Yoshio Hosaka

CFE-1, a Novel Plasmid-Encoded AmpC β-Lactamase with an ampR Gene Originating from Citrobacter freundii

ABSTRACT A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded β-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a β-lactamase could be transferred by either conjugation or transformation with plasmid pKU601 into E. coli ML4947. The substrate and inhibition profiles of this enzyme resembled those of the AmpC β-lactamase. The resistance gene of pKU601, which was cloned and expressed in E. coli, proved to contain an open reading frame showing 99.8% DNA sequence identity with the ampC gene of Citrobacter freundii GC3. DNA sequence analysis also identified a gene upstream of ampC whose sequence was 99.0% identical to the ampR gene from C. freundii GC3. In addition, a fumarate operon (frdABCD) and an outer membrane lipoprotein (blc) surrounding the ampR-ampC genes in C. freundii were identified, and insertion sequence (IS26) elements were observed on both sides of the sequences identified (forming an IS26 composite transposon); these results confirm the evidence of the translocation of a β-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC β-lactamase, CFE-1, with an ampR gene derived from C. freundii.

Characterization of ermB Gene Transposition by Tn1545 and Tn917 in Macrolide-Resistant Streptococcus pneumoniae Isolates

ABSTRACT In Streptococcus pneumoniae, the ermB gene is carried by transposons, such as Tn917 and Tn1545. This study investigated the relationship between macrolide resistance and the presence of the ermB gene on Tn917 or Tn1545 in 84 Japanese pneumococcal isolates. Macrolide-resistant strains were classified into two groups as follows. Group 1 (19 strains) showed a tendency to high resistance to erythromycin (MIC at which 50% of isolates are inhibited, 4 mg/liter; MIC at which 90% of isolates are inhibited [MIC90], 128 mg/liter) but susceptibility to rokitamycin (MIC90, 1 mg/liter), with the ermB gene located on Tn1545. Group 2 (65 strains) showed a tendency to high resistance to both antibiotics (MIC90s for both erythromycin and rokitamycin, >128 mg/liter), with the ermB gene located on Tn917. There were no strains with constitutive macrolide resistance in either group. All of the strains in group 2 had a deletion in the promoter region of ermB and an insertion of the TAAA motif in the leader peptide. The results of pulsed-field gel electrophoresis and serogrouping showed that Tn1545 spread clonally while Tn917 spread both horizontally and clonally. In conclusion, in Japanese macrolide-resistant S. pneumoniae isolates, the ermB gene is carried and spread primarily by Tn917.

Antimicrobial host defense in the upper gastrointestinal tract

Background With the exception of fungi, microbial infections are rare in the oesophagus. Herein, we aimed to systematically assess the distribution and quantity of different antimicrobial host factors as well as, for thefirst time, functional mucosal antimicrobial activity in the upper gastrointestinal tract. Methods We investigated biopsies from the healthy oesophagus, three different locations in the stomach and the duodenum in a total of 12 individuals. Using real-time PCR with external standards, we compared absolute expression of mRNA encoding antimicrobial peptides including defensins, cathelicidin, bactericidal/permeability-increasing protein, psoriasin, and elafin. In addition, we performed immunostaining for human-&bgr;-defensin-1 (HBD1), elafin, and psoriasin. To test functional relevance, we assessed antimicrobial as well as antifungal activity of cationic extracts from biopsies against E. coli ATCC 25922 and a clinical isolate of Candida albicans. Results In contrast to HBD1 which was similarly expressed in all tissues, inducible &bgr;-defensins in the healthy oesophagus were much higher compared with the stomach and duodenum (for HBD2–4: P<0.01). In addition, the antiproteases elafin and psoriasin were also predominantly expressed in the oesophagus (P<0.005). In contrast, LL-37 and bactericidal/permeability-increasing protein were only marginally expressed. Cationic tissue extracts from both the oesophagus as well as the stomach showed potent antibacterial activity against E. coli. Consistent with susceptibility to Candida infection, the esophageal extracts exhibited a weaker activity against C. albicans (P=0.026). Conclusion Despite dominant expression of antimicrobial host peptides, oesophageal tissue shows a weakened potency to kill C. albicans. These data suggest an important role of yet unknown antimicrobial molecules.

Gastric Antimicrobial Peptides Fail to Eradicate Helicobacter pylori Infection Due to Selective Induction and Resistance

Background Although antimicrobial peptides protect mucus and mucosa from bacteria, Helicobacter pylori is able to colonize the gastric mucus. To clarify in which extend Helicobacter escapes the antimicrobial defense, we systematically assessed susceptibility and expression levels of different antimicrobial host factors in gastric mucosa with and without H. pylori infection. Materials and Methods We investigated the expression levels of HBD1 (gene name DEFB1), HBD2 (DEFB4A), HBD3 (DEFB103A), HBD4 (DEFB104A), LL37 (CAMP) and elafin (PI3) by real time PCR in gastric biopsy samples in a total of 20 controls versus 12 patients colonized with H. pylori. Immunostaining was performed for HBD2 and HBD3. We assessed antimicrobial susceptibility by flow cytometry, growth on blood agar, radial diffusion assay and electron microscopy. Results H. pylori infection was associated with increased gastric levels of the inducible defensin HBD2 and of the antiprotease elafin, whereas the expression levels of the constitutive defensin HBD1, inducible HBD3 and LL37 remained unchanged. HBD4 was not expressed in significant levels in gastric mucosa. H. pylori strains were resistant to the defensins HBD1 as well as to elafin, and strain specific minimally susceptible to HBD2, whereas HBD3 and LL37 killed all H. pylori strains effectively. We demonstrated the binding of HBD2 and LL37 on the surface of H. pylori cells. Comparing the antibacterial activity of extracts from H. pylori negative and positive biopsies, we found only a minimal killing against H. pylori that was not increased by the induction of HBD2 in H. pylori positive samples. Conclusion These data support the hypothesis that gastric H. pylori evades the host defense shield to allow colonization.

Characterization of pKU701, a 2.5-kb plasmid, in a Japanese Helicobacter pylori isolate.

A cryptic plasmid of Helicobacter pylori, pKU701 (accession number AB078638), was isolated and the complete nucleotide sequence was determined. No drug resistance properties were mediated by pKU701. The 2454b pKU701 sequence, which had a 38% content of G-C residues, generated one polypeptide from a single open reading frame (ORF1). Extensive sequence homology was evident between pKU701 and ORF1 of H. pylori plasmid pHPO100 (repA, 88.8% identity) as well as ORF3 of plasmid pHPS1 (repB, 80.2% identity), but pKU701 showed only 46.3% homology with ORF1 of plasmid pHPK255 (repA). Tandem direct repeats of a 33-bp segment were found in pKU701 outside ORF1, but there were no inverted repeat ends such as those found in typical insertion sequences. The ability of drug resistance plasmids to replicate in H. pylori is probably limited, so chromosomal mutation may be a more likely cause of resistance.

Use of the restriction enzyme EcoRI for pulsed-field gel electrophoretic analysis of Helicobacter pylori.

ABSTRACT Pulsed-field gel electrophoretic (PFGE) analysis of Helicobacter pylori isolates is not commonly employed because of the inability to compare the typing with other typing systems. We adapted the PFGE analysis for H. pylori by using EcoRI and slightly modified our laboratory methods to improve the typing of isolates (typeability was 97%).