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In vivo development of decreased susceptibility to vancomycin in clinical isolates of methicillin-resistant Staphylococcus aureus
Diagnostic Microbiology and Infectious Disease 38 (2000) 159 –167
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In vivo development of decreased susceptibility to vancomycin in clinical isolates of methicillin-resistant Staphylococcus aureus Yasuteru Suginoa, Yoshitsugu Iinumab,*, Satoshi Ichiyamac, Yuji Itoa, Saburo Ohkawad, Nobuo Nakashimab, Kaoru Shimokatae, Yoshinori Hasegawaa a
The first Department of Internal Medicine, Nagoya University School of Medicine Departments of Clinical Laboratory, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan c Department of Clinical Laboratory Medicine, Kyoto University Hospital, 54 Kawahara-cho, Syogoin, Sakyo-ku, Kyoto 606-8507, Japan d Nippon Becton Dickinson Co., Ltd., 8-5-26 Akasaka, Minato-ku, Tokyo 107-0052, Japan e Clinical Preventive Medicine, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan b
Abstract To investigate the possibility of in vivo development of decreased vancomycin susceptibility, the vancomycin susceptibilities of 12 methicillinresistant Staphylococcus aureus (MRSA) isolates serially recovered from six patients with vancomycin therapy were tested by standard MIC determination method and population analysis. While all of the MRSA isolates were susceptible to vancomycin (MICs, 1-2 g/ml) by standard method, population analysis showed the upward shifts indicating decreased vancomycin susceptibility among serial isolates from two patients. These bacteria with decreased vancomycin susceptibility could be selected by using vancomycin selection of pre-therapy isolates under laboratory conditions. Furthermore, the reversion phenomenon of decreased vancomycin susceptibility was confirmed after 20 serial passages of the post-therapy isolates on drug-free agar. These data suggest that in vivo isolates may develop decreased vancomycin susceptibility that is not of such magnitude to cross a breakpoint threshold. This resistance may be unstable, and appears to result from a selective or inducible process that occurs in MRSA clinical strains during vancomycin therapy. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction For nearly four decades, vancomycin has been widely and successfully used for the treatment of serious infections caused by Gram-positive bacterial pathogen. In recent years, however, reduced susceptibilities to glycopeptides among some clinical isolates of coagulase-negative staphylococci have been reported (Aubert et al., 1990; Cercenado et al., 1996; Schwalbe et al., 1987). More recently, Hiramatsu and colleagues (Hiramatsu et al., 1997) reported the first clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with decreased susceptibility to vancomycin (MIC, 8 g/ml). Subsequently, similar isolates with decreased susceptibility to vancomycin have been reported in the United States and France (Centers for Disease Control and Prevention 1997a; Centers for Disease Control and Prevention 1997b; Ploy et al., 1998). These MRSA isolates exhibited low-level vancomycin resistance mediated through novel and as-yet-undefined mechanisms that
differ from those responsible for enterococcal vancomycin resistance (Hanaki et al., 1998a; Tenover et al., 1998). In vitro selection of glycopeptide-resistant staphylococcal mutants has been demonstrated, and the microbiological properties of these mutants have been investigated in detail (Daum et al., 1992; Kaatz et al., 1990; Sieradzki et al., 1996; Sieradzki et al., 1998). However, it is unclear how in vivo resistance to vancomycin could develop in MRSA clinical strains in relation to vancomycin therapy. In order to study the possibility and process of in vivo development of decreased vancomycin susceptibility, we examined the vancomycin susceptibilities of 12 MRSA isolates serially recovered from six patients, who received intravenous vancomycin therapy for MRSA infections, by standard MIC determination method and population analysis.
2. Patients and methods 2.1. Patients
* Corresponding author. Yoshitsugu Iinuma, Department of Clinical Laboratory, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan. Tel.: ⫹81-52-744-2615; fax: ⫹81-52-744-2613. E-mail address: [email protected] (Y. Iinuma).
At Nagoya University Hospital, a 1,035-bed tertiary care and university teaching hospital, nearly 30 patients are annually treated with vancomycin for MRSA infections. We
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retrospectively reviewed the hospital records of patients treated with intravenous vancomycin for MRSA infections between 1992 and 1997; MRSA infection was defined on the basis of clinical significance, if the patients showed both an acute systemic inflammatory response (e.g., fever and elevation of C-reactive protein levels) and local infectious signs or symptoms (e.g., purulent sputum and pyuria). Six patients from whom serial MRSA isolates had been successfully recovered and stored frozen before and during vancomycin therapy were studied. These six patients had never received any prior treatment with glycopeptides. The following information was obtained for each patient: demographics, underlying diseases, medical conditions, pertinent laboratory data, treatment, and outcome. 2.2. MRSA isolates We studied 12 MRSA isolates obtained serially from six individual patients: 6 were isolated before vancomycin therapy (isolates 1591, 2203, 3793, 4510, 4863, and 5658) and the others were isolated during vancomycin therapy (isolates 1905, 2313, 3822, 4568, 4902, and 5677). All isolates were tested for coagulase reaction and susceptibility to oxacillin before inclusion in the study. MRSA was defined as S. aureus with oxacillin of 4 g/ml or above by the micro-broth dilution MIC method performed and interpreted according to the recommendations from the National Committee on Clinical Laboratory Standards (NCCLS) (National Committee for Clinical Laboratory Standards 1997). The isolates were stored as frozen stocks in skim milk (Difco, Detroit) at ⫺70°C until use. Tryptic soy broth (Difco, Detroit) were used for the cultivation of bacterial cultures, which were grown at 37°C with aeration. 2.3. Chromosomal DNA analysis by pulsed-field gel electrophoresis (PFGE) Clonal identity between serially recovered isolates was confirmed by PFGE. Preparation of chromosomal DNA for PFGE and separation of SmaI-restricted fragments in a contour-clamped homogeneous electric field system (Pulsaphor Plus; Pharmacia LKB Biotechnology, Uppsala, Sweden) were carried out as reported previously (Ichiyama et al., 1991). The electrophoresis conditions were 200 V for 20 h, with pulse times ranging from 5 to 60 s for SmaI digestion. The DNA banding pattern was photographed under UV light after ethidium bromide staining, and classified according to the number and size of the restriction fragments. 2.4. Antimicrobial susceptibility The MICs of oxacillin and vancomycin for 12 MRSA isolates were determined by the micro-broth dilution method of NCCLS with cation-adjusted Mueller-Hinton broth (Difco, Detroit). The MIC was the lowest concentra-
tion of antibiotic that yielded no visible growth after incubation at 37°C for 24 h. Standard antimicrobial powders for laboratory use were obtained from the respective manufactures: oxacillin, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan; and vancomycin, Shionogi Co., Ltd., Osaka, Japan. 2.5. Population analysis profiles (PAPs) Resistant subpopulations of bacteria (Hiramatsu et al., 1997; Tomasz et al., 1991) were analyzed by spreading 50 l of the starting cell suspension (prepared by diluting overnight cultures to an optical density [OD] of 0.3 at 540 nm) and serial diluents of this suspension on to brain-heart infusion (BHI) agar plates (Eiken Chemical, Tokyo) containing various concentrations of vancomycin. The plates were incubated at 37°C for 48 h, and the number of mature colonies was counted. The number of resistant cells contained in 50 l of the starting cell suspension was calculated and plotted on a semi-logarithmic graph. PAPs were determined in duplicate with each isolate. 3. Results 3.1. Patients We studied six patients who had received intravenous vancomycin therapy for MRSA infections. Clinical and microbiologic features of the patients are summarized in Table 1. All patients had underlying disease(s); four had malignant neoplasms. Five patients were mainly treated with imipenem in addition to vancomycin, and one (patient 5) received combination therapy with cefepime and vancomycin. The duration of vancomycin therapy ranged from 5 to 28 days, and the cumulative dosage ranged from 4,160 to 32,000 mg. Among the three patients who died, one (patient 4) died from pyothorax and pneumonia caused by MRSA, and two (patients 1 and 6) died from their underlying disease. 3.2. Chromosomal DNA analysis by PFGE Six distinctive restriction patterns, tentatively designated A to E’ in Table 1, were observed in the 12 MRSA isolates tested in this study. Each of the paired isolates obtained from the five individual patients (patients 1, 2, 3, 4, and 6) showed an identical PFGE pattern (Fig. 1). The paired isolates from patient 5 also showed similar band patterns with only one band difference which was considered to be closely related (Tenover et al., 1995). Therefore, each of paired isolates from a given patient had an identical genetic background. 3.3. Antimicrobial susceptibility The MICs of vancomycin for the 12 MRSA isolates are summarized in Table 1. All of the MRSA isolates were
T 6.32 6,000 VCM, IPM/CS
5
32,000 VCM, CFPM
16
4,160 VCM, IPM/CS
28
T 6.81 P 37.97 T 18.9 P 40.35 ND 12 12,000
ND 7 7,000
67 6
M
63 5
M
Lung cancer
Pneumonia Pyothorax Perianal abscess Pneumonia Tetralogy of Fallot Leukemia 2 4
M
Pneumonia M 62 3
M 53 2
a M, male patient; F, female patient. b UTI, urinary tract infection. c VCM, vancomycin; IPM/CS, imipenem/cilastatin; FOM, fosfomycin; ABK, arbekacin; CFPM, cefepime. d ND, not done; T, trough; P, peak. e Died (⫺), died from underlying disease; Died (⫹), died from MRSA infection.
Died(⫺)
Alive
Died(⫹)
Unknown
Died(⫺)
A A B B C C D D E E’ C C 18
1591 1905 2203 2313 3793 3822 4510 4568 4863 4902 5658 5677 Recurrent UTI Peritonitis 79 1
F
Bladder cancer Diabetes mellitus Lung cancer
12/1/92 3/1/93 9/6/93 9/21/93 4/10/95 4/24/95 12/13/95 1/14/96 4/23/96 5/10/96 1/28/97 2/4/97
Urine Urine Abscess Abscess Sputum Sputum Sputum Sputum Abscess Abscess Sputum Sputum
VCM, IPM/CS, FOM VCM, IPM/CS, ABK VCM, IPM/CS
10,000
ND
1 2 1 1 2 2 1 2 1 1 2 2
Alive
Outcomee Culture source Isolation date (mo/day/yr) Isolate no. Infectionb Underlying condition Sexa Age (yr) Patient no.
Table 1 Characteristics of six patients infected with MRSA and properties of MRSA isolates.
Treatmentc
Cumulative vancomycin dosage (mg)
Duration of vancomycin therapy (days)
Serum vancomycin level (g/ml)d
Vancomycin susceptibility MIC (g/ml)
PFGE pattern
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susceptible to vancomycin (MICs, 1-2 g/ml). The vancomycin MICs for two isolates obtained during vancomycin therapy (isolates 1905 and 4568) were slightly elevated from 1 to 2 g/ml, but remained unchanged for the other four isolates during vancomycin therapy. All MRSA isolates had an MIC of oxacillin ranging from 8 to 1,024 g/ml and were thus classifiable as MRSA by NCCLS criteria. 3.4. Population analysis profiles (PAPs) Fig. 2 shows the vancomycin susceptibility profiles by population analysis for each of the two MRSA isolates serially isolated from the six individual patients. The upward shifts in PAPs indicating decreased vancomycin susceptibility (Figs. 2A and D) were observed among serial isolates from two patients (patients 1 and 4), who had received longer courses of vancomycin therapy than the other four patients. The broth dilution vancomycin MICs for these two isolates with decreased vancomycin susceptibility in PAPs slightly increased from 1 to 2 g/ml within the susceptible range by NCCLS criteria. Isolate 4568 from patient 4 who died from MRSA infection showed growth on agar plates containing 8 g of vancomycin per ml at a frequency of approximately 10⫺6. This pattern of PAPs was the same as those of previously reported vancomycin-heteroresistant S. aureus strains (Hiramatsu et al., 1997). Isolate 4510, the parental isolate for isolate 4568 from patient 4, had subpopulations growing on agar plates containing 4 g of vancomycin per ml, while the upward shift of PAPs occurred even in isolate 1591 which had no colonies capable of growing on agar plates containing 4 g of vancomycin per ml. For two of the pairs, the second isolates had shapes of PAPs unchanged in comparison to the respective parental isolates. The other serial isolates from four patients (patients 2, 3, 5 and 6) showed no upward shifts during vancomycin therapy (Figs. 2B, C, E and F). For post-therapy isolates 3822 and 5677, there was a 10- to 1,000-fold increase in subpopulations growing on the 2-g/ml-vancomycin plates, but isolates showed no increase of subpopulations which grew on vancomycin concentrations ⱖ4 g/ml. 3.5. Selection of subpopulations with decreased vancomycin susceptibility in vitro It was relatively easy to increase the proportion of these subpopulations under laboratory conditions. A colony of isolate 1591 from the 2-g/ml-vancomycin plate (isolate 1591-2R) and a colony of isolate 4510 from the 4-g/mlvancomycin plate (isolate 4510-4R) were picked, diluted in drug-free tryptic soy broth, grown overnight, and subsequently plated for population analysis. Isolates 1591-2R and 4510-4R exhibited the upward shifts in PAPs very similar to those of the post-therapy isolates 1905 and 4568 with decreased vancomycin susceptibility in vivo, respectively
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Fig. 1. PFGE patterns of 12 MRSA isolates recovered from six patients who had received vancomycin therapy. Chromosomal DNA was prepared, SmaI restricted, and separated by PFGE. S denotes lambda molecular-size standards; lane 1, isolate 1591 from patient 1; lane 2, isolate 1905 from patient 1; lane 3, isolate 2203 from patient 2; lane 4, isolate 2313 from patient 2; lane 5, isolate 3793 from patient 3; lane 6, isolate 3822 from patient 3; lane 7, isolate 4510 from patient 4; lane 8, isolate 4568 from patient 4; lane 9; isolate 4863 from patient 5; lane 10, isolate 4902 from patient 5; lane 11, isolate 5658 from patient 6; and lane 12, isolate 5677 from patient 6.
(Figs. 2A and D). Such in vitro selection also occurred in other four pre-therapy isolates (data not shown). In an additional experiment, isolate 4568, which contained subpopulations capable of growing on agar supplemented with 8 g (frequency, 10⫺6) of vancomycin per ml was tested whether to yield further resistant subclones under laboratory conditions. A colony of isolate 4568 from the 8-g/ml-vancomycin plate (isolate 4568-8R) was picked and analyzed by population analysis as above-mentioned method. In consequence, isolate 4568-8R exhibited a distinct upward shift in PAPs with subpopulations (isolate 4568-16R) capable of growing on 16 g (frequency, 10⫺5) of vancomycin per ml. Upon replating for population analysis of isolate 4568-16R, all the cells in this culture could grow on agar supplemented with 8 g of vancomycin per ml and contained bacteria capable of growing on 16 g (frequency, 10⫺2) of vancomycin per ml. This culture became more homogeneous with respect to vancomycin resistance (Fig. 3). The vancomycin MICs for these subclones were: 4568-8R 8 g/ml and 4568-16R 16 g/ml by NCCLS standards.
3.6. Stability of decreased vancomycin susceptibility in vitro Cultures of two isolates (isolates 1905 and 4568) with decreased vancomycin susceptibility in PAPs were repeatedly grown on BHI agar plate without vancomycin, and population analysis was reassessed after 10 and 20 serial passages to evaluate the stability of decreased vancomycin susceptibility. PAPs for the two isolates and serial passaged isolates are shown in Fig. 4. The subpopulations with decreased vancomycin susceptibility were unstable for both isolates. For isolate 1905, there was a 10-fold decrease in subpopulations growing on the 4-g/ml-vancomycin plates after 10 passes (Fig. 4A). For isolate 4568, there was also a 10-fold decrease in subpopulations growing on the 4-g/ ml-vancomycin plates, and no colonies grew on the 8-g/ ml-vancomycin plates after 10 passes (Fig. 4B). Both PAPs for the 20th passaged isolates (isolates 1905[P20] and 4568 [P20]) virtually reverted to those of the pre-therapy isolates (isolates 1591 and 4510). The vancomycin MICs for isolates 1905[P20] and 4568[P20] decreased to 1 g/ml by NCCLS standards.
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Fig. 2. Population analysis profiles (PAPs) for vancomycin susceptibility of 12 MRSA isolates from six individual patients: patient 1 (A), patient 2 (B), patient 3 (C), patient 4 (D), patient 5 (E), and patient 6 (F). Open circles show PAPs of the isolates obtained before vancomycin therapy, and solid squares show PAPs of the isolates obtained during vancomycin therapy. Isolates 1591-2R and 4510-4R (open squares) were derivatives obtained by selecting isolate 1591 with the 2-g/ml-vancomycin plate and isolate 4510 with the 4-g/ml-vancomycin plate, respectively (arrows). Isolate numbers and symbols are identified in each panel.
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Fig. 4. PAPs for post-therapy isolates 1905 and 4568, and their serial passaged isolates on nonselective medium without vancomycin. “[P10]” and “[P20]” refer to the isolates obtained after 10 and 20 serial passages of the post-therapy isolate, respectively. Isolate numbers and symbols are identified in each panel.
4. Discussion The recent reports on MRSA isolates with decreased susceptibility to vancomycin (Centers for Disease Control and Prevention 1997a; Centers for Disease Control and Prevention 1997b; Hiramatsu et al., 1997; Ploy et al., 1998)
Fig. 3. Enrichment of vancomycin-heteroresistant MRSA (isolate 4568) for the subpopulations of bacteria for which vancomycin MICs were elevated. Isolate 4568-8R was derivative obtained by selecting isolate 4568 with the 8-g/ml-vancomycin plate. Isolate 4568-16R was obtained by selecting isolate 4568-8R with the 16-g/ml-vancomycin plate. Population analysis was tested for vancomycin susceptibility of three isolates: solid squares show isolate 4568; open circles, isolate 4568-8R; and open squares, isolate 4568-16R.
have renewed concern about the emergence of glycopeptide-resistant MRSA strains for which there may be no effective antimicrobial therapy. As well, decreased susceptibility to glycopeptide has been reported among clinical isolates of coagulase-negative methicillin-resistant staphylococci (Sieradzki et al., 1998). More recently, the first report of in vivo development of intermediate vancomycinresistant MRSA during prolonged vancomycin therapy has been published (Sieradzki et al., 1999a). However, the precise mechanisms and process of in vivo development of vancomycin resistance are unidentified as yet. Our study clearly shows that in vivo, the development of decreased vancomycin susceptibility of which level is below the breakpoint by standard MIC determination method occurs in MRSA clinical strains during vancomycin therapy. We found the decreased vancomycin susceptibility in PAPs among serial isolates from two patients during vancomycin therapy. It should be notable that in vivo development of decreased vancomycin susceptibility occurred even in a MRSA isolate which had no colonies capable of growing on agar plates containing 4 g of vancomycin per ml. The potential for the development of vancomycin resistance in staphylococci has been studied for well over a decade. In vitro study, it has been reported that vancomycin-resistant mutants of S. aureus can be selected by a stepwise procedure (Sieradzki et al., 1996), and subclones equivalent to S. aureus with intermediate vancomycin resistance can be obtained by selection with vancomycin-heteroresistant strains (Hiramatsu et al., 1997). Among MRSA clinical isolates in our study, it was relatively easy to increase the proportion of subpopulations with decreased vancomycin susceptibility after a repeated passage on vancomycin-containing agar. Actually, the subpopulations (isolates 1591-2R and 4510-
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4R) derived from the pre-therapy isolates (isolates 1591 and 4510) showed very similar PAPs to those of the posttherapy isolates (isolates 1905 and 4568) with decreased vancomycin susceptibility in vivo. Furthermore, we confirmed the reversion phenomenon of decreased vancomycin susceptibility in these two isolates (isolates 1905 and 4568) by serially passing them on nonselective medium and testing PAPs of the 20th passaged isolates. These findings suggest that in vivo development of vancomycin resistance occurs by stepwise fashion. The stepwise nature of reversion phenomenon has been recognized in vancomycin-intermediate S. aureus (VISA) (Aeschlimann et al., 1999; Boyle-Vavra S et al., 2000). Therefore, decreased vancomycin susceptibility in vivo may result from the selection of cells which have adapted to survive in the presence of the drug and may decrease upon removal of the selective pressure. The exact mechanism of vancomycin resistance in Staphylococci eludes us, although considerable progress has been made to date and we know that they make abnormal cell walls that can bind vancomycin, hyperexpress penicillin-binding proteins, and have other alterations that likely contribute to vancomycin resistance (Hanaki et al., 1998b; Sieradzki et al., 1997; Sieradzki et al., 1999a). The fact that some of the cell wall changes observed were unstable and were reversed on repeated subculture suggests that they may represent an adaptation to vancomycin exposure through the changes of structural gene expression regulating cell wall synthesis rather than may reflect stable genetically determined factors mediating vancomycin resistance. Our observations on the development and reversion of decreased vancomycin susceptibility are important especially on clinical settings and needed to be considered during future study of vancomycin resistance in Staphylococci. Isolate 4568 in this study has similar PAPs to those of previously reported vancomycin-heteroresistant S. aureus strains (Hiramatsu et al., 1997). It also has been reported that coagulase-negative staphylococci with decreased susceptibility to vancomycin have heterogeneous vancomycinsusceptibility profiles (Sieradzki et al., 1998; Sieradzki et al., 1999b). However, the origin of vancomycin-heteroresistant MRSA is not completely understood. It is supposed that the finding of vancomycin-heteroresistant MRSA may be a more common problem than has generally been recognized and that this event may be inherent in the Staphylococcus genus (Ariza et al., 1999); on the other hand, it has been reported that prior use of vancomycin may serve to induce vancomycin-heteroresistance in Staphylococci, including MRSA, among patients with clinically significant bacteremia (Wong SSY et al., 1999). Our data show that vancomycin therapy is selecting for the isolates with elevated MICs. In consequence, vancomycin-heteroresistant MRSA with subpopulations capable of growing on agar supplemented with 8 g (frequency, 10⫺6 or higher) of vancomycin per ml may be inducible during vancomycin therapy. Moreover, it is of great interest that the vancomy-
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cin-heteroresistant MRSA from patient 4 who died from MRSA infection despite adequate serum vancomycin level, may have contributed to vancomycin therapeutic failure. This is the first report providing in vivo evidence that the development of vancomycin-heteroresistance in MRSA, possibly affecting the efficacy of vancomycin, occurs during vancomycin therapy. Further selection of isolate 4568 with serial passages on vancomycin-containing agar produced more resistant subpopulations to vancomycin (MICs 8-16 g/ml). This in vitro study demonstrates that vancomycin-heteroresistant MRSA strains can be potential reservoirs of MRSA with intermediate or full vancomycin resistance. Given the relative ease with which vancomycin resistant MRSA can be selected in the laboratory, there have not been more reports of these organisms from clinical specimens in the world. The reversion phenomenon we observed may explain why VISA isolates are rarely isolated from patients receiving vancomycin therapy. We speculate that most MRSA isolates revert rapidly in the absence of vancomycin but that the identified VISA isolates may have developed a mechanism to stabilize the vancomycin resistance phenotype for a longer period. The observations described in this study suggest that the reversion phenomenon may also occur in vancomycin-heteroresistant MRSA strains, and the heteroresistant phenotype may be unstable on removal of the drug pressure. The prevalence and clinical relevance of vancomycin-heteroresistant MRSA strains are still unknown. These strains are currently difficult to detect (Howe et al., 1999), as indicated by the poor specificity and reproducibility of the original method proposed by Hiramatsu et al. First of all, a reliable and simplified method should be established as soon as possible, because population analysis which can identify the heterogeneity of vancomycin susceptibility is too time-consuming. As well, a prospective study should be needed to ascertain the true significance of infections due to vancomycin-heteroresistant MRSA. It is unclear why in vivo development of decreased susceptibility to vancomycin did not occur in four MRSA isolates. We can speculate that in vivo development of vancomycin resistance may be depended on the different environmental conditions. There are certain common clinical features among previously reported patients with infections due to VISA isolates. First, repeated and prolonged vancomycin therapy is probably a major risk factor for decreasing vancomycin susceptibility. It appears that under prolonged vancomycin exposure, MRSA for which vancomycin MICs are increased could originate from the subpopulations of bacteria. Second, the presence of prosthetic materials may lead to an increase in the degree of vancomycin resistance of MRSA. Relatively poor access of antibacterial agents to staphylococci adhering to artificial surfaces leads to decreased susceptibility to antibiotic killing (Chuard et al., 1997; Khardori et al., 1995; Sieradzki et al., 1999b). In our study, the post-therapy isolates with decreased vancomycin susceptibility in PAPs were isolated
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from two patients (patients 1 and 4), who had received longer courses of vancomycin therapy than the other four patients. Patient 4 also had many devices with prosthetic materials placed such as a thoracheostomy tube, a percutaneous gastrostomy catheter, and a central venous catheter. In addition to these factors, therapeutic failures with vancomycin therapy versus MRSA may be attributed to such factors as a slower killing rate versus -lactam antibiotics, the dosage regimen used, the decreased penetration to infected tissue sites, or the underlying compromised health status of the patient (Houlihan et al., 1997; Sieradzki et al., 1998). The mechanisms of staphylococcal glycopeptide resistance have mainly been studied in vitro until now. Elucidation of in vivo mechanisms for glycopeptide resistance is needed in the light of environmental factors under clinical conditions. In conclusion, our study shows that in vivo, the development of decreased susceptibility to vancomycin in MRSA clinical isolates appears to be unstable, but a selective or inducible process that increased during vancomycin therapy. This phenomenon is likely to occur in MRSA clinical strains for which MICs are susceptible to vancomycin by standard MIC determination method. Whether MRSA strains with decreased vancomycin susceptibility do or do not represent a new plague of antibiotic resistance remains to be determined, but under no circumstances should they be ignored. To prevent further emergence of MRSA strains with decreased vancomycin susceptibility, the prudent use of vancomycin must be needed, and active surveillance for these strains should be implemented particularly in populations at high risk, such as patients in whom vancomycin therapy is unsuccessful. Acknowledgments We are grateful to Hidehiko Saito, the First Department of Internal Medicine, Nagoya University School of Medicine, for encouragement. We also thank Mie Tadokoro for expert technical assistance. References Aeschlimann, J. R., Hershberger, E., & Rybak, M. J. (1999). Analysis of vancomycin population susceptibility profiles, killing activity, and postantibiotic effect against vancomycin-intermediate Staphylococcus aureus. Antimicrob Agents Chemother 43, 1914 –1918. Ariza, J., Pujol, M., Cabo, J., Pena, C., Fernandez, N., Linares, J., Ayats, J., & Gudiol, F. (1999). Vancomycin in surgical infections due to methicillin-resistant Staphylococcus aureus with heterogeneous resistance to vancomycin. Lancet 353, 1587–1588. Aubert, G., Passot, S., Lucht, F., & Dorche, G. (1990). Selection of vancomycin- and teicoplanin-resistant Staphylococcus haemolyticus during teicoplanin treatment of Staphylococcus epidermidis infection. J Antimicrob Chemother 25, 491– 493. Boyle-Vavra, S., Berke, S. K., Lee, J. C., & Daum, R. S. (2000). Reversion of the Glycopeptide Resistance Phenotype in Staphylococcus aureus Clinical Isolates. Antimicrob Agents Chemother 44, 272–277.
Centers for Disease Control and Prevention. (1997a). Staphylococcus aureus with reduced susceptibility to vancomycin—United States, 1997. MMWR 46, 765–766. Centers for Disease Control and Prevention. (1997b). Update: Staphylococcus aureus with reduced susceptibility to vancomycin—United States, 1997. MMWR 46, 813– 815. Cercenado, E., Garcı´a-Leoni, M. E., Dı´az, M. D., Sanchez-Carrillo, C., Catalan, P., De Quiros, J. C., & Bouza, E. (1996). Emergence of teicoplanin-resistant coagulase-negative staphylococci. J Clin Microbiol 34, 1765–1768. Chuard, C., Vaudaux, P. E., Proctor, R. A., & Lew, D. P. (1997). Decreased susceptibility to antibiotic killing of a stable small colony variant of Staphylococcus aureus in fluid phase and on fibronectin-coated surfaces. J Antimicrob Chemother 39, 603– 608. Daum, R. S., Gupta, S., Sabbagh, R., & Milewski, W. M. (1992). Characterization of Staphylococcus aureus isolates with decreased susceptibility to vancomycin and teicoplanin: isolation and purification of a constitutively produced protein associated with decreased susceptibility. J Infect Dis 166, 1066 –1072. Hanaki, H., Kuwahara-Arai, K., Boyle-Vavra, S., Daum, R. S., Labischinski, H., & Hiramatsu, K. (1998a). Activated cell-wall synthesis is associated with vancomycin resistance in methicillin-resistant Staphylococcus aureus clinical strains Mu3 and Mu50. J Antimicrob Chemother 42, 199 –209. Hanaki, H., Labischinski, H., Inaba, Y., Kondo, N., Murakami, H., & Hiramatsu, K. (1998b). Increase in glutamine-non-amidated muropeptides in the peptidoglycan of vancomycin-resistant Staphylococcus aureus strain Mu50. J Antimicrob Chemother 42, 315–320. Hiramatsu, K., Aritaka, N., Hanaki, H., Kawasaki, S., Hosoda, Y., Hori, S., Fukuchi, Y., & Kobayashi, I. (1997). Dissemination in Japanese hospitals of strains of Staphylococcus aureus heterogeneously resistant to vancomycin. Lancet 350, 1670 –1673. Houlihan, H. H., Mercier, R. C., & Rybank, M. J. (1997). Pharmacodynamics of vancomycin alone and in combination with gentamicin at various dosing intervals against methicillin-resistant Staphylococcus aureus-infected fibrin-platelet clots in an in vitro infection model. Antimicrob Agents Chemother 41, 2497–2501. Howe, R. A., Wooton, M., Walsh, T. R., Bennett, P. M., & MacGowan, A. P. (1999). Expression and detection of hetero-vancomycin resistance in Staphylococcus aureus. J Antimicrob Chemother 44, 675– 678. Ichiyama, S., Ohta, M., Shimokata, K., Kato, N., & Takeuchi, J. (1991). Genomic DNA fingerprinting by pulsed-field gel electrophoresis as an epidemiological marker for study of nosocomial infections caused by methicillin-resistant Staphylococcus aureus. J Clin Microbiol 29, 2690 –2695. Kaatz, G. W., Seo, S. M., Dorman, N. J., & Lerner, S. A. (1990). Emergence of teicoplanin resistance during therapy of Staphylococcus aureus endocarditis. J Infect Dis 162, 103–108. Khardori, N., Yassien, M., & Wilson, K. (1995). Tolerance of Staphylococcus epidermidis grown from indwelling vascular catheters to antimicrobial agents. J Ind Microbiol 15, 148 –151. National Committee for Clinical Laboratory Standards (NCCLS). (1997). Methods for dilution antimicrobial susceptibility tests for bacteria that grow aerobically, fourth edition: approved standard, M7–A4. Villanova, Pennsylvania: NCCLS. Ploy, M. C., Gre´laud, C., Martin, C., de Lumley, L., & Denis, F. (1998). First clinical isolate of vancomycin-intermediate Staphylococcus aureus in a French hospital. Lancet 351, 1212. Schwalbe, R. S., Stapleton, J. T., & Gilligan, P. H. (1987). Emergence of vancomycin resistance in coagulase-negative staphylococci. N Engl J Med 316, 927–931. Sieradzki, K., & Tomasz, A. (1996). A highly vancomycin-resistant laboratory mutant of Staphylococcus aureus. FEMS Microbiol Lett 142, 161–166. Sieradzki, K., & Tomasz, A. (1997). Inhibition of cell wall turnover and autolysis by vancomycin in a highly vancomycin-resistant mutant of Staphylococcus aureus. J Bacteriol 179, 2557–2566.
Yasuteru Sugino et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 159 –167 Sieradzki, K., Villari, P., & Tomasz, A. (1998). Decreased susceptibilities to teicoplanin and vancomycin among coagulase-negative methicillinresistant clinical isolates of staphylococci. Sieradzki, K., Roberts, R. B., Haber, S. W., & Tomasz, A. (1999a). The development of vancomycin resistance in a patient with methicillinresistant Staphylococcus aureus infection. N Engl J Med 340, 517–523. Sieradzki, K., Roberts, R. B., Serur, D., Hargrave, J., & Tomasz, A. (1999b). Heterogeneously vancomycin-resistant Staphylococcus epidermidis strain causing recurrent peritonitis in a dialysis patient during vancomycin therapy. J Clin Microbiol 37, 39 – 44. Tenover, F. C., Arbeit, R. D., Goering, R. V., Mickelsen, P. A., Murray, B. E., Persing, D. H., & Swaminathan, B. (1995). Interpreting chromosomal DNA restriction patterns produced by pulsed-field gel elec-
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trophoresis: criteria for bacterial strain typing. J Clin Microbiol 33, 2233–2239. Tenover, F. C., Lancaster, M. V., Hill, B. C., Steward, C. D., Stocker, S. A., Hancock, G. A., O’Hara, C. M., McAllister, S. K., Clark, N. C., & Hiramatsu, K. (1998). Characterization of staphylococci with reduced susceptibilities to vancomycin and other glycopeptides. J Clin Microbiol 36, 1020 –1027. Tomasz, A., Nachman, S., & Leaf, H. (1991). Stable classes of phenotypic expression in methicillin-resistant clinical isolates of staphylococci. Antimicrob Agents Chemother 35, 124 –129. Wong, S. S. Y., Ho, P. L., Woo, P. C. Y., & Yuen, K. Y. (1999). Bacteremia caused by Staphylococci with inducible vancomycin heteroresistance. Clin Infect Dis 29, 760 –767.
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In vivo development of decreased susceptibility to vancomycin in clinical isolates of methicillin-resistant Staphylococcus aureus Yasuteru Suginoa, Yoshitsugu Iinumab,*, Satoshi Ichiyamac, Yuji Itoa, Saburo Ohkawad, Nobuo Nakashimab, Kaoru Shimokatae, Yoshinori Hasegawaa a
The first Department of Internal Medicine, Nagoya University School of Medicine Departments of Clinical Laboratory, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan c Department of Clinical Laboratory Medicine, Kyoto University Hospital, 54 Kawahara-cho, Syogoin, Sakyo-ku, Kyoto 606-8507, Japan d Nippon Becton Dickinson Co., Ltd., 8-5-26 Akasaka, Minato-ku, Tokyo 107-0052, Japan e Clinical Preventive Medicine, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan b
Abstract To investigate the possibility of in vivo development of decreased vancomycin susceptibility, the vancomycin susceptibilities of 12 methicillinresistant Staphylococcus aureus (MRSA) isolates serially recovered from six patients with vancomycin therapy were tested by standard MIC determination method and population analysis. While all of the MRSA isolates were susceptible to vancomycin (MICs, 1-2 g/ml) by standard method, population analysis showed the upward shifts indicating decreased vancomycin susceptibility among serial isolates from two patients. These bacteria with decreased vancomycin susceptibility could be selected by using vancomycin selection of pre-therapy isolates under laboratory conditions. Furthermore, the reversion phenomenon of decreased vancomycin susceptibility was confirmed after 20 serial passages of the post-therapy isolates on drug-free agar. These data suggest that in vivo isolates may develop decreased vancomycin susceptibility that is not of such magnitude to cross a breakpoint threshold. This resistance may be unstable, and appears to result from a selective or inducible process that occurs in MRSA clinical strains during vancomycin therapy. © 2000 Elsevier Science Inc. All rights reserved.
1. Introduction For nearly four decades, vancomycin has been widely and successfully used for the treatment of serious infections caused by Gram-positive bacterial pathogen. In recent years, however, reduced susceptibilities to glycopeptides among some clinical isolates of coagulase-negative staphylococci have been reported (Aubert et al., 1990; Cercenado et al., 1996; Schwalbe et al., 1987). More recently, Hiramatsu and colleagues (Hiramatsu et al., 1997) reported the first clinical isolate of methicillin-resistant Staphylococcus aureus (MRSA) with decreased susceptibility to vancomycin (MIC, 8 g/ml). Subsequently, similar isolates with decreased susceptibility to vancomycin have been reported in the United States and France (Centers for Disease Control and Prevention 1997a; Centers for Disease Control and Prevention 1997b; Ploy et al., 1998). These MRSA isolates exhibited low-level vancomycin resistance mediated through novel and as-yet-undefined mechanisms that
differ from those responsible for enterococcal vancomycin resistance (Hanaki et al., 1998a; Tenover et al., 1998). In vitro selection of glycopeptide-resistant staphylococcal mutants has been demonstrated, and the microbiological properties of these mutants have been investigated in detail (Daum et al., 1992; Kaatz et al., 1990; Sieradzki et al., 1996; Sieradzki et al., 1998). However, it is unclear how in vivo resistance to vancomycin could develop in MRSA clinical strains in relation to vancomycin therapy. In order to study the possibility and process of in vivo development of decreased vancomycin susceptibility, we examined the vancomycin susceptibilities of 12 MRSA isolates serially recovered from six patients, who received intravenous vancomycin therapy for MRSA infections, by standard MIC determination method and population analysis.
2. Patients and methods 2.1. Patients
* Corresponding author. Yoshitsugu Iinuma, Department of Clinical Laboratory, Nagoya University Hospital, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan. Tel.: ⫹81-52-744-2615; fax: ⫹81-52-744-2613. E-mail address: [email protected] (Y. Iinuma).
At Nagoya University Hospital, a 1,035-bed tertiary care and university teaching hospital, nearly 30 patients are annually treated with vancomycin for MRSA infections. We
0732-8893/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved. PII: S 0 7 3 2 - 8 8 9 3 ( 0 0 ) 0 0 1 8 6 - 3
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retrospectively reviewed the hospital records of patients treated with intravenous vancomycin for MRSA infections between 1992 and 1997; MRSA infection was defined on the basis of clinical significance, if the patients showed both an acute systemic inflammatory response (e.g., fever and elevation of C-reactive protein levels) and local infectious signs or symptoms (e.g., purulent sputum and pyuria). Six patients from whom serial MRSA isolates had been successfully recovered and stored frozen before and during vancomycin therapy were studied. These six patients had never received any prior treatment with glycopeptides. The following information was obtained for each patient: demographics, underlying diseases, medical conditions, pertinent laboratory data, treatment, and outcome. 2.2. MRSA isolates We studied 12 MRSA isolates obtained serially from six individual patients: 6 were isolated before vancomycin therapy (isolates 1591, 2203, 3793, 4510, 4863, and 5658) and the others were isolated during vancomycin therapy (isolates 1905, 2313, 3822, 4568, 4902, and 5677). All isolates were tested for coagulase reaction and susceptibility to oxacillin before inclusion in the study. MRSA was defined as S. aureus with oxacillin of 4 g/ml or above by the micro-broth dilution MIC method performed and interpreted according to the recommendations from the National Committee on Clinical Laboratory Standards (NCCLS) (National Committee for Clinical Laboratory Standards 1997). The isolates were stored as frozen stocks in skim milk (Difco, Detroit) at ⫺70°C until use. Tryptic soy broth (Difco, Detroit) were used for the cultivation of bacterial cultures, which were grown at 37°C with aeration. 2.3. Chromosomal DNA analysis by pulsed-field gel electrophoresis (PFGE) Clonal identity between serially recovered isolates was confirmed by PFGE. Preparation of chromosomal DNA for PFGE and separation of SmaI-restricted fragments in a contour-clamped homogeneous electric field system (Pulsaphor Plus; Pharmacia LKB Biotechnology, Uppsala, Sweden) were carried out as reported previously (Ichiyama et al., 1991). The electrophoresis conditions were 200 V for 20 h, with pulse times ranging from 5 to 60 s for SmaI digestion. The DNA banding pattern was photographed under UV light after ethidium bromide staining, and classified according to the number and size of the restriction fragments. 2.4. Antimicrobial susceptibility The MICs of oxacillin and vancomycin for 12 MRSA isolates were determined by the micro-broth dilution method of NCCLS with cation-adjusted Mueller-Hinton broth (Difco, Detroit). The MIC was the lowest concentra-
tion of antibiotic that yielded no visible growth after incubation at 37°C for 24 h. Standard antimicrobial powders for laboratory use were obtained from the respective manufactures: oxacillin, Banyu Pharmaceutical Co., Ltd., Tokyo, Japan; and vancomycin, Shionogi Co., Ltd., Osaka, Japan. 2.5. Population analysis profiles (PAPs) Resistant subpopulations of bacteria (Hiramatsu et al., 1997; Tomasz et al., 1991) were analyzed by spreading 50 l of the starting cell suspension (prepared by diluting overnight cultures to an optical density [OD] of 0.3 at 540 nm) and serial diluents of this suspension on to brain-heart infusion (BHI) agar plates (Eiken Chemical, Tokyo) containing various concentrations of vancomycin. The plates were incubated at 37°C for 48 h, and the number of mature colonies was counted. The number of resistant cells contained in 50 l of the starting cell suspension was calculated and plotted on a semi-logarithmic graph. PAPs were determined in duplicate with each isolate. 3. Results 3.1. Patients We studied six patients who had received intravenous vancomycin therapy for MRSA infections. Clinical and microbiologic features of the patients are summarized in Table 1. All patients had underlying disease(s); four had malignant neoplasms. Five patients were mainly treated with imipenem in addition to vancomycin, and one (patient 5) received combination therapy with cefepime and vancomycin. The duration of vancomycin therapy ranged from 5 to 28 days, and the cumulative dosage ranged from 4,160 to 32,000 mg. Among the three patients who died, one (patient 4) died from pyothorax and pneumonia caused by MRSA, and two (patients 1 and 6) died from their underlying disease. 3.2. Chromosomal DNA analysis by PFGE Six distinctive restriction patterns, tentatively designated A to E’ in Table 1, were observed in the 12 MRSA isolates tested in this study. Each of the paired isolates obtained from the five individual patients (patients 1, 2, 3, 4, and 6) showed an identical PFGE pattern (Fig. 1). The paired isolates from patient 5 also showed similar band patterns with only one band difference which was considered to be closely related (Tenover et al., 1995). Therefore, each of paired isolates from a given patient had an identical genetic background. 3.3. Antimicrobial susceptibility The MICs of vancomycin for the 12 MRSA isolates are summarized in Table 1. All of the MRSA isolates were
T 6.32 6,000 VCM, IPM/CS
5
32,000 VCM, CFPM
16
4,160 VCM, IPM/CS
28
T 6.81 P 37.97 T 18.9 P 40.35 ND 12 12,000
ND 7 7,000
67 6
M
63 5
M
Lung cancer
Pneumonia Pyothorax Perianal abscess Pneumonia Tetralogy of Fallot Leukemia 2 4
M
Pneumonia M 62 3
M 53 2
a M, male patient; F, female patient. b UTI, urinary tract infection. c VCM, vancomycin; IPM/CS, imipenem/cilastatin; FOM, fosfomycin; ABK, arbekacin; CFPM, cefepime. d ND, not done; T, trough; P, peak. e Died (⫺), died from underlying disease; Died (⫹), died from MRSA infection.
Died(⫺)
Alive
Died(⫹)
Unknown
Died(⫺)
A A B B C C D D E E’ C C 18
1591 1905 2203 2313 3793 3822 4510 4568 4863 4902 5658 5677 Recurrent UTI Peritonitis 79 1
F
Bladder cancer Diabetes mellitus Lung cancer
12/1/92 3/1/93 9/6/93 9/21/93 4/10/95 4/24/95 12/13/95 1/14/96 4/23/96 5/10/96 1/28/97 2/4/97
Urine Urine Abscess Abscess Sputum Sputum Sputum Sputum Abscess Abscess Sputum Sputum
VCM, IPM/CS, FOM VCM, IPM/CS, ABK VCM, IPM/CS
10,000
ND
1 2 1 1 2 2 1 2 1 1 2 2
Alive
Outcomee Culture source Isolation date (mo/day/yr) Isolate no. Infectionb Underlying condition Sexa Age (yr) Patient no.
Table 1 Characteristics of six patients infected with MRSA and properties of MRSA isolates.
Treatmentc
Cumulative vancomycin dosage (mg)
Duration of vancomycin therapy (days)
Serum vancomycin level (g/ml)d
Vancomycin susceptibility MIC (g/ml)
PFGE pattern
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susceptible to vancomycin (MICs, 1-2 g/ml). The vancomycin MICs for two isolates obtained during vancomycin therapy (isolates 1905 and 4568) were slightly elevated from 1 to 2 g/ml, but remained unchanged for the other four isolates during vancomycin therapy. All MRSA isolates had an MIC of oxacillin ranging from 8 to 1,024 g/ml and were thus classifiable as MRSA by NCCLS criteria. 3.4. Population analysis profiles (PAPs) Fig. 2 shows the vancomycin susceptibility profiles by population analysis for each of the two MRSA isolates serially isolated from the six individual patients. The upward shifts in PAPs indicating decreased vancomycin susceptibility (Figs. 2A and D) were observed among serial isolates from two patients (patients 1 and 4), who had received longer courses of vancomycin therapy than the other four patients. The broth dilution vancomycin MICs for these two isolates with decreased vancomycin susceptibility in PAPs slightly increased from 1 to 2 g/ml within the susceptible range by NCCLS criteria. Isolate 4568 from patient 4 who died from MRSA infection showed growth on agar plates containing 8 g of vancomycin per ml at a frequency of approximately 10⫺6. This pattern of PAPs was the same as those of previously reported vancomycin-heteroresistant S. aureus strains (Hiramatsu et al., 1997). Isolate 4510, the parental isolate for isolate 4568 from patient 4, had subpopulations growing on agar plates containing 4 g of vancomycin per ml, while the upward shift of PAPs occurred even in isolate 1591 which had no colonies capable of growing on agar plates containing 4 g of vancomycin per ml. For two of the pairs, the second isolates had shapes of PAPs unchanged in comparison to the respective parental isolates. The other serial isolates from four patients (patients 2, 3, 5 and 6) showed no upward shifts during vancomycin therapy (Figs. 2B, C, E and F). For post-therapy isolates 3822 and 5677, there was a 10- to 1,000-fold increase in subpopulations growing on the 2-g/ml-vancomycin plates, but isolates showed no increase of subpopulations which grew on vancomycin concentrations ⱖ4 g/ml. 3.5. Selection of subpopulations with decreased vancomycin susceptibility in vitro It was relatively easy to increase the proportion of these subpopulations under laboratory conditions. A colony of isolate 1591 from the 2-g/ml-vancomycin plate (isolate 1591-2R) and a colony of isolate 4510 from the 4-g/mlvancomycin plate (isolate 4510-4R) were picked, diluted in drug-free tryptic soy broth, grown overnight, and subsequently plated for population analysis. Isolates 1591-2R and 4510-4R exhibited the upward shifts in PAPs very similar to those of the post-therapy isolates 1905 and 4568 with decreased vancomycin susceptibility in vivo, respectively
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Fig. 1. PFGE patterns of 12 MRSA isolates recovered from six patients who had received vancomycin therapy. Chromosomal DNA was prepared, SmaI restricted, and separated by PFGE. S denotes lambda molecular-size standards; lane 1, isolate 1591 from patient 1; lane 2, isolate 1905 from patient 1; lane 3, isolate 2203 from patient 2; lane 4, isolate 2313 from patient 2; lane 5, isolate 3793 from patient 3; lane 6, isolate 3822 from patient 3; lane 7, isolate 4510 from patient 4; lane 8, isolate 4568 from patient 4; lane 9; isolate 4863 from patient 5; lane 10, isolate 4902 from patient 5; lane 11, isolate 5658 from patient 6; and lane 12, isolate 5677 from patient 6.
(Figs. 2A and D). Such in vitro selection also occurred in other four pre-therapy isolates (data not shown). In an additional experiment, isolate 4568, which contained subpopulations capable of growing on agar supplemented with 8 g (frequency, 10⫺6) of vancomycin per ml was tested whether to yield further resistant subclones under laboratory conditions. A colony of isolate 4568 from the 8-g/ml-vancomycin plate (isolate 4568-8R) was picked and analyzed by population analysis as above-mentioned method. In consequence, isolate 4568-8R exhibited a distinct upward shift in PAPs with subpopulations (isolate 4568-16R) capable of growing on 16 g (frequency, 10⫺5) of vancomycin per ml. Upon replating for population analysis of isolate 4568-16R, all the cells in this culture could grow on agar supplemented with 8 g of vancomycin per ml and contained bacteria capable of growing on 16 g (frequency, 10⫺2) of vancomycin per ml. This culture became more homogeneous with respect to vancomycin resistance (Fig. 3). The vancomycin MICs for these subclones were: 4568-8R 8 g/ml and 4568-16R 16 g/ml by NCCLS standards.
3.6. Stability of decreased vancomycin susceptibility in vitro Cultures of two isolates (isolates 1905 and 4568) with decreased vancomycin susceptibility in PAPs were repeatedly grown on BHI agar plate without vancomycin, and population analysis was reassessed after 10 and 20 serial passages to evaluate the stability of decreased vancomycin susceptibility. PAPs for the two isolates and serial passaged isolates are shown in Fig. 4. The subpopulations with decreased vancomycin susceptibility were unstable for both isolates. For isolate 1905, there was a 10-fold decrease in subpopulations growing on the 4-g/ml-vancomycin plates after 10 passes (Fig. 4A). For isolate 4568, there was also a 10-fold decrease in subpopulations growing on the 4-g/ ml-vancomycin plates, and no colonies grew on the 8-g/ ml-vancomycin plates after 10 passes (Fig. 4B). Both PAPs for the 20th passaged isolates (isolates 1905[P20] and 4568 [P20]) virtually reverted to those of the pre-therapy isolates (isolates 1591 and 4510). The vancomycin MICs for isolates 1905[P20] and 4568[P20] decreased to 1 g/ml by NCCLS standards.
Yasuteru Sugino et al. / Diagnostic Microbiology and Infectious Disease 38 (2000) 159 –167
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Fig. 2. Population analysis profiles (PAPs) for vancomycin susceptibility of 12 MRSA isolates from six individual patients: patient 1 (A), patient 2 (B), patient 3 (C), patient 4 (D), patient 5 (E), and patient 6 (F). Open circles show PAPs of the isolates obtained before vancomycin therapy, and solid squares show PAPs of the isolates obtained during vancomycin therapy. Isolates 1591-2R and 4510-4R (open squares) were derivatives obtained by selecting isolate 1591 with the 2-g/ml-vancomycin plate and isolate 4510 with the 4-g/ml-vancomycin plate, respectively (arrows). Isolate numbers and symbols are identified in each panel.
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Fig. 4. PAPs for post-therapy isolates 1905 and 4568, and their serial passaged isolates on nonselective medium without vancomycin. “[P10]” and “[P20]” refer to the isolates obtained after 10 and 20 serial passages of the post-therapy isolate, respectively. Isolate numbers and symbols are identified in each panel.
4. Discussion The recent reports on MRSA isolates with decreased susceptibility to vancomycin (Centers for Disease Control and Prevention 1997a; Centers for Disease Control and Prevention 1997b; Hiramatsu et al., 1997; Ploy et al., 1998)
Fig. 3. Enrichment of vancomycin-heteroresistant MRSA (isolate 4568) for the subpopulations of bacteria for which vancomycin MICs were elevated. Isolate 4568-8R was derivative obtained by selecting isolate 4568 with the 8-g/ml-vancomycin plate. Isolate 4568-16R was obtained by selecting isolate 4568-8R with the 16-g/ml-vancomycin plate. Population analysis was tested for vancomycin susceptibility of three isolates: solid squares show isolate 4568; open circles, isolate 4568-8R; and open squares, isolate 4568-16R.
have renewed concern about the emergence of glycopeptide-resistant MRSA strains for which there may be no effective antimicrobial therapy. As well, decreased susceptibility to glycopeptide has been reported among clinical isolates of coagulase-negative methicillin-resistant staphylococci (Sieradzki et al., 1998). More recently, the first report of in vivo development of intermediate vancomycinresistant MRSA during prolonged vancomycin therapy has been published (Sieradzki et al., 1999a). However, the precise mechanisms and process of in vivo development of vancomycin resistance are unidentified as yet. Our study clearly shows that in vivo, the development of decreased vancomycin susceptibility of which level is below the breakpoint by standard MIC determination method occurs in MRSA clinical strains during vancomycin therapy. We found the decreased vancomycin susceptibility in PAPs among serial isolates from two patients during vancomycin therapy. It should be notable that in vivo development of decreased vancomycin susceptibility occurred even in a MRSA isolate which had no colonies capable of growing on agar plates containing 4 g of vancomycin per ml. The potential for the development of vancomycin resistance in staphylococci has been studied for well over a decade. In vitro study, it has been reported that vancomycin-resistant mutants of S. aureus can be selected by a stepwise procedure (Sieradzki et al., 1996), and subclones equivalent to S. aureus with intermediate vancomycin resistance can be obtained by selection with vancomycin-heteroresistant strains (Hiramatsu et al., 1997). Among MRSA clinical isolates in our study, it was relatively easy to increase the proportion of subpopulations with decreased vancomycin susceptibility after a repeated passage on vancomycin-containing agar. Actually, the subpopulations (isolates 1591-2R and 4510-
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4R) derived from the pre-therapy isolates (isolates 1591 and 4510) showed very similar PAPs to those of the posttherapy isolates (isolates 1905 and 4568) with decreased vancomycin susceptibility in vivo. Furthermore, we confirmed the reversion phenomenon of decreased vancomycin susceptibility in these two isolates (isolates 1905 and 4568) by serially passing them on nonselective medium and testing PAPs of the 20th passaged isolates. These findings suggest that in vivo development of vancomycin resistance occurs by stepwise fashion. The stepwise nature of reversion phenomenon has been recognized in vancomycin-intermediate S. aureus (VISA) (Aeschlimann et al., 1999; Boyle-Vavra S et al., 2000). Therefore, decreased vancomycin susceptibility in vivo may result from the selection of cells which have adapted to survive in the presence of the drug and may decrease upon removal of the selective pressure. The exact mechanism of vancomycin resistance in Staphylococci eludes us, although considerable progress has been made to date and we know that they make abnormal cell walls that can bind vancomycin, hyperexpress penicillin-binding proteins, and have other alterations that likely contribute to vancomycin resistance (Hanaki et al., 1998b; Sieradzki et al., 1997; Sieradzki et al., 1999a). The fact that some of the cell wall changes observed were unstable and were reversed on repeated subculture suggests that they may represent an adaptation to vancomycin exposure through the changes of structural gene expression regulating cell wall synthesis rather than may reflect stable genetically determined factors mediating vancomycin resistance. Our observations on the development and reversion of decreased vancomycin susceptibility are important especially on clinical settings and needed to be considered during future study of vancomycin resistance in Staphylococci. Isolate 4568 in this study has similar PAPs to those of previously reported vancomycin-heteroresistant S. aureus strains (Hiramatsu et al., 1997). It also has been reported that coagulase-negative staphylococci with decreased susceptibility to vancomycin have heterogeneous vancomycinsusceptibility profiles (Sieradzki et al., 1998; Sieradzki et al., 1999b). However, the origin of vancomycin-heteroresistant MRSA is not completely understood. It is supposed that the finding of vancomycin-heteroresistant MRSA may be a more common problem than has generally been recognized and that this event may be inherent in the Staphylococcus genus (Ariza et al., 1999); on the other hand, it has been reported that prior use of vancomycin may serve to induce vancomycin-heteroresistance in Staphylococci, including MRSA, among patients with clinically significant bacteremia (Wong SSY et al., 1999). Our data show that vancomycin therapy is selecting for the isolates with elevated MICs. In consequence, vancomycin-heteroresistant MRSA with subpopulations capable of growing on agar supplemented with 8 g (frequency, 10⫺6 or higher) of vancomycin per ml may be inducible during vancomycin therapy. Moreover, it is of great interest that the vancomy-
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cin-heteroresistant MRSA from patient 4 who died from MRSA infection despite adequate serum vancomycin level, may have contributed to vancomycin therapeutic failure. This is the first report providing in vivo evidence that the development of vancomycin-heteroresistance in MRSA, possibly affecting the efficacy of vancomycin, occurs during vancomycin therapy. Further selection of isolate 4568 with serial passages on vancomycin-containing agar produced more resistant subpopulations to vancomycin (MICs 8-16 g/ml). This in vitro study demonstrates that vancomycin-heteroresistant MRSA strains can be potential reservoirs of MRSA with intermediate or full vancomycin resistance. Given the relative ease with which vancomycin resistant MRSA can be selected in the laboratory, there have not been more reports of these organisms from clinical specimens in the world. The reversion phenomenon we observed may explain why VISA isolates are rarely isolated from patients receiving vancomycin therapy. We speculate that most MRSA isolates revert rapidly in the absence of vancomycin but that the identified VISA isolates may have developed a mechanism to stabilize the vancomycin resistance phenotype for a longer period. The observations described in this study suggest that the reversion phenomenon may also occur in vancomycin-heteroresistant MRSA strains, and the heteroresistant phenotype may be unstable on removal of the drug pressure. The prevalence and clinical relevance of vancomycin-heteroresistant MRSA strains are still unknown. These strains are currently difficult to detect (Howe et al., 1999), as indicated by the poor specificity and reproducibility of the original method proposed by Hiramatsu et al. First of all, a reliable and simplified method should be established as soon as possible, because population analysis which can identify the heterogeneity of vancomycin susceptibility is too time-consuming. As well, a prospective study should be needed to ascertain the true significance of infections due to vancomycin-heteroresistant MRSA. It is unclear why in vivo development of decreased susceptibility to vancomycin did not occur in four MRSA isolates. We can speculate that in vivo development of vancomycin resistance may be depended on the different environmental conditions. There are certain common clinical features among previously reported patients with infections due to VISA isolates. First, repeated and prolonged vancomycin therapy is probably a major risk factor for decreasing vancomycin susceptibility. It appears that under prolonged vancomycin exposure, MRSA for which vancomycin MICs are increased could originate from the subpopulations of bacteria. Second, the presence of prosthetic materials may lead to an increase in the degree of vancomycin resistance of MRSA. Relatively poor access of antibacterial agents to staphylococci adhering to artificial surfaces leads to decreased susceptibility to antibiotic killing (Chuard et al., 1997; Khardori et al., 1995; Sieradzki et al., 1999b). In our study, the post-therapy isolates with decreased vancomycin susceptibility in PAPs were isolated
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from two patients (patients 1 and 4), who had received longer courses of vancomycin therapy than the other four patients. Patient 4 also had many devices with prosthetic materials placed such as a thoracheostomy tube, a percutaneous gastrostomy catheter, and a central venous catheter. In addition to these factors, therapeutic failures with vancomycin therapy versus MRSA may be attributed to such factors as a slower killing rate versus -lactam antibiotics, the dosage regimen used, the decreased penetration to infected tissue sites, or the underlying compromised health status of the patient (Houlihan et al., 1997; Sieradzki et al., 1998). The mechanisms of staphylococcal glycopeptide resistance have mainly been studied in vitro until now. Elucidation of in vivo mechanisms for glycopeptide resistance is needed in the light of environmental factors under clinical conditions. In conclusion, our study shows that in vivo, the development of decreased susceptibility to vancomycin in MRSA clinical isolates appears to be unstable, but a selective or inducible process that increased during vancomycin therapy. This phenomenon is likely to occur in MRSA clinical strains for which MICs are susceptible to vancomycin by standard MIC determination method. Whether MRSA strains with decreased vancomycin susceptibility do or do not represent a new plague of antibiotic resistance remains to be determined, but under no circumstances should they be ignored. To prevent further emergence of MRSA strains with decreased vancomycin susceptibility, the prudent use of vancomycin must be needed, and active surveillance for these strains should be implemented particularly in populations at high risk, such as patients in whom vancomycin therapy is unsuccessful. Acknowledgments We are grateful to Hidehiko Saito, the First Department of Internal Medicine, Nagoya University School of Medicine, for encouragement. We also thank Mie Tadokoro for expert technical assistance. References Aeschlimann, J. R., Hershberger, E., & Rybak, M. J. (1999). Analysis of vancomycin population susceptibility profiles, killing activity, and postantibiotic effect against vancomycin-intermediate Staphylococcus aureus. Antimicrob Agents Chemother 43, 1914 –1918. Ariza, J., Pujol, M., Cabo, J., Pena, C., Fernandez, N., Linares, J., Ayats, J., & Gudiol, F. (1999). Vancomycin in surgical infections due to methicillin-resistant Staphylococcus aureus with heterogeneous resistance to vancomycin. Lancet 353, 1587–1588. Aubert, G., Passot, S., Lucht, F., & Dorche, G. (1990). Selection of vancomycin- and teicoplanin-resistant Staphylococcus haemolyticus during teicoplanin treatment of Staphylococcus epidermidis infection. J Antimicrob Chemother 25, 491– 493. Boyle-Vavra, S., Berke, S. K., Lee, J. C., & Daum, R. S. (2000). Reversion of the Glycopeptide Resistance Phenotype in Staphylococcus aureus Clinical Isolates. Antimicrob Agents Chemother 44, 272–277.
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