Mutant selection window of four quinolone antibiotics against clinical isolates of Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis.

Community-acquired pneumonia and otitis media are caused by several bacterial species, including Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis. For the treatment of these diseases, various quinolones are frequently used. We determined the mutant prevention concentration (MPC) of four quinolones, levofloxacin, sitafloxacin, tosufloxacin, and garenoxacin, using 92 clinical isolates and evaluated each mutant selection window (MSW). Furthermore, the DNA sequence of the quinolone resistance-determining region (QRDR) for the resistant mutant selected based on the MSW was determined. The MIC90 and MPC90 of levofloxacin were 0.781 µg/mL and 6.250 µg/mL for S. pneumoniae and 0.049 µg/mL and 1.563 µg/mL for M. catarrhalis and were higher than those for the other three quinolones. In addition, 5 strains of 30 S. pneumoniae (16.7%) selected based on the MSW of levofloxacin acquired resistance to only levofloxacin. In these 5 strains, a mutation of gyrA and/or parC was detected. In this study, no resistant mutant was selected in the MSW of any of the other three quinolones. On the other hand, clinical isolates of H. influenzae showed no resistance by all quinolone exposure. Finally, The MIC value and the mutation status in the QRDR did not change after 14 passages in antibiotic-free medium. In conclusion, our findings suggest that the increased use of levofloxacin may contribute to the increased quinolone-resistance of S. pneumoniae and M. catarrhalis.

[Production of an Anti-PIVKA-II Recombinant Single-Chain-Antibody in Escherichia Coli].

Protein induced by vitamin K absence or antagonist-II (PIVKA-II) is an abnormal prothrombin lacking gamma-carboxylation of the 10 glutamic-acid residues in the N-terminal region. PIVKA-II has been used as an effective biomarker of hepatocellular carcinoma (HCC) since the PIVKA-II level is not correlated with that of alpha-fetoprotein (AFP), which is another effective biomarker for HCC. Monoclonal antibodies for the clinical biomarker test are usually expensive because of their high production costs. Recently, many studies involving the expression of the recombinant globulin molecules in bacterial cells and plants have been con- ducted. These studies have enabled us to produce recombinant monoclonal antibodies at much lower costs. In this study, we first produced a hybridoma expressing a monoclonal antibody against PIVKA-II, and then we constructed and produced a single-chain fragment-variable antibody (scFv), created by the linking of variable regions of light- and heavy-chains of the PIVKA-II monoclonal antibody with a peptide linker of triplicated GGGGS. The scFv was expressed in E. coli and exhibited high specificity for PIVYKA-II binding, while its binding titer was low. [Review].

Antimicrobial efficacy of combined clarithromycin plus daptomycin against biofilms-formed methicillin-resistant Staphylococcus aureus on titanium medical devices.

In vitro efficacy of combined eradication therapy with clarithromycin and daptomycin against biofilm-formed methicillin-resistant Staphylococcus aureus on the orthopedic titanium devices was evaluated. The bactericidal effect of this antibiotic was investigated by a re-culture test, the scanning electron microscopy, and fluorescence microscopy using a double-staining dyes. Clarithromycin decreased the amount to half in 24 h. Although MRSA biofilms were not eradicated with clarithromycin or daptomycin alone, clarithromycin combined with daptomycin was useful to sterilize titanium devices within 72 h. This in vitro study showed that combined treatment with clarithromycin plus daptomycin is useful to eradicate staphylococcal biofilms formed on orthopedic devices.

Screening method for trimethoprim/sulfamethoxazole-resistant small colony variants of Staphylococcus aureus.

OBJECTIVES: Trimethoprim/sulfamethoxazole (SXT) is used to treat Staphylococcus aureus infections. However, the effect of treatment with SXT is sometimes not sufficient and there are patients whose treatment has to be prolonged. There are few reports of isolated strains of SXT-resistant S. aureus, but it is possible that some resistant strains cannot be detected by current testing methods We have therefore developed a tool to identify these resistant strains. METHODS: The mutant selection window (MSW) of SXT for 40 clinical isolates of S. aureus, including 20 methicillin-resistant S. aureus (MRSA), was determined. The optimum concentration of SXT and thymidine in agar for detecting SXT-resistant small colony variants (SCVs) of S. aureus was investigated. RESULTS: The MSW50 and MSW90 of SXT, presented as a multiple of the minimum inhibitory concentration (MIC), were 16× MIC and >256× MIC, respectively. SCVs were detected within the MSW in 32 (80%) of the 40 clinical isolates studied. To maintain the morphology of SCVs, the most suitable concentrations of SXT and thymidine for screening were 4mg/L and 0.01µg/mL, respectively. All 32 SCVs were resistant to SXT (MIC >32mg/L). The sensitivity and specificity of this screening method was 100% and 88.9%, respectively. CONCLUSIONS: SXT-resistant SCVs are not usually detected by routine laboratory tests performed in hospitals. However, the screening test described here can easily distinguish SXT-resistant SCVs among S. aureus isolated from specimens. This newly developed screening test could become an important tool to prevent inappropriate use of SXT.