in 2006.  In our study, MRSA was susceptible to vancomycin by disc diffusion method, but the MIC of 18 isolates was ≥ 4 μgm/ml (VISA). These VISA isolates had escaped detection by routine disc diffusion method.
Recently, the Centers for Disease Control and Prevention (CDC) issued specific recommendations intended to reduce the development and transmission of S. aureus with intermediate glycopeptide resistance. First, laboratory personnel should use a quantitative method based on the minimal inhibitory concentration to detect S. aureus isolates with intermediate glycopeptide resistance. Vancomycin disk diffusion does not reliably identify S. aureus isolates with decreased susceptibility to glycopeptides. Secondly, programs to educate health care personnel about infection-control precautions against S. aureus with intermediate glycopeptide resistance should be developed, and infection-control specialists should monitor compliance with these precautions. Third, infection-control and laboratory personnel should implement active surveillance for S. aureus with intermediate glycopeptide resistance, particularly in populations at high risk, such as patients on dialysis and patients in whom vancomycin therapy is unsuccessful.  If an S. aureus isolate with potential intermediate resistance to glycopeptides is identified, prompt notification of the state health department and the CDC is critical so that epidemiologic and laboratory support can be provided. 
The observations of the present study and various other studies infer that staphylococcal infections are common among hospitalized patients and continue to be a growing 'thorn' for the medical personnel. Despite the recent reports that gram negative bacteria have overtaken staphylococci as the leading cause of nosocomial infections, MRSA continues to be the main threat in the health care setting. Extensive surgical procedures, indiscriminate use of antibiotics, lack of barrier nursing practices and prolonged hospitalization paves way for the staphylococci to cause infections and to develop drug resistance.
The emergence and spread of resistance to vancomycin is a threat to the already challenging therapy of MRSA. Staphylococci with reduced susceptibility were observed in the present study. So, as a precautionary measure should be ensured before starting the patient on vancomycin; the clinicians should seek the help of clinical microbiologist to determine the MIC of such strains so that VISA is not missed and emergence of vancomycin resistance can be prevented. All the clinical microbiology laboratories should routinely test the MIC of vancomycin for methicillin-resistant staphylococci for appropriate treatment of patients and implementation of infection control measures to prevent the spread of resistance.
Antimicrobial resistance to penicillin, methicillin, or vancomycin is an unavoidable consequence of the selective pressure of antibiotic exposure. Minimizing the antibiotic pressure is essential to control the emergence of resistant strains in the hospital and in the community.
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