Novel roles of osteopontin and CXC chemokine ligand 7 in the defence against mycobacterial infection.

Granulocyte-macrophage colony-stimulating factor (GM-CSF)-induced human monocyte-derived macrophage (GM-Mphi) or macrophage CSF (M-CSF)-induced human monocyte-derived Mphi (M-Mphi) are distinct in terms of the resistance to Mycobacterium tuberculosis. To elucidate the role of molecules involved in the functional differences between these Mphis, we investigated the gene expression profiles using microarray. After culture of CD14+ monocytes with CSFs, Mphis were cultured with or without bacillus Calmette-Guérin (BCG) (GM-Mphi-BCG and M-Mphi-BCG). The gene expression profiles from these cells were compared. Chemokines highly expressed in M-Mphis were selected and evaluated for anti-mycobacterial activity and superoxide production. FN1 and FCGR2B were the most up-regulated genes in GM-Mphi and M-Mphi, respectively. After stimulation with BCG, three chemokine genes (Osteopontin (SPP1), CXC chemokine ligand 7 (CXCL7) and CC chemokine ligand 11 (CCL11)) were highly expressed in M-Mphi-BCG when compared to those in GM-Mphi-BCG. A significantly increased resistance to M. tuberculosis H37Ra was observed after the stimulation of GM-Mphi with SPP1 or CXCL7. Superoxide production levels of SPP1- or CXCL7-stimulated GM-Mphis were higher than those of GM-Mphis without stimulation. These results indicate that both SPP1 and CXCL7 might have a role in the resistance against mycobacteria, at least in part, through augmenting reactive oxygen intermediate production in Mphis.

The type of carbon dioxide absorbent has no relation to the concentration of carbon monoxide in the breathing circuit during low-flow isoflurane anaesthesia in smoking and non-smoking subjects.

The present study was designed to investigate the concentrations of carbon monoxide (CO) in the anaesthetic circuit and of arterial carboxyhaemoglobin (COHb) during low-flow isoflurane anaesthesia in smoking and non-smoking subjects using three kinds of cardon dioxide (CO2) absorbent. Thirty smoking and 30 non-smoking subjects were selected for this study, and these two groups were each divided into three groups according to the type of CO2 absorbent used (Wakolime A, Drägersorb Free, and Amsorb). Anaesthesia was maintained with 1.0% isoflurane and nitrous oxide (1. 0 l min(-1))/oxygen (1.0 l min(-1)). Concentrations of CO in the inspired breathing circuit and concentrations of arterial COHb were measured at 0, 1, 2, 3, and 4 hours after exposure to isoflurane. In the smoking groups there were no significant differences in CO concentrations in the circuit between the groups and the CO concentrations did not change significantly during the study period. There were also no significant differences in the arterial COHb values between the groups and the COHb concentrations remained constant. There was a significant linear correlation between the concentrations of CO and COHb (r=0.86, n =30, P<0.001). In the non-smoking groups all of the parameters remained constant at low levels that were independent of the type of CO2 absorbents tested. The major source for increased intraoperative CO exposure is related to the patient's smoking status, and the type of CO2 absorbent used has no relation to an increase in CO concentration in the breathing circuit.

Shifting serotypes, plasmid profile analysis and antimicrobial resistance pattern of shigellae strains isolated from Kolkata, India during 1995-2000.

One hundred and sixty-six shigellae strains, isolated from stool samples of paediatric patients (< 5 years old) at a Childrens' Hospital in Kolkata, India during the period of 1995-2000 were examined for serotyping, drug resistance pattern and plasmid profiles. Sh. flexneri (58 %) was found to be commonest isolate of total shigellae, followed by Sh. sonnei (28 %), Sh. boydii (9%) and Sh. dysenteriae (5%). This profile of species was in sharp contrast to the picture obtained before 1995, when Sh. dysenteriae 1 predominated over Sh. flexneri. In Sh. flexneri strains, Sh. flexneri 2a (35%) was the most prevalent serotype, following Sh. flexneri 3a (31%), Sh. flexneri 6 (14%), Sh. flexneri 2b (11%) and Sh. flexneri 4 (9%). Resistance patterns of the strains to 12 commonly used antimicrobial agents and minimum inhibitory concentrations (MICs) of the antibiotics were also tested. All strains were found uniformly susceptible to norfloxacin, but more than 90% strains were resistant to tetracycline, co-trimoxazole and 67% strains were resistant to ampicillin. Resistance to amoxicillin, chloramphenicol and nalidixic acid was found in 55% (range 45-74%), 46% (range 40-60%) and 29% (range 15-40%) strains respectively. Overall, shigellae strains showed statistically significant increase in resistance against tetracycline, nalidixic acid and furazolidone (P < 0.05) over the years of this study. This indicates decreased efficacy of furazolidone, cotrimoxazole and nalidixic acid for the empirical treatment of shigellosis in Kolkata. Although a few strains showed intermediate susceptibility to ciprofloxacin (4%) and cefotaxime (10%) by disk diffusion test, but the MICs of those antibiotics were within the normal limits. Almost 57% of the strains were resistant to four or more drugs with high MICs of the antibiotics. Plasmid profile analysis revealed presence of large plasmid of 220 kb in majority of the strains except in Sh. sonnei and a correlation between presence of smaller plasmids and shigellae serotypes. Hence this study reports epidemiological change of shigellae species in Kolkata, India with regard to serotypes and antibiotic resistance patterns.

Toxicity of Shiga toxin 1 in the central nervous system of rabbits.

The action of Shiga toxin (Stx) on the central nervous system was examined in rabbits. Intravenous Stx1 was 44 times more lethal than Stx2 and acted more rapidly than Stx2. However, Stx1 accumulated more slowly in the cerebrospinal fluid than did Stx2. Magnetic resonance imaging demonstrated a predominance of Stx1-dependent lesions in the spinal cord. Pretreatment of the animals with anti-Stx1 antiserum intravenously completely protected against both development of brain lesions and mortality.

Delayed onset of systemic bacterial dissemination and subsequent death in mice injected intramuscularly with Streptococcus pyogenes strains.

Streptococcus pyogenes causes severe invasive diseases in humans, including necrotizing fasciitis, sepsis, and streptococcal toxic shock syndrome (STSS). We found that mice infected intramuscularly (i.m.) with S. pyogenes strains developed bacteremia and subsequent sudden death after at least 10 days of a convalescent period. Mostly, it occurred more than 21 days after muscle infection. We provisionally designate this phenomenon as "delayed death." Just after muscle infection, all the mice lost weight and activity, but recovered completely within 3 days. They had kept good activity and a fine coat of fur till one or two days before their death. Some of the dead mice were found to have soft-tissue necrosis. There was no correlation between the virulence leading to the delayed death and the severity of diseases from which strains were isolated. It was also found that the production of neither streptococcal pyrogenic exotoxin (SPE) A nor B correlated to the virulence leading to delayed death. The bacteria obtained from the organs of the mice with delayed death expressed capsule. We suggest that the mice with delayed onset of systemic bacterial dissemination and subsequent death after muscle infection with S. pyogenes are the animal models of STSS, because the pathophysiology is extremely similar to that of human STSS.

Subtyping of Haemophilus influenzae strains by pulsed-field gel electrophoresis.

A total of 200 isolates of Haemophilus influenzae were analyzed by serotyping, biotyping, and pulsed-field gel electrophoresis (PFGE). A total of 178 epidemiologically unrelated strains of H. influenzae demonstrated a variety of genome patterns by PFGE, and 165 genotypes were thus obtained in this study. PFGE typing proved to have a much stronger discriminatory power than either serotyping or biotyping. Six serotype b strains were all classified into discrete genotypes. A PFGE analysis of 18 strains obtained from the nasopharynx, blood, and cerebrospinal fluid of patients with meningitis also supported the hypothesis that invasive H. influenzae disseminates from the nasopharynx to the bloodstream and then subsequently to other body sites. PFGE typing of 10 other strains isolated from household contacts of patients with H. influenzae infection revealed that the strain that caused the H. influenzae infection often colonized the nasopharynges of household contacts. Our findings suggest that PFGE analysis is useful for the epidemiological study of H. influenzae infection, even when the invasive disease is caused by serotype b strains.

Isolation and characterization of rugose form of Vibrio cholerae O139 strain MO10.

An extracellular exopolysaccharide (slime) is produced by Vibrio cholerae O139 MO10 in response to nutrient starvation. The presence of this slime layer on the cell surface and its subsequent release have been shown to be associated with biofilm formation and the change from a normal smooth colony morphology to a rugose one. An immunoelectron microscopic examination demonstrated that there is an epitope common to the exopolysaccharide antigen of V. cholerae O1 and that of O139 MO10.

Vibrio cholerae O1 strain TSI-4 produces the exopolysaccharide materials that determine colony morphology, stress resistance, and biofilm formation.

Vibrio cholerae O1 strain TSI-4 (El Tor, Ogawa) can shift to a rugose colony morphology from its normal translucent colony morphology in response to nutrient starvation. We have investigated differences between the rugose and translucent forms of V. cholerae O1 strain TSI-4. Electron microscopic examination of the rugose form of TSI-4 (TSI-4/R) revealed thick, electron-dense exopolysaccharide materials surrounding polycationic ferritin-stained cells, while the ferritin-stained material was absent around the translucent form of TSI-4 (TSI-4/T). The exopolysaccharide produced by V. cholerae TSI-4/R was found to have a composition of N-acetyl-D-glucosamine, D-mannose, 6-deoxy-D-galactose, and D-galactose (7.4:10.2:2.4:3.0). The expression of an amorphous exopolysaccharide promotes biofilm development under static culture conditions. Biofilm formation by the rugose strain was determined by scanning electron microscopy, and most of the surface of the film was colonized by actively dividing rod cells. The corresponding rugose and translucent strains were compared for stress resistance. By having exopolysaccharide materials, the rugose strains acquired resistance to osmotic and oxidative stress. Our data indicated that an exopolysaccharide material on the surface of the rugose strain promoted biofilm formation and resistance to the effects of two stressing agents.

Filamentous bacteriophages of Vibrio parahaemolyticus as a possible clue to genetic transmission.

We have previously reported the isolation and characterization of two filamentous bacteriophages of Vibrio parahaemolyticus, designated Vf12 and Vf33. In this study, to understand the potential of these phages as tools for genetic transmission, we investigated the gene structures of replicative-form (RF) DNAs of their genomes and the distribution of these DNAs on chromosomal and extrachromosomal DNAs. The 7,965-bp nucleotide sequences of Vf12 and Vf33 were determined. An analysis of the overall gene structures revealed that Vf12 and Vf33 had conserved regions and distinctive regions. The gene organization of their conserved regions was similar to that of CTX phage of Vibrio cholerae and coliphage Ff of Escherichia coli, while their distinctive regions were characteristic of Vf12 and Vf33 phage genomes. Southern blot hybridization testing revealed that the filamentous phage genomes integrated into chromosomal DNA of V. parahaemolyticus at the distinctive region of the phage genome and were also distributed on some plasmids of V. parahaemolyticus and total cellular DNAs of one Vibrio damsela and one nonagglutinable Vibrio strain tested. These results strongly suggest the possibilities of genetic interaction among the bacteriophage Vf12 and Vf33 genomes and chromosomal and plasmid-borne DNAs of V. parahaemolyticus strains and of genetic transmission among strains through these filamentous phages.

Entry and intracellular localization of Legionella dumoffii in Vero cells.

Organisms of some Legionella species are known to internalize and multiply within epithelial cell lines. During the study on interaction between Legionella spp. and Vero cells, we found that L. dumoffii Tex-KL (ATCC 33343) can enter into Vero cells approximately four to 20 times more often than five other strains of four species of legionella. The mode of entry between L. dumoffii Tex-KL and L. pneumophila Philadelphia-1 was compared and studied by treating Vero cells with reagents which inhibit phagocytosis and endocytosis. Monodansylcadaverine, cytochalasin D and nocodazol were used as inhibitors of receptor-mediated endocytosis, microfilament-dependent phagocytosis and polymerization of microtubules, respectively. The uptake of L. dumoffii Tex-KL required receptor-mediated endocytosis by Vero cells, while the uptake of L. pneumophila Philadelphia-1 used mainly microfilament-dependent phagocytosis. Polymerization of microtubules was necessary for Vero cells for the uptake of both strains of legionella. An electron microscopic examination revealed that some organisms of the L. dumoffii strain Tex-KL escaped from endosomal vacuoles into cytoplasm in the early stage of infection, and proliferated in the cytoplasm. At that period, most of the bacteria were surrounded by rough endoplasmic reticula. In contrast, L. pneumophila Philadelphia-1 proliferated only within ribosome-lined endosome. We suggest that L. dumoffii Tex-KL internalize and proliferate in Vero cells in a different way to L. pneumophila Philadelphia-1, and that there is a variety of the mode of interaction between Legionella spp. and epithelial cells.

Molecular analysis of kanamycin and viomycin resistance in Mycobacterium smegmatis by use of the conjugation system.

We examined the molecular mechanisms of resistance to kanamycin and viomycin in Mycobacterium smegmatis. All of the M. smegmatis strains with high-level kanamycin resistance had a nucleotide substitution from A to G at position 1389 of the 16S rRNA gene (rrs). This position is equivalent to position 1408 of Escherichia coli, and mutation at this position is known to cause aminoglycoside resistance. Mutations from G to A or G to T at position 1473 of the M. smegmatis rrs gene were found in viomycin-resistant mutants which had been designated vicB mutants in our earlier studies. Using the M. smegmatis conjugation system, we confirmed that these mutations indeed contributed to kanamycin and viomycin resistance, and kanamycin susceptibility was dominant over resistance in a heterogenomic strain. Additional experiments showed that three of four Mycobacterium tuberculosis strains with high-level kanamycin resistance had a mutation from A to G at position 1400, which was equivalent to position 1389 of M. smegmatis.

Marked inhibition of the intracellular multiplication of Legionella pneumophila in monocytes isolated from carriers of human T lymphotrophic virus type I.

Intracellular growth patterns of Legionella pneumophila were examined in monocytes obtained from carriers of human T-lymphotropic virus type I (HTLV-1) and controls who were HTLV-1 seronegative. All subjects were seronegative for antibodies against L. pneumophila. Bacterial growth was determined 0, 1, 2, and/or 3 days after infecting peripheral blood mononuclear cells (PBMCs) with the bacteria. The intracellular growth of L. pneumophila was markedly inhibited in HTLV-1 carriers compared with normal controls. When the lymphocytes were depleted from the HTLV-1 carrier PBMC cultures before infection, this inhibition was abolished. Inhibition reappeared, however, when the 72-h culture supernatants of PBMCs from HTLV-1 carriers were added to the lymphocyte-depleted cultures. Culture supernatants of infected and uninfected PBMCs from HTLV-1 carriers exhibited markedly increased levels of interferon-gamma (IFN-gamma) and tumor necrosis factor alpha (TNF-alpha) compared with the HTLV-1 seronegative controls. In the HTLV-1 carriers, IFN-gamma was produced by the CD4+ lymphocytes, whereas TNF-alpha was secreted by the monocytes. Addition of anti-IFN-gamma or anti-TNF-alpha antibodies to the HTLV-1 carrier PBMC cultures diminished the inhibition of intracellular growth of L. pneumophila. Results suggest that the monocytes are activated in HTLV-1 carriers. These findings may explain why an opportunistic infection by certain intracellular pathogens is rarely seen among HTLV-1 carriers.

Bacteriostatic activity of BCG/PPD-activated macrophages against Mycobacterium fortuitum does not involve reactive nitrogen or oxygen intermediates.

Mycobacteria preferentially reside in resident macrophages whereas activated macrophages are presumed to eliminate the bacteria effectively. The aim of the present study was to determine the antibacterial activities of resident and activated murine peritoneal macrophages against Mycobacterium fortuitum and the intracellular mechanisms involved. After phagocytosis M. fortuitum could not be killed by either BCG/PPD-activated and IFN-gamma-activated macrophages and resident macrophages. The mycobacteria did not multiply in BCG/PPD-activated macrophages and the rate of proliferation of M. fortuitum in IFN-gamma-activated macrophages was only slightly inhibited compared to that in resident macrophages. Experiments with selective inhibitors of the production of reactive nitrogen intermediates (RNI) and reactive oxygen intermediates (ROI) demonstrated that these factors are not essential for the mycobacteriostatic activity of BCG/PPD-activated macrophages. After phagocytosis of M. fortuitum, BCG/PPD-activated and IFN-gamma-activated macrophages produced substantial amounts of both RNI and ROI. No correlation was found between the levels of these intermediates and the proliferation of M. fortuitum in the macrophages. In conclusion, BCG/PPD-activated macrophages are bacteriostatic, but not bacteriocidal for M. fortuitum and the former does not involve reactive nitrogen and oxygen intermediates.

Protective effects of a leukotriene inhibitor and a leukotriene antagonist on endotoxin-induced mortality in carrageenan-pretreated mice.

The leukotrienes and tumor necrosis factor (TNF) play an important role in the pathophysiology of septic shock, in which hypotension, leukopenia, thrombocytopenia, and hemoconcentration are observed. This study was performed to examine the effects of a 5-lipoxygenase inhibitor (AA-861), a selective leukotriene receptor antagonist (ONO-1078), and a cyclooxygenase inhibitor (indomethacin) on endotoxin-induced mortality and TNF production in mice. Mice were injected intraperitoneally with carrageenan (5 mg per mouse), which we previously reported as an effective priming agent for lipopolysaccharide (LPS)-induced TNF production and mortality (M. Ogata, S. Yoshida, M. Kamochi, A. Shigematsu, and Y. Mizuguchi, Infect. Immun. 59:679-683, 1991). The indicated doses of AA-861, ONO-1078, indomethacin, or controls were administrated subcutaneously 30 min before LPS (50 micrograms per mouse) provocation. The mortality of mice was significantly decreased by pretreatment with AA-861 (P less than 0.001) or ONO-1078 (P less than 0.01) but not by pretreatment with indomethacin. The 50% lethal dose of LPS in the mice treated with dimethyl sulfoxide or ethanol was 32 or 33 micrograms, respectively, and it increased to 83 micrograms with AA-861 or 59 micrograms with ONO-1078, respectively. Neither AA-861 nor ONO-1078 suppressed LPS-induced TNF production in sera. Treatment with AA-861 significantly decreased the leukopenia and thrombocytopenia, and ONO-1078 significantly decreased the hemoconcentration and thrombocytopenia. The role of endogenous TNF was also examined in the carrageenan-pretreated mice. Treatment with 2 x 10(5) U of rabbit anti TNF-alpha antibody intravenously 2 h before LPS challenge significantly suppressed the LPS-induced TNF activity and decreased the mortality. Therefore, both leukotrienes and TNF play important roles in endotoxin-induced shock and mortality.

Gonorrhoea caused by a mixture of penicillinase-positive and -negative gonococci: a case report.

A 23-year-old unmarried man was infected with gonorrhoea from a prostitute in Fukuoka City and was treated with ampicillin which resulted in failure. By a rapid iodometric test it was found that the isolates before the treatment were penicillinase-negative. After the ampicillin treatment, however, the isolates turned to penicillinase-positive but were, in fact, a mixture of penicillinase-positive and penicillinase-negative gonococcal strains. Treatment by spectinomycin and doxycycline resulted in failure but the gonorrhoea was cured by ribostamycin.

Epidemiology and treatment of gonorrhoea caused by penicillinase-producing strains of Neisseria gonorrhoeae in Fukuoka, Japan.

Thirty strains of penicillinase-producing Neisseria gonorrhoeae (PPNG) were detected by a rapid iodometric method out of 206 strains isolated from patients with gonorrhoea between January and December 1981. Of the 30 patients, five of the nine women were prostitutes and 14 of the 21 men were infected by prostitutes in or around Fukuoka City, with the exception of one who was infected in Formosa. Treatment with ampicillin or amoxycillin resulted in failure whereas treatment with spectinomycin was successful.