Toshio Chida

Molecular analysis of Clostridium difficile at a university teaching hospital in Japan: a shift in the predominant type over a five-year period

Clostridium difficile isolates recovered from patients admitted to a teaching hospital in Japan over a 5-year period were analyzed. Two molecular typing systems, PCR ribotyping and pulsed-field gel electrophoresis (PFGE) analysis, were used. Twenty-six PCR ribotypes were found among the 148 isolates. The predominant type at our hospital appeared to shift during the study period, from PCR ribotype a in 2000 (15/33, 45%) to PCR ribotype f in 2004 (18/28, 64%). By using PFGE with thiourea added to both the gel and running buffer, all 148 Clostridium difficile isolates were successfully classified into 37 types and 61 subtypes. The PCR ribotype f isolates were further classified into four types and 11 subtypes by PFGE. The PFGE patterns of the 11 subtypes differed from each other by only 1 to 4 bands, suggesting that these differences might reflect genetic changes during patient-to-patient transmission over the 5-year period analyzed, and that PCR ribotype f isolates might be outbreak-related. In addition, the PCR ribotype f was identical to the PCR ribotype designated smz, which is reported to have caused multiple outbreaks in Japan. These results confirmed that PCR ribotype f (type smz) has specific virulence or survival factors that make it more likely to cause nosocomial outbreaks at Japanese hospitals. PCR ribotype 027, which has been reported to have caused recent outbreaks in North America and Europe, was recovered from one patient in this study; however, this strain was community-acquired. Our findings emphasize the importance of monitoring specific strains to control and prevent nosocomial infection.

Suppression of human immunodeficiency virus type 1 replication in macrophages by commensal bacteria preferentially stimulating Toll-like receptor 4

Protection from primary human immunodeficiency virus type 1 (HIV-1) infection has not yet been accomplished by vaccines inducing HIV-1-specific acquired immunity. Nevertheless, it has been reported that a small subgroup of women remain resistant to HIV-1 infection under natural conditions. If similar conditions can be induced in uninfected individuals, it will contribute the first line of protection against HIV-1 infection, and also improve the effects of anti-HIV-1 vaccines. We reasoned that innate immunity may be involved in the resistance to HIV-1 infection, and investigated the effects of various Toll-like receptor (TLR) ligands and commensal bacteria on HIV-1 replication in macrophages, one of the initial targets of HIV-1 infection and also the main mediators of innate immunity. We established the HIV-1 reporter monocytic cell line, THP-1/NL4-3luc, which could be differentiated into macrophage-like cells in vitro. In these cells, stimulation of TLR3 and TLR4 by their ligands suppressed HIV-1 expression partly through type I interferon (IFN). Among the commensal bacteria tested, Escherichia coli, Veillonella parvula and Neisseria mucosa suppressed HIV-1 expression, whereas Lactobacillus acidophilus, Prevotella melaninogenica, P. bivia and Mycobacterium smegmatis enhanced it. The bacteria with suppressive effects preferentially stimulated TLR4, whereas the ones with enhancing effects stimulated TLR2. Neutralizing antibodies against TLR4 and IFN-α/β receptor abrogated bacterially mediated HIV-1 suppression. Suppressive effects of E. coli, V. parvula and N. mucosa on HIV-1 replication were reproducible in primary monocyte-derived macrophages following acute HIV-1 infection. These findings suggest that certain commensal bacteria preferentially stimulating TLR4 potentially produce local environments resistant to HIV-1 infection.

Cytotoxic Component(s) of Klebsiella oxytoca on HEp-2 Cells

A cytotoxic component(s) was detected in culture filtrates of Klebsiella oxytoca isolated from patients with antibiotic‐associated hemorrhagic colitis. Eleven of 12 isolates exhibited cytotoxicity on HEp‐2 cells. The cytotoxic activity of K. oxytoca strain MH43‐1 was stable for the treatment of 60 C for 30 min, but inactivated by the treatment of 100 C for 15 min. This cytotoxicity was not destroyed by the treatment with trypsin or pronase, and the component was filtrable through a membrane filter which cut off molecular weight 5,000.

The Complete DNA Sequence of the O Antigen Gene Region of Plesiomonas shigelloides Serotype O17 Which Is Identical to Shigella sonnei Form I Antigen

We cloned and determined the sequence of a DNA region of approximately 15‐kb containing the cluster of genes required for O17 antigen expression in the Escherichia coli K‐12 strain from the chromosome of Plesiomonas shigelloides serotype O17:H2 strain. The sequencing analysis revealed that the minimum essential region of the P. shigelloides O17 antigen gene cluster had a size of approximately 11.5‐kb and contained 9 contiguous open reading frames (ORFs), which were almost identical to the corresponding ORFs of Shigella sonnei form I antigen gene region, except for IS630 sequence, at the DNA as well as amino acid levels. The putative function of most of the ORFs could be determined on the basis of amino acid sequence similarities and characteristics. In addition, the G + C content of the P. shigelloides O17 antigen genes was lower than that of the chromosomal DNA of P. shigelloides and S. sonnei, suggesting that both P. shigelloides O17 and S. sonnei form I antigen genes had been derived from the same origin with a low G+C content.

Antimicrobial susceptibility and mechanism of quinolone resistance in Campylobacter jejuni strains isolated from diarrheal patients in a hospital in Tokyo

We determined the minimum inhibitory concentrations of six types of antimicrobial agents for 523 strains of Campylobacter jejuni that were isolated from diarrheal patients in a general hospital in Tokyo during the period between 2003 and 2005. It was revealed that 20.2%, 22.9%, 6.7%, and 0.6% of all the C. jejuni strains tested were resistant to ciprofloxacin (CPFX), nalidixic acid, ampicillin, and fosfomycin, respectively. All the strains were susceptible to clarithromycin and erythromycin. To elucidate the mechanism of quinolone resistance, in a total of 55 strains selected randomly, we carried out sequence determination and analysis of the quinolone-resistance determining regions (QRDRs) of their gyrA and gyrB genes. Amino-acid substitution at codon 86 (Thr → IIe) of GyrA was found in all the 37 CPFX-resistant strains. There was no amino-acid substitution in the QRDR of the gyrB gene. All of the genomic DNAs of these 55 strains showed distinct pulsed-field gel electrophoresis patterns. Taken together, these results suggested that the quinolone resistance of C. jejuni was attributable mainly to the mutation at codon 86 (Thr → IIe) in the QRDR of GyrA, and that this particular mutation and other silent mutations could be found not only in a certain clone of C. jejuni but also universally in a wide variety of strains.

Molecular characterizations of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida

To analyze the genetic mechanisms of carbapenem and ciprofloxacin resistance in clinical isolates of Pseudomonas putida, 27 clinical isolates (comprising 11 carbapenem- and ciprofloxacin-resistant strains, 13 carbapenem-resistant and ciprofloxacin-susceptible strains, and 3 carbapenem- and ciprofloxacin-susceptible strains) were collected from different patients. Carbapenem resistance was examined by polymerase chain reaction (PCR) and DNA sequencing for metallo-β-lactamase (MBL) and integrase genes (IntI-1 and IntI-3), and by reverse transcriptase-PCR (RT-PCR) for expression of the porin gene (oprD). Ciprofloxacin resistance was characterized by PCR and DNA sequencing for mutations in the quinoloneresistance determining regions of the gyrA and parC genes. The blaIMP-1 MBL and intI-1 and/or intI-3 genes were detected in all carbapenem-resistant strains, and decreased expression of the oprD gene as compared to carbapenemsusceptible strains was observed in several strains. All the 11 strains with ciprofloxacin minimal inhibitory concentrations (MICs) of ≥64 mg/l had substitution in GyrA (Thr83Ile), and one (ciprofloxacin MIC of 512 mg/l) of these strains also had substitution in ParC (Ser87Leu). Overproduction of the efflux pump was observed in 10 of the 11 ciprofloxacin-resistant strains. We concluded that the production of IMP-1 type MBL was the most critical factor in developing high-level resistance to carbapenems, and mutations in the target proteins and overproduction of the efflux pump synergistically contribute to the acquisition of high-level resistance to ciprofloxacin in clinical isolates of P. putida.

Chemical Analysis of Lipopolysaccharides of Shigella sonnei Form II Strains Expressed by Cloned Form I Antigen Genes

A compositional sugar analysis was carried out on lipopolysaccharide (LPS) from Shigella sonnei form II in which a plasmid with cloned form I antigen genes had been introduced. The recipient form II strains contained galactose, glucose, heptose, glucosamine, and 2‐keto‐3‐deoxyoctonic acid (KDO) (2: 3: 1: 2: 2) in its LPS, while the transformant form I LPS contained, besides these sugars, N‐acetyl‐L‐altrosaminouronic acid as an additional sugar constituent, which is known to be one of the antigenic determinants of form I antigen.

Moraxella catarrhalis Does Not Grow on Nutrient Agar without Sodium Chloride Supplementation

None of the 58 Moraxella catarrhalis strains grew on nutrient agar without sodium chloride supplementation, whereas 49 of 51 commensal Neisseria spp. strains tested did. Growth on nutrient agar without sodium chloride supplementation could be used for screening between M. catarrhalis and commensal Neisseria spp.