INTRODUCTION:

Staphylococcus aureus is a major cause of hospital acquired infections, causing high morbidity and mortality across the world. The proportion of MRSA has increased worldwide during the last two decades. The treatment of choice for multiresistant MRSA areglycopeptides, particularly vancomycin.^[1]Since the emergence of vancomycin resistance in enterococci in 1988 and its ability to transfer the resistance genes (Van A and Van B) to other bacterial species including S.aureus, emergence of vancomycin resistance in staphylococci has become a great concern.^[2] Clinicians are being challenged by infections caused by S. aureus. this pathogen is serve as a major cause of both community acquired and health care associated infections, but the treatment of suspected S. aureus infections is becoming increasingly more complicated^.[3]

Low level vancomycin resistance in coagulase negative staphylococci has also been reported. In 1997 the first strain with reduced susceptibility to vancomycin and teicoplanin followed by another strain were reported from Japan.^[1]Soon thereafter a report of two additional cases from the United States was documented.^[4]

Recently there have been further reports of VRSA and hetero VRSA (hVRSA) from France, Spain and UK.^[5]Vancomycin resistance in S.aureus is difficult to determine mainly due to the methodological problems in their detection.^[6] As per National Committee for Clinical Laboratory Standards (NCCLS) staphylococci with MIC of vancomycin< 4µg/mL is susceptible, while for which the MIC is 8-16µg/mL are intermediate and those with MIC > 32µg/mL are resistant.^[4]VISA isolates are not reliably distinguished from vancomycin susceptible isolates by rapid automated methods such as Microscan. NCCLS disk diffusion method and the stokes method are not accurate predictors of reduced vancomycin susceptibility in S. aureus.^[4] A screening strategy has been suggested to rapidly detect VISA or VRSA strains in clinical laboratory settings. ^[5] With this background, our study was undertaken to detect the VRSA status in our clinical isolates of S. aureus.

MATERIALS AND METHODS:

Bacterial Isolates:

A total of 20 clinical isolates of S. aureus were collected from different clinical specimens of patients attending Saveetha Medical Collage and hospital. They were processed for a battery of standard biochemical tests and confirmed.Isolates were preserved in semisolid trypticase soy medium and stored at 4ºC until further use.

Antibiotic Susceptibility Test:

Antibiotic susceptibility testing was determined for these isolates to the following antibiotics such as penicillin, erythromycin, clindamycin, ciprofloxacin, tetracycline, cotrimoxazole and linezolid. These antibiotics were procured from Himedia, Mumbai. This was performed by Kirby-bauer disc diffusion method as per CLSI guidelines.^[7]

Detection of vancomycin resistance:

Overnight grown cultures of S. aureus were adjusted to 0.5 Mc Farland standard. 10 µl of culture suspension was spot inoculated on Mueller Hinton Agar containing 6µg/ml of vancomycin. Plates were incubated at 37ºC for 24 hours. Strains were found to be resistant if there is a growth in the spot inoculation.^[8]

RESULTS:

Samplewise Distribution of Clinical Isolates of S. aureus

Of 20 clinical isolates of S. aureus, 8/20 (40%) were obtained from pus, 6/20 (30%) were from wound, 4/20 (20%) and 2/20 (10%) were from blood and sputum respectively (Figure 1).

Figure 1: Pie chart showing the sample wise distribution of S. aureus

Antibiotic Susceptibility Pattern:

We have observed a varied pattern of sensitivity among one S. aureus isolates. There was complete resistance observed for penicillin (100%), 9/20(45%) isolates were shown to the resistant to erythromycin, 6/20(30%) were to cotrimoxazole, 4/20(20%) were to linezolid followed by 3/20(15%) were resistant to ciprofloxacin and clindamycin respectively (Table 1) (Figure 2).

Table 1: Results of antibiotic susceptibility pattern of S.aureus

Antibiotics	Sensitive (%)	Intermediate (%)	Resistant (%)
Penicillin	0	0	20(100)
Erythromycin	14(70)	4(20)	2(10)
Clindamycin	15(75)	2(10)	3(15)
Ciprofloxacin	9(45)	8(40)	3(15)
Tetracyclin	14(70)	4(20)	2(10)
Cotrimoxazole	10(50)	4(20)	6(30)
Linezolid	10(50)	6(30)	4(20)

Figure 2: Representative picture showing antibiotic sensitivity pattern of S.aureus

Results of vancomycin resistance in S. aureus:

Isolates that showed resistance to vancomycin in disc diffusion method was detected for the same by agar screening method using vancomycin powder. Using this method, 3/20 (15%) isolates were found to be resistant to the concentration of 6µg/ml, which indicates that there were Vancomycin Resistant Staphylococcus aureus (VRSA) in our clinical isoaltes.

Fig 3: Agar screening plates with vancomycin showed 3 VRSA isolates

DISCUSSION:

Isolates of vancomycin resistant S.aureus have emerged in several parts of the world. When these isolates appear to be resistance to vancomycin that leads to treatment failure. At present, the proportion of MRSA with reduced susceptibility to vancomycin is well known. Only 21 strains have so far been reported in literature. The first VRSA reported from Japan^.[9] The methods proposed for detection of heteroVRSA by Hiramatshu et al ^[10]is similar to the method used by Daum et al^[11]to detect S.aureus resistant to vancomycin. It remains unclear whether this method detects vancomycin resistance or selects for it.^[12]

Vancomycin resistance can be difficult to detect in clinical microbiology laboratory. Disk diffusion sensitivity testing by standard 30µg vancomycin frequently misclassifies intermediately susceptible isolates as fully susceptible isolate. Presently MIC determinations by broth or agar dilution or by E test are the gold standard for determining vancomycin susceptibility,^[13]but these methods are not generally suitable for routine use in the diagnostic laboratories.

Hiramatshu et al suggested to use BHI agar with 4µg/mL vancomycin. Bierbaum et.al ^[14] reported that 23 of 25 isolates showing growth on this medium were susceptible by CLSI guidelines.^[4] According to Tenover et al ^[4]growth of number of S.aureus strains on 4µg/mL vancomycin containing agar did not have elevated MICs. In agreement with the above reports the present study showed that 3/20 (15%) strains growing on 4µg/mL vancomycin plates were infact susceptible by CLSI criteria.

CONCLUSION:

We would recommend the diagnostic laboratories to screen S. aureus isolates by the CDC agar screening method and submit strains growing on these plates to reference laboratories for confirmation of vancomycin resistance by determining the MIC. Clinicians should continue to exercise caution in their use of vancomycin in order to preserve this useful antibiotic and prolong its therapeutic usefulness.

ACKNOWLEDGEMENT:

We thank Dr. Kalyani, Professor and Head of the Department of Microbiology, Saveetha Medical College, Chennai for kindly providing the clinical isolates to carry out our research work

REFERENCES:

1. Wootton M, Howe RA, Hillman R, Walsh TR, Bennett PM, MacGowan AP. A modified population analysis (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J Antimicrobchemother 2001;47:399-403.

2. Tenover CF, Lancaster MV, Hill CB, Steward CD, Stocker SA, Hancock GA, O’ hara CM, Clark NC, Hiramatshu K. Characterization of Staphylococci with reduced susceptibilities to vancomycin and other glycopeptides. J Clin Microbiol 1998; 34(4): 1020-1027.

3. Fridkin SK. Vancomycin intermediate and resistant Staphylococcus aureus: What the infectious diseases specialist needs to know. Clin Infect Dis 2001; 32:108-115.

4. Tenover CF, Biddle JW, Lancaster MV. Increasing resistance to vancomycin and other glycopeptides in Staphylococcus aureus. Emerging Infect Dis 2001; 7(2):327-332.

5. Huber SK, Mohammed JM, Fridkin SK, Gaynes RP, McGowan JE, Tenover FC. Glycopeptide intermediate Staphylococcus aureus: Evaluation of a novel screening methods and results of a survey of selected U.S hospitals. J ClinMicrobiol 1999; 37(11): 3590-3593.

6. Srinivasan A, Dick JD, Perl TM. Vancomycin resistance in Staphylococci. ClinMicrobiol Rev 2002;15(3):430-438.

7. Clinical Laboratory Standards Institution: Performance standards for antimicrobial susceptibility testing. In NCCLS approved standard M2-A8. Wayne, PA USA: CLSI, 2004.

8. Wootton M, Howe RA, Hillman R, Walsh TR, Bennett PM, MacGowan AP. A modified population analysis (PAP) method to detect hetero-resistance to vancomycin in Staphylococcus aureus in a UK hospital. J Antimicrobchemother 2001; 47:399-403.

9. Hiramatshu K. Vancomycin resistance in staphylococci. Drugs Resistance Updates 1998; 1: 135-150.

10. Hiramatshu K, Aritaka N, Hanaki H, Kawasaki S, Hosoda Y, Hori S. Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 1997; 350:1670-1673.

11. Daum RS, Gupta S, Sabbagh R, Milewski WM. Characterization of Staphylococcus aureus isolates with decreased susceptibility to vancomycin and teicoplanin; Isolation and purification of constitutively produced proteins associated with decreased susceptibility. J Infect Dis 1992; 166:1066-1072.

12. Howe RA, Wootton M, Walsh TR, Bennett PM, Macgowan AP. Expression and detection of heterovancomycin resistance in Staphylococcus aureus. J Antimicrob Chemother 1999; 44:675-678.

13. Clinical Laboratory Standards Institution: Performance standards for antimicrobial susceptibility testing. In NCCLS approved standard M2-A8. Wayne, PA USA: CLSI, 2004.

14. Bierbaum G, Fuchs K, Lenz W, Szekat C, Sahl HG. Presence of Staphylococcus aureus with reduced susceptibility to vancomycin in Germany. Eur J clinmicrobiol Infect Dis 1999; 18:691-696.

Received on 05.09.2017 Modified on 06.10.2017

Research J. Pharm. and Tech 2018; 11(2):621-623.

DOI: 10.5958/0974-360X.2018.00115.4