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Objectives: Molecular analysis of meropenem-resistant mechanisms in mutants emerging from long-term in vitro meropenem exposure to borderline meropenem-susceptible carbapenemase-producing Enterobacterales (CPE) and non-CPE.Methods: Escherichia coli TUM13867 harbouring both blaIMP-6- and blaCTX-M-2-carrying IncN plasmid and Citrobacter koseri TUM13189 with blaCTX-M-2-carrying chromosome were used. Meropenem MIC was 1 mg/L against both strains. Each strain was cultured in the hollow-fibre infection model (HFIM) to approximately 1 × 106 colony formation unit (cfu)/mL, and meropenem 1 g q8h treatment was initiated. Then, changes in total and meropenem-resistant populations were observed for 124 h. Meropenem resistance mechanisms were analysed using full-length whole-genome sequencing (WGS), reverse-transcription quantitative PCR and digital PCR.Results: Meropenem reduced TUM13867 and TUM13189 to approximately 5 and 2 log10 cfu/mL, respectively, at 2 h after initiation, but regrowth was observed at 24 h. The meropenem-resistant mutant emergence frequency at 120 and 124 h was 4.4 × 10-4 for TUM13867 and 7.6 × 10-1 for TUM13189. Meropenem MIC of the mutants derived from TUM13867 (TUM20902) and TUM13189 (TUM20903) increased 4- and 16-fold, respectively. TUM20902, which harboured pMTY20902_IncN plasmid with a 27 505-bp deletion that included blaCTX-M-2, and blaIMP-6 showed 4.21-fold higher levels of transcription than the parental strain. TUM20903 had a 49 316-bp deletion that included ompC and a replicative increase of blaCTX-M-2 to three copies.Conclusions: Molecular analysis including full-length WGS revealed that the resistance mechanisms of meropenem-resistant mutants that emerged during long-term in vitro meropenem exposure were increased blaIMP-6 transcripts in CPE and increased blaCTX-M-2 transcripts due to gene triplication and OmpC loss resulting from ompC deletion in non-CPE. [ABSTRACT FROM AUTHOR]

Background: There is no comprehensive study on PAM-like MBLs.Objectives: Our aim was to characterize novel B3 MBL variants, PAM-2 and PAM-3, from Pseudomonas tohonis clinical isolates.Methods: We evaluated the antimicrobial susceptibility and the MBL gene composition of three novel P. tohonis clinical isolates identified at a Japanese hospital, using the broth microdilution method and WGS, respectively. We characterized the PAM-2 and PAM-3 proteins using recombinant protein expression and biochemical evaluations.Results: Low carbapenem MICs (meropenem MIC = 0.125-1 mg/L) were observed for all three P. tohonis isolates; however, the isolates produced MBLs. We identified blaPAM-2 and blaPAM-3 as potential genes, belonging to a novel subclass of B3 MBLs. Their genomic sequence was similar to that of blaPAM-1 from Pseudomonas alcaligenes. PAM-2 and PAM-3 comprised 287 amino acids and exhibited 90% amino acid identity with PAM-1, 73% identity with POM-1 from Pseudomonas otitidis and 61% identity with L1 from Stenotrophomonas maltophilia. Biochemical evaluations of recombinant PAM-2 and PAM-3 revealed similar kcat/Km ratios and demonstrated catalytic activity against all the tested β-lactams, except for aztreonam. In addition, the kcat/Km ratio for imipenem was 40-fold lower than that for meropenem.Conclusions: P. tohonis harbours a species-specific PAM-family MBL gene. This enzyme has higher hydrolytic activity against meropenem compared with that against imipenem. [ABSTRACT FROM AUTHOR]

Objectives: To investigate the spread of ceftriaxone-resistant Neisseria gonorrhoeae lineages similar to strains H041 (2009) and FC428 (2015), we characterized 55 strains collected in 2013 from hospitals across Japan.Methods: Susceptibility testing and whole-genome sequencing.Results: Susceptibility rates were 58% for cefixime and 98% for ceftriaxone. The 55 strains were whole-genome sequenced and classified into nine MLST-STs. MLST-ST1901 was the most prevalent (n = 19) followed by MLST-ST7363 (n = 12) and MLST-ST7359 (n = 11). The most prevalent penA [encoding penicillin binding protein 2 (PBP2)] mosaic types, based on the N. gonorrhoeae sequence typing for antimicrobial resistance (NG-STAR) scheme, were 10.001 (n = 20) followed by 34.001 (n = 13). The H041 and FC428 strains were not detected; however, a single ceftriaxone-resistant strain (TUM15748) with a MIC of 0.5 mg/L ceftriaxone was identified. The TUM15748 strain belonged to MLST-ST7359 and N. gonorrhoeae multiantigen sequence typing-ST6771, and had a novel PBP2 (PBP2TUM15748, penA type 169.001). The amino acid sequence of PBP2TUM15748 showed partial similarity to that of PBP2 from N. gonorrhoeae GU140106 and commensal Neisseria perflava and Neisseria cinerea. Natural transformation and recombination experiments using full-length TUM15748 penA showed that the ceftriaxone MICs of transformants increased 16-fold or more compared with the parental ceftriaxone-susceptible recipient strain (NG9807, belonging to MLST-ST7363). No ceftriaxone-resistant MLST-ST7359 strains have previously been reported.Conclusions: We showed here that a ceftriaxone-susceptible lineage acquired a mutant PBP2 mosaic type, integrating partial PBP2 sequences from commensal Neisseria species, resulting in the emergence of ceftriaxone-resistant strains. [ABSTRACT FROM AUTHOR]

Background: Clostridioides difficile is the most common cause of antimicrobial-associated diarrhoea in high-income countries. Fluoroquinolone resistance enabled the emergence and intercontinental spread of the epidemic ribotype (RT) 027 strain of C. difficile in the early 2000s. Despite frequent inappropriate antimicrobial use in Asia, RT 027 is rarely isolated in the region, but the often fluoroquinolone- and clindamycin-resistant RT 017 strain predominates.Objectives: This study evaluated the antimicrobial activity of ridinilazole, a novel antimicrobial agent with highly specific activity for C. difficile, against clinical strains of C. difficile from Asia.Methods: C. difficile strains from Japan (n = 64), South Korea (n = 32) and China (n = 44) were tested by the agar dilution method for susceptibility to ridinilazole, metronidazole, vancomycin, clindamycin, moxifloxacin, rifaximin and fidaxomicin.Results: All strains were susceptible to ridinilazole, with low MICs (0.03-0.25 mg/L). Several strains showed multiresistance profiles, particularly RT 017 (100% clindamycin resistant, 91.3% moxifloxacin resistant, 82.6% rifaximin resistant) and RT 369 (94.4% clindamycin resistant, 100% moxifloxacin resistant). Rifaximin resistance was absent in all strains from Japan. Multiresistance to clindamycin, moxifloxacin and rifaximin was found in 19 RT 017 strains (from China and South Korea), 2 RT 001 strains (South Korea) and 1 RT 046 strain (South Korea).Conclusions: Ridinilazole showed potent activity against a range of Asian C. difficile strains, which otherwise frequently displayed resistance to several comparator antimicrobial agents. Ongoing surveillance of antimicrobial resistance profiles is required to monitor and control the spread of resistant strains. [ABSTRACT FROM AUTHOR]

Objectives: Detection of carbapenem-hydrolysing class D β-lactamase (CHDL)-producing Acinetobacter spp. is critical for understanding antibiotic resistance. In this study, we compared the available detection techniques derived from the carbapenem inactivation method (CIM), using CHDL-producing Acinetobacter spp., and developed a modified method that uses bacterial lysate (lysate CIM; LCIM).Methods: A total of 159 Acinetobacter spp. (102 carbapenemase producers and 57 non-producers) and 14 Pseudomonas spp. (7 carbapenemase producers and 7 non-producers) were tested. Modified CIM, simplified CIM, CIMTris, Triton-CIM and LCIM were compared using these strains. Distinct from the CIM, LCIM includes a longer incubation period (4 h) with 2.0% Triton X-100 (v/v) in 20 mM MOPS buffer instead of water.Results: The sensitivity/specificity of the modified CIM, simplified CIM, CIMTris, Triton-CIM and LCIM were 71.6%/100%, 66.1%/89.1%, 88.1%/95.3%, 80.7%/100% and 97.2%/100%, respectively. LCIM was the most sensitive and specific.Conclusions: Use of bacterial lysate and MOPS increased the sensitivity of the CIM in detecting CHDL-producing Acinetobacter spp. [ABSTRACT FROM AUTHOR]

Objectives Ventilator-associated pneumonia (VAP) is an important cause of morbidity and mortality in critical care settings. Acinetobacter has become a leading cause of VAP. In particular, the appearance and spread of multidrug-resistant Acinetobacter is of great concern. In this study, we examined the effect of the antioxidant procysteine on Acinetobacter murine pneumonia in hyperoxic conditions in order to simulate VAP. Methods Acinetobacter was administered intranasally to BALB/c mice kept in hyperoxic conditions. At designated timepoints, bacterial number, cytokine production and histopathological findings in the lungs were examined. The effects of procysteine on survival rates, lung bacterial burdens and the phagocytic activities of alveolar macrophages were evaluated. Results Drastic decreases in survival were observed when the infected mice were kept in hyperoxic conditions (P < 0.001). Significant differences in pulmonary bacterial number and neutrophil accumulation were observed between mice kept in hyperoxic or normoxic conditions on day 3. Although all mice infected with Acinetobacter spp. and kept in hyperoxic conditions died by day 3, procysteine treatment significantly improved survival (60% survival on day 7, P < 0.01). Procysteine treatment decreased the lung bacterial burden on days 2 and 3. Finally, improved uptake of FITC-labelled beads by alveolar macrophages from mice treated with procysteine and kept in hyperoxic conditions was noted. Conclusions These results suggest that hyperoxia increases mortality in mice with Acinetobacter pneumonia and that procysteine improves survival by increasing the phagocytic activity of alveolar macrophages in mice kept in hyperoxic conditions. [ABSTRACT FROM AUTHOR]

Objectives The β-barrel assembly machinery (BAM) complex plays a critical role in outer membrane protein (OMP) biogenesis. The outer membrane (OM) of Pseudomonas aeruginosa is centrally involved in mechanisms of antibiotic resistance. This study aimed to identify effects of a synthetic peptide based on conserved sequences in the putative BamA-binding region of BamD, focusing on antibiotic susceptibility and OMP characteristics in P. aeruginosa. Methods We synthesized a peptide FIRL (Phe-Ile-Arg-Leu-CONH2) with a sequence related to that of the BamD protein. We assessed antibiotic susceptibility of P. aeruginosa PAO1 using the chequerboard method and a time–kill assay. Changes in OMPs and in OM permeability were examined using SDS-PAGE, western blot analysis and nitrocefin assays. The combined effects of the peptide and antibiotics were investigated using a mouse pneumonia model. Results Although the peptide alone exerted no antimicrobial effect, it reduced the MICs of colistin, levofloxacin, erythromycin, vancomycin and rifampicin for P. aeruginosa PAO1 by 4-fold or more. Time–kill tests revealed bacterial numbers were significantly reduced after 2 h of incubation with the peptide plus colistin or levofloxacin. Moreover, in the presence of the peptide, expression of OprM was reduced by a third, and OM permeability was increased. The combination of the peptide (2.08 mg/kg) and colistin (1.25 mg/kg) significantly reduced P. aeruginosa by more than 1 log cfu/mL in a mouse pneumonia model. Conclusions We show, for the first time, that a synthetic peptide based on homologous sequences of BamD can potentiate antibiotic susceptibility of P. aeruginosa. [ABSTRACT FROM PUBLISHER]

Candidemia remains a major cause of morbidity and mortality, especially in immunocompromised patients. A strategy of reducing virulence and virulence factors of Candida spp. is an attractive approach for the treatment of serious infections caused by these yeasts. Recently, farnesol has been reported to be a quorum-sensing autoinducer, as well as a virulence factor of C. albicans. In the present study, we examined the effects of pravastatin, a 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitor on the in vitro production of farnesol. In addition, the synergistic effects of pravastatin with fluconazole (FLC) were examined in a mouse model of systemic infections. In vitro experiments demonstrated that pravastatin had synergistic activity with FLC as judged by fractional inhibitory concentration index (FICI) and suppression of farnesol production at sub-minimum inhibitory concentrations. Furthermore, significant improvement of survival in systemic infection models was shown with pravastatin supplementation. The survival benefits of pravastatin were correlated with reductions of fungal burden. These data suggest the potential of pravastatin as a supportive therapy against C. albicans infections. Synergistic antifungal activity and suppression of HMG-CoA reductase-associated Candida virulence factors, including farnesol, may explain, at least in part, the in vivo efficacy of pravastatin. [ABSTRACT FROM AUTHOR]

Objectives In vitro and in vivo antibacterial activities of modithromycin against Streptococcus pneumoniae, Streptococcus pyogenes and Haemophilus influenzae were examined. Methods MICs were determined by the broth microdilution method. Experimental infection of epithelial cell line A549 was performed to compare the intracellular activity and lasting effects of the antimicrobial agents. To evaluate in vivo efficacy, the rat pulmonary infection model was used. Results Modithromycin had MICs of ≤1 mg/L against all the clinical strains of both streptococci, including erythromycin-resistant strains. In particular, the MICs of modithromycin for erm(B)- or mef(A)-carrying S. pyogenes were 16–32 times or 2–4 times lower than those of telithromycin, respectively. The MIC90 of modithromycin for H. influenzae was 8 mg/L, which was 4 times higher than that of telithromycin. Modithromycin, as well as azithromycin, showed a lasting inhibitory effect on bacterial growth of cell-associated H. influenzae compared with telithromycin and levofloxacin after removal of the agents from the apical medium. In the pulmonary infection model, modithromycin showed greater or comparable efficacy against erm(B)-carrying S. pneumoniae and H. influenzae, respectively, than telithromycin, regardless of having an MIC that was 2 or 4 times higher for these strains. Conclusions Modithromycin has the most potent anti-S. pyogenes activity of the antimicrobial agents tested. Modithromycin also has the better in vivo efficacy against S. pneumoniae and H. influenzae, which might be due to its lasting intracellular activity. [ABSTRACT FROM PUBLISHER]

Objectives Clostridium difficile produces toxins and is an aetiological organism of pseudomembranous colitis. Immunoglobulin is one of the treatment strategies against fulminant C. difficile infections, but the clinical evidence is still limited. We examined the efficacy of intravenous immunoglobulin (IVIg) in C. difficile toxin (CDT)-mediated lethality and cellular injury in mice. Methods Mice were intraperitoneally injected with 0.2 mL of filter-sterilized C. difficile culture supernatant (CDT preparation). The IVIg preparation was intravenously administered at several timepoints. We also examined alteration of intestinal permeability and an apoptosis marker in the gut. In in vitro experiments, HEp-2 cells were incubated with a CDT preparation in the presence or absence of the IVIg preparation, after which cell viability and lactate dehydrogenase release were examined. Results All control mice died by day 2 after injection of the CDT preparation. The maximum effects of IVIg (100% survival) were observed when the mice were treated with IVIg at the same time as injection of the CDT preparation. The IVIg effects were closely associated with improvement of intestinal vascular permeability and mucosal damage in the gut. In addition, reduction of an apoptosis marker (histone-associated DNA fragments) was demonstrated in the mice treated with IVIg. Interestingly, a smaller increase in histone-associated DNA fragments was observed in FasL-deficient mice treated with the CDT preparation compared with wild-type. Conclusions These data demonstrated that IVIg may be protective against CDT-mediated lethality, when administered at the appropriate time. The present data also suggest an increase in intestinal permeability, probably through exaggeration of Fas/FasL-mediated apoptosis, as a key mechanism of C. difficile-mediated diseases. [ABSTRACT FROM PUBLISHER]

The present study was designed to compare in vitro antibacterial activities of linezolid and vancomycin against vancomycin-susceptible Enterococcus faecalis (VSEF) and vancomycin-resistant enterococci (VRE) isolated in Japan with those of quinupristin-dalfopristin, teicoplanin and minocycline, and the in vitro short time bactericidal activity and the in vivo activities of linezolid and vancomycin against vancomycin-susceptible and -resistant E. faecalis. The MIC90s of linezolid, quinupristin-dalfopristin, vancomycin, teicoplanin and minocycline for VSEF and VRE were both 2 mg/L, both 2 mg/L, 2 and >128 mg/L, 0.25 and >128 mg/L, and both 32 mg/L, respectively. The efficacy of linezolid for mice with bacteraemia caused by VSEF was similar to that of vancomycin, but the elimination ratio of viable organisms from the blood of mice treated with vancomycin was significantly higher than in linezolid-treated and untreated mice at 2 h post-administration, and those of the two groups at 4 and 6 h were significantly higher than in untreated mice. Moreover, linezolid was highly active in mice with bacteraemia caused by vancomycin-resistant E. faecalis because this drug had potent in vitro activity against the organisms. Our results indicate that linezolid is suitable for the treatment of VRE and VSEF bacteraemia, and vancomycin is suitable for VSEF bacteraemia. [ABSTRACT FROM AUTHOR]

Objective: Accumulating data have demonstrated that macrolide antibiotics suppress Pseudomonas aeruginosa virulence, which may explain the efficacy of macrolides in clinical settings. We examined the virulence of macrolide-treated bacteria in vivo.Methods: P. aeruginosa PAO-1 was grown for 24 h on agar containing sub-MIC antibiotics, and then mice were challenged intranasally with 10(7) cfu of bacteria.Results and Conclusions: The mortality rate of mice inoculated with bacteria grown in the presence of clarithromycin (10 mg/L), erythromycin (10 mg/L) or azithromycin (5 mg/L) was 80%, 80% and 100%, respectively. In contrast, none of the mice inoculated with non-treated bacteria or bacteria treated with other antibiotics died. Lung weight and protein concentration in bronchoalveolar lavage fluid (BALF) were significantly higher in the clarithromycin group between 3 and 9 h. Moreover, we detected higher levels of tumour necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) in the BALF of these mice. These data demonstrate that macrolide-treated P. aeruginosa induced paradoxically strong responses, such as elevation of TNF-alpha, NO and permeability in the lungs. [ABSTRACT FROM AUTHOR]

The in vivo activity of olamufloxacin (HSR-903), a new fluoroquinolone, was evaluated and compared with ciprofloxacin, sparfloxacin and levofloxacin. Olamufloxacin was active against systemic infection in mice inoculated with both Gram-positive and -negative bacteria. Olamufloxacin had equal efficacy for experimental urinary tract infections in mice caused by Pseudomonas aeruginosa. [ABSTRACT FROM AUTHOR]

The in vitro activities (MIC and MBC) of telithromycin (HMR 3647) against clinical isolates in Japan were investigated in comparison with those of erythromycin A, clarithromycin, azithromycin, amoxycillin, cefdinir and levofloxacin. Telithromycin was more potent than the reference compounds against erythromycin A-susceptible or resistant Streptococcus pneumoniae isolates possessing either mef (mefA or mefE) genes or the ermB gene. Against erythromycin A-susceptible or inducibly resistant Staphylococcus aureus and erythromycin A-susceptible and intermediate Enterococcus faecalis, telithromycin was highly active. [ABSTRACT FROM PUBLISHER]

The MIC90 of glycylcycline (< or =0.06 mg/L) against 55 strains of Streptococcus pneumoniae was 100-fold lower than that of minocycline or tetracycline. In a mouse model of penicillin-resistant S. pneumoniae (PRSP) pneumonia, glycylcycline (10 mg/kg) decreased bacterial counts in the lungs from 10(6) cfu to <10(2) cfu, whereas no apparent reduction of bacterial numbers was observed with minocycline or penicillin G. Pharmacokinetic studies showed that the half-life and area under the curve of glycylcycline were superior to those of minocycline and penicillin G in the lungs. These results show a preferential distribution of glycylcycline in the lungs and potent in vivo bactericidal activity in PRSP pneumonia. [ABSTRACT FROM AUTHOR]

We have previously reported that erythromycin at sub-inhibitory concentrations enhanced the serum sensitivity of some Pseudomonas aeruginosa strains. To explore the mechanism of this effect, we have now examined the influence of macrolide antibiotics on outer membrane proteins and lipopolysaccharide (LPS) of P. aeruginosa. The strains S-6 and PAO-1 of P. aeruginosa were used as susceptible and resistant strains respectively to assess enhancement of serum sensitivity by erythromycin. The strain S-6 became more serum-sensitive when grown on agar with sub-MICs of erythromycin or azithromycin, but not of josamycin, whereas no change was observed in the serum sensitivity of the strain PAO-1 after growth with any of these antibiotics. The analysis of outer membrane proteins showed that erythromycin treatment resulted in a reduction in the amount of the 38 kDa protein (OprF) and in a prominent increase of 41 kDa protein band in the strain S-6, but not in the strain PAO-1. By an immunoblotting assay, this 41 kDa protein was shown to be highly reactive to the immune serum against untreated P. aeruginosa. LPS of the strain S-6 were examined by SDS acrylamide gel electrophoresis. The treatment with erythromycin or azithromycin, but not with josamycin, reduced the amounts of LPS species with lower molecular weights although the levels of LPS species with high molecular weights were similar to those of untreated bacteria. These results suggest that the enhanced serum sensitivity of P. aeruginosa by erythromycin is associated with changes in bacterial surface components, such as outer membrane proteins and LPS. [ABSTRACT FROM AUTHOR]

This murine model of nontypeable (unencapsulated) Haemophilus influenzae (NTHI) bronchopneumonia used organisms bound to mouse fetal lung (MFL) cells as an inoculum. Pretreatment of the mice with 40 µ-L of 1% formalin 3 days before intranasal instillation of the bacteria was necessary to allow infection. The number of NTHI recovered from the lungs plus trachea on day 7 after instillation was >100 times the number originally inoculated. Later, however, the number of recovered bacteria diminished gradually, and by day 14 it was almost identical to the original inoculum size. Serum IgM also peaked on day 7 after infection, after which IgG increased while IgM decreased. Histologically, bronchoalveolar infiltration of neutrophils was observed on day 3 after inoculation and continued at least for the following 4 days. The present experiment demonstrates that MFL cells can protect bacteria that have invaded the cells from the opsonizing and killing activities of host humoral defense mechanisms. [ABSTRACT FROM PUBLISHER]