Low-intensity ultrasound inhibits melanoma cell proliferation in vitro and tumor growth in vivo.

PURPOSE: To determine the effect of low-intensity ultrasound on cancer cell proliferation in vitro and tumor growth in vivo. METHODS: In vitro, several cancer cell lines were exposed to low-intensity ultrasound at 0.11 W/cm2 for 2 min. Of the cell lines screened, melanoma C32 is one of the cell lines that showed sensitivity to growth inhibition by ultrasound and was therefore used in succeeding experiments. In vivo, under the same ultrasound conditions used in vitro, C32 tumors in mice were exposed to ultrasound daily for 2 weeks, and the tumor volumes were monitored weekly using sonography. RESULTS: In vitro, C32 cell growth was inhibited, attaining 43.2% inhibition on the 3rd day. In vivo, tumor growth was significantly inhibited, with the treated tumors exhibiting 2.7-fold slowed tumor growth vs. untreated tumors at week 2. Such inhibition was not associated with increased cell death. Several genes related to the cell cycle and proliferation were among those significantly regulated. CONCLUSION: These findings highlight the potential of low-intensity ultrasound to inhibit tumor growth in a noninvasive, safe, and easy-to-administer way. In addition, this may suggest that the mechanical stress induced by ultrasound on C32 cells may have affected the intrinsic biomolecular mechanism related to the cell growth of this particular cell line. Further research is needed to identify which of the regulated genes played key roles in growth inhibition.

Macrolides Inhibit Capsule Formation of Highly Virulent Cryptococcus gattii and Promote Innate Immune Susceptibility.

Cryptococcus gattii is a fungal pathogen, endemic in tropical and subtropical regions, the west coast of Canada, and the United States, that causes a potentially fatal infection in otherwise healthy individuals. Because the cryptococcal polysaccharide capsule is a leading virulence factor due to its resistance against innate immunity, the inhibition of capsule formation may be a promising new therapeutic strategy for C. gattii Macrolides have numerous nonantibiotic effects, including immunomodulation of mammalian cells and suppression of bacterial (but not fungal) pathogenicity. Thus, we hypothesized that a macrolide would inhibit cryptococcal capsule formation and improve the host immune response. Coincubation with clarithromycin (CAM) and azithromycin significantly reduced the capsule thickness and the amount of capsular polysaccharide of both C. gattii and C. neoformans CAM-treated C. gattii cells were significantly more susceptible to H2O2 oxidative stress and opsonophagocytic killing by murine neutrophils. In addition, more C. gattii cells were phagocytosed by murine macrophages, resulting in increased production of tumor necrosis factor alpha (TNF-α) by CAM exposure. After CAM exposure, dephosphorylation of Hog1, one of the mitogen-activated protein kinase (MAPK) signaling pathways of Cryptococcus, was observed in Western blot analysis. In addition, CAM exposure significantly reduced the mRNA expression of LAC1 and LAC2 (such mRNA expression is associated with cell wall integrity and melanin production). These results suggest that CAM may aid in inhibiting capsular formation via the MAPK signaling pathway and by suppressing virulent genes; thus, it may be a useful adjunctive agent for treatment of refractory C. gattii infection.

Intraspecies variation in the efficacy of adjunctive recombinant interferon-γ therapy against cryptococcal meningoencephalitis in mice.

The efficacy of recombinant interferon γ (rIFN-γ) for cryptococcal meningoencephalitis has been poorly understood. Compared to Cryptococcus gattii, rIFN-γ significantly improved the survival in experimental meningoencephalitis due to Cryptococcus neoformans. The number of phagocytic macrophages and the levels of inflammatory cytokines production for ex vivo co-incubation with C. neoformans were increased after rIFN-γ stimulation but not C. gattii. Intraspecies differences of phagocytosis by the rIFN-γ-activated macrophages might be associated to the severity of cryptococcal infection.

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.

Interferon-γ promotes phagocytosis of Cryptococcus neoformans but not Cryptococcus gattii by murine macrophages.

Among invasive fungal infections, cryptococcosis caused by inhalation of Cryptococcus neoformans or Cryptococcus gattii is particularly dangerous because it can disseminate to the central nervous system and cause life-threatening meningitis or meningoencephalitis. Previous reports described significant differences in the histopathological features of C. neoformans and C. gattii infection, such as greater pathogen proliferation and a limited macrophage response in mouse lung infected by C. gattii. To elucidate the difference in pathogenicity of these two Cryptococcus species, we investigated the interaction of C. neoformans and C. gattii with murine macrophages, the first line of host defense, by confocal laser microscopy. Only thin-capsulated, and not thick-capsulated C. neoformans and C. gattii were phagocytosed by macrophages. Preactivation with interferon-γ increased the phagocytic rate of thin-capsulated C. neoformans up to two-fold, but did not promote phagocytosis of thin-capsulated C. gattii. Lipopolysaccharide preactivation or Aspergillus fumigatus conidia co-incubation had no effect on internalization of thin-capsulated C. neoformans or C. gattii by macrophages. Phagocytosis of live thin-capsulated C. neoformans, but not that of live thin-capsulated C. gattii, induced interleukin-12 release from macrophages. However, phagocytosis of heat-killed or paraformaldehyde-fixed thin-capsulated C. neoformans did not increase IL-12 release, showing that the internalization of live yeast is important for initiating the immune response during C. neoformans-macrophage interactions. Our data suggest that macrophage response to C. gattii is limited compared with that to C. neoformans and that these results may partially explain the limited immune response and the greater pathogenicity of C. gattii.

Antibiotic susceptibility survey of blood-borne MRSA isolates in Japan from 2008 through 2011.

We conducted an antibiotic susceptibility survey of 830 blood-borne methicillin resistant Staphylococcus aureus collected from nationwide hospitals in Japan over a three-year period from January 2008 through May 2011. Antibiotic susceptibility was judged according to the criteria recommended by the Clinical Laboratory Standard Institute. Over 99% of the MRSA showed to be susceptible to teicoplanin, linezolid, sulfamethoxazole/trimethoprim and vancomycin, and over 97% of them were susceptible to daptomycin, arbekacin and rifampin. The majority of the MRSA strains showed resistant to minocycline, meropenem, imipenem, clindamycin, ciprofloxacin, cefoxitin, and oxacillin in the rates of 56.6, 72.9, 73.7, 78.7, 89.0, 99.5, and 99.9%, respectively. Among the MRSA strains, 72 showed reduced susceptibility to vancomycin, including 8 strains (0.96%) of vancomycin-intermediate S. aureus (VISA), 54 (6.51%) of heterogeneous vancomycin-intermediate S. aureus (hVISA), and 55 (5.63%) of ß-lactam antibiotics-induced vancomycin resistant S. aureus (BIVR). Unexpectedly, among the 54 hVISA and 55 BIVR, 45 isolates (83.3% and 81.8%, respectively) showed both hVISA and BIVR phenotypes. A new trend of vancomycin resistance found in this study was that VISA strains were still prevalent among the bacteremic specimens. The high rates of the hVISA/BIVR two-phenotypic vancomycin resistance, and the prevalence of VISA in the bloodborne MRSA call attention in the MRSA epidemiology in Japan.

Low level ß-lactamase production in methicillin-resistant staphylococcus aureus strains with ß-lactam antibiotics-induced vancomycin resistance.

BACKGROUND: A class of methicillin-resistant Staphylococcus aureus (MRSA) shows resistance to vancomycin only in the presence of ß-lactam antibiotics (BIVR). This type of vancomycin resistance is mainly attributable to the rapid depletion of free vancomycin in the presence of ß-lactam antibiotics. This means that ß-lactam antibiotics remain active or intact in BIVR culture, although most MRSA cells are assumed to produce ß-lactamase. We hypothesised that the BIVR cells either did not harbour the ß-lactamase gene, blaZ, or the gene was quiescent. We tested this hypothesis by determining ß-lactamase activity and conducting PCR amplification of blaZ. RESULTS: Five randomly selected laboratory stock BIVR strains showed an undetectable level of ß-lactamase activity and were blaZ-negative. Five non-BIVR stock strains showed an average ß-lactamase activity of 2.59 ± 0.35 U. To test freshly isolated MRSA, 353 clinical isolates were collected from 11 regionally distant hospitals. Among 25 BIVR strains, only 16% and 8% were blaZ positive and ß-lactamase-positive, respectively. In contrast, 95% and 61% of 328 non-BIVR strains had the blaZ gene and produced active ß-lactamase, respectively. To know the mechanism of low ß-lactamase activity in the BIVR cells, they were transformed with the plasmid carrying the blaZ gene. The transformants still showed a low level of ß-lactamase activity that was several orders of magnitude lower than that of blaZ-positive non-BIVR cells. Presence of the ß-lactamase gene in the transformants was tested by PCR amplification of blaZ using 11 pairs of primers covering the entire blaZ sequence. Yield of the PCR products was consistently low compared with that using blaZ-positive non-BIVR cells. Nucleotide sequencing of blaZ in one of the BIVR transformants revealed 10 amino acid substitutions. Thus, it is likely that the ß-lactamase gene was modified in the BIVR cells to downregulate active ß-lactamase production. CONCLUSIONS: We concluded that BIVR cells gain vancomycin resistance by the elimination or inactivation of ß-lactamase production, thereby preserving ß-lactam antibiotics in milieu, stimulating peptidoglycan metabolism, and depleting free vancomycin to a level below the minimum inhibitory concentration of vancomycin.

Limited detectability of linezolid-resistant Staphylococcus aureus by the Etest method and its improvement using enriched media.

The aim of this study was to evaluate Etest for detectability of linezolid-resistant meticillin-resistant Staphylococcus aureus (MRSA). The MIC of linezolid obtained by the Etest method in 18 linezolid-resistant strains of MRSA was compared with that obtained using standard agar and broth dilution methods according to Clinical and Laboratory Standards Institute guidelines. The mean linezolid MIC obtained by Etest in 18 linezolid-resistant strains of MRSA using Mueller-Hinton (MH) agar was 12.6-fold lower than that obtained by the agar dilution method, with the result that 78 % of the linezolid-resistant strains were incorrectly classified as linezolid-susceptible. The MIC of linezolid by Etest on brain-heart infusion (BHI) agar had a mean value 2.5-fold lower than that obtained by the agar dilution method, suggesting that replacing MH agar with BHI agar considerably improved the detectability of linezolid-resistant MRSA. Use of blood agar (MH agar supplemented with 5 % sheep blood) and 48 h of incubation resulted in 100 % agreement with the agar and broth dilution methods. Thus, this study revealed that the Etest on MH agar and BHI agar yielded false-negative results in a significant fraction of the linezolid-resistant MRSA. Hence, the use of blood agar and prolonged incubation is highly recommended for the accurate detection of linezolid-resistant MRSA using Etest.

Emergence of linezolid-resistant mutants in a susceptible-cell population of methicillin-resistant Staphylococcus aureus.

Methicillin-resistant Staphylococcus aureus with a MIC of linezolid of 4 µg/ml, isolated from a patient who had undergone unsuccessful linezolid therapy, yielded linezolid-resistant mutants in blood agar at 48 h of incubation. The resistant clones showed a MIC of linezolid ranging from 8 to 64 µg/ml and accumulated the T2500A mutation(s) of the rRNA genes. Emergence of these resistant clones appears to be facilitated by a cryptic mutation or mutations associated with chloramphenicol resistance.

Trends in empirical chemotherapy of bacterial meningitis in children aged more than 4 months in Japan: a survey from 1997 through 2008.

Bacterial meningitis is a serious problem in pediatric clinics and, therefore, needs urgent and empirical chemotherapy. We investigated 1,116 cases of empirical chemotherapy of patients aged older than 4 months from 1997 through 2008 by sending questionnaires. A single antibiotic treatment was carried out in less than 30% of the cases throughout the years, whereas the combination of two antibiotics had been practiced in more than 70% of the cases. The main antibiotics used were cephalosporins, carbapenems, and ampicillin. Combinatory use of ampicillin and cephalosporin was carried out in 74.7-82.7% of cases in 1997-2000, but sharply declined thereafter to 0-13.8% in 2004-2008. However, the combination of carbapenem and cephalosporin compensated for the decline, increasing from 3.8-6.6% in 1998-1999 to 79.5-89.9% in 2005-2008. The breakdown in the use of cephalosporins, carbapenems, and ampicillin in two-drug combinatory therapy was as follows. (i) Use of cefotaxime was 61.8-75.3% in 1997-2001, but decreased to nearly 50%, equivalent to the level of ceftriaxone use in 2003-2008. (ii) Use of ampicillin dropped from 74.7-92.3% in 1997-2000 to 4.6% in 2008, and this decreased level was compensated for by the use of carbapenems. Overall, combinatory chemotherapy of the third-generation cephalosporins and carbapenems seems to be practical. The discussion in this report includes the difference between Japan and the United States in the prevalence of the causative agents and the use of antibiotics. These studies provide information on trends in the treatment of children's meningitis in Japan and will be useful for the design of future empirical chemotherapy.

Synergistic effect of ultrasound and antibiotics against Chlamydia trachomatis-infected human epithelial cells in vitro.

To investigate whether or not the combined ultrasound and antibiotic treatment is effective against chlamydial infection, a new ultrasound exposure system was designed to treat chlamydia-infected cells. First, the minimum inhibitory concentrations of antibiotics against Chlamydia trachomatis were determined. Infected cultures were treated with antibiotics then sonicated at intensity of 0.15 or 0.44 W/cm(2) with or without Bubble liposomes. After 48 or 72 h after infection, chlamydial inclusions were stained and examined by fluorescence microscopy. The internalization of dextran-fluorescein conjugates by ultrasound irradiation with Bubble liposomes was observed by fluorescence microscopy. The results showed that application of nanobubble-enhanced ultrasound caused no significant effect on cell viability and chlamydial infectivity. However, Doxycycline (1/2 MIC) or CZX (1.0 µg/ml) in combination with nanobubble-enhanced ultrasound dramatically reduced the number of inclusions compared with that administered with antibiotics only. Bubble dose-dependent synergy was also observed. After ultrasound irradiation at intensity of 0.44 W/cm(2) on the presence of Bubble liposomes, 10% of HeLa cells were observed to have internalized the dextran molecules. This study suggests the possibility of using nanobubble-enhanced ultrasound to deliver antibiotic molecules into cells to eradiate intracellular bacteria, such as chlamydiae, without causing much damage to the cells itself.

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.

Emergence of the community-acquired methicillin-resistant Staphylococcus aureus USA300 clone in a Japanese child, demonstrating multiple divergent strains in Japan.

In 2008 we isolated methicillin-resistant Staphylococcus aureus (MRSA) from an 11-month-old Japanese girl who lived in Saitama, Japan, and suffered from cellulitis of the lower thigh and sepsis. The MRSA (strain NN47) belonged to multilocus sequence type (ST) 8 and exhibited spa363 (t024), agr1, staphylococcal cassette chromosome mec (SCCmec) type IVa, and coagulase type III. It was positive for Panton-Valentine leukocidin (PVL) and the arginine catabolic mobile element (ACME). Pulsed-field gel electrophoresis (PFGE) demonstrated that the MRSA was the USA300 clone, which is the predominant community-acquired MRSA (CA-MRSA) in the US. Strain NN47 was divergent, in terms of the spa type and patterns of PFGE and plasmids, from the USA300-0114 type strain or USA300 strain NN36, previously isolated from a visitor (Indian girl) from the US. Strain NN47 was resistant to erythromycin, in addition to beta-lactam agents (e.g., oxacillin). These data demonstrate the first emergence of the USA300 clone in Japanese children who have never been abroad and have had no contact with foreigners (and therefore, the first USA300 spread in Japan), and also emergence of multiple divergent strains of the USA300 clone in Japan. Because the USA300 clone is highly transmissible and virulent, surveillance of the USA300 clone is needed.

Effects of two basidiomycete species on interleukin 1 and interleukin 2 production by macrophage and T cell lines.

Two basidiomycete species, Lentinus edodes mycelia (LEM) and Cordyceps sinensis (CS) were examined for induction of cytokines in murine macrophage cell line R309 (R309) and T cell line LBRM-33 1A5 (1A5). When lipopolysaccharide (LPS)-activated R309 were exposed to the extracts of basidiomycetes, R309 induced significant levels of interleukin 1 (IL-1). Interleukin 2 (IL-2) induction was recognized in 1A5 cultures in the presence of IL-1 and phytohemagglutinin (PHA). However, no enhancement of IL-2 production by these basidiomycetes was discerned in 1A5 cultures with IL-1 and PHA, i.e., direct action of basidiomycetes was not found on IL-2 production of 1A5. PHA-stimulated 1A5 exposed to basidiomycetes induced IL-2 without IL-1 when co-cultured with LPS-activated R309 as a source of IL-1. Effects of basidiomycetes on IL-2 production in 1A5 seemed to be caused through their action on macrophages. The induction of IL-2, Th1 type cytokine in T lymphocyte, is a significant finding since basidiomycetes, taken as a dietary supplement for immuno-suppressed patients, especially cancer patients, would be helpful in improving their immune activity against cancer.

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.

Sonodynamic therapy.

Recently, there have been numerous reports on the application of non-thermal ultrasound energy for treating various diseases in combination with drugs. Furthermore, the introduction of microbubbles and nanobubbles as carriers/enhancers of drugs has added a whole new dimension to therapeutic ultrasound. Non-thermal mechanisms for effects seen include various forms of energy due to cavitation, acoustic streaming, micro jets and radiation force which increases possibilities for targeting tissue with drugs, enhancing drug effectiveness or even chemically activating certain materials. Examples such as enhancement of thrombolytic agents by ultrasound have proven to be beneficial for acute stroke patients and peripheral arterial occlusions. Non-invasive low intensity focused ultrasound in conjunction with anti-cancer drugs may help to reduce tumor size and lessen recurrence while reducing severe drug side effects. Chemical activation of drugs by ultrasound energy for treatment of atherosclerosis and tumors is another new field recently termed as "Sonodynamic therapy". Lastly, advances in molecular imaging have aroused great expectations in applying ultrasound for both diagnosis and therapy simultaneously. Microbubbles or nanobubbles targeted at the molecular level will allow medical doctors to make a final diagnosis of a disease using ultrasound imaging and then immediately proceed to a therapeutic ultrasound treatment.

The antagonistic effects of a combination of vancomycin and minocycline in Staphylococcus aureus with heterogeneous resistance to vancomycin.

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.