Toyotaka Sato

Fluoroquinolone resistance mechanisms in an Escherichia coli isolate, HUE1, without quinolone resistance-determining region mutations.

Fluoroquinolone resistance can cause major clinical problems. Here, we investigated fluoroquinolone resistance mechanisms in a clinical Escherichia coli isolate, HUE1, which had no mutations quinolone resistance-determining regions (QRDRs) of DNA gyrase and topoisomerase IV. HUE1 demonstrated MICs that exceeded the breakpoints for ciprofloxacin, levofloxacin, and norfloxacin. HUE1 harbored oqxAB and qnrS1 on distinct plasmids. In addition, it exhibited lower intracellular ciprofloxacin concentrations and higher mRNA expression levels of efflux pumps and their global activators than did reference strains. The genes encoding AcrR (local AcrAB repressor) and MarR (MarA repressor) were disrupted by insertion of the transposon IS3-IS629 and a frameshift mutation, respectively. A series of mutants derived from HUE1 were obtained by plasmid curing and gene knockout using homologous recombination. Compared to the MICs of the parent strain HUE1, the fluoroquinolone MICs of these mutants indicated that qnrS1, oqxAB, acrAB, acrF, acrD, mdtK, mdfA, and tolC contributed to the reduced susceptibility to fluoroquinolone in HUE1. Therefore, fluoroquinolone resistance in HUE1 is caused by concomitant acquisition of QnrS1 and OqxAB and overexpression of AcrAB–TolC and other chromosome-encoded efflux pumps. Thus, we have demonstrated that QRDR mutations are not absolutely necessary for acquiring fluoroquinolone resistance in E. coli.

The Role of Flies in Spreading the Extended-Spectrum β-lactamase Gene from Cattle

The spreading of antimicrobial-resistant bacteria and genes from food-producing animals to humans has been a subject of increasing concern. To clarify the role of flies in spreading the extended-spectrum β-lactamase (ESBL) gene from food-producing animals to humans, we isolated and characterized a third-generation cephalosporin-resistant Escherichia coli strain from flies and cattle feces from a cattle barn. Cephalosporin-resistant strains were isolated from 14.3% (13/91) of houseflies, 10.3% (7/68) of false stable flies, and 7.5% (7/93) of cattle feces. Twenty-seven cephalosporin-resistant strains were tested for the presence of antimicrobial resistance genes. Of the 27 samples, 22 isolates from 11 houseflies, 5 false stable flies, and 6 cattle feces samples harbored the blaCTX-M-15 gene. All blaCTX-M-15-harboring isolates belonged to phylogenetic group D and the ST38 clonal group. Analysis of pulsed-field gel electrophoresis showed that these isolates were divided into two clusters, indicating that flies carried several of the same clones that were detected in cattle feces. All blaCTX-M-15 gene-harboring plasmids were transferable and were members of incompatibility group FIB. These results suggest that transferable plasmids encoding ESBL were prevalent among flies and cattle. As vectors, flies may play an important role in spreading ESBL-producing bacteria from food-producing animals to humans.

Prevalence of Fluoroquinolone-Resistant Escherichia coli O25:H4-ST131 (CTX-M-15-Nonproducing) Strains Isolated in Japan

Background: Fluoroquinolone-resistant and extended-spectrum β-lactamase (ESBL)-carrying multidrug-resistant Escherichia coli have become severely problematic. In particular, a lineage of multilocus sequence-type ST131 which belongs to O25:H4 and carries ESBL CTX-M-15 has spread worldwide. Methods: Fluoroquinolone-resistant E. coli strains were isolated from various clinical specimens in a commercial clinical laboratory in 2008 and 2009 in Hokkaido Prefecture, Japan. Results: Among 478 clinical isolates, 112 strains (23.4%) showed levofloxacin (LVX) resistance. About 80% of the fluoroquinolone-resistant strains (88 strains) showed common features, namely O25:H4-ST131, phylogenetic group B and the same mutation pattern in quinolone resistance-determining regions. Pulsed field gel electrophoresis patterns suggested numerous lineages of O25:H4-ST131. The fluoroquinolone-resistant strains, including strains of O25:H4-ST131 and other types, more frequently shared CTX-type ESBL genes than did fluoroquinolone-susceptible strains. The ESBL genes fell into the CTX-M-9 and CTX-M-2 groups. CTX-M-15 (CTX-M-1 group) was not found among any of the strains isolated in this study. Sitafloxacin showed markedly potent activity against E. coli isolates compared with LVX, ciprofloxacin and ulifloxacin. Conclusion: The most prevalent fluoroquinolone-resistant strains of E. coli isolated in Hokkaido Prefecture, Japan, are O25:H4-ST131. However, similar to other areas of Japan, the ST131 clones represent distinct lineages from the general worldwide dispersal of multidrug-resistant clones which carry CTX-M-15.

A Fluoroquinolone-Resistant Escherichia coli Clinical Isolate without Quinolone Resistance-Determining Region Mutations Found in Japan

Fluoroquinolones are broad-spectrum and highly bactericidal antimicrobials agents that are used to treat various bacterial infections. Escherichia coli infections, especially in the urinary tract, are frequently treated with fluoroquinolones, and fluoroquinolone resistance has increased in the

Association of veterinary third-generation cephalosporin use with the risk of emergence of extended-spectrum-cephalosporin resistance in Escherichia coli from dairy cattle in Japan.

The use of extended-spectrum cephalosporins in food animals has been suggested to increase the risk of spread of Enterobacteriaceae carrying extended-spectrum β-lactamases to humans. However, evidence that selection of extended-spectrum cephalosporin–resistant bacteria owing to the actual veterinary use of these drugs according to criteria established in cattle has not been demonstrated. In this study, we investigated the natural occurrence of cephalosporin-resistant Escherichia coli in dairy cattle following clinical application of ceftiofur. E. coli isolates were obtained from rectal samples of treated and untreated cattle (n = 20/group) cultured on deoxycholate-hydrogen sulfide-lactose agar in the presence or absence of ceftiofur. Eleven cefazoline-resistant isolates were obtained from two of the ceftiofur-treated cattle; no cefazoline-resistant isolates were found in untreated cattle. The cefazoline-resistant isolates had mutations in the chromosomal ampC promoter region and remained susceptible to ceftiofur. Eighteen extended-spectrum cephalosporin–resistant isolates from two ceftiofur-treated cows were obtained on ceftiofur-supplemented agar; no extended-spectrum cephalosporin–resistant isolates were obtained from untreated cattle. These extended-spectrum cephalosporin–resistant isolates possessed plasmid-mediated β-lactamase genes, including bla CTX-M-2 (9 isolates), bla CTX-M-14 (8 isolates), or bla CMY-2 (1 isolate); isolates possessing bla CTX-M-2 and bla CTX-M-14 were clonally related. These genes were located on self-transmissible plasmids. Our results suggest that appropriate veterinary use of ceftiofur did not trigger growth extended-spectrum cephalosporin–resistant E. coli in the bovine rectal flora; however, ceftiofur selection in vitro suggested that additional ceftiofur exposure enhanced selection for specific extended-spectrum cephalosporin–resistant β-lactamase-expressing E. coli clones

Clarithromycin prevents human respiratory syncytial virus-induced airway epithelial responses by modulating activation of interferon regulatory factor-3.

Macrolide antibiotics exert immunomodulatory activity by reducing pro-inflammatory cytokine production by airway epithelial cells, fibroblasts, vascular endothelial cells, and immune cells. However, the underlying mechanism of action remains unclear. Here, we examined the effect of clarithromycin (CAM) on pro-inflammatory cytokine production, including interferons (IFNs), by primary human nasal epithelial cells and lung epithelial cell lines (A549 and BEAS-2B cells) after stimulation by Toll-like receptor (TLR) and RIG-I-like receptor (RLR) agonists and after infection by human respiratory syncytial virus (RSV). CAM treatment led to a significant reduction in poly I:C- and RSV-mediated IL-8, CCL5, IFN-β and -λ production. Furthermore, IFN-β promoter activity (activated by poly I:C and RSV infection) was significantly reduced after treatment with CAM. CAM also inhibited IRF-3 dimerization and subsequent translocation to the nucleus. We conclude that CAM acts a crucial modulator of the innate immune response, particularly IFN production, by modulating IRF-3 dimerization and subsequent translocation to the nucleus of airway epithelial cells. This newly identified immunomodulatory action of CAM will facilitate the discovery of new macrolides with an anti-inflammatory role.

Mechanism of Reduced Susceptibility to Fosfomycin in Escherichia coli Clinical Isolates

In recent years, multidrug resistance of Escherichia coli has become a serious problem. However, resistance to fosfomycin (FOM) has been low. We screened E. coli clinical isolates with reduced susceptibility to FOM and characterized molecular mechanisms of resistance and reduced susceptibility of these strains. Ten strains showing reduced FOM susceptibility (MIC ≥ 8 μg/mL) in 211 clinical isolates were found and examined. Acquisition of genes encoding FOM-modifying enzyme genes (fos genes) and mutations in murA that underlie high resistance to FOM were not observed. We examined ability of FOM incorporation via glucose-6-phosphate (G6P) transporter and sn-glycerol-3-phosphate transporter. In ten strains, nine showed lack of growth on M9 minimum salt agar supplemented with G6P. Eight of the ten strains showed fluctuated induction by G6P of uhpT that encodes G6P transporter expression. Nucleotide sequences of the uhpT, uhpA, glpT, ptsI, and cyaA shared several deletions and amino acid mutations in the nine strains with lack of growth on G6P-supplemented M9 agar. In conclusion, reduction of uhpT function is largely responsible for the reduced sensitivity to FOM in clinical isolates that have not acquired FOM-modifying genes or mutations in murA. However, there are a few strains whose mechanisms of reduced susceptibility to FOM are still unclear.

Phylogenetic association of fluoroquinolone and cephalosporin resistance of D-O1-ST648 Escherichia coli carrying blaCMY-2 from faecal samples of dogs in Japan.

This study aimed to investigate the genetic association between fluoroquinolone (FQ) and/or cephalosporin (CEP) resistance in Escherichia coli isolates from dogs, and the risk to human health. We characterized E. coli clinical isolates, derived from faecal samples of dogs attending veterinary hospitals, using phylogenetic grouping, determination of virulence factor (VF) prevalence, multilocus sequence typing (MLST) and O serotyping. The D group was the dominant phylogenetic group among strains resistant to FQ and/or CEP. In contrast, the dominant group among susceptible strains was group B2. Group D strains showed a significantly higher prevalence of VFs than strains belonging to groups A and B1, and were resistant to significantly more antimicrobials than group B2 strains. The phylogenetic distribution of FQ-CEP-resistant E. coli groups (FQ-CEPRECs) and FQ-resistant groups was significantly correlated (r = 0.98), but FQ-CEPRECs and CEP-resistant E. coli groups were not correlated (r = 0.58). Data from PFGE, O serotype and MLST analyses indicated that the majority of FQ-resistant strains derived from a particular lineage of phylogenetic group D: serotype O1 and sequence type (ST) 648. Some D-O1-ST648 strains carried blaCMY-2, showed multidrug resistance and possessed a higher prevalence of the VFs kspMT, ompT and PAI compared with other group D strains. Our data indicate that the emergence of FQ-CEP-resistant E. coli is based primarily on FQ-resistant E. coli. Moreover, as strains of the D-O1-ST648 lineage have been found in clinical isolates derived from humans at a relatively high frequency, our findings indicate that the spreading of D-O1-ST648 strains may cause serious difficulties in both veterinary and human clinical fields in the future.

Novel antimicrobial activities of a peptide derived from a Japanese soybean fermented food, Natto, against Streptococcus pneumoniae and Bacillus subtilis group strains

We recently isolated a tumoricidal peptide from Natto, a Japanese traditional fermented food. In the present study, antimicrobial activity of the Natto peptide was examined. The peptide consisted of 45 amino acid residues, and its structure was predicted to be rich in α-helix. It excreted antimicrobial activity only against Streptococcus pneumoniae and Bacillus subtilis group (B. subtilis, Bacillus pumilus, and Bacillus licheniformis). Lesser antimicrobial activity was observed for Streptococcus species other than S. pneumoniae. Hemolysate or hemin was required for the antimicrobial activity of the peptide. The Natto peptide damages the cell membrane of B. subtilis. On the other hand, chain morphology was induced in S. pneumoniae, which is naturally diplococcus, during the early phases of the Natto peptide treatment; following that the cells were rapidly lysed. This suggested that the Natto peptide displayed a novel narrow spectrum of bactericidal activity and inhibited cell separation during cell division of S. pneumoniae.

Mumps Virus Induces Protein-Kinase-R-Dependent Stress Granules, Partly Suppressing Type III Interferon Production

Stress granules (SGs) are cytoplasmic granular aggregations that are induced by cellular stress, including viral infection. SGs have opposing antiviral and proviral roles, which depend on virus species. The exact function of SGs during viral infection is not fully understood. Here, we showed that mumps virus (MuV) induced SGs depending on activation of protein kinase R (PKR). MuV infection strongly induced interferon (IFN)-λ1, 2 and 3, and IFN-β through activation of IFN regulatory factor 3 (IRF3) via retinoic acid inducible gene-I (RIG-I) and the mitochondrial antiviral signaling (MAVS) pathway. MuV-induced IFNs were strongly upregulated in PKR-knockdown cells. MuV-induced SG formation was suppressed by knockdown of PKR and SG marker proteins, Ras-GTPase-activating protein SH3-domain-binding protein 1 and T-cell-restricted intracellular antigen-1, and significantly increased the levels of MuV-induced IFN-λ1. However, viral titer was not altered by suppression of SG formation. PKR was required for induction of SGs by MuV infection and regulated type III IFN (IFN-λ1) mRNA stability. MuV-induced SGs partly suppressed type III IFN production by MuV; however, the limited suppression was not sufficient to inhibit MuV replication in cell culture. Our results provide insight into the relationship between SGs and IFN production induced by MuV infection.