1 Sera and Vaccines Central Research Laboratory, ul. Chemska 30/34, 00-725 Warsaw; 2 The Maria Sk
odowska-Curie Memorial Centre and Institute of Oncology, ul. Roentgena 5, 02-185 Warsaw, Poland
Received 25 March 2002; returned 8 August 2002; revised 30 August 2002; accepted 16 September 2002
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Abstract |
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Introduction |
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In this study, we characterized the first hVISA isolates to be identified in Poland.
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Materials and methods |
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Twenty-nine MRSA isolates were analysed in this study; selected clinical data concerning the isolates are presented in Table 1. The analysis was commenced with eight isolates (isolates 18) collected from a single patient between June and August 2000 in an oncology centre in Warsaw (centre WA I). The next isolate (isolate 9) was recovered in June 2001 from a patient in a paediatric hospital in Warsaw (centre WA II). Twenty additional MRSA isolates (isolates 1029), all from the collection of strains in the Sera and Vaccines Central Research Laboratory, were included in the study for comparative epidemiological analysis.
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MICs of vancomycin and teicoplanin were evaluated in accordance with NCCLS guidelines4 and using Etest (AB Biodisk, Solna, Sweden), by both the standard procedure and the macromethod.5 The screening test and population analysis of vancomycin susceptibility were performed as recommended by Hiramatsu et al.1,3 S. aureus ATCC 29213 and S. aureus Mu3 (hVISA) and Mu50 (VISA)1 were used as reference strains.
PFGE
PFGE analysis of the isolates was performed using a CHEF DRII apparatus (Bio-Rad, Hercules, CA, USA) as described by Chung et al.6 PFGE patterns were compared with the use of Molecular Analyst software, version 1.12 (Bio-Rad).
Typing by ClaI restriction fragment length polymorphism (RFLP) analysis of mecA and Tn554 loci
RFLP analysis of mecA and Tn554 loci was performed as reported by Leski et al.7 Hybridization patterns were classified into ClaImecA and ClaITn554 types by comparison with the types described previously.7
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Results |
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MRSA isolates 18 from centre WA I were collected from a 45-year-old male patient with a stomach cancer. Three days after oesophagogastrectomy, the patient developed peritonitis and was transferred to the intensive care unit (ICU), where he spent 73 days undergoing numerous invasive procedures, including intubation, tracheostomy, intravenous central catheterization and total parenteral nutrition. The patient developed several infections caused by various microorganisms and was intensively treated with antibiotics. The glycopeptide treatment consisted of two courses of vancomycin (41 days altogether) and one course of teicoplanin (21 days). The patient improved following quinupristin/dalfopristin therapy.
MRSA isolates were recovered from various specimens at several time points over the whole period of treatment (Table 1). The first two isolates were cultured soon after his admission to the ICU. The next three isolates were identified 3 days after the first or during the second course of vancomycin therapy. The last three isolates were recovered several days after teicoplanin therapy, before and just at the beginning of treatment with quinupristin/dalfopristin.
Table 1 and Figure 1 show the results of the analysis of isolates 18. Regarding MICs, the isolates behaved as susceptible to glycopeptides;4 however, in the screening test, they produced colonies on plates containing vancomycin 4 mg/L, with a frequency of 106. The population study revealed that cultures of the isolates contained fractions of cells that grew at a vancomycin concentration of 7 mg/L (
107). According to the criteria of Hiramatsu et al.,1 they were classified as hVISA. PFGE typing demonstrated that isolates 18 were either indistinguishable or closely related to each other. The predominant pattern was identified in the database of Polish MRSA as subtype D6 (J. Krzyszto
-Russjan & W. Hryniewicz, unpublished results). All the isolates were characterized by ClaImecA RFLP type I and ClaITn554 RFLP type E. Therefore, they were specified by the combined RFLP type I::E::D, where I and E are international designations of ClaImecA and ClaITn554 types,8 and D is a PFGE type designation in the context of the clonal structure of MRSA in Poland.
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Isolate 9 from centre WA II was recovered from a throat swab of an asymptomatic newborn at the time of its admission from another hospital, WA III. The isolate was classified as putative hVISA by the WA II laboratory, which routinely tests MRSA on brainheart infusion (BHI) agar plates with vancomycin 4 mg/L. Table 1 and Figure 1 show the results of the analysis of the isolate. The glycopeptide susceptibility testing revealed that it behaved similarly to isolates 18 and could be interpreted as hVISA.1 Moreover, the isolate was closely related to isolates 18 in typing and was classified into the I::E::D RFLP type, subtype I::E::D6.
Isolates 1022
Results of the analysis of isolates 18 and 9 indicated the necessity of a wider study, including additional MRSA isolates from centres WA I and WA III. Isolates 1019 were collected from different patients in WA I between 1998 and 2001. Results of their analysis are shown in Table 1. The glycopeptide susceptibility data demonstrated that the majority of the isolates, including isolate 10 from 1998, were of the hVISA phenotype,1 and all these could be classified into the I::E::D RFLP type, mostly subtype I::E::D6. The remaining non-hVISA isolates represented another RFLP type, III::B/DD::B.
Similar data were obtained for isolates 2022 from January 2000, which were the only MRSA isolates available from centre WA III (Table 1 and Figure 1). They demonstrated the hVISA phenotype1 and belonged to the I::E::D RFLP type, including subtype I::E::D6.
Isolates 2329
Identification of the hVISA phenotype in MRSA isolates of the I::E::D clone in separate Warsaw hospitals was the reason for a retrospective study of other isolates of the same clone. Seven MRSA isolates, collected from 1994 to 1998 in four different hospitals (WA IV, WA V, SI and BB), were selected from the database of Polish MRSA (J. Krzyszto-Russjan & W. Hryniewicz, unpublished results). They represented PFGE subtype D6 or closely related subtypes (Table 1) and belonged to ClaImecA type I and, except for one isolate, to ClaITn554 type E. None of the isolates was positive in the screening test for reduced susceptibility to vancomycin; therefore, they could not be classified as hVISA.
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Discussion |
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The hVISA phenotype correlated with the specific MRSA clone, as all the hVISA isolates analysed here were closely related to each other. They all represented variants of a strain of the combined I::E::D RFLP type, mostly subtype I::E::D6. PFGE type D MRSA isolates have been observed in Polish hospitals, mainly in Warsaw, since 19947 (subtype D6 since 1996), and they have usually represented the ClaImecA type I and ClaITn554 type E7 (J. Krzyszto-Russjan & W. Hryniewicz, unpublished results). Recently, the Polish PFGE type D MRSA clone was identified as belonging to the Iberian international clone.8 The hVISA phenotype has not been an imminent characteristic of the I::E::D clone in Poland, as indicated by retrospective analysis of isolates identified in four hospitals from 1994 to 1998.
The hVISA phenotype was originally identified in 1996 in the Mu3 MRSA strain in Japan, and its isolation coincided with the clinical failure of vancomycin therapy.1 It has since been observed in MRSA isolates in other countries,2,3 the majority of which were identified in retrospective studies, and it has been impossible to define clearly the clinical significance of the phenotype because of the lack of full clinical data.9,10 What seems to be widely accepted is that hVISA may represent an intermediary stage in the evolution of VISA strains, which have been convincingly documented as non-responding to glycopeptide therapy.1,2 Therefore, the precise monitoring of hVISA occurrence seems to be of high importance.
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Acknowledgements |
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Footnotes |
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References |
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