Differential growth of epidemic methicillin-resistant Staphylococcus aureus in vancomycin

ABSTRACT

This research examines the ability of isolates representing the 17 epidemic methicillin-resistant strains of Staphylococcus aureus to become greater [i]or[/i] larger in increasing levels of vancomycin. alone EMRSAs 1, 2, 8, 11 12 and 15 showed any increase and were designated EMRSAs 1A, 2A, 8A, 11A, 12A and 15A. upon population analysis, these strains all produc clone that grew upon 32 ?µg/mL. vancomycin, while EMRSA 12A and 15A grew at 128 ?µg/mL This was associated with increased resistance to lysostaphin and teicoplanin, a los of phage sensitivity and an increase in small room wall diameter. Typing by pulsed-field gel electrophoresis following SmaI digestion showed no change in EMRSA 8A and 15A, while the other EMRSAs all missing or gained at least single band. EMRSAs 1A, 2A and 15A became more resistant to methicillin, and EMRSAs 8A, 11A and 12A became les resistant to methicillin. These follows suggest that more than individual mechanism is responsible for this phenomenon.

KEY WORDS: Methicillin resistance.

Resistance factors.

Staphylococcus aureus.

Vancomycin.

Introduction

Staphylococcus aureus has drawn out been recognised as the greatest in quantity common cause of significant infection, with a high mortality in hospitalised patients.1 This has achieved smooth greater importance with the increasing resistance to the ??-lactams and also the hetero-resistance to glycopeptides that has now been described.2-4 In the UK 17 epidemic methicillin-resistant s aureus (EMRSA 1-17) strains have been documented.5

In a British hospital, EMRSA 15 was dominant between 1994 and 1998 In 42 cases of septicaemia, vancomycin monotherapy carried a mortality rate of 78% while combination with rifampicin reduc this figure to 4% All isolates were susceptible to vancomycin by means of conventional laboratory testing, but this was not to be found by passaging the strain in vancomycin, during which a minimal inhibitory concentration (MIC) of 8 ?µg/mL was achieved. This prov stable upon subculture. The genetic fingerprint of the strain remained constant on the other hand changes to the phenotype included los of phage sensitivity and a thickened small room wall.6 These findings have also been observ in other vancomycinresistant isolates.2-4, 7-8

This close attention aims to ascertain if other EMRSA strains can increase in increasing concentrations of vancomycin, and whether or not this is associated with consistent changes in phenotype. The importance of this is that clinical failures have been reported with vancomycin therapy alone,9,10 and this may throw back antibiotic failure in the absence of antibiotic resistance defined by dint of routine clinical testing. This may be a strain-specific phenomenon that correlates with, and perhaps is characterised by means of an ability to grow in sequentially increasing concentrations of drug

Materials and methods

Induction of vancomycin resistance

The 17 parental strains (EMRSA 1-17) were inoculated in 10-mL nutrient soup (Oxoid, Basingstoke, UK) containing vancomycin at 1 ?µg/mL and incubated at 37?°C with shaking. When the soup became turbid, 200 ?µL was used to inoculate another 10-mL soup containing 2 ?µg/mL vancomycin. This was repeated with increasing concentrations of vancomycin at 1 ?µg/mL intervals until no development was apparent after five days' incubation. Isolates were subcultur upon Mueller-Hinton agar for further characterisation.

Population analysis profiles of the vancomycin-resistant isolates

A population analysis profile of vancomycin-resistant isolates was determined by means of plating overnight cultures of the above in nutrient soup diluted to a turbidity of 05 (McFarland standard), equivalent to ?‰? 10^sup 10^ colony-forming units (cfu)/mL upon Mueller-Hinton agar plates. The agar contained antibiotic concentrations of vancomycin ranging from naught to 128 mg/mL. Plates were incubated at 37?°C for 48 h Three colonies of the pair the original isolate and the succeeding isolate with the highest resistance to vancomycin were typ by dint of pulsed-field gel electrophoresis (PFGE) following SmaI digestion.11

Phenotype changes in the vancomycin-resistant isolates

Antibiotic sensitivities (penicillin, rifampicin, chloramphenicol, gentamycin, erythromycin, clindamycin, ciprofloxacin, fusidic acid, trimethroprim, tetracycline and mupirocin) were determined upon all vancomycin-resistant isolates by standard disc testing.12 Methicillin, vancomycin and teicoplanin sensitivity proofs were performed by E-test (AB Biodisk, Piscataway, NJ USA) according to the manufacturer's instructions.

The MIC of lysostaphin was determined in Mueller-Hinton soup with lysostaphin (Sigma, Poole, Dorset) suspended in buffing-apparatus (0.15 mol/L NaCl, 50 mmol/L Tris [pH 76]) The lysostaphin dilution ranged from 0125 to 32 ?µg/mL with an initial inoculum of 10^sup 5^ cfu and incubation at 37?°C for 48 h13

Phage susceptibilities were determined by the agency of the method of Blair and Williams,14 using the generally received set of international typing phages (PHL Laboratory, southerly Manchester, UK). Cell wall changes were demonstrated by means of transmission electron microscopy, as previously described.6