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Vancomycin-intermediately resistant Staphylococcus aureus (VISA) and heterogeneous VISA (hVISA) are associated with treatment failure. hVISA contains only a subpopulation of cells with increased minimal inhibitory concentrations, and its detection is problematic because it is classified as vancomycin-susceptible by standard susceptibility testing and the gold-standard method for its detection is impractical in clinical microbiology laboratories. Recently, a research group developed a machine-learning classifier to distinguish VISA and hVISA from vancomycin-susceptible S. aureus (VSSA) according to matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) data. Nonetheless, the sensitivity of hVISA classification was found to be 76%, and the program was not completely automated with a graphical user interface. Here, we developed a more accurate machine-learning classifier for discrimination of hVISA from VSSA and VISA among MRSA isolates in Japanese hospitals by means of MALDI-TOF MS data. The classifier showed 99% sensitivity of hVISA classification. Furthermore, we clarified the procedures for preparing samples and obtaining MALDI-TOF MS data and developed all-in-one software, hVISA Classifier, with a graphical user interface that automates the classification and is easy for medical workers to use; it is publicly available at https://github.com/bioprojects/hVISAclassifier. This system is useful and practical for screening MRSA isolates for the hVISA phenotype in clinical microbiology laboratories and thus should improve treatment of MRSA infections.

We previously reported a novel phenotype of vancomycin-intermediate Staphylococcus aureus (VISA), i.e., "slow VISA," whose colonies appear only after 72 h of incubation. Slow-VISA strains can be difficult to detect because prolonged incubation is required and the phenotype is unstable. To develop a method for detection of slow-VISA isolates, we studied 23 slow-VISA isolates derived from the heterogeneous VISA (hVISA) clinical strain Mu3. We identified single nucleotide polymorphisms (SNPs) in genes involved in various pathways which have been implicated in the stringent response, such as purine/pyrimidine synthesis, cell metabolism, and cell wall peptidoglycan synthesis. We found that mupirocin, which also induces the stringent response, caused stable expression of vancomycin resistance. On the basis of these results, we developed a method for detection of slow-VISA strains by use of 0.032 μg/ml mupirocin (Yuki Katayama, 7 March 2017, patent application PCT/JP2017/008975). Using this method, we detected 53 (15.6%) slow-VISA isolates among clinical methicillin-resistant S. aureus (MRSA) isolates. In contrast, the VISA phenotype was detected in fewer than 1% of isolates. Deep-sequencing analysis showed that slow-VISA clones are present in small numbers among hVISA isolates and proliferate in the presence of vancomycin. This slow-VISA subpopulation may account in part for the recurrence and persistence of MRSA infection., (Copyright © 2017 Katayama et al.)

It was reported that some methicillin-resistant Staphylococcus aureus (MRSA) show resistance to vancomycin (VCM) and beta-lactam antibiotics; thus, they are termed beta-lactam antibiotic-induced VCM-resistant MRSA (BIVR). The VCM resistance of MRSA is induced by the administration of beta-lactam antibiotics, but this phenomenon can be difficult to detect in the clinical laboratory. We detected the BIVR strain in a 64-year-old man who had had a ventilator tube inserted directly into the windpipe during long-term VCM therapy. The patient was diagnosed with MRSA pneumonia and septicemia on July 5, 2007, and sulbactam/ampicillin (SBT/ABPC) was administered for 5 days. However, the fever recurred, and administration of VCM was resumed for 7 days from July 19. Fever developed again, and VCM was administered again for 14 days from September 30. BIVR and VCM-low-sensitive MRSA were isolated from blood on October 18 and 22, although the VCM trough concentration was 10.2 microg/ml. On October 27, we changed to a combination of fosfomycin (FOM) and arbekacin (ABK), and thereafter the fever quickly decreased and the clinical symptoms abated. We isolated five MRSA strains from the blood of the patient, three strains of VCM-sensitive MRSA, one strain of BIVR, and one strain of a VCM-low-sensitive MRSA. The DNA band patterns determined by pulsed-field gel electrophoresis were completely identical except for the VCM-low-sensitive MRSA, which was missing one band. Furthermore, the VCM-low-sensitive MRSA became sensitive to beta-lactam antibiotics. Our results indicate the possibility that long-term VCM therapy is one of the factors that allow BIVR or VCM-low-sensitive MRSA to emerge, and this allows VCM therapy for MRSA to fail.

A class of methicillin-resistant Staphylococcus aureus strains shows vancomycin resistance in the presence of beta-lactam antibiotics (beta-lactam-induced VAN-resistant methicillin-resistant S. aureus [BIVR]). Two possible explanations may be offered: (i) vancomycin in culture medium is depleted, and (ii) the D-Ala-D-Ala terminal of the peptidoglycan network is replaced with D-Ala-D-lactate. We tested these hypotheses by quantifying free vancomycin in the medium through the course of cell growth and by PCR amplification of the van genes. Growth of the BIVR cells to an absorption level of approximately 0.3 at 578 nm required about 24 h in the presence of vancomycin alone at the MIC (4.0 microg/ml). However, growth was achieved in only about 10 h when 1/1,000 to 1/2,000 the MIC of beta-lactam antibiotic was added 2 h prior to the addition of vancomycin, suggesting that the beta-lactams shortened the time to recovery from vancomycin-mediated growth inhibition. Free vancomycin in the culture medium decreased to 2.3 microg/ml in the first 8 h in the culture containing vancomycin alone, yet cell growth was undetectable. When the vancomycin concentration dropped below approximately 1.5 microg/ml at 24 h, the cells began to grow. In the culture supplemented with the beta-lactam 2 h prior to the addition of vancomycin, the drug concentration continuously dropped from 4 to 0.5 microg/ml in the first 8 h, and the cells began to grow at a vancomycin concentration of approximately 1.7 microg/ml or at 4 h of incubation. The gene encoding the enzyme involved in D-Ala-D-lactate synthesis was undetectable. Based on these results, we concluded that BIVR is attributable mainly to a rapid depletion of vancomycin in the medium triggered or promoted by beta-lactam antibiotics.

Some methicillin-resistant Staphylococcus aureus (MRSA) strains in which combinations of vancomycin (VCM) and beta-lactam antibiotics show antagonism have recently emerged, and these strains are called beta-lactam-induced VCM-resistant MRSA (BIVR). We examined whether various antibiotics exhibited an antagonistic effect with VCM when used against Mu3 and Fu10 (representative BIVR strains), using a simple agar disc method. Chloramphenicol, tetracyclines, macrolides, and lincosamides showed an antagonistic effect with VCM. We attempted to elucidate the antagonistic mechanism of a combination of VCM and minocycline (MINO) in BIVR strains. We determined the rates of autolysis, autolytic activities, and the change in morphology of Mu3 treated with a combination of VCM and MINO. We observed that Mu3 grown in a combination of VCM and MINO showed increasing rates of autolysis, and lower minimal bacteriolytic enzyme dose (MBD) values compared with Mu3 grown in VCM alone, but no cell wall thickening was observed. Taken together, these results suggest that cell wall thickening may not be essential in the increased resistance of BIVR strains. Our present data therefore suggest that these combination therapies of VCM with tetracyclines should be adopted with great care in order to prevent VCM treatment failure.

The Clinical and Laboratory Standards Institute (CLSI) amended the criteria for vancomycin susceptibility and resistance of Staphylococcus aureus in 2006. The earlier criteria had established that S. aureus with minimum inhibitory concentrations (MICs) of vancomycin of < or =4 microg/ml, 8 to 16 microg/ml, and > or =32 microg/ml were vancomycin-susceptible, -intermediate-resistant and -resistant, respectively. The revised recommendation states that bacteria showing vancomycin MICs of < or =2 microg/ml, 4 to 8 microg/ml, and > or =16 microg/ml are -susceptible, -intermediate-resistant, and -resistant, respectively. We examined, based on these new criteria, the vancomycin susceptibility of methicillin-resistant S. aureus (MRSA) strains isolated in Japan from 1978 through 2005 at 17 general hospitals. The results showed that, among 2446 MRSA isolates tested, 8 were classified as intermediate-vancomycin-resistant (VISA). Re-examination of vancomycin susceptibility in these 8 strains in 2006 revealed that 6 strains showed a vancomycin MIC of 4 microg/ml, as tested by the agar dilution method, broth dilution methods, and E-test; the 2 other strains had lost the vancomycin resistance. Pulsed-field gel electrophoresis (PFGE) of the chromosomal DNA of these strains exhibited five unique profiles; 2 strains isolated from the same hospital were identical. These results revealed that at least five different types of VISA strains could be identified in Japan so far according to the new CLSI criteria. All these VISA strains had type II staphylococcal cassette chromosome, mec. This study revealed, for the first time in Japan, the presence of intermediate vancomycin-resistant MRSA in this country.

The concomitant use of low concentration beta-lactam antibiotics antagonizes the activity of vancomycin against some strains of MRSA. These strains, called beta-lactam antibiotic-induced vancomycin resistant MRSA (BIVR), have been increasing for a number of years. We previously reported that the combination of VCM and ceftizoxime displayed this antagonism not only in vitro, but also in vivo, in a systemic infection caused by BIVR in mice. In the present study, we validated the antagonism in combinations of VCM with other beta-lactams, i.e., flomoxef (FMOX), ampicillin (AMPC), azthreonam (AZT) and imipenem/cilastatin (IPM/CS), in systemic infections and pneumoniain in mice. The survival rate of the mice with systemic infections caused by BIVR treated with combinations of FMOX, AMPC, AZT, and IPM/CS with VCM were significantly lower than with VCM monotherapy, and the number of residual viable cells in the kidneys of mice treated with combinations of FMOX and IPM/CS with VCM were significantly higher than with VCM monotherapy The number of residual viable cells in the lungs of mice with pneumonia caused by BIVR treated with the combination of IPM/CS and VCM was significantly higher than with VCM monotherapy. On the other hand, the survival rate with combination therapy consistings IPM/CS plus teicoplanin (T EIC) was significantly higher, and the number of residual viable cells in the kidney was significantly lower, than with TEIC monotherapy alone. In the mice with pneumonia, the number of residual viable cells in the lung after combination therapy with IPM/CS and TEIC was significantly lower than with TEIC monotherapy. Combination therapy with beta-lactams plus VCM showed antagonistic in models of systemic infection and pulmonary infection caused by BIVR, whereas combination therapy consisting of a beta-lactam plus TEIC had a synergistic effect in the same models, even though VCM and TEIC are member of the same glycopeptide antibiotic class.

Recently, beta-lactam antibiotic induced vancomycin-resistant MRSA (BIVR) has been reported increasingly in Japan. Between 1998 and 2002, we tried to detect BIVR from 500 strains of MRSA in a cancer hospital. And the difference of the detection rate under condition of pre-culture with or without ceftizoxime was compared. The detection rate of BIVR under condition of pre-culture with 1.0 mg/L of ceftizoxime was 20.4% (102/500), and without ceftizoxime was 9% (45/500). That of preculture with 1.0 mg/L of ceftizoxime was higher than those without ceftizoxime with the significant difference. (p < 0.001; McNemar-t examination). In comparing each department, the detection rate of BIVR from Chemotherapy, Head & Neck, and Urology department was 33.3%, 27.0%, and 20.0%, respectively. These results mean that addition of beta-lactam as ceftizoxime in pre-culture induces the ability of resistance to vancomycin for MRSA having a capacity as BIVR.

Despite the fact that the combination of vancomycin and a beta-lactam antibiotic are known to act synergistically on vancomycin-susceptible Staphylococcus aureus (VSSA), some MRSA have emerged showing antagonism to the combination of vancomycin and a beta-lactam antibiotic. These MRSA are called beta-lactam antibiotic-induced vancomycin resistant MRSA (BIVR). A method based on this antagonistic phenomenon has been devised to detect BIVR strains. The method inhibits the VSSA strain but allows the BIVR strain to grow. Forty-six commercially available beta-lactam antibiotics induced the vancomycin-resistance. Using this detection method, 717 MRSA clinical isolates obtained from eight institutes throughout Japan were thus screened and 6.3% of these were detected as BIVR when judged at 48 h.

The combination of vancomycin and beta-lactam antibiotic synergistically is known to act on vancomycin-susceptible Staphylococcus aureus (VSSA). But some MRSA with the antagonism in the combination of vancomycin and beta-lactam antibiotic was identified in Japan. We called the MRSA "beta-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR)", to distinguish it from hetero-VISA. The percentage of hetero-VISA isolated in Japan that Hiramatsu et al. reported in The Lancet in 1997 was that of "candidate-hetero-VISA" because it did not satisfy the criteria for detection of hetero-VISA that they proposed. Therefore, except for Mu3, a strictly defined hetero-VISA strain has never been detected in Japan. However, BIVR is certainly detectable in Japan. We performed a retrospective study of BIVR in 189 MRSA strains isolated from blood between 1978 and 1999 at the same institution. To performed a retrospective study, 189 MRSA strains were divided such as 1978-1984 (45strains), 1985-1989 (45strains), 1990-1994 (49strains), 1995-1999 (50strains). MIC90 of anti-MRSA drugs according to above chronological transition were 2, 2, 2, 1 as vancomycin, 2, 1, 1, 1 as teicoplanin, and 8, 8, 1, 1 microgram/mL as arbekacin, respectively, and then the detection rate of BIVR was 2.2, 2.2, 6.1, 10.4%, respectively. The BIVR detection rate in MRSA isolated from blood at 14 institutions was 14.8% (12/81) in 1999-2002, and that of non-blood was 4.6% (42/905) (p < 0.001; chi 2-t examination). Of particular importance is that the percentage of BIVR isolated from blood is higher than that from non-blood, and the detection rate of BIVR from blood increases annually.

Mu3 strain with heterogeneous intermediated-resistance to vancomycin (hetero-VISA) reported in 1997, also have possessed a character of an antagonistic effect of beta-lactam antibiotics and vancomycin. Mu3 is only strain which satisfies the definition of hetero-VISA in Japan. But, MRSA with antagonistic effects of beta-lactam antibiotics and vancomycin, was reported by many institutions. To separate hetero-VISA, we called "beta-lactam antibiotic induced vancomycin-resistant MRSA (BIVR)". But the detection rate of clinical isolated BIVR in Japan is unknown, we reported on the detection method and the epidemiological investigation for BIVR. Mu 3 agar containing 4 micrograms/mL of vancomycin is used to detect BIVR. Mu3 strains were spread on the agar, BIVR can grow around the paper disc impregnated with ceftizoxime or grow on the whole surface on Mu 3 agar after incubation. The detection rate of BIVR was 45 in 717 (6.3%) clinical isolated strains. In detected strains as BIVR, the number of strains with grown on the whole surface of Mu3 agar showing a high resistance to vancomycin were 10 strains (1.4%). Besides, from 106 strains of blood isolates MRSA, BIVR were detected 16 strains (15.1%), from 611 strains of non-blood isolates MRSA, BIVR were detected 29 strains (4.7%) (P < 0.0001). In BIVR strains grown on the whole surface of Mu3 agar, the number of BIVR strains isolated from blood were 8 in 106 (7.5%), from non-blood were 2 in 611 (0.3%) (P < 0.0001). On one side, hetero-VISA were not detected from all of BIVR growing on the whole surface of Mu3 agar. As a result, detection method and the definition of BIVR were quite different from those of hetero-VISA. An existence of BIVR in Japan was confirmed, we thought that the high detection rate of BIVR isolated from blood compared with that of non-blood showed the pathogenecity of BIVR which contribute to MRSA infections.

Objectives: Staphylococcus aureus with low-level resistance to vancomycin (VLSA) which could develop into vancomycin-resistant S. aureus (VRSA) is most important. However, VLSA is difficult to detect by standard laboratory methods. We describe here improved methods to detect VLSA., Methods: Three methicillin-resistant S. aureus (MRSA) strains, designated Fu6, Fu10, and Fu18, were sequentially isolated from the burn wound site of a patient, during vancomycin therapy. The properties of these strains were compared with those of reference strains Mu3 and Mu50 (previous resistant isolates from other patients)., Results: The isolated strains, Fu10 and Fu18, had identical phenotypes and genotypes. The vancomycin resistance of Fu10 was equivalent to that of strain Mu3, whereas Fu18 had much higher vancomycin resistance than Fu10 and Mu3, although reaching the level of Mu50. Fu18 showed similar growth to Mu50 on gradient gels and on Mu3 medium., Conclusions: Our data indicate that the VLSA developed vancomycin resistance during exposure to vancomycin in vivo. The population analysis of tested VLSA and vancomycin intermediately resistant S. aureus (VISA) indicates that a penem at relatively low concentrations induced a significant increase in the number of vancomycin-resistant subpopulations. Furthermore, we confirmed that gradient gel analysis and Mu3 medium are simple and useful methods for the detection of VLSA judged as VSSA by its conventional MIC alone.

We tested the combined activity of vancomycin and seven beta-lactam antibiotics against Staphylococcus aureus clinical strain Mu3, which displays heterogeneous resistance to vancomycin. When combined with vancomycin, four of the seven tested beta-lactams exhibited an additive effect at or near their MICs, while all showed an antagonistic effect at lower, sub-MIC levels. This study implicated the unpredictable nature of combination therapy of beta-lactams and vancomycin against S. aureus with reduced susceptibility to vancomycin.

Two hundred and thirteen clinical strains of coagulase-negative staphylococci isolated in Japan between 1980 and 1997 were analyzed for glycopeptide susceptibility by determining MIC using both Mueller-Hinton agar (MHA) and Brain Heart Infusion agar (BHIA) plates. Of 37 Staphylococcus epidermidis strains isolated between 1980 and 1981, all were susceptible to vancomycin and teicoplanin on both MHA and BHIA. However, of 122 isolates of Staphylococcus epidermidis isolated between 1994 and 1997, 1 (0.8%) was intermediate to vancomycin on MHA and 39 (32%) were intermediate on BHIA, while 3 (2.5%) and 27 (22.1%) were intermediate or resistant to teicoplanin on MHA and BHIA, respectively. It was demonstrated that the susceptibilities of the strains in 1990s to vancomycin and teicoplanin were significantly decreased compared with those in 1980s. Population analysis was performed with six strains each of Staphylococcus epidermidis and Staphylococcus haemolyticus (three with vancomycin MIC > or = 8 micrograms/ml and three with vancomycin MIC < or = 4 micrograms/ml using BHIA). The population curves of the Staphylococcus epidermidis strains showed a homogeneous pattern of susceptibility. Whereas, those for two Staphylococcus haemolyticus strains (vancomycin MIC = 8 micrograms/ml using BHIA) showed a typical heterogeneous pattern. Vancomycin-resistant mutants (MIC > or = 32 micrograms/ml) were obtained with a high frequency of 10(-4)-(-5) from the strains by one-step selection with 16 micrograms/ml of vancomycin.

The peptidoglycan compositions of vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (MIC 8 mg/L) and hetero-VRSA strain Mu3 (MIC 3 mg/L) were compared in order to understand the mechanism of vancomycin resistance. As compared with Mu3, the cell wall of Mu50 had increased amounts of glutamine-non-amidated muropeptides and decreased cross-linking of peptidoglycan with a greatly decreased dimer/monomer ratio of muropeptides. In agreement with this observation, the peptidoglycan of Mu50 bound 1.4 times more vancomycin than that of Mu3. The increase in non-amidated muropeptides and the reduced cross-linking of the cell-wall peptidoglycan may contribute to the vancomycin resistance by increasing the consumption of vancomycin by the pre-existing cell wall of Mu50 and reducing the amount of vancomycin reaching the cytoplasmic membrane where the vital targets of the antibiotic are situated.

We have developed a new method of detecting clinical S. aureus strains possessing heterogeneous resistance to vancomycin (hetero-VRSA). The method exploits characteristic antagonism between vancomycin and beta-lactam antibiotics against the strain Mu3, a representative hetero-VRSA. The method comprises of the following procedures: 1) overnight culture of bacteria is streaked on brain heart infusion agar containing 4 micrograms/ml of vancomycin, 2) paper discs impregnated with any one of beta-lactam antibiotics are placed onto the plate, and 3) the plate is incubated at 37 degrees C and the cell growth was observed after 24 h and 48 h of incubation. The Mu3 cells were grown only around the beta-lactam discs due to the antagonism between vancomycin and beta-lactam against Mu3 cells. A total of 321 MRSA clinical strains isolated in 1995 and 1996 from 12 medical facilities in Japan were tested with this method. A total of 39 strains (12.1%; 24 h) and 67 strains (20.96%; 48 h) grew around the beta-lactam discs. All the 10 strains (representing 10 facilities), tested with the analysis of resistant subpopulations to vancomycin, showed similar heterogeneous patterns as observed with Mu3. The method was considered as a convenient and rapid substitute for the population analysis, the authentic method for the detection of hetero-VRSA strains.

We have previously reported methicillin-resistant Staphylococcus aureus clinical strains, Mu50 and Mu3, representing two categories of vancomycin resistance: Mu50 representing vancomycin-resistant S. aureus (VRSA) with MICs > or = 8 mg/L, and Mu3 representing hetero-VRSA with MICs < or = 4 mg/L using standard MIC determination methods. The mechanisms of vancomycin resistance in these strains were investigated. These strains did not carry the enterococcal vancomycin-resistance genes, vanA, vanB, or vanC1-3, as tested by PCR using specific primers. However, both strains produced three to five times the amount of penicillin-binding proteins (PBPs) 2 and 2' when compared with vancomycin-susceptible S. aureus control strains with or without methicillin resistance; the amounts of PBP2 produced in Mu3 and Mu50 were comparable to those in the vancomycin-resistant S. aureus mutant strains selected in vitro. Incorporation of 14C-labelled Nacetyl-glucosamine into the cell was three to 20 times increased in Mu50 and Mu3, and release of the radioactive cell wall material was increased in Mu3 (and also in Mu50, though to a lesser extent), compared with control strains. The amounts of intracellular murein monomer precursor in these strains were three to eight times greater than those found in control strains. Transmission electron microscopy showed a doubling in the cell wall thickness in Mu50 compared with the control strains. Mu3 did not show obvious cell wall thickening. These data indicate that activated synthesis and an increased rate of cell wall turnover are common features of Mu3 and Mu50 and may be the prerequisite for the expression of vancomycin resistance in S. aureus.

The mechanism of resistance was studied with vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50. It was demonstrated that the incorporation of 14C-N-acetylglucosamine into the cell wall of Mu50 was not suppressed in the presence of 8 microliters/ml of vacomycin, whereas it was completely suppressed in vancomycin-susceptible strains FDA209P and H-1. Increased binding of vancomycin to the wall of Mu50 was observed compared to the control strains: 1.7 x 10(16) (Mu50), 6.1 x 10(15) (209P), and 6.7 x 10(15) (H-1) vancomycin molecules/mg cell wall, respectively. Remarkable proportion of the cell-wall component muropeptides were non-amidated in the cell wall of Mu50. In concordance with this phenomena, peptidoglycan cross-linkage decreased strikingly in the Mu50 strain. Free D-Ala-D-Ala residues at the end of muropeptides in the pre-existing cell wall generated by decreased cross-linkage seems to account for increased vancomycin binding. The increase of vancomycin-resistance level is presumably caused by sequestration of vancomycin molecules from primary target point on cell membrane. It was considered that at least two phenotypic changes are required for the vancomycin resistance in the Mu50 strain. First, as we have described previously, is the activated cell wall synthesis, and second, the reduction of cross-linkage of peptidoglycan by production of non-amidated muropeptide precursors.

The mechanism of vancomycin resistance in methicillin-resistant Staphylococcus aureus (MRSA) Mu50 was investigated. More than 3 times increase of the incorporation of 14C-GluNAc into the cell wall of Mu50 was observed compared to those of vancomycin-susceptible strains FDA209P, H-1, LR5P1. The amount of cytoplasmic murein-monomer precursor increased more than 3 times in Mu50 compared to those of control strains. There was an increased production of PBP1, PBP2, and PBP2', which were 1.51, 17.2, and 7.06 times greater, respectively, in Mu50 than those in H-1, and 2.38, 4.46, and 1.96 times greater respectively, than those in LR5P1. By transmission electromicrograph, it was shown that the cell wall of Mu50 was twice thicker than that of LR5P1. Increase of tightly-bound vancomycin to the cell wall fraction was observed in Mu50 when compared to those in FDA209P and H-1 strains. From these results, the increase of the vancomycin targets, free D-Ala-D-Ala residues in the cell wall, in number, due to the activated cell wall synthesis, and/or decrease of the cross-linkage of the cell wall was suggested to be the mechanism of vancomycin resistance in the Mu50 strain.

Background: Since the discovery of the vancomycin-resistant Staphylococcus aureus (VRSA) strain Mu50 (minimum inhibitory concentration [MIC] 8 mg/L), there has been concern about the potential spread of such strains throughout Japanese hospitals. Two important questions need to be answered: (1) what is the prevalence of VRSA, and (2) by what mechanism does vancomycin resistance occur., Methods: The vancomycin susceptibilities of three methicillin-resistant S aureus (MRSA) strains (Mu50, Mu3, and H1) and the methicillin-susceptible S aureus type strain FDA209P were compared by MIC determinations and population analysis. Mu3 (MIC 3 mg/L) was isolated from the sputum of a patient with pneumonia after surgery who had failed vancomycin therapy. H1 (MIC 2 mg/L), which is a representative vancomycin-susceptible MRSA strain, was isolated from a patient with pneumonia who responded favourably to vancomycin therapy. Subclones of Mu3 with increased resistance against vancomycin were selected with serial concentrations of vancomycin and their MICs were determined. The prevalence of VRSA and Mu3-like strains in Japanese hospitals was estimated by population analysis from 1149 clinical MRSA isolates obtained from 203 hospitals throughout Japan. The genetic traits of the Mu3 and Mu50 strains were compared with clonotypes of MRSA from around the world., Findings: Mu3 and Mu50 had an identical pulsed-field gel electrophoresis banding pattern. When grown in a drug-free medium, Mu3 produced subpopulation of cells with varying degrees of vancomycin resistance, thus demonstrating natural heterogeneity, or variability, in susceptibility to vancomycin. In the presence of vancomycin, Mu3 produced subclones with resistance roughly proportional to the concentrations of vancomycin used. Selection of Mu3 with 8 mg/L or more of vancomycin gave rise to subclones with vancomycin resistance equal to that of Mu50 (MIC 8 mg/L) at a frequency of 1/1,000,000. During screening of Japanese MRSA strains, no strain of VRSA additional to Mu50 was found. The prevalence of MRSA isolates heterogeneously resistant to vancomycin was 20% in Juntendo University Hospital, 9.3% in the other seven university hospitals, and 1.3% in non-university hospitals or clinics., Interpretation: Heterogeneously resistant VRSA is a preliminary stage that allows development into VRSA upon exposure to vancomycin. Heterogeneously resistant VRSA was found in hospitals throughout Japan. This finding could explain, at least partly, the frequent therapeutic failure of MRSA infection with vancomycin in Japan.

We evaluated a clinical strain of methicillin-resistant Staphylococcus aureus (MRSA), Mu50, for vancomycin susceptibility. Mu50 was isolated from a patient with infection of a surgical incision site which resisted vancomycin therapy. Mu50 showed a decrease in susceptibility to vancomycin, but this strain did not carry vanA or vanB or vanC genes as judged from PCR amplification. MICs of vancomycin against Mu50 and vancomycin-susceptible S. aureus FDA209P, S. aureus ATCC-29213, and MRSA H-1 were 8, 1, 1, and 1 microgram/ml, respectively by agar dilution and macro-broth dilution methods according to NCCLS. MIC values with agar dilution method using MHA + 20% horse serum, HIA, and BHIA agreed with the MIC values with micro- and macro-broth dilution method. Population analysis revealed that vancomycin concentration required for inhibition of ca. 10(7) cells of Mu50, S. aureus FDA209P, S. aureus ATCC29213, and MRSA H-1 were 36, 2, 2, and 2 micrograms/ml, respectively. These results showed that the activity of vancomycin against Mu50 was at least 8-fold decreased compared to that against S. aureus FDA209P, S. aureus ATCC29213, and MRSA H-1.