Tomoo Saga

Chromosomally Encoded blaCMY-2 Located on a Novel SXT/R391-Related Integrating Conjugative Element in a Proteus mirabilis Clinical Isolate

ABSTRACT Integrating conjugative elements (ICEs) are mobile genetic elements that can transfer from the chromosome of a host to the chromosome of a new host through the process of excision, conjugation, and integration. Although SXT/R391-related ICEs, originally demonstrated in Vibrio cholerae O139 isolates, have become prevalent among V. cholerae isolates in Asia, the prevalence of the ICEs among Gram-negative bacteria other than Vibrio spp. remains unknown. In addition, SXT/R391-related ICEs carrying genes conferring resistance to extended-spectrum cephalosporins have never been described. Here we carried out a genetic analysis of a cefoxitin-resistant Proteus mirabilis clinical isolate, TUM4660, which revealed the presence of a novel SXT/R391-related ICE, ICEPmiJpn1. ICEPmiJpn1 had a core genetic structure showing high similarity to that of R391 and carried xis and int genes completely identical to those of R391, while an IS10-mediated composite transposon carrying blaCMY-2 was integrated into the ICE. A nucleotide sequence identical to the 3′ part of ISEcp1 was located upstream of the blaCMY-2 gene, and other genes observed around blaCMY-2 in earlier studies were also present. Furthermore, the nucleotide sequences of hot spot 2 and hot spot 4 in ICEPmiJpn1 showed high similarity to that of hot spot 2 in SXTMO10 and with a part of the nucleotide sequence found in P. mirabilis ATCC 29906, respectively. ICEPmiJpn1 was successfully transferred to Escherichia coli, Klebsiella pneumoniae, Salmonella enterica serovar Typhimurium, and Citrobacter koseri in conjugation experiments. These observations suggest that ICEs may contribute to the dissemination of antimicrobial resistance genes among clinically relevant Enterobacteriaceae, which warrants careful observation of the prevalence of ICEs, including SXT/R391-related ICEs.

Roles of Interleukin-17 in an Experimental Legionella pneumophila Pneumonia Model

ABSTRACT Interleukin-17 (IL-17) is a key factor in T helper type 17 (Th17) lineage host responses and plays critical roles in immunological control of a variety of infectious diseases. Although Legionella pneumophila, an intracellular bacterium found widely in the environment, often causes a serious and life-threatening pneumonia in humans, the contribution of IL-17 to immune function during Legionella pneumonia is unknown. In the present study, we used an experimental Legionella pneumonia infection to clarify the role of IL-17 in the resulting immune response. We observed robust production of pulmonary IL-17A and IL-17F (IL-17A/F), peaking on day 1 and declining thereafter. Upregulated production of tumor necrosis factor alpha (TNF-α), IL-6, and IL-1β, but not monocyte chemotactic protein 1 (MCP-1), was observed in Legionella-infected bone marrow-derived macrophages from BALB/c mice that had been stimulated with IL-17A or IL-17F. A significant decrease in the production of proinflammatory cytokines IL-6 and TNF-α was observed in IL-17A/F-deficient mice (BALB/c background) infected with L. pneumophila. Moreover, we found impaired neutrophil migration and lower numbers of chemokines (KC, LIX, and MIP-2) in IL-17A/F-deficient mice. IL-17A/F-deficient mice also eliminated L. pneumophila more slowly and were less likely to survive a lethal challenge. These results demonstrate that IL-17A/F plays a critical role in L. pneumophila pneumonia, probably through induction of proinflammatory cytokines and accumulation of neutrophils at the infection site.

Efficacy of Calcium-EDTA as an Inhibitor for Metallo-β-Lactamase in a Mouse Model of Pseudomonas aeruginosa Pneumonia

ABSTRACT In this study, we have evaluated the efficacy of calcium-EDTA (Ca-EDTA) as an inhibitor of bacterial metalloenzymes, such as metallo-β-lactamase (MBL) and other proteases, in a mouse model of Pseudomonas aeruginosa pneumonia. The simultaneous presence of Ca-EDTA (32 μg/ml) reduced the MICs of imipenem (IPM) in all MBL-producing P. aeruginosa isolates (IMP-1, -2, -7, and -10 and VIM-2) but not non-MBL-producing strains. In the pneumonia model, mice were intranasally infected with MBL-producing P. aeruginosa and then kept under conditions of hyperoxia to mimic ventilator-associated pneumonia. With both intranasal and subcutaneous administrations, Ca-EDTA significantly potentiated survival benefits of IPM compared to those of IPM alone. Ca-EDTA combination therapy induced a significant reduction of the bacterial burden in the lungs (P < 0.05). Furthermore, the inhibition activity of Ca-EDTA against MBL activity was confirmed by using the purified IMP-1 enzyme, which was characterized by a 50% inhibitory concentration (IC50) of 55 ± 8.2 μM. Finally, the protective effects of Ca-EDTA were demonstrated by culture supernatant-induced epithelial cell damage and acute lung injury in mice. These data suggest the therapeutic potential of Ca-EDTA not only by the blocking of MBLs but also by neutralizing tissue-damaging metalloproteases in P. aeruginosa infections.

Molecular characterization of carbapenem-non-susceptible Acinetobacter spp. in Japan: predominance of multidrug-resistant Acinetobacter baumannii clonal complex 92 and IMP-type metallo-β-lactamase-producing non- baumannii Acinetobacter species

We conducted an epidemiological study concerning carbapenem-non-susceptible clinical isolates of Acinetobacter spp. in Japan by molecular procedures including carbapenemase gene identification and amplified ribosomal DNA restriction analysis. Among 598 clinically isolated Acinetobacter spp. in 2007, 27 (4.5%) were non-susceptible to carbapenems. Most carbapenem-non-susceptible Acinetobacter baumannii (13/14) belonged to clonal complex (CC) 92, harbored blaOXA-51-like genes, including novel blaOXA-206, downstream of ISAba1, and were recovered mainly from the Kanto region. Carbapenem-non-susceptible A. baumannii CC92 isolates were further divided by pulsed-field gel electrophoresis into two groups, one of which was characterized by the presence of blaOXA-23. One A. baumannii CC276 isolate carried blaIMP-1 and blaOXA-58. Almost all non-baumannii Acinetobacter isolates (12/13), including Acinetobacter pittii (formerly Acinetobacter genomic species 3) and Acinetobacter nosocomialis (formerly Acinetobacter genomic species 13TU), produced IMP-type metallo-β-lactamases, and were recovered from various regions in Japan. This is the first report describing the nationwide molecular epidemiology of carbapenem-non-susceptible Acinetobacter spp. with genomic species-level identification in Japan.

Virulence-Suppressing Effects of Linezolid on Methicillin-Resistant Staphylococcus aureus: Possible Contribution to Early Defervescence

ABSTRACT In the present study, immunomodulatory effects of linezolid (LZD) on methicillin-resistance Staphylococcus aureus (MRSA) infections were evaluated. We have retrospectively reviewed treatment effects of LZD on 52 patients with severe MRSA infections. Sixty-four percent of the febrile patients demonstrated significant defervescence within 3 days, despite the presence of positive culture results. We speculated that this finding might be due to early anti-inflammatory effects of LZD, and to investigate this further we initiated in vivo experiments using mice MRSA pneumonia models. Mice were treated with either LZD or vancomycin (VCM) immediately after intranasal administration of MRSA. Bacterial numbers and levels of inflammatory cytokines in the lungs were determined. Although the bacterial burden in the lungs was not apparently different between the two groups, LZD but not VCM treatment significantly reduced induction of inflammatory cytokines in the lungs (P < 0.05). To evaluate whether this anti-inflammatory response was due to suppression of virulence factor expression, filter-sterilized supernatants of MRSA incubated in broth overnight with sub-MICs of LZD were subcutaneously administered to mice. To clarify whether LZD possesses direct host-modulating activity, cytokine responses to the supernatants were examined in mice pretreated with LZD. Interestingly, MRSA solutions prepared in the presence of sub-MICs of LZD revealed significant suppression of interleukin 6 (IL-6) in a dose-dependent manner (P < 0.05), but pretreatment of mice with LZD revealed no changes in cytokines. These findings suggest that sub-MICs of LZD might suppress virulence factors of MRSA, which may be associated with a reduction in endogenous pyrogens. These data may explain at least in part early defervescence observed in LZD-treated individuals.

Chromosomal Integration and Location on IncT Plasmids of the blaCTX-M-2 Gene in Proteus mirabilis Clinical Isolates

ABSTRACT Analysis of five CTX-M-2-producing Proteus mirabilis isolates in Japan demonstrated that blaCTX-M-2 was located on the chromosome in four isolates and on IncT plasmids in three isolates, including two isolates that also carried the gene on the chromosome. In all four isolates with chromosomal blaCTX-M-2, ISEcp1 was responsible for the integration of the gene into the chromosome. Three different sites in the P. mirabilis genomic sequence were utilized as integration sites.

Molecular analysis of the integrons of metallo-β-lactamase-producing Pseudomonas aeruginosa isolates collected by nationwide surveillance programs across Japan

BackgroundWe investigate the evolving molecular epidemiology of metallo-β-lactamase (MBL)-producing Pseudomonas aeruginosa isolates collected in a 100 institution, nationwide surveillance study in Japan from 2004 to 2006.ResultsMBL-producers were detected in 23/996 isolates (2.3%) in 2004 and 21/992 (2.1%) in 2006. Antimicrobial resistance (specifically, carbapenem resistance) rates between two periods did not differ significantly. MBL-producers were more prevalent in urinary tract isolates. blaIMP-1 group was the most predominant (38 isolates, 80%), followed by 3 blaIMP-7, 2 blaIMP-11 group, and 1 blaVIM-1. All MBL genes were identified in 16 different class 1 integrons, most of which were novel to INTEGRALL database. A total of 17 isolates of sequence type (ST) 235, a recognized worldwide drug-resistant lineage, were distributed in 5 geographic regions across Japan. ST235 isolates included a sublineage associated with In113-like integron. ST357 was identified in 14 isolates, 9 of which harboring a sole blaIMP-1 gene cassette (In994) were recovered from Chugoku region in 2004. ST357 isolates with blaIMP-11 group or ST235 with blaIMP-7 emerged in 2006. We also report for the first time the presence of novel fosI gene cassette in strains other than Mycobacterium spp.ConclusionsOur data give an important “snapshot” of the molecular characteristics and dynamics of MBL-producing lineages in P. aeruginosa in Japan. The significant association of specific genotypes and integrons implies that dissemination and transmission of the preexisting resistant lineage, rather than horizontal gene transfer in situ, might largely explain their endemicity.

ESBL-producing Enterobacteriaceae in environmental water in Dhaka, Bangladesh.

Pathogens encoding extended-spectrum β-lactamase (ESBL) genes represent a threat for failure of empirical antibiotic therapy and are associated with high mortality, morbidity and expenses. We examined surface water in Dhaka, capital of Bangladesh and isolated ESBL-producing Escherichia coli, Klebsiella pneumoniae and Enterobacter cloacae, suggesting the potential role of water for the dissemination and transmission of resistant genes among microorganisms. E. coli found most prevalent among isolated Enterobacteriaceae from environmental water. Molecular and genetic analysis revealed CTX-M-type and SHV-type ESBL genes in isolates that may influence the spread of multidrug resistant pathogenic bacteria causing human and animal infections in Bangladesh.

Pravastatin inhibits farnesol production in Candida albicans and improves survival in a mouse model of systemic candidiasis

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.

Characterization of qnrB-Like Genes in Citrobacter Species of the American Type Culture Collection

ABSTRACT Among five American Type Culture Collection (ATCC) Citrobacter strains, qnrB60 in Citrobacter freundii ATCC 6879, an isolate from the preantibiotic era, and qnrB61 in Citrobacter braakii ATCC 51113T, a type strain belonging to the C. freundii complex, were identified. Meanwhile, a truncated qnrB-like pseudogene was identified in C. freundii ATCC 8090T and ATCC 43864. No qnrB-like sequence was found in Citrobacter koseri ATCC 27028T. These findings underscore the close relationship between this species and qnrB.