Staphylococcus aureus has long been recognized as a major pathogen of hospital acquired infections. Over the last decade, methicillin resistant S. aureus (MRSA) strains have become endemic in hospitals worldwide. In addition, it is now incipient community pathogen in many geographical regions 1. MRSA is important because, in addition to being methicillin resistant, most strains are also resistant to other β-lactam antibiotic, with the exception of glycopeptide antibiotics 23. In 1980s, because of widespread occurrence of MRSA, empiric therapy for staphylococcal infections (particularly nosocomial sepsis) was changed to vancomycin in many health care institutions. Vancomycin use in United States also increased during this period because of the growing numbers of infections with Clostridium difficile and coagulase negative staphylococci (CoNS) in health care institutions 45. Thus, the early 1990s have shown a discernible increase in vancomycin use. As a consequence, selective pressure was established that eventually lead to the emergence of strains of S. aureus and other species of staphylococci with decreased susceptibility to vancomycin and other glycopeptides 6. In 1997, the first strain of S. aureus with reduced susceptibility to vancomycin and teicoplanin was reported from Japan 7. Shortly after, two additional cases were reported from United States 8. However, first clinical isolate of vancomycin resistant S. aureus (VRSA) was reported from United States in 2002 9. More recently some workers have reported vancomycin resistant staphylococcal stains from Brazil 10 and Jordan 11.

Strains of Vancomycin Intermediate S. aureus (VISA) with vancomycin MIC of 8 μg/ml have been reported from Japan 7, United States 121314, France 15, United Kingdom 16 and Germany 17. Most of these isolates appear to have developed from preexisting MRSA infections. Assadullah et al from India have reported reduced susceptibility of vancomycin against MRSA and CoNS 18. Keeping this in view we performed extensive longitudinal study of total 1681 staphylococcal strains for the assessment of current situation of vancomycin resistance in northern part of India. This is the first report of VRSA emergence from a tertiary care hospital from this part of world.

Out of 1681 staphylococcal isolates, 761 isolates were slide coagulase positive, 772 were slidex staph plus positive, and 783 were tube coagulase positive. Thus, 783 strains were confirmed as S. aureus and remaining 898 strains as CoNS.

MIC of 783 S. aureus strains and 93 CoNS against oxacillin had shown that 318 S. aureus stains and 51 CoNS strains were resistant to oxacillin (MIC ≥ 4 μg/ml for S. aureus and MIC ≥ 0.5 μg/ml for CoNS). Out of 318 oxacillin resistant S. aureus strains, total two strains were found to be VRSA strains. One strain had shown vancomycin and teicoplanin MIC 32 μg/ml and the other strain with vancomycin and teicoplanin MIC 64 μg/ml. Out of 51 oxacillin resistant CoNS, one strain had shown vancomycin and teicoplanin MIC 32 μg/ml. Six strains of S. aureus were to be found vancomycin intermediate S. aureus (two strains with MIC of 16 μg/ml and four strains with MIC of 8 μg/ml). Two S. aureus strains were to be teicoplanin intermediate (MIC 16 μg/ml). The detailed description of all vancomycin intermediate/resistant S. aureus strains are given in table 1.

All oxacillin resistant S. aureus and CoNS strains had shown presence of mecA gene be PCR methods. All VISA, VRSA and vancomycin intermediate and resisistant CoNS strains were grown on BHI vancomycin screen agar. Disc diffusion test revealed that all vancomycin resistant staphylococcal isolates have shown antimicrobial resistance to most of the commonly used antimicrobials (table 2). However, none of theses strains could demonstrate the presence of vanA and or vanB gene by PCR.

In the routine microbiology laboratory prompt identification of the staphylococcal strains up to species level were done by catalase, coagulase and other standard biochemical test. However during routine screening by slide coagulase test many strains of S. aureus are missed due to their poor sensitivity and falsely reported as coagulase negative staphylococci during routine screening process. In our study we performed tube coagulase tests of all 1681 staphylococcal strains. Therefore the main criterion used for the S. aureus identification was tube coagulase test. However, the slidex staph plus test was also found to be good to differentiate S. aureus and CoNS.

MICs of oxacillin, vancomycin and teicoplanin against S. aureus and CoNS revealed that there is a rise in the resistance. S. aureus and CoNS have shown 40.61 % and 54.83 % oxacillin resistance, respectively. We have found two MRSA strains which were showing vancomycin resistance (VRSA). These VRSA strains have been isolated from the pus of two different patients who were admitted in the different wards. One VRSA (vancomycin MIC 64 μg/ml) strain was isolated from pus of a 56 years old patient who was admitted in Medicine ward and; the other VRSA (vancomycin MIC 32 μg/ml) strain was isolated from pus of a 49 years old patient who was admitted in Skin and VD ward of our hospital (table 1). Both the isolates were also found to be resisistant to several other antimicrobials such as gentamicin, tobramycin, amikacin, norfloxacin, ciprofloxacin, erythromycin, tetracycline, trimethoprim/sulfamethoxazole and cefoperazone/sulbactam (table 2). While going through the detailed history of these two patients it was found that they were already treated with glycopeptides for more than 20 days. These patients were not responding to further vancomycin therapy and later died due to complication of the disease.

The present study also encountered one vancomycin resistant CoNS strain with vancomycin and teicoplanin MIC 32 μg/ml. The strain was later identified as Staphylococcus epidermidis. This strain was isolated from culture of tip of endo-trachial tube of a patient who was admitted in the intensive care unit of teaching hospital.

Of total six VISA strains, four had their MIC 8 μg/ml and two strains showed vancomycin and teicoplanin MIC of 16 μg/ml. Out of six VISA strains, two were isolated from the pus specimens of the patient visiting outpatient department and remaining four were from the pus specimens of patients admitted in post-operative ward.

Out of two strains of CoNS (Staphylococcus epidermidis) with vancomycin and teicoplanin MIC 16 μg/ml, one was isolated from the blood of a patient who was admitted in orthopedic ward, and the other was isolated from the urine specimen of a patient visiting the endocrinology outpatient department.

In this study we have found almost similar pattern of MIC of vancomycin and teicoplanin. The present study shows that there is a significant rise of reduced susceptibility of oxacillin, vancomycin and teicoplanin. The emergence of the glycopeptide resistance is of great concern. Though first case of VRSA was reported in 2002 in USA 9 but few other countries have reported the reduced susceptibility of S. aureus and CoNS against glycopeptides 6781011131415161718. Recently Pallazo et al 10 have also reported some vancomycin resistant strains of CoNS in Brazil. More recently Bathaineh has reported VRSA strains from Jordan 11. Ashdulla et al 18 have reported some strains of vancomycin intermediate S. aureus (VISA) from India. Song et al, 2004 have also been reported the emergence of heterogeneous vancomycin resistant S. aureus strains from India and its neighboring countries 24. The current vancomycin resistant staphylococci in hospital as well as in community are alarming situation to the clinicians. The development of antibiotic resistance in developing countries like ours seems to be very much related to the irrational antibiotic usage due to it's easy availability at the drug store without prescription, injudicious use in hospitals and uncontrolled use in agriculture, animal husbandry and fisheries 25. This emergence of VRSA/VISA may be due to building of selective pressure of vancomycin. Vancomycin, a glycopeptide is currently the main antimicrobial agent available to treat life-threatening infections with MRSA. Until recently vancomycin resistance among gram-positive bacteria had been thought to be uncommon but the confirmed reports of vancomycin resistance in Enterococcus spp; S. aureus and CoNS have been reported from various part of world. Widespread use of vancomycin to treat infections caused by MRSA and other gram-positive cocci has led to the emergence of vancomycin resistance. The large scale of development and subsequent spread of resistance to vancomycin has been perceived as a fearsome threat to the already challenging therapy of MRSA.

The true mechanism of vancomycin resistance in S. aureus is not known. It was initially feared that S. aureus would acquire the van gene that code for vancomycin resistance in Enterococcus spp; this phenomenon was successfully accomplished in the laboratory 26. Further, Showsh et al, 2001 27 have demonstrated the presence of sex pheromone in S. aureus that promotes plasmid transfer in Enterococcus spp. Release of these pheromones by S. aureus with proximity to vancomycin-resistant enterococci causes the transfer of plasmids encoding van gene to the S. aureus.

In current study all strains of VRSA and VISA strains were negative for vanA/vanB gene by PCR. Therefore the absence of vanA/B genes in the present isolates dose not rule out that these strains are not VRSA or VISA. There is another hypothesis which says that cell wall thickening is responsible for the development of vancomycin resistance. The mechanism of vancomycin resistance has been extensively studied with the first clinical VRSA strain, Mu50 282930. Biochemical and transmission electron microscopy (TEM) examination of the Mu50 cell, suggested that it produces increased amounts of peptidoglycan. More murein monomers and more layers (probably 30–40 layers as judged by cell-wall thickness observed with TEM) of peptidoglycan are considered to be present in the cell wall. As a result, more vancomycin molecules are trapped in the peptidoglycan layers before reaching the cytoplasmic membrane where peptidoglycan synthesis occurs. Moreover, a higher concentration of vancomycin would be required to saturate all the murein monomers that are supplied at an increased rate in Mu50. Besides the vancomycin-trapping mechanism, designated "affinity trapping," 313233, Hiramatsu has suggested that dense accumulation of vancomycin molecules within the thickened cell-wall significantly delays the timing of complete inhibition of cell-wall synthesis by not allowing efficient penetration of vancomycin molecules through the thickened cell-wall layers 30.

The thickened cell wall of VRSA strains become thinner with the loss of vancomycin resistance during the drug free passage and again become thick in resistant mutants. Pallazo et al 10 have also demonstrated the thickening of cell wall in vancomycin resistant staphylococci. This could be the possible mechanism behind the vancomycin resistant staphylococcal isolates that we have reported though we could not perform the test for the demonstration of cell wall thickening in these isolates.

Our study for the first time has exposed the emergence of VRSA and VISA in North India. Although we confined our study within North India, the emergence of VRSA/VISA might also be prevalent in other parts of India as antibiotic misuse is equally common there. Hence, there should be an immediate response from the concerned authorities to check further emergence and spreading of these notorious VRSA strains. A strict regulation on irrational antibiotic usages might be an appropriate and effective approach in this direction. Moreover, nationwide surveillance program should be carried out to map the vancomycin susceptibility pattern in this country. For this, all strains with vancomycin MIC 4 μg/ml should be earmarked and sent to the reference laboratory for the further characterization. This will help to identify the potential areas which are already under the major threat of VRSA/VISA emergence and therefore draw more focused attention of Government for prompt tackling of this problem.

The author(s) declare that they have no competing interests.

HK has conceived and designed the work and performed strain isolation and identification, antibiotic susceptibility testing, PCR tests and prepared the manuscript. MR has participated in the designing of the work and correction of manuscript. Both the authors read and approved the final manuscript.