Molecular characterization of extraintestinal Escherichia coli isolates in Japan: relationship between sequence types and mutation patterns of quinolone resistance-determining regions analyzed by pyrosequencing.

Infection from fluoroquinolone-resistant Enterobacteriaceae is an increasing health problem worldwide. In the present study, we developed a pyrosequencing-based high-throughput method for analyzing the nucleotide sequence of the quinolone resistance-determining regions (QRDRs) of gyrA and parC. By using this method, we successfully determined the QRDR sequences of 139 out of 140 clinical Escherichia coli isolates, 28% of which were nonsusceptible to ciprofloxacin. Sequence results obtained by the pyrosequencing method were in complete agreement with those obtained by the Sanger method. All fluoroquinolone-resistant isolates (n = 35; 25%) contained mutations leading to three or four amino acid substitutions in the QRDRs. In contrast, all isolates lacking a mutation in the QRDR (n = 81; 57%) were susceptible to ciprofloxacin, levofloxacin, and nalidixic acid. The qnr determinants, namely, the qnrA, qnrB, and qnrS genes, were not detected in the isolates, and the aac(6')-Ib-cr gene was detected in 2 (1.4%) of the isolates. Multilocus sequence typing of 34 randomly selected isolates revealed that sequence type 131 (ST131) (n = 7; 20%) is the most prevalent lineage and is significantly resistant to quinolones (P < 0.01). The genetic background of quinolone-susceptible isolates seemed more diverse, and interestingly, neighboring STs of ST131 in the phylogenetic tree were all susceptible to ciprofloxacin. In conclusion, our investigation reveals the relationship between fluoroquinolone resistance caused by mutations of QRDRs and the population structure of clinical extraintestinal E. coli isolates. This high-throughput method for analyzing QRDR mutations by pyrosequencing is a powerful tool for epidemiological studies of fluoroquinolone resistance in bacteria.

Use of culture-independent analysis to reveal alteration of intestinal microflora by heat-killed Lactobacillus pentosus in a mouse model of endogenous sepsis.

In this study we evaluated alteration of intestinal microflora by terminal-restriction fragment length polymorphism (T-RFLP) analysis and quantitative PCR (qPCR) for specific microbes. The effects of orally administered heat-killed Lactobacillus pentosus strain b240 (HK-b240) in immunosuppressed mice with endogenous Pseudomonas aeruginosa sepsis was estimated. By T-RFLP analysis, 5 dominant operational taxonomic units (OTUs) including Bacteroides spp. (OTU460) and Lactobacillus spp. (OTU657) were consistently observed, irrespective of treatment, at all time points. A significantly higher population of segmented filamentous bacteria (SFB) was observed by qPCR after 3 weeks of HK-b240 administration; thereafter, the difference was not sustained during immunosuppression and progression of sepsis. Although not significant, Lactobacillus spp. accounted for a larger population in the HK-b240-treated group. In conclusion, this study demonstrated successful application of culture-independent assays for evaluating biological agents by detecting changes in microflora even if the protection was not sufficient to result in significant survival change.

Role of infected grandmothers in transmission of Helicobacter pylori to children in a Japanese rural town.

AIM: Although the prevalence of Helicobacter pylori (H. pylori) increases with age and the main period of acquisition is childhood, the route of transmission of H. pylori infection remains unclear. This study aims to evaluate the relationship between prevalence of children and grandparents. METHODS: A total of 838 consecutive children who attended the Urita clinic and whose blood was taken for work up were enrolled in the present study. They were 449 boys and 389 girls, with a mean age of 12.4 years. H. pylori serology of their family members who were living together in one house was picked up to analyse intra-familial clustering of H. pylori infection. The family members of these children consisted of 448 fathers, 597 mothers, 205 grandfathers, 361 grandmothers and 589 siblings. RESULTS: The seropositive rates of mothers, grandmother and siblings in seropositive children were significantly higher than those in seronegative children. H. pylori infection in mothers and grandmothers was a marked risk factor for infection in the index children. Larger family size was not a risk factor for H. pylori infection. In contrast, having an infected father or grandfather was not an independent predictor for children infection. CONCLUSIONS: Our data demonstrate that not only mother-to-child transmission but also grandmother-to-child transmission is an important mechanism for the spread of H. pylori in a three-generation household.

Prognostic significance of the serum phosphorus level and its relationship with other prognostic factors in multiple myeloma.

We studied the serum phosphorus (P) level of 110 patients with multiple myeloma (MM) (age range 42-83 years, median 62 years) and evaluated the relationship between that and other prognostic factors. Serum P level significantly correlated with the prognostic factors that are relevant to renal dysfunction: serum creatinine (P<0.00000001), serum beta2-microglobulin (P=0.00000088), serum uric acid (P=0.0000014), and corrected serum calcium (cCa P=0.000067). Although it also correlated with the percentage of plasma cells in bone marrow nucleated cells (BMPC%) and the hemoglobin (Hb) and leukocyte counts, the significance was less than for the other four prognostic factors. Serum creatinine, BMPC%, leukocyte count, serum uric acid, bone lesions, beta2-microglobulin, and serum cCa were all significantly higher and Hb significantly was lower in the MM patients with hyperphosphatemia (serum P>3.8 mg/dl). The survival time was significantly shorter in these patients (P=0.000087). Multivariate analysis (Cox's proportional hazards regression model) showed that the serum P level is a significant negative prognostic factor in MM patients.