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.

Absence of adverse effects of oseltamivir on sleep: a double-blind, randomized study in healthy volunteers in Japan.

Influenza-associated neuropsychiatric symptoms include parasomnias such as sleepwalking which is a common sleep disturbance in childhood. Oseltamivir is a widely used antiviral drug for influenza. Recently, sleepwalking-like events have been reported in patients with influenza receiving oseltamivir. We investigated whether oseltamivir itself has effects on sleep. In this crossover study, healthy Japanese male volunteers were randomized into two treatment groups, each of which comprised two double-blind 4-day treatment periods. In the first period, group A received 75 mg oseltamivir (evening dose) on day 3, followed by 75 mg b.i.d. on day 4, and placebo in the second period. Group B received the same treatments, but in reverse order. Polysomnographic assessments were performed on all four nights of each treatment period. Pharmacokinetics were assessed during a 2-day open-label phase beginning on day 12. Thirty-one volunteers aged 20-24 years were enrolled. No volunteer had electroencephalographic abnormalities, and no abnormal behaviour was observed. Sleep parameters measured over the whole night and during early- and late sleep periods (first and last thirds of the night) were very similar for oseltamivir and placebo, although the amount of stage 2 sleep in the middle sleep period was slightly greater with oseltamivir. Pharmacokinetics for oseltamivir phosphate in groups A and B were very similar, but for oseltamivir carboxylate, AUC and C(max) values were higher in group B, probably because this group received oseltamivir on the evening of day 11. Oseltamivir was well tolerated. Oseltamivir did not produce clinically relevant changes on nocturnal polysomnographic variables in young Japanese men.

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 the first 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-beta-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 beta-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.

Investigation about the homogeneity of nasopharyngeal microflora at the different location of nasopharynx of children with acute otitis media.

OBJECTIVE: Nasopharynx is thought to be a very important site as becterial reservoir for acute otitis media (AOM). In this study, we investigated on the homogeneity of nasopharyngeal microflora at the different location of nasopharynx of children with AOM. METHODS: Thirty nasopharyngeal samples of 15 children with AOM, two samples harvested from both nostrils of each child, were cultured and analyzed by patterns of antibiotic susceptibility and pulsed-field gel electrophoresis analysis (PFGE). RESULTS: A total of 30 nasopharyngeal samples were cultured and 19 isolates of Streptococcus pneumoniae from 10 children (66.7%), 8 isolates of Haemophilus influenzae from 4 children (26.7%), and 12 isolates of Moraxella catarrhalis from 7 children (46.7%) were obtained. In all children except three, the nasopharyngeal microflora at right and left orifice of the eustachian tubes showed no obvious differences in the bacterial species and quantities. Furthermore, in children with the same species of were cultured from right and left orifice of the eustachian tubes at the same time, all nine couples of S. pneumoniae isolates, four couples of H. influenzae isolates, and five couples of M. catarrhalis isolates showed about the same susceptibility and PFGE patterns. CONCLUSION: These results suggest that the microflora at the different location of nasopharynx of children with AOM is almost homogeneous, irrespective of the clinical signs.

In vitro investigation of the indirect pathogenicity of beta-lactamase-producing microorganisms in the nasopharyngeal microflora.

OBJECTIVE: Nasopharyngeal microflora contains some beta-lactamase-producing microorganisms. In this study, we investigated in vitro on the indirect pathogenicities of Haemophilus parainfluenzae (H. parainfluenzae) and Moraxella catarrhalis (M. catarrhalis) against the antipneumococcul activities of some beta-lactams. METHODS: We compared the antimicrobial and bactericidal activities of beta-lactams against penicillin-susceptible Streptococcus pneumoniae (PSSP) with or without presence of the enzymes of two species of beta-lactamase-producing microorganisms, H. parainfluenzae and M. catarrhalis. RESULTS: When adding the enzymes extracted from these two beta-lactamase-producing microorganisms in equivalent amounts of 10(7) CFU/spot, the minimum inhibitory concentrations of amoxicillin (AMPC) and cefaclor (CCL) increased to >64 microg/mL. Even third-generation cephalosporins, such as cefditren (CDTR) and ceftriaxone (CTRX) showed marked increases with the enzyme of M. catarrhalis. In time-kill kinetics, same phenomenon was observed in mixed culture indicating the indirect pathogenicities of distinct bacteria, not extracted enzymes, on the cidal activities of beta-lactams against PSSP. Clavulanic acid (CVA)/AMPC, faropenem (FRPM), and imipenem (IPM) were not affected by these beta-lactamase-producing strains with respect to their activities against PSSP. However, these two beta-lactamase-producing strains and their enzymes did not show any significant influence on the antipneumococcul activities of beta-lactams, until the number of bacterial cells reached >10(8) CFU/mL. CONCLUSION: Our results suggest that these two species of beta-lactamase-producing microorganisms in the nasopharyngeal microflora may act as indirect pathogens on the antipneumococcul activities of beta-lactams with reflecting their substrate profiles, but this is dependent on sufficient amounts of enzyme for their influence as indirect pathogens.

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

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%).

Characterization of ermB gene transposition by Tn1545 and Tn917 in macrolide-resistant Streptococcus pneumoniae isolates.

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 [MIC(90)], 128 mg/liter) but susceptibility to rokitamycin (MIC(90), 1 mg/liter), with the ermB gene located on Tn1545. Group 2 (65 strains) showed a tendency to high resistance to both antibiotics (MIC(90)s 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.

CFE-1, a novel plasmid-encoded AmpC beta-lactamase with an ampR gene originating from Citrobacter freundii.

A clinical isolate of Escherichia coli from a patient in Japan, isolate KU6400, was found to produce a plasmid-encoded beta-lactamase that conferred resistance to extended-spectrum cephalosporins and cephamycins. Resistance arising from production of a beta-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 beta-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 beta-lactamase-associated gene region from the chromosome to a plasmid. Finally, we describe a novel plasmid-encoded AmpC beta-lactamase, CFE-1, with an ampR gene derived from C. freundii.

Inhibition of peptic ulcer relapse by ranitidine and ecabet independently of eradication of Helicobacter pylori: a prospective, controlled study versus ranitidine.

BACKGROUND/AIMS: The recent increase in resistant strains of Helicobacter pylori has become a serious problem. Ecabet is a novel anti-ulcer agent that acts directly on the gastric mucosa, has bactericidal activity, and inhibits adhesion of Helicobacter pylori to the gastric mucosa. These actions result from inhibition of urease and ATPase in Helicobacter pylori, a mechanism distinct from that of antibiotics. METHODOLOGY: Sixty-three patients positive for Helicobacter pylori who had been cured of gastric ulcers and duodenal ulcers were randomly assigned to receive maintenance therapy with ranitidine alone or a combination of ranitidine and ecabet. Ulcer relapse was studied in these patients. RESULTS: The cumulative relapse rates in the ranitidine group and the ecabet plus ranitidine group were respectively, 29.6% and 4.4% after 1 year of treatment and 66.1% and 13.0%, after 2 years. These differences were significant (p = 0.006). Multivariate analysis of factors potentially related to relapse showed that outcome was significantly related only to treatment (p = 0.020) and not to other characteristics, such as age, diagnosis, or sex. CONCLUSIONS: We conclude that maintenance therapy with a combination of ranitidine and ecabet prevents ulcer relapse in Helicobacter pylori-positive patients. Controlled studies comparing ulcer relapse rates between eradication treatment and maintenance therapy with ranitidine and ecabet are awaited.

Prominent induction of cyclin B1 in G2/M renal cancer cells with butyrolactone 1.

BACKGROUND: Butyrolactone 1 (BL) is a cyclin dependent kinase (CDK) inhibitor derived from Aspergillus terreus. None of the present drugs are effective for the treatment of renal cell carcinoma. The use of BL is expected to promote a new type therapy of renal cancer. METHODS: We investigated three human renal cancer cell lines: ACHN, OS-RC-2 and RCC10RGB, using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay and two-color flow cytometry. Simultaneous measurements of DNA content and cyclin expression allowed us to perform cell cycle specific analysis. Western blot analysis was performed using ACHN to represent cell lines. RESULTS: BL inhibited cell proliferation and caused cell accumulation at G2/M phase associated with the emergence of the third peak. Moreover, BL induced cyclin B1 over-expression in G2/M cells. These changes were quite definite, whereas cyclins D1, E and A showed no changes at all. Cyclin B1 accumulation was confirmed by western blot analysis. The chronological observation revealed that the emergence of the third peak preceded the regression of the increased cyclin B1 positive G2/M cells. These results suggested that BL accelerated cyclin B1 accumulation in G2/M cells, which then shifted to G1 phase without cell division. New G1 cells started DNA synthesis most likely as endoreduplication to form the third peak and the mechanism of cyclin B1 accumulation converted into down-regulation. CONCLUSION: BL induced significant cell kinetic interference in the tested human renal carcinoma cell lines. This might indicate the possibility of a new medical treatment modality for renal cancer.

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.

Power Doppler ultrasonography-directed prostate biopsy in men with elevated serum PSA levels: an evaluation of the clinical utility and limitations.

OBJECTIVES: To determine the utility and limitations of power Doppler ultrasonography (PDU)-directed prostate biopsy in patients with elevated serum prostate-specific antigen (PSA) levels. METHODS: A total of 108 men (mean age 67.7 years, range 50 to 86) with serum PSA levels of greater than 4.0 ng/mL were assessed using digital rectal examination (DRE), gray-scale transrectal ultrasonography (TRUS), and PDU. Prostate vasculature identified on PDU was judged using a grading system. Subsequently, these patients underwent systematic six-core transperineal biopsy and additional biopsies for positive sites on DRE, gray-scale TRUS, and PDU. RESULTS: A hypervascular site and prostate cancer on PDU was identified in 43 (40%) and 40 (37%) cases, respectively. PDU-directed and systematic six-core biopsies could independently detect 36 and 30 cancer cases, respectively. The sensitivity of PDU for cancer detection was 90%, specificity 90%, positive predictive value 84%, negative predictive value 94%, and accuracy 90%. High test performance was also observed in 53 cases with serum PSA levels of 4.1 to 10 ng/mL (sensitivity 77%, specificity 88%, positive predictive value 67%, negative predictive value 92%, and accuracy 85%). These values were superior or comparable to those of DRE and gray-scale TRUS. Inflammatory reactions and prostatic calculi were probable major causes of false-positive and false-negative results on PDU, respectively. CONCLUSIONS: PDU can identify appropriate sites for biopsy and improve the cancer detection rate. However, PDU-directed biopsy does not appear to identify prostate cancer with sufficient accuracy to omit the systematic biopsy, and combined use of these methods should be preferable.

Helicobacter pylori may survive ampicillin treatment in the remnant stomach.

Helicobacter pylori (H. pylori) is a Gram-negative curved rod-like or spiral bacterium that chronically infects the human gastric mucosa, and is a major risk factor for gastritis, gastric and duodenal ulcer and adenocarcinoma of the stomach. After partial gastrectomy, some patients may have persistent H. pylori infection for five years or more. In this study, we detected three bacteria, i.e., Klebsiella pneumoniae, Enterobacter aerogenes, and Escherichia coli, in the gastric juice of patients with a remnant stomach. Some of these bacteria produced beta-lactamase. These findings are potentially important since such bacteria could provide H. pylori with the chance to acquire drug resistance and to transfer drug resistance genes. This could be one reason why H. pylori is difficult to eradicate in the remnant stomach.

[Identification and classification of beta-lactamases in clinical isolates of bacteria].

Emergence of bacterial resistance has rendered ineffective a number of previously valuable antibiotic treatments and now threatens the effectiveness of others. beta-Lactam resistance is no longer predominantly a hospital-treated problem; it has now become an important issue in community medicine. More than 100-beta-lactamases have been identified and classified according to their structure, substrate specificity, and whether they are chromosomal or plasmid-mediated. beta-Lactamase production is rare among Gram-positive pathogens, important exceptions being Staphylococcus aureus and Enterococcus faecalis. By contrast, many Gram-negative pathogens are beta-lactamase-positive; inducible and/or hyper-productive strains are particularly challenging in the clinical setting. Surveillance programs have shown that, in general, beta-lactam resistance is on the increase, and that the plasmid-mediated beta-lactamase have developed rapidly over past decade such as ESBLs and carbapenemases.