1 Service de Microbiologie, 2 Unité dEpidémiologie et dHygiène Hospitalière and 3 Service de Pneumologie, Hôpital Erasme, Université Libre de Bruxelles, 808 route de Lennik, 1070 Brussels, Belgium; 4 Hôpital Edouard Herriot, Université Claude Bernard, Lyon, France
Received 13 May 2002; accepted 10 June 2002
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Abstract |
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Keywords: Staphylococcus aureus, VISA, Belgium, vancomycin
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Introduction |
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Since 1997, infections caused by MRSA strains with intermediate susceptibility to vancomycin (VISA) (MIC 816 mg/L) have been reported from Japan, France, the United States, Korea and Germany.410 These strains were recovered from patients who failed therapy with vancomycin that had often been used for prolonged periods of time. Other strains, named hetero-VISA, appear to be borderline susceptible to vancomycin (MIC 24 mg/L) but exhibit low-level subpopulations (106 cells) able to grow at vancomycin concentrations of 48 mg/L.11 Such strains have been described in Europe, Asia and Brazil.1220 Hetero-VISA strains could represent first-step mutants that are precursors of VISA strains in patients receiving prolonged courses of vancomycin. Both VISA and hetero-VISA strains belong to a restricted range of epidemic MRSA genotypes.1015,1720
We performed a prospective study to assess the incidence and characteristics of VISA strains recovered from patients admitted to a Belgian University hospital and we report the clinical course of VISA infections.
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Materials and methods |
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VISA was defined as an S. aureus strain with: (i) reproducible growth on brainheart infusion (BHI) agar containing 6 mg/L vancomycin; (ii) a vancomycin MIC > 4 mg/L by Etest on MuellerHinton (MH) agar and by broth microdilution; (iii) a population analysis profile similar to homogeneous VISA control strains Mu50 and HIP5827.4,6
Hetero-VISA was defined as an S. aureus strain with: (i) a vancomycin MIC 4 mg/L by Etest on MH agar or by broth microdilution; (ii) a population analysis profile similar to hetero-VISA strain Mu3 with detectable subpopulation growing at 4 mg/L vancomycin.11
Bacterial strains
Between 1 January 1999 and 31 December 1999, all clinical isolates (n = 2145) of S. aureus from patients admitted to Erasme University hospital, an 858 bed tertiary care teaching hospital, were studied. Identification of S. aureus was performed by coagulase test with human plasma. MRSA strains were confirmed by multiplex PCR for mecA and nuc genes as described previously.21
Antimicrobial susceptibility testing
All isolates were tested by a vancomycin screen agar method.22 Briefly, 10 µL of 0.5 McFarland suspension of the first pure subculture was inoculated on to BHI agar supplemented with 6 mg/L vancomycin (BBL, Becton Dickinson, USA). Isolates showing growth after 24 or 48 h on this medium were subcultured on BHI agar supplemented with 1 mg/L vancomycin before determination of the MIC of vancomycin.
The MIC of vancomycin was determined by a broth microdilution method according to NCCLS guidelines and by two Etest methods (AB Biodisk, Solna, Sweden). In the first protocol, 3 mL of McFarland 0.5 suspension was flooded on to MH agar and incubated for 24 h at 35°C.23 In the second, BHI agar was inoculated with 100 µL of a McFarland 2 suspension and incubated for 48 h at 35°C.24 MICs were interpreted according to NCCLS breakpoints.
Strains with vancomycin MICs > 4 mg/L were characterized by population analysis.11 Briefly, 100 µL of an overnight suspension (McFarland 2) was spread on to agar plates supplemented with 0, 2, 4 and 8 mg/L vancomycin. The number of colonies was counted after incubation for 48 h at 35°C. Each assay included the following control strains: vancomycin-susceptible S. aureus strain ATCC 29213, hetero-VISA strain Mu3 and VISA strains Mu50 and HIP5827.4,6,11
Susceptibility to other antimicrobials was determined by a disc diffusion method (Rosco, NeoSensitab, Taastrup, Denmark) using interpretative inhibitory zone size according to NCCLS breakpoints. The antibiotics tested included penicillin G (P), rifampicin (R), erythromycin (E), clindamycin (Cl), minocycline (M), doxycycline (D), fusidic acid (F), gentamicin (G), amikacin (A), ciprofloxacin (C), co-trimoxazole (SXT) and mupirocin (M). Oxacillin (O) was tested on oxacillin screen agar (6 mg/L) according to NCCLS recommendations. Oxacillin MICs were determined by Etest on MH agar supplemented with 2% NaCl incubated for 24 h at 35°C.
VISA strains were examined by multiplex PCR for the presence of vanA, vanB and vanC-1/2/3 genes coding for vancomycin resistance in enterococci.25
Electron microscopy
All cultures were grown to an OD600 of 0.6 in BHI broth prior to processing for electron microscopic examination, as described by Mani et al.26 The strains were cultured at 37°C in 10 mL of BHI broth in two series of tubes containing for each strain either no antibiotic or 2 mg/L vancomycin. Cells were harvested in the post-exponential growth phase (1824 h OD600 of 0.6) and fixed in 2% glutaraldehyde in 0.05 M cacodylate buffer (pH 7.2)MgSO4 0.05% for 1.5 h, then rinsed twice in cacodylate buffer 0.1 M. They were treated with 1% osmium tetroxide in 0.2 M cacodylate buffer for 2 h at 4°C. Cells were dehydrated with graded concentrations of ethanol and embedded in Epon A+B. Ultra-thin sections were stained with uranyl acetate and lead citrate, then examined with a transmission electron microscope (TEM) (JEOL 1200MX) at different magnifications and photographed.
Pulsed-field gel electrophoresis (PFGE)
Macrorestriction (SmaI) and PFGE analysis were performed as described previously.21 SmaI patterns were normalized and compared using the Dice coefficient and UPGMA clustering method with BioNumerics software version 1.0 (Applied Maths, Ghent, Belgium). Patterns differing by one to six DNA fragments were considered as subtypes (designated by a letter suffix) and those distinguished by seven or more DNA fragments were labelled as distinct types (designated by numeral).21
Infection control and epidemiological investigation
The isolation procedures for patients colonized with MRSA followed Belgian national guidelines.27 Briefly, carriers were placed in single rooms. Healthcare workers wore disposable gloves and gowns before entering the patients room. In addition, they wore face masks for aerosol-generating procedures. Hand disinfection with hydro-alcoholic solution was undertaken after glove removal. In addition, patients with VISA colonization/infection were cared for by dedicated nursing staff. Compliance with the isolation procedures was strictly monitored by infection control personnel. Patients and healthcare workers in contact with patients colonized with VISA were screened for colonization to assess potential transmission of VISA. Swabs obtained from anterior nares and hands were inoculated on to sheep blood agar and into MRSA selective broth medium containing colistin (32 mg/L), oxacillin (2 mg/L) and 2.5% NaCl. S. aureus isolates were identified by the coagulase test and tested for susceptibility to oxacillin and vancomycin by agar screen methods.
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Results |
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Of 2145 isolates tested, of which 881 (41.1%) were oxacillin resistant, 146 (4.9%) S. aureus (of which 107 were MRSA) isolated from 106 patients grew on vancomycin screen agar after 24 or 48 h (Figure 1). By the heavy inoculum Etest procedure on BHI agar, 73 (50%) of these strains showed decreased susceptibility to vancomycin (MIC 616 mg/L). By Etest with a standard inoculum on MH agar, only four strains showed decreased susceptibility to vancomycin (MIC 68 mg/L). By the heavy inoculum Etest method on BHI agar, the distribution of MICs for the 146 strains showed a four-fold greater modal MIC value (8 versus 2 mg/L; P < 0.001) than that determined by standard inoculum on MH agar. By broth microdilution, four strains showed intermediate susceptibility to vancomycin (MIC 816 mg/L) (Table 1). No VISA strains carried van resistance genes.
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The proportion of hetero-VISA strains was 0.1% of S. aureus and 0.4% of MRSA strains, whereas the proportion of VISA strains was 0.1% of S. aureus and 0.3% of MRSA strains.
Antimicrobial susceptibility of VISA and hetero-VISA strains
All strains with reduced susceptibility to vancomycin possessed the mecA gene and exhibited multiple resistance to other antimicrobials (Table 1). Hetero-VISA and two VISA strains, P2V136 and P3V156, were susceptible to minocycline, doxycycline, fusidic acid and co-trimoxazole (Table 1). VISA P1V44 strain was susceptible to clindamycin, gentamicin, minocycline, doxycycline, fusidic acid, co-trimoxazole and rifampicin. This isolate was borderline susceptible to oxacillin (MIC 2 mg/L) but possessed the mecA gene by PCR. This VISA strain derived from a high-level oxacillin-resistant MRSA (MIC > 256 mg/L) progenitor strain P39575 isolated previously from patient 1.
PFGE analysis
By PFGE, VISA and hetero-VISA strains belonged to either of the two multidrug-resistant MRSA types A and D, both of which are endemic in our hospital. These PFGE types were distinct from VISA strains from Japan and the USA (Mu50 and HIP5827). VISA isolate P1V44 and hetero-VISA isolate P1V69 recovered from patient 1 in 1999 were indistinguishable by PFGE from MRSA strain P39575, a fully vancomycin-susceptible strain isolated during a previous hospitalization in December 1998.
TEM analysis
Three MRSA strains isolated consecutively from patient 1 (vancomycin-susceptible MRSA strain P39575, hetero-VISA strain P1V69 and VISA strain P1V44) were examined by TEM (Figure 3). MRSA strain P39575 and hetero-VISA strain P1V69 showed normal cell wall thickness but an uneven cell surface, irregular cell shape and abnormal septation (Figure 3c and d). These abnormalities were more pronounced in hetero-VISA strain P1V69. Strains P1V69 and P39575 showed increased cell wall thickness when grown in 2 mg/L vancomycin. P1V44 strain showed a thickened cell wall (Figure 3b), irregular cell shape and abnormal septation in contrast to the thin and regular cell wall morphology of S. aureus ATCC 29213 strain (Figure 3a). When grown in the presence of vancomycin (2 mg/L), the abnormalities of strain P1V44 were slightly enhanced, whereas strain ATCC 29213 showed no morphological change (not shown).
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Patient 1. An 18-year-old woman with cystic fibrosis, colonized with MRSA since 1993, presented three successive pulmonary exacerbations attributed to MRSA (strain P39575) and Pseudomonas aeruginosa during the winter 19981999. Each episode required hospital admission and intravenous (iv) treatment with a combination of vancomycin, ceftazidime and tobramycin. In September 1999, she was hospitalized for pulmonary exacerbation. Sputum showed Gram-positive cocci and culture grew P. aeruginosa and S. aureus (strain P1V44) that was susceptible to oxacillin and intermediately susceptible to vancomycin (Table 1 and Figure 1). She received a 10 day course of iv oxacillin in combination with ceftazidime and tobramycin. She was discharged after only a moderate improvement. When she was readmitted 2 weeks later for a new deterioration, sputum culture grew an MRSA strain with a subpopulation hetero-resistant to vancomycin (strain P1V69). Intermittent vancomycin (2 g/day) was administered for 12 days in combination with anti-pseudomonal treatment. After a transient improvement, she was again readmitted a few days later. She received high doses (3 g/day) of vancomycin by continuous infusion, in combination with ceftazidime and tobramycin for another 21 days. Two weeks after discharge, she was again readmitted for the fourth time in 3 months. As a hetero-VISA strain was the only organism isolated again from a sputum sample that showed numerous Gram-positive cocci on direct examination, the relapse was attributed to persisting infection with S. aureus of decreased vancomycin susceptibility. High doses of vancomycin (3 g/day) were administered by continuous infusion in combination with fusidic acid (1.5 g/day) for 11 days. Serum levels of vancomycin ranged between 14.3 and 33.8 mg/L with a median of 27.8 mg/L. The iv treatment was followed by oral treatment with minocycline (200 mg/day) and fusidic acid (1.5 g/day) for 3 months. Her clinical progression was good and she remained exacerbation-free for 8 months thereafter. During the next 24 months, only vancomycin-susceptible MRSA strains were isolated from sputum.
Patient 2. A 54-year-old woman was admitted to the intensive care unit (ICU) in November 1999, for multiple trauma with hepatic and pulmonary contusions. On day 11, she developed a primary MRSA bacteraemia. She was given intermittent vancomycin for 11 days adapted to serum levels, which were all in the expected range (trough, 510 mg/L; peak 3040 mg/L). She improved but fever and bacteraemia recurred on day 22. Rifampicin was added to intermittent vancomycin for 10 more days. Because of the emergence of rifampicin resistance, rifampicin was then discontinued and replaced by iv fusidic acid (1.5 g/day). During this treatment (day 47), she presented with a second recurrence of bacteraemia due to an MRSA strain that was susceptible to vancomycin. Concomitantly, a VISA strain (P2V136) was isolated from a decubitus ulcer. From day 56 to 112, she was given continuous infusion of vancomycin (23 g/day) in combination with fusidic acid (1.5 g/day). During continuous infusion, serum levels of vancomycin ranged between 24.2 and 51.2 mg/L, with a median of 33.3 mg/L. She presented on day 233 with a new episode of bacteraemia due to an MRSA strain that was susceptible to vancomycin (MIC 1 mg/L) attributed to pulmonary infection. Sepsis resolved rapidly under treatment with continuous vancomycin infusion combined with fusidic acid. She died suddenly on day 243 of a non-infectious cause.
Patient 3. A 46-year-old man underwent a liver transplant for alcoholic cirrhosis in January 1999. The post-operative course was complicated by splenic haemorrhage, acute renal failure and graft rejection. On day 14, he developed a deep-seated surgical infection and bacteraemia due to MRSA. This strain was susceptible to vancomycin (MIC 3 mg/L). Intermittent vancomycin was administered from day 14 to 21 and dosing was adapted to serum levels. His clinical state deteriorated and surgical drainage was performed on day 20. Treatment was altered thereafter to continuous iv infusion of vancomycin (1.5 g/day) combined with rifampicin (900 mg/day). During continuous infusion, serum levels of vancomycin ranged between 18.5 and 54.6 mg/L with a median of 38 mg/L. The patient developed multiple organ failure and died 33 days after transplantation. Post-mortem examination disclosed generalized cytomegalovirus infection and lung abscess (4 cm diameter). A VISA strain (P3V156) was isolated from a post-mortem bronchial swab.
Epidemiological investigation and infection control intervention
Screenings were performed in the ICU and pulmonary ward after patients 1 and 2 were identified. No VISA or hetero-VISA were detected among patients from those wards (n = 30) and healthcare workers (n = 69). Infection control procedures as described above were applied to patients 1 and 2 during their entire hospital stay. No transmission to patients or healthcare workers occurred during their hospital stay.
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Discussion |
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In contrast to the rarity of reported cases of VISA infection, the proportion of S. aureus with heterogeneous expression of reduced susceptibility to vancomycin ranges widely between surveys worldwide, including those from the European region: Spain 56%, France 20%, Germany 214%, Italy 1%, Japan 526%, Brazil 4%, England 016%, Greece 1%.10,11,1315,17,18,20,24,28,29 The results from these studies are difficult to compare because most investigations used a retrospective study design and many restricted their analyses to a limited number of strains, usually from selected groups of patients. More importantly, definitions of VISA and methods for MIC determination of vancomycin differ markedly between investigators. For example, hetero-VISA strains are variously defined by different authors as S. aureus strains that exhibit a cell subpopulation growing at either 4 or 8 mg/L vancomycin.11,13,24,30 Using more restricted criteria and in an unselected population, we observed a low incidence of 0.3% VISA and 0.4% hetero-VISA among the patients in our hospital colonized or infected by MRSA.
The low-level resistance of VISA strains to vancomycin (MIC 8 mg/L) makes them difficult to detect by standard susceptibility test methods.23 A reversible vancomycin resistance phenotype observed with some VISA strains may compound this difficulty.31 Tenover et al.23 have shown that the disc diffusion method is inadequate to detect VISA strains. In our study, we used BHI agar supplemented with 6 mg/L vancomycin for VISA screening as recommended by the NCCLS, but with prolonged incubation (48 h) instead of the recommended 24 h.22 Using this modified technique, vancomycin agar screening had a low specificity in our study, since 95% of strains that grew after 48 h on screen agar were not confirmed as VISA. Other authors have reported artefactual growth of occasional vancomycin-susceptible S. aureus isolates on a vancomycin agar screen when using media that were prepared in-house.23,32 Our finding of low specificity using this screening method is in contrast to the report of Walsh et al.,33 who found it specific. The Etest method using a heavy inoculum on BHI agar with prolonged incubation also proved to be of low specificity in the present study (90% of false positive). This was also reported by Aucken et al.29 but it is again at variance with the findings of Walsh et al.33 In addition to technical differences, these discrepancies between studies could be explained by differences in study design. Our study looked at consecutive S. aureus clinical isolates, whereas Walsh et al.33 examined a selected collection of strains referred for reduced susceptibility to glycopeptides. Hetero-VISA strains are even more difficult to detect than VISA strains. They do not grow reproducibly on vancomycin screen agar and appear to be susceptible by Etest on MH agar with a standard inoculum and by microdilution MIC testing. The population analysis profile is at present the only reliable confirmation method but is too time consuming for routine use.
Many VISA and hetero-VISA strains are related to epidemic gentamicin-resistant MRSA clones that are disseminated in hospitals in Europe, Brazil and Japan.1015,17,18,24,34 The VISA and hetero-VISA isolates in the present study belonged to two different MRSA types that are endemic in our hospital and unrelated to the US and Japanese VISA strains. One of these genotypes has been widely disseminated in western Europe since the mid-1980s and has been reported in more than 80% of Belgian hospitals.21 Outbreaks of S. aureus with reduced susceptibility to glycopeptides have been described in French hospitals among patients who had not received glycopeptides.30,35 It appears that these strains are hetero-VISA. In our study, the three patients with VISA infection or colonization showed no spacetime clustering. According to local policy, the patients were already cared for using MRSA isolation precautions, including placement in a private room and use by healthcare personnel of gowns, gloves and masks.27 When the VISA strains were identified, nursing staff were increased in number for the care of VISA carriers to consolidate compliance with the isolation precautions according to the published recommendations.36
The mechanism of decreased susceptibility of S. aureus to glycopeptides is not yet well understood. Like other VISA strains described previously, the VISA strains studied here lacked the vanA and vanB genes that are responsible for glycopeptide resistance in enterococci. The strains that we examined by TEM showed the same abnormality of cell wall structure that has been described in other strains.4,67,9,37 Strains Mu50 and Mu3 have an accelerated cell wall turnover with an increased proportion of glutamine non-amidated muropeptides and overexpression of penicillin-binding proteins 2 and 2'.3840 These abnormalities suggest increased production of D-Ala-D-Ala residues acting as false targets, which trap the antibiotic away from its lethal target site of cell wall synthesis adjacent to the membrane.38 Moreover, all VISA strains have a loss of detectable PBP4 activity, which leads to a decrease in cell wall cross-linking.41 Interestingly, in one of the VISA clinical isolates detected in our study, strain P1V44, the decreased susceptibility to vancomycin was accompanied by a parallel loss of expression of oxacillin resistance. This phenomenon has been reported previously to occur in vivo by Sieradzki et al.42 It was reproduced in vitro by selection of a mutant VISA strain from an MRSA strain exposed to increasing vancomycin concentrations.
In conclusion, our study describes the first cases of infection and colonization with VISA and hetero-VISA strains in Belgium. These VISA strains emerged during vancomycin therapy for infections due to MRSA strains that are endemic in Belgian hospitals. Although these strains appear to be rare, they were associated with therapeutic difficulties, as reported previously. Therefore, it is desirable to improve the methods of routine detection of VISA strains to ensure their epidemiological surveillance. The optimal management of patients infected with VISA strains is not well defined. We concur with the recommendation that isolation precautions used for control of MRSA nosocomial transmission should be strictly enforced. Therapy with high-dose vancomycin administered by continuous infusion in combination with other anti-staphylococcal drugs may be a therapeutic option worth investigating in the case of VISA infection, but the removal of any foreign body or undrained collections should always be the first and foremost intervention.
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Acknowledgements |
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Footnotes |
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