Fumiko Kirikae

Detection of Multidrug Resistance in Mycobacterium tuberculosis

ABSTRACT We developed a DNA sequencing-based method to detect mutations in the genome of drug-resistant Mycobacterium tuberculosis. Drug resistance in M. tuberculosis is caused by mutations in restricted regions of the genome. Eight genome regions associated with drug resistance, including rpoB for rifampin (RIF), katG and the mabA (fabG1)-inhA promoter for isoniazid (INH), embB for ethambutol (EMB), pncA for pyrazinamide (PZA), rpsL and rrs for streptomycin (STR), and gyrA for levofloxacin, were amplified simultaneously by PCR, and the DNA sequences were determined. It took 6.5 h to complete all procedures. Among the 138 clinical isolates tested, 55 were resistant to at least one drug. Thirty-four of 38 INH-resistant isolates (89.5%), 28 of 28 RIF-resistant isolates (100%), 15 of 18 EMB-resistant isolates (83.3%), 18 of 30 STR-resistant isolates (60%), and 17 of 17 PZA-resistant isolates (100%) had mutations related to specific drug resistance. Eighteen of these mutations had not been reported previously. These novel mutations include one in rpoB, eight in katG, one in the mabA-inhA regulatory region, two in embB, five in pncA, and one in rrs. Escherichia coli isolates expressing individually five of the eight katG mutations showed loss of catalase and INH oxidation activities, and isolates carrying any of the five pncA mutations showed no pyrazinamidase activity, indicating that these mutations are associated with INH and PZA resistance, respectively. Our sequencing-based method was also useful for testing sputa from tuberculosis patients and for screening of mutations in Mycobacterium bovis. In conclusion, our new method is useful for rapid detection of multiple-drug-resistant M. tuberculosis and for identifying novel mutations in drug-resistant M. tuberculosis.

Polymorphisms of interferon-inducible genes OAS-1 and MxA associated with SARS in the Vietnamese population

Abstract We hypothesized that host antiviral genes induced by type I interferons might affect the natural course of severe acute respiratory syndrome (SARS). We analyzed single nucleotide polymorphisms (SNPs) of 2′,5′-oligoadenylate synthetase 1 (OAS-1), myxovirus resistance-A (MxA), and double-stranded RNA-dependent protein kinase in 44 Vietnamese SARS patients with 103 controls. The G-allele of non-synonymous A/G SNP in exon 3 of OAS-1 gene showed association with SARS (p =0.0090). The G-allele in exon 3 of OAS-1 and the one in exon 6 were in strong linkage disequilibrium and both of them were associated with SARS infection. The GG genotype and G-allele of G/T SNP at position −88 in the MxA gene promoter were found more frequently in hypoxemic group than in non-hypoxemic group of SARS (p =0.0195). Our findings suggest that polymorphisms of two IFN-inducible genes OAS-1 and MxA might affect susceptibility to the disease and progression of SARS at each level.

Lipopolysaccharide (LPS)‐Induced Intra‐Uterine Fetal Death (IUFD) in Mice Is Principally Due to Maternal Cause but Not Fetal Sensitivity to LPS

The present study deals with whether lipopolysaccharide (LPS)‐induced intra‐uterine fetal death (IUFD) is related to LPS‐susceptibility of either mother or fetus and how LPS or LPS‐induced TNF causes IUFD. LPS‐susceptible C3H/HeN or ‐hypo‐susceptible C3H/HeJ pregnant mice and the mice mated reciprocally with these mice were used on days 14 to 16 of gestation for experiments. All of fetuses in pregnant C3H/HeN mice mated with either C3H/HeN males [HeN(HeN)] or C3H/HeJ males [HeN(HeJ)] were killed within 24 hr when injected intravenously (i.v.) with 50 or 100 μg of LPS. On the other hand, the majority of fetuses in C3H/HeJ females mated with either C3H/HeJ males [HeJ(HeJ)] or C3H/HeN males [HeJ(HeN)] survived when injected i.v. with even 400 μg of LPS. These findings indicate that LPS‐induced IUFD depends on the maternal LPS‐responsiveness. LPS injected into mothers could pass through placenta to fetuses, since an injection with 125I‐labeled LPS or IgG into pregnant mice resulted in considerable levels of radioactivity in fetuses as well as placenta. Cultured peritoneal macrophages derived from F1 mice of HeJ(HeN) or HeN(HeJ) mice, produced nitric oxide (NO) and tumor necrosis factor (TNF) in response to LPS, although the levels of NO and TNF were lower in comparison with those of C3H/HeN macrophage cultures, suggesting a possibility that the fetus as well as F1 cells might be responsible to LPS. LPS‐induced IUFD was not blocked by treatment with anti‐TNF antibody which inhibited LPS‐induced TNF production in pregnant females, although an injection of recombinant TNFα instead of LPS could induce IUFD, suggesting that the cause of IUFD cannot be attributed to mother‐derived TNF alone. The roles of LPS passed through placenta and LPS‐induced mediators on IUFD were discussed.

Development and Evaluation of a Line Probe Assay for Rapid Identification of pncA Mutations in Pyrazinamide-Resistant Mycobacterium tuberculosis Strains

ABSTRACT Resistance of Mycobacterium tuberculosis to pyrazinamide (PZA) derives mainly from mutations in the pncA gene. We developed a reverse hybridization-based line probe assay with oligonucleotide probes designed to detect mutations in pncA. The detection of PZA resistance was evaluated in 258 clinical isolates of M. tuberculosis. The sensitivity and specificity of PZA resistance obtained by this new assay were both 100%, consistent with the results of conventional PZA susceptibility testing. This assay can be used with sputa from tuberculosis patients. It appears to be reliable and widely applicable and, given its simplicity and rapid performance, will be a valuable tool for diagnostic use.

Evidence of immunostimulating lipoprotein existing in the natural lipoteichoic acid fraction

ABSTRACT Lipoteichoic acid (LTA) is a cell surface glycoconjugate of gram-positive bacteria and is reported to activate the innate immune system. We previously reported that purified LTA obtained from Enterococcus hirae has no immunostimulating activity, but a subfraction (Eh-AF) in an LTA fraction possesses activity. In this study, we established a mouse monoclonal antibody neutralizing the activity of Eh-AF and investigated its inhibitory effects. Monoclonal antibody (MAbEh1) was established by the immunization of BALB/c mice with Eh-AF, followed by hybridoma screening based on its inhibitory effect for the production of interleukin-6 (IL-6) induced by Eh-AF. MAbEh1 neutralized the production of IL-6 by LTA fraction from not only E. hirae but also Staphylococcus aureus, while it failed to block that of lipopolysaccharide, suggesting that the antibody recognized a common active structure(s) in LTA fractions. Synthetic glycolipids in these LTAs did not induce cytokine production, at least in our system. Interestingly, the antibody was found to inhibit the activity of immunostimulating synthetic lipopeptides, Pam3CSK4 and FSL-1. These results suggest that MAbEh1 neutralizes the activity of lipoprotein-like compounds which is responsible for the activity of the LTA fraction of E. hirae and S. aureus.

Structural significance of the acyl group at the C-10 position and the A ring of the taxane core of paclitaxel for inducing nitric oxide and tumor necrosis factor production by murine macrophages.

The antitumor agent, paclitaxel (Taxol®), mimics the actions of lipopolysaccharide (LPS) on murine macrophages (Mφ). Various synthetic analogs of paclitaxel were examined for their potencies to induce nitric oxide (NO) and tumor necrosis factor (TNF) production by murine peritoneal Mφ, and by human peripheral blood cells. The benzoyl group at C‐2, the hydroxy group at C‐7 and the acetyl group at C‐10 were found to be critically important sites to activate murine Mφ. Nor‐seco‐taxoid analogs lacking the A ring of the taxane core of paclitaxel were inactive, but inhibit paclitaxel‐ or LPS‐induced NO production. All the compounds tested did not induce TNF production by human blood cells.

Use of immunoglobulin enriched bovine colostrum against oral challenge with enterohaemorrhagic Escherichia coli O157:H7 in mice.

An immunoglobulin enriched bovine colostrum preparation, IMMULAC™ (New Zealand Dairy Group, Cambridge, New Zealand), contains antibodies against various bacterial antigens. In the present study, we investigated the protective effects of a commercial bovine colostrum preparation against infections with enterohaemorrhagic Escherichia coli (EHEC) O157:H7 in a murine model. Balb/c mice were given drinking water containing streptomycin for 3 days before and following oral challenge with streptomycin‐resistant EHEC O157:H7 strain (O157‐SMR). In mice pretreated with streptomycin, EHEC O157:H7 maintained stable levels of bacterial colonization in the intestines for the 3‐week experimental time period. Oral administration of colostrum resulted in rapid decrease in the bacteria numbers compared with administration of skim‐milk. Colostrum showed no direct in vitro bactericidal properties against either EHEC O157:H7. When sections prepared from cecum walls of streptomycin‐pretreated mice were incubated in vitro with EHEC O157:H7, the colostrum significantly prevented the attachment of the organisms to the sections when compared with skim‐milk. These results indicate that oral administration of bovine colostrum effectively protects mice against food‐borne infections by inhibiting bacterial attachment to the intestinal mucous membrane, colonization and growth in the intestinal tract.

Endotoxin contamination in fetal bovine serum and its influence on tumor necrosis factor production by macrophage-like cells J774.1 cultured in the presence of the serum

Trace amounts of endotoxin (lipopolysaccharide: LPS) are assumed to contaminate commercially available fetal bovine serum (FBS) for tissue or cell culture during the manufacturing process. We examined how cultured cells were affected by the endotoxin and how much endotoxin was in the FBS. Macrophage-like J774.1 cells maintained in RPMI1640 medium supplemented with FBS containing low doses of LPS for 15 or 21 days showed less TNF production in response to LPS than the cells maintained under LPS-free conditions, and the affected responses of the cells were not recovered by an additional 21 day culture in medium with LPS-free FBS. Concentrations of LPS in 40 lots of FBS obtained from 13 international manufacturers were measured by a highly sensitive and LPS-specific chromogenic limulus assay. The median of endotoxin levels in these lots was 46 ng/ml and the maximum was 38.8 ng/ml. Relatively higher concentrations of LPS (> 1 ng/ml) or lower levels (< 10 pg/ml) were found in 9 and 6 lots, respectively. The majority of the FBS lots contained various levels of (1-->3)-beta-D-glucan, and all lots contained high-density lipoprotein (HDL). However, no correlation was found between LPS and (1-->3)-beta-D-glucan or HDL level in the lots. Each FBS was added to macrophage-like J774.1 cells which had been maintained in LPS-free medium. Five lots of FBS induced significant TNF production by the cells without addition of any stimulant. These active 5 FBS contained relatively higher levels of LPS and pretreatment of the FBS with polymyxin B eliminated their ability to induce TNF production. No correlation was found between (1-->3)-beta-D-glucan levels in FBS and the TNF-inducing capability of FBS. These results show that considerable lots of FBS contain significant levels of LPS, which must affect cell culture.

Characterization of a Trinucleotide Repeat Sequence (CGG)5 and Potential Use in Restriction Fragment Length Polymorphism Typing of Mycobacterium tuberculosis

ABSTRACT The genomes of 28 bacterial strains, including mycobacterial species Mycobacterium tuberculosis and Mycobacterium bovis, were analyzed for the presence of a special class of microsatellite, that of trinucleotide repeat sequences (TRS). Results of a search of all 10 possible TRS motifs (i.e., CCT, CGG, CTG, GAA, GAT, GTA, GTC, GTG, GTT, and TAT) with five or more repeating units showed that (CGG)5 was highly represented within the genomic DNA of M. tuberculosis and M. bovis. Most of the (CGG)5 repeats in the genome were within the open reading frames of two large gene families encoding PE_PGRS and PPE proteins that have the motifs Pro-Glu (PE) and Pro-Pro-Glu (PPE). (CGG)5-probed Southern hybridization showed that some mycobacterial species, such as Mycobacterium marinum, Mycobacterium kansasii, and Mycobacterium szulgai, possess many copies of (CGG)5 in their genomes. Analysis of clinical isolates obtained from Tokyo and Warsaw with both IS6110 and (CGG)5 probes showed that there is an association between the fingerprinting patterns and the geographic origin of the isolates and that (CGG)5 fingerprinting patterns were relatively more stable than IS6110 patterns. The (CGG)5 repeat is a unique sequence for some mycobacterial species, and (CGG)5 fingerprinting can be used as an epidemiologic method for these species as well as IS6110 fingerprinting can. If these two fingerprinting methods are used together, the precise analysis of M. tuberculosis isolates will be accomplished. (CGG)5-based fingerprinting is particularly useful for M. tuberculosis isolates with few or no insertion elements and for the identification of other mycobacterial species when informative probes are lacking.

Non-standard biological activities of lipopolysaccharide from Helicobacter pylori

As assessed by the lipopolysaccharide (LPS)-specific chromogenic Limulus amoebocyte lysate (LAL) assay, Helicobacter pylori LPS extracted by the phenol-water procedure showed full potency to coagulate LAL, as did LPS from Salmonella minnesota and Escherichia coli. However, pretreatment of H. pylori LPS with polymyxin B, which easily destroys the endotoxic activity of enterobacterial LPS/lipid A, had little effect on the LAL coagulation activity, although the same treatment of E. coli LPS markedly diminished its activity. The H. pylori LPS induced very weak production of nitric oxide (NO) or tumour necrosis factor (TNF) by murine macrophages and TNF by human peripheral whole blood in vitro in comparison with S. minnesota LPS. These findings indicate that H. pylori LPS has the unique endotoxic characteristic of retaining full LAL coagulation activity with polymyxin B resistance, despite losing its endotoxic potencies such as the ability to induce NO and TNF production.