Emergence of heterogeneous intermediate vancomycin resistance in Staphylococcus aureus isolates in the Dusseldorf area

J Antimicrob Chemother 1999; 43: 846–848 Sir, Hiramatsu et al.1,2,3 recently described the emergence in Japanese hospitals of heterogeneous intermediate resistance to vancomycin in clinical isolates of Staphylococcus aureus (hetero-VISA, also referred to as ‘hetero-VRSA’ ). (A hetero-VISA strain is one that is susceptible to vancomycin, i.e. MIC ≤ 4 mg/L, according to breakpoints recommended by the National Committee for Clinical Laboratory Standards (NCCLS), 4 but which contains subpopulations of cells, at frequencies ≥ 10 − 6, 1 that exhibit intermediate susceptibility, i.e. MICs 8- 16 mg/L.) These investigators reported the incidences of such isolates to be 20% in Juntendo University Hospital, 9.3% in seven other university hospitals and 1.3% in non-university hospitals. 1,2 All of the strains possessed the same pulsed-field gel electrophoresis (PFGE) pattern, II-A— an observation that suggests that they belonged to a single clone. Homogeneous VISA isolates have also been identified in the USA. 5 Hiramatsu et al.1,3 suggested that hetero-VISA isolates are precursors of S. aureus strains that are resistant to vancomycin (MICs ≥ 32 mg/L). For this reason, it is important that the prevalences of VISA and hetero-VISA strains in the S. aureus population are closely monitored. In order to determine the prevalences of VISA and hetero-VISA isolates locally, we studied 85 methicillin-resistant S. aureus (MRSA) strains randomly selected from 210 non-replicate isolates recovered between 1992 and 1998 and referred to the Institute of Medical Microbiology and Virology, University Hospital Düsseldorf, by 11 regional hospitals. The isolates were analysed by PFGE, the most discriminatory molecular technique for typing MRSA strains. 6 Carriage of the mecA and coa genes was confirmed with a multiplex PCR. Susceptibility to vancomycin was determined by a microbroth dilution method recommended by the NCCLS, 4 with cation-adjusted Mueller- Hinton broth, and by the Etest method (AB Biodisk North America Inc., Piscataway, NJ, USA). The isolates were also screened by a method of detecting hetero-VISA previously described by Hiramatsu et al.1 Each strain was inoculated on to Brain Heart Infusion (BHI) agar containing vancomycin in a concentration of 4 mg/L. The plates were incubated at 37°C for 48 h and examined after 24 h and 48 h. If there was no growth after 48 h, a strain was considered susceptible to vancomycin. If confluent growth was observed after 24 h the strain was considered to be potentially resistant to vancomycin, whereas if 1- 30 discrete colonies were observed within the 48 h incubation period it was designated as potentially hetero-VISA. Hetero-VISA status was confirmed if a strain produced subclones with vancomycin MICs ≥ 8 mg/L following subculture in the presence of increasing concentrations of vancomycin and if the MIC remained stable for <9 days after daily subculture in a drug-free medium. Of the 85 MRSA isolates tested, none was classified as VISA according to the above criteria. However, seven (8.2%) isolates fulfilled the criteria of Hiramatsu et al. for hetero-VISA; 1,2,3 variants exhibiting reduced susceptibility occurred at frequencies of 10 − 3- 10 − 4. As determined by the microbroth dilution method, the MICs of vancomycin and teicoplanin for all seven strains were 1 mg/L and 4 mg/L, respectively. Thus, these strains would have been classified as susceptible to the glycopeptides on the basis of NCCLS criteria. 4 Hanaki et al.1,3 demonstrated that strains exhibiting reduced susceptibility to vancomycin (one each of VISA and hetero-VISA) produced three-fold to five-fold greater amounts of penicillin-binding proteins (PBPs) 2 and 2′ and three-fold to eight-fold greater amounts of intracellular murein monomer precursor, compared with the vancomycin-susceptible S. aureus control strains. These investigators also showed that amidation of glutamine residues in cell-wall muropeptides and reduced crosslinking of cell-wall peptidoglycan might contribute to reduced susceptibility to vancomycin. 7 All of these alterations produce a so-called ‘ trapping effect’ , whereby increased amounts of vancomycin are bound to the pre-existing modified cell wall, thus reducing the amount of drug that reaches its vital targets— the lipid II precursors of new cell-wall synthesis which are found in the outer leaflet of the cytoplasmic membrane beneath the pre-existing cell wall. In order to determine whether similar cell-wall modifications were present in our isolates, one strain was subjected to muropeptide analysis by high-performance liquid chromatography (HPLC), as described by Hanaki et al.7 The cell wall was isolated, the teichoic acids were removed, and the remaining peptidogycan was digested by a muramidase. The resulting muropeptides were separated by reverse-phase HPLC. Peak assignments were made by comparison with previous samples in which the peaks were identified by amino acid analysis and mass spectroscopy. The cell wall of the strain contained increased amounts of glutamine-non-amidated muropeptides (although the numbers of these molecules fell following repeated subculture). The amounts of the corresponding muropeptides were even greater than those found in Mu50, the first homogeneous VISA strain described by Hiramatsu et al.,1,3,7 and were greater than those of the standard amidated precursors. The strain also exhibited reduced crosslinking of cell-wall peptidoglycan. All of these data suggest that the mechanisms responsible for heterogeneous intermediate resistance to vancomycin in our S. aureusisolates are very similar, if not identical, to those identified in the Japanese isolates. The seven hetero-VISA strains that we isolated were referred from three different hospitals in the Düsseldorf area. Five strains were isolated from patients on two different wards of the same hospital (three from one and two from another) and two of the five patients occupied the same room before the MRSA strains were isolated, suggesting nosocomial transmission (and, possibly, cross-infection) of these strains; a relationship in terms of acquisition/transmission could not be demonstrated for the other three isolates. PFGE revealed the seven hetero-VISA strains to be indistinguishable and to be identical to the north German epidemic strain of MRSA which is the most prevalent strain in both the Düsseldorf area and northern Germany. The distribution of hetero-VISA strains among three geographically distinct hospitals in the Düsseldorf area suggests the potential for wider dissemination. All seven hetero-VISA isolates appear to have belonged to a single clone that was first detected in 1998, no hetero-VISA strains having been isolated in this region between 1992 and 1997; the emergence of heterogeneous intermediate resistance to vancomycin in S. aureus is therefore a recent development. All patients from whose blood cultures or wound swabs hetero-VISA strains were isolated were critically ill with either carcinomatosis or severe postoperative complications. Two of the seven were treated with vancomycin before hetero-VISA strains were isolated and three received vancomycin after the strains were isolated. Thus, the isolation of a hetero-VISA strain was not clearly linked to previous vancomycin therapy and the glycopeptide appears to have been active both in vitro and in vivo against the hetero-VISA isolates. All seven patients were cured following treatment with either vancomycin or another appropriate agent. Nonetheless, isolates with reduced susceptibilities to vancomycin can still be associated with treatment failures and, as has been suggested previously, hetero-VISA strains might be precursors of S. aureus strains that are resistant to vancomycin. 1,3 These alone are adequate reasons to justify prospective surveillance for the purpose of identifying S. aureusisolates with reduced susceptibilities to vancomycin.