Kazunori Maebashi

Classification of OprD sequence and correlation with antimicrobial activity of carbapenem agents in Pseudomonas aeruginosa clinical isolates collected in Japan

A total of 99 clinical isolates of metallo‐ß‐lactamase‐negative Pseudomonas aeruginosa collected in Japan between 1998 and 2001 were studied for their susceptibilities to carbapenem agents and corresponding oprD gene mutations. The OprD sequence of each strain was grouped into two major classes, based on the pattern of alterations. Eighty strains (80.8%) were so‐called ‘full length type’, whose OprD proteins were fully encoded. The remaining 19 strains (19.2%) were so‐called ‘defective type’, which possessed deletions or major alterations that might cause conformational changes in the OprD porin protein. The changes in ‘defective type’ strains led to 15‐, 17‐ and 23‐fold increases in the geometric mean MIC for imipenem, meropenem and biapenem compared with ‘full length type’ strains, respectively. ‘Full length type’ strains were further classified into six carbapenem susceptible types with the exception of four carbapenem‐resistant subtypes with additional amino acid substitutions at D43, G183, R154, G314, G316. However, ‘defective type’ strains were classified into four types as follows: 10 strains which contained a stop codon within the coding region; six strains which contained IS; one strain with a short deletion near the C‐terminal domain; and two strains without a stop codon in the sequenced region. Western blot analysis using OprD antibody showed that binding abilities of OprD proteins against ‘full length type’ strains were normal, whereas those against ‘defective type’ strains were lost without exception. These results indicate that OprD structure and antimicrobial activities for carbapenem agents proved to be highly correlated in P. aeruginosa

A novel model of cutaneous candidiasis produced in prednisolone-treated guinea-pigs

In an attempt to develop an animal model of cutaneous candidiasis useful for the pre-clinical evaluation of antifungal drugs, experimental cutaneous Candida albicans infections were produced in mature and immature guinea-pigs treated with prednisolone. The morbidity of this model in terms of the extent and the duration of the superficial infection was compared with that of two other reported models: those produced by alloxan treatment, or the use of occlusive dressings. Infected animals were also included which received neither of these treatments. The Candida infection continued steadily for 14 days or more in both mature and immature animal groups treated subcutaneously with prednisolone and appeared to be more suitable than that produced with alloxan treatment or under occlusive dressings. In prednisolone-induced infection, most of the micro-organisms were confined to the epidermis and there was no evidence of their dermal penetration during the 14 day experimental period. In view of these facts, we attempted a more quantitative method by counting viable Candida in order to evaluate antimycotics in a shorter therapy period during which animals would receive less exposure to the vehicle. These findings appear to indicate that the experimental model of cutaneous C. albicans infection produced in prednisolone-treated mature and immature guinea-pigs would be useful for studies on the therapeutic efficacy of antifungal agents against cutaneous candidiasis.

A novel mechanism of fluconazole resistance associated with fluconazole sequestration in Candida albicans isolates from a myelofibrosis patient.

A series of 10 strains of Candida albicans, from TIMM 3309 to TIMM 3318, were repeatedly isolated in one myelofibrosis‐complicated patient with recurrent candidemia. The latter five isolates, from TIMM 3314 to TIMM 3318, became suddenly resistant to fluconazole during the 10 to 16 weeks after antimycotic therapy. We investigated the resistant mechanism of fluconazole using one susceptible isolate and two of the five resistant isolates in the series. The ergosterol synthesis by cell‐free extracts from the two resistant isolates was less susceptible to fluconazole partly as a result of a decreased affinity of cytochrome P‐450. Unexpectedly, these two resistant isolates showed higher levels of an intracellular accumulation of [3H]fluconazole than the susceptible isolate and the control strain of C. albicans ATCC 10231. In the resistant isolate, TIMM 3318, most intracellular incorporated fluconazole was distributed in the 12,000 × g pellet (P‐120) fraction by centrifugation unlike the two susceptible strains. An observation of the ultrastructure of TIMM 3318 showed the most notable alteration to be the characteristic appearance of numerous vesicular vacuoles (diameter, 150 to 400 nm); these vacuoles were not observed, however, in either of the susceptible strains. A direct observation of the subcellular fraction prepared from TIMM 3318 by the electron microscopy negative‐staining method suggests that most of the vesicular vacuoles were recovered in the P‐120 fraction. These results suggest that fluconazole sequestration caused by vesicular vacuoles of the resistant isolate might act as a novel mechanism of fluconazole resistance besides the decreased affinity of cytochrome P‐450.

Activity of carbapenems with ME1071 (disodium 2,3-diethylmaleate) against Enterobacteriaceae and Acinetobacter spp. with carbapenemases, including NDM enzymes

OBJECTIVESnME1071 is a maleic acid that inhibits metallo-β-lactamases (MBLs). We examined its ability to potentiate different carbapenems against MBL-producing Enterobacteriaceae in relation to its inhibition kinetics.nnnMETHODSnEnterobacteriaceae and Acinetobacter isolates with IMP, VIM and NDM MBLs were tested; bacteria with other types of carbapenem resistance were used as controls. Chequerboard titrations were performed by CLSI agar dilution, carbapenemases were cloned into pET-28a(+) and purified by column chromatography, and kinetic parameters were determined by spectrophotometry.nnnRESULTSnThe key findings were: (i) the MICs of carbapenems varied widely among isolates with the same carbapenemase, but those with the NDM types were generally the most resistant; (ii) biapenem was the carbapenem least compromised by all MBL types, owing to weaker kinetic efficiency (k(cat)/K(m)) for hydrolysis, contingent on lower affinity (higher K(m)); (iii) MBLs were the only carbapenemases inhibited by ME1071, confirming its specificity of action; and (iv) irrespective of the partner carbapenem, synergy with ME1071 was least for organisms with NDM MBLs and most for those with IMP types, correlating with ME1071 having weakest affinity (highest K(i)) for NDM-1 and strongest affinity for IMP-1.nnnCONCLUSIONSnME1071 reduced the MICs of carbapenems for bacteria with NDM-1 enzyme though synergy was weaker than for bacteria with IMP and VIM metallo-enzymes; this correlated with ME1071 having weaker affinity for NDM-1 than IMP-1 and VIM-2. As the weakest MBL substrate carbapenem, biapenem was the easiest to protect.

Characterization of Mechanisms of Fluconazole Resistance in a Candida albicans Isolate from a Japanese Patient with Chronic Mucocutaneous Candidiasis

We examined the mechanisms of fluconazole resistance in a fluconazole‐resistant Candida albicans isolate from a Japanese patient with chronic mucocutaneous candidiasis. It was demonstrated that the highly resistant phenotype of this strain was associated with combined mechanisms of the energy‐dependent reduced intracellular accumulation of fluconazole, presumably due to the increased expression of the ATP‐binding cassette efflux pump CDR gene(s), and the reduced affinity of the target enzyme, Erg11p, to fluconazole. In particular, the reduced affinity of Erg11p was considered to contribute largely to the fluconazole resistance in the TIMM3209 strain. Biochemical studies indicated that the Erg11p from the TIMM3209 strain showed reduced susceptibility both to fluconazole and itraconazole of cell‐free ergosterol biosynthesis, and cytochrome P‐450 also showed reduced affinity to fluconazole in the carbon monoxide‐cytochrome P‐450 complex formation assay. We identified two amino acid substitutions, Y132H and G448V, in Erg11p from the TIMM3209 strain. We found that the cytochrome P‐450 from the TIMM3209 strain decayed during incubation at 37 C without fluconazole although it is unknown whether or not the phenomenon is linked to the resistant phenotype. These mutations are thought to confer the above‐mentioned characteristics to Erg11p.

Proliferation of Intracellular Structure Corresponding to Reduced Affinity of Fluconazole for Cytochrome P‐450 in Two Low‐Susceptibility Strains of Candida albicans Isolated from a Japanese AIDS Patient

Three Candida albicans isolates, TIMM 3164, 3165 and 3166 with reduced fluconazole susceptibility, were isolated from two Japanese AIDS patients. We earlier reported that a reduced intracellular accumulation of fluconazole in these isolates played an important role in the resistance mechanism of fluconazole, but we did not exclude the involvement of other factors. We here examined characteristics related to cytochrome P‐450 (CYP), especially sterol 14α‐demethylase encoded by the ERG11 gene which is the target molecule for fluconazole. In TIMM 3164 and 3165, the ergosterol synthesis by cell‐free extracts was somewhat less susceptible to fluconazole, due to a decrease in fluconazole affinity for CYP. The nucleotide substitutions in the ERG11 gene were identified to result in three amino acid changes of K143R, E266D and V488I in TIMM 3164, and of E266D, V404L and V488I in TIMM 3165. These amino acid substitutions might contribute to the decreased affinity for CYP in both isolates. However, a single amino acid change, E266D, observed in TIMM 3166 was unrelated to the decreased affinity for CYP. The most prominent finding on the ultrastructure of TIMM 3164 and 3165 was the development of mesh membrane structures of the endoplasmic reticula, which is a location related to sterol synthesis. This phenomenon was not observed in the cells of TIMM 3166 or the susceptible control strains of ATCC 90028 and 10231. In addition to the reduced intracellular accumulation, the decreased affinity of fluconazole for CYP in TIMM 3164 and 3165 is assumed to be associated with the fluconazole‐resistance phenotype.

Pharmacokinetic and pharmacodynamic properties of biapenem, a carbapenem antibiotic, in rat experimental model of severe acute pancreatitis.

Objectives: It is known that prophylaxis with imipenem reduces the risk of infection accompanying severe acute pancreatitis. In this study,we modified a rat experimental model of severe acute pancreatitis for antibiotic evaluation, and the effect of biapenem was compared with that of imipenem to determine the usefulness of biapenem. Methods: Severe acute pancreatitis was induced by 5% sodium taurocholate. Antibiotics were subcutaneously administered at 3 and 6 hours and evaluated at 12 hours after the pancreatitis induction. For pharmacokinetic evaluation, antibiotics were subcutaneously administered at 3 hours after the pancreatitis induction. Results: From 3 hours after the induction, bacteria were detected from the pancreas. The total bacterial count increased in a time-dependent manner for 12 hours. Biapenem administration reduced the total bacterial count in the pancreas, as observed in imipenem administration. The plasma concentration of biapenem was almost equivalent to that of imipenem; however, the pancreatic penetration of biapenem was approximately twice that of imipenem in this model. Conclusions: Biapenem was suggested to be effective in prophylactic treatment of infectious complications as much as imipenem because of its superior penetration to the pancreas in severe acute pancreatitis.

In vitro Activities of Oral Cephem and Telithromycin Against Clinical Isolates of Major Respiratory Pathogens in Japan

The in vitro antibacterial activities of oral cephem antibiotics and ketolide telithromycin against major respiratory pathogens possessing β-lactam-resistant mutations (within the pbp gene) and/or macrolide-resistant genes (erm and mef) were examined in clinical isolates collected at 66 institutes in all over the Japan between 2002 and 2003. Telithromycin showed the strongest antibacterial activity against methicillin-susceptible Staphylococcus aureus strains with and without macrolide-resistant genes, such as ermA or ermC gene. All the cephem antibiotics showed potent antibacterial activity against Streptococcus pyogenes, with minimum inhibitory concentrations (MICs) of 0.015 mg/L or lower. Cefdinir had a much higher MIC90 against genotypic penicillin-resistant Streptococcus pneumoniae (gPRSP) than cefditoren and cefcapene (8 mg/L cefdinir vs. 1 mg/L cefditoren and cefcapene). The majority of gPRSP harbored either ermB or mefA, and the antibacterial activity of telithromycin against these strains was decreased however some susceptibility was still sustained. Cefditoren exerted the strongest antibacterial activity against β-lactamase-negative ampicillin-resistant Haemophilus influenzae, with an MIC90 of 0.5 mg/L. These results underline the importance of checking the susceptibility and selecting an appropriate antibiotic against target pathogens.