Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan.

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64 µg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 µg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.

Linezolid-resistant Staphylococcus aureus isolated from 2006 through 2008 at six hospitals in Japan.

Limited use of linezolid for treating methicillin-resistant Staphylococcus aureus (MRSA) infection was approved in Japan in 2006. We report here the status of linezolid-resistant MRSAs in Japan. Eleven linezolid-resistant clinical isolates from 11 patients at six hospitals were collected from 2006 through 2008. The minimal inhibitory concentration (MIC) of linezolid in these strains varied from 8 to 64 µg/ml. All strains had at least one G2576T mutation in the chromosomal gene(s) encoding domain V of the 23S ribosomal RNA (rRNA). Chromosomal DNA encoding five copies of the domain V region was analyzed by polymerase chain reaction (PCR). Strains with the linezolid MICs of 64, 32, 16, and 8 µg/ml had the G2576T mutation(s) in four, three (or four), two, and one copy of the 23S rRNA genes, respectively. These results suggest that the level of linezolid resistance seems to be roughly correlated with the number of mutations in the genes encoding 23S rRNA. DNA samples from all 11 strains were subjected to pulsed-field gel electrophoresis and were classified into seven independent clones having >92% identity. Among the 11 patients, five had been treated with linezolid and the remainder, in two hospitals, had no history of prior linezolid use. The results suggested possible nosocomial infections by linezolid-resistant MRSA.

Identification of the active component that induces vancomycin resistance in MRSA.

A fraction of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin (VCM) in the presence of ß-lactam antibiotics (BIVR) at low concentrations. We hypothesized that the BIVR phenomenon might be exerted by a peptidoglycan derivative(s) generated as a consequence of ß-lactam antibiotic action. To verify this hypothesis, we isolated the fraction that mimicked the effect of ß-lactam antibiotics by the enzymatic treatment of the crude cell wall. The active components were purified by a combination of reverse phase chromatographies, mass spectrum and amino-acid analyses, and were identified to be a muropeptide with the following formula: N-acetyglucosamyl-N-acetylmuramyl--Ala-D-isoGln-L-Lys-(ɛ-NH-4Gly)-D-Ala-2Gly. This is the very first identification of the active component, which induces VCM resistance in MRSA. We found that the BIVR cells are highly sensitive to this compound rendering the cells resistant to VCM compared with non-BIVR MRSA.

Occurrence and molecular characteristics of methicillin-resistant Staphylococcus aureus and methicillin-resistant Staphylococcus pseudintermedius in an academic veterinary hospital.

Recently, methicillin-resistant Staphylococcus aureus (MRSA) and methicillin-resistant Staphylococcus pseudintermedius (MRSP) have been increasingly isolated from veterinarians and companion animals. With a view to preventing the spread of MRSA and MRSP, we evaluated the occurrence and molecular characteristics of each in a veterinary college. MRSA and MRSP were isolated from nasal samples from veterinarians, staff members, and veterinary students affiliated with a veterinary hospital. Using stepwise logistic regression, we identified two factors associated with MRSA carriage: (i) contact with an identified animal MRSA case (odds ratio [OR], 6.9; 95% confidence interval [95% CI], 2.2 to 21.6) and (ii) being an employee (OR, 6.2; 95% CI, 2.0 to 19.4). The majority of MRSA isolates obtained from individuals affiliated with the veterinary hospital and dog patients harbored spa type t002 and a type II staphylococcal cassette chromosome mec (SCCmec), similar to the hospital-acquired MRSA isolates in Japan. MRSA isolates harboring spa type t008 and a type IV SCCmec were obtained from one veterinarian on three different sampling occasions and also from dog patients. MRSA carriers can also be a source of MRSA infection in animals. The majority of MRSP isolates (85.2%) carried hybrid SCCmec type II-III, and almost all the remaining MRSP isolates (11.1%) carried SCCmec type V. MRSA and MRSP were also isolated from environmental samples collected from the veterinary hospital (5.1% and 6.4%, respectively). The application of certain disinfection procedures is important for the prevention of nosocomial infection, and MRSA and MRSP infection control strategies should be adopted in veterinary medical practice.

Linezolid-resistant methicillin-resistant Staphylococcus aureus isolated after long-term, repeated use of linezolid.

We report a 54-year-old male patient with an infection caused by linezolid-resistant methicillin-resistant Staphylococcus aureus (MRSA), isolated after long-term, repeated use of linezolid. Five MRSA strains isolated from our patient were preserved and submitted to bacteriological analysis. All five of these strains were found to have identical genotypes by pulsed-field gel electrophoresis. Two strains isolated in the early hospital period were sensitive to linezolid, while three isolated in the late hospital period were resistant. These three strains that had acquired resistance to linezolid were found to have a G2576T point mutation in the 23SrRNA domain V. Linezolid-resistant MRSA is rare, but may occur with the long-term, repeated administration of linezolid.

Characterization of beta-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR) in a patient with septicemia during long-term vancomycin administration.

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.

Rapid depletion of free vancomycin in medium in the presence of beta-lactam antibiotics and growth restoration in Staphylococcus aureus strains with beta-lactam-induced vancomycin resistance.

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.

Neutralization of staphylococcal exotoxins in vitro by human-origin intravenous immunoglobulin.

Human-origin intravenous immunoglobulin (IVIG) collected from healthy individuals was tested for its neutralizing activity against the hemolysin, toxic shock syndrome toxin-1 (TSST-1), and enterotoxins, produced by laboratory strains of methicillin-resistant Staphylococcus aureus (MRSA). The hemolytic activity of the culture supernatant against sheep red blood cells was reduced from 100% to 5.5% relative hemolysis in the presence of 0.156 mg protein/ml of IVIG. The maximum dilution endpoint of the culture supernatant for TSST-1-mediated latex aggregation was 32-fold. This level of TSST-1 activity was reduced to eightfold dilution by 0.78 mg protein/ml of IVIG, and the latex aggregation activity of undiluted TSST-1 in the culture supernatant was inhibited by 1.56 mg protein/ml of IVIG. Similarly, the enterotoxin A-mediated latex aggregation titer appeared to be a 320-fold dilution. This toxin activity was reduced to an 80-fold dilution and below a tenfold dilution by 0.049 through 0.78 and 1.56 mg protein/ml, respectively, of IVIG. These results show that IVIG has powerful neutralizing activity against hemolysin, TSST-1, and enterotoxin A. Therefore, IVIG may be useful for passive immunization therapy in patients suffering from diseases caused by MRSA exotoxins.

Occurrence of vancomycin-intermediate-resistant Staphylococcus aureus in Japan.

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.

Nosocomial infection of beta-lactam antibiotic-induced vancomycin-resistant Staphylococcus aureus (BIVR).

We report here an outbreak of beta-lactam-induced vancomycin-resistant methicillin-resistant Staphylococcus aureus (MRSA; BIVR) at one of the Cancer-Institute-affiliated hospitals in Tokyo. We examined a total of 500 strains (100 per year) of clinically isolated MRSA from 1998 to 2002. The detection rates of BIVR in the years 1998, 1999, 2000, 2001, and 2002 were 10%, 9%, 49%, 15%, and 19%, respectively. To investigate the cause of the high incidence of BIVR detection in the year 2000, we carried out pulsed-field gel electrophoresis (PFGE) of the SmaI-digested chromosomal DNA of BIVR and MRSA. The results showed that 96% of the BIVR strains isolated in 2000 were classified as an identical DNA type "A", while only 47% of the MRSA strains were classified as this type. We concluded, based on these results, that this hospital had a nosocomial infection of BIVR in the year 2000. An important message given by this study would be that nosocomial BIVR infection could occur in any hospital where MRSA infection is treated with vancomycin and beta-lactam antibiotics.

Investigation of beta-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR).

We could not detect hetero-vancomycin-intermediate resistant Staphylococcus aureus (hetero-VISA), according to the definition of hetero-VISA, from the clinical isolates of 140 methicillin-resistant S. aureus (MRSA) strains. However, 15 beta-lactam antibiotic-induced vancomycin-resistant MRSA (BIVR) strains were detected from the same strains. We screened 1882 MRSA clinical isolates obtained in 2002 from 21 institutes throughout Japan. The detection rate of blood-isolated BIVR was 12.6% (19/151), and that of nonblood-isolated BIVR was 4.9% (85/1731; P < 0.001; chi2 test). Uridine-diphosphate-N-acetylmuramyl-L: -alanyl-D: -isoglutamyl-L: -lysine, used as the peptidoglycan material of S. aureus, showed the same results as beta-lactam antibiotics in BIVR.

[Combination effect of pazufloxacin and anti-mrsa drugs against beta-lactam antibiotic induced vancomycin-resistant MRSA (BIVR)].

The in vitro combination effects of pazufloxacin (PZFX) with an anti-MRSA drug such as vancomycin (VCM), teicoplanin (TEIC), arbekacin (ABK), minocycline (MINO), rifampicin (RFP) and sulfamethoxazole-trimethoprim (ST) were investigated against 26 strains of beta-lactam antibiotic induced vancomycin-resistant MRSA (BIVR) by the checkerboard method. The additive and synergistic effects were observed with the combination of PZFX and VCM (50%, 13/26 strains), PZFX and TEIC (96%, 25/26 strains), PZFX and ABK (65%, 17/26 strains), PZFX and MINO (46%, 12/26 strains), PZFX and ST (54%, 14/26 strains). The synergistic effects were observed with the combination of PZFX and TEIC (4%, 1/26 strains), PZFX and ABK or MINO (15%, 4/26 strains). The antagonistic effects were observed with only PZFX and MINO (12%, 3/26 strains), others were all indifference.