Kiyotaka Yoshiie

Isolation of a Coxiella burnetii Strain That Has Low Virulence for Mice from a Patient with Acute Q Fever

Coxiella burnetii was isolated from a patient with Q fever. It could not be determined whether this was an imported case or an indigenous one. Identification of the isolate was made by electron microscopic morphology and the indirect fluorescent antibody test with convalescent‐phase serum from a Q fever patient having a known titer of antibody to C. burnetii. The isolated strain, named TK‐1, caused no symptoms in ddY and BALB/c mice except when the mice were treated with cyclophosphamide.

A subcloned human esophageal squamous cell carcinoma cell line with low thrombomodulin expression showed increased invasiveness compared with a high thrombomodulin-expressing clone – thrombomodulin as a possible candidate for an adhesion molecule of squamous cell carcinoma

Thrombomodulin (TM) is an endothelial cell surface glycoprotein which converts thrombin from a procoagulant protease to an anticoagulant. We have previously reported that TM is a useful marker for immunohistochemical diagnosis of angiogenic tumors and also have reported that TM is expressed on squamous cell carcinoma (SCC) of the human esophagus. In addition, the expression of TM is significantly decreased in metastatic foci in lymph nodes compared with that in primary lesions. In order to reveal the biological significance of TM in SCC, we subcloned and established two different cell lines, i.e. TM-high-expressing (TE3HTM) cells and TM-low-expressing (TE3LTM) cells, from a human SCC cell line, TE3, using fluorescence-activated cell sorter (FACS) and examined the biological characteristics of these variant cell lines. These tumor cells revealed very similar morphological figures in ordinary cultured conditions and showed almost equal growth rates under various cultured conditions. By the invasion assay of these tumor cells using matrigel, we found that TE3LTM cells showed significantly increased invasive ability compared with that of TE3HTM cells. Characteristic intercellular localization of TM and a different manner of invasiveness between TE3LTM cells and TE3HTM cells suggest that TM may act as a cell-to-cell interaction molecule.

Serological evidence that the Q fever agent (Coxiella burnetii) has spread widely among dairy cattle of Japan.

Serological examination of bovine and human sera for antibodies against Coxiella burnetii was carried out by the immunofluorescence technique. Twenty to 30%o of the cows examined were antibody‐positive. Sera from two veterinarians also had antibody against C. burnetii. These results suggest an increase in the number of infected cows with C. burnetii in Japan since 1954, and also imply the possibility of the prevalence of acute Q fever in the human population, which had been underestimated and undiagnosed for the last three decades.

A heat-stable component of Bartonella henselae upregulates intercellular adhesion molecule-1 expression on vascular endothelial cells

Bartonella henselae upregulated the expression of intercellular adhesion molecule‐1 (ICAM‐1) on human umbilical vein endothelial cells (HUVECs). The induction level of ICAM‐1 depended on the inoculation bacterial dose. ICAM‐1 expression began increasing 4 h after infection and reached a sustained peak beginning at 12 h after B. henselae infection; this time course was similar to that of lipopolysaccharide (LPS) of Escherichia coli. The stimulatory effect was abolished when live B. henselae were separated from HUVECs by a filter membrane. The nonpiliated strain, which is unable to invade endothelial cells, induced ICAM‐1 expression to the same extent as the piliated strain. Inactivation of B. henselae by ultraviolet (UV) irradiation, heat (56 °C, 30 min), or sonication did not alter its stimulatory activity. Polymyxin B, which strongly inhibited the effect of LPS, did not exert any influence on the stimulatory activity of B. henselae. Furthermore, the effect of sonicated B. henselae was not inhibited even by boiling, which was also the case with LPS. Our data suggest that some heat‐stable component of B. henselae binds to the endothelial cell surface, inducing ICAM‐1 expression. Though the participation of LPS could not be completely ruled out, we suppose that some unidentified heat‐stable proteins, lipids, or polysaccharides may be the stimulatory factor(s). The ability of B. henselae to enhance the expression of adhesion molecules on endothelial cells may be an important mechanism in the pathogenesis of B. henselae infection.

Environmental mutagens may be implicated in the emergence of drug-resistant microorganisms

The emergence of drug-resistant microorganisms is an important medical and social problem. Drug-resistant microorganisms are thought to grow selectively in the presence of antibiotics. Most clinically isolated drug-resistant microorganisms have mutations in the target genes for the drugs. While any of the many mutagens in the environment may cause such genetic mutations, no reports have yet described whether these mutagens can confer drug resistance to clinically important microorganisms. We investigated how environmental mutagens might be implicated in acquired resistance to antibiotics in clinically important microorganisms, which causes human diseases. We selected mutagens found in the environment, in cigarette smoke, or in drugs, and then exposed Pseudomonas aeruginosa to them. After exposure, the incidence of rifampicin- and ciprofloxacin-resistant P. aeruginosa strains markedly increased, and we found mutations in genes for the antibiotic-target molecule. These mutations were similar to those found in drug-resistant microorganisms isolated from clinical samples. Our findings show that environmental mutagens, and an anticancer drug, are capable of inducing drug-resistant P. aeruginosa similar to strains found in clinical settings.

CD14-Mediated Induction of Interleukin-8 and Monocyte Chemoattractant Protein-1 by a Heat-Resistant Constituent of Porphyromonas gingivalis in Endothelial Cells

Viable and inactivated Porphyromonas gingivalis dose‐dependently induced interleukin‐8 (IL‐8) and monocyte chemoattractant protein‐1 (MCP‐1) secretion in human umbilical vein endothelial cells (HUVECs). The inactivated P. gingivalis, in comparison with viable bacteria, tended to enhance the production of both chemokines more strongly. The production of MCP‐1 protein began increasing immediately after stimulation by P. gingivalis, and there was a nearly linear increase from 0 to 8 h of incubation, whereas IL‐8 production showed a linear increase between 4 and 12 h of incubation. The IL‐8 and MCP‐1 mRNA expressions in HUVECs as determined by reverse transcriptase‐polymerase chain reaction (RT‐PCR) or Quantikine mRNA colorimetric quantification kits were found to be enhanced by P. gingivalis. Furthermore, the time courses of IL‐8 and MCP‐1 mRNA expressions were in accordance with those of protein production. Addition of polymyxin B or boiling did not weaken the stimulatory effect of P. gingivalis, which inhibited the effect of Escherichia coli lipopolysaccharide (E. coli LPS) and tumour necrosis factor‐α (TNF‐α), respectively. In contrast, the induction of IL‐8 and MCP‐1 by P. gingivalis was significantly reduced by anti‐CD14 antibody. Our results suggest that some heat‐stable component of P. gingivalis, including LPS, may be responsible for the induction of IL‐8 and MCP‐1 in HUVECs by a CD14‐dependent mechanism. These effects might be involved in the accumulation and activation of neutrophils and monocytes at an early stage of the periodontal pathogenesis.

Phylogenetic Analysis of Enteroaggregative Escherichia coli (EAEC) Isolates from Japan Reveals Emergence of CTX-M-14-Producing EAEC O25:H4 Clones Related to Sequence Type 131

ABSTRACT Enteroaggregative Escherichia coli (EAEC) causes acute or persistent diarrhea. The aggR gene is widely used as a marker for typical EAEC. The heterogeneity of EAEC is well known; however, there are few reports on the phylogenetic relationships of EAEC. Recently, CTX-M extended-spectrum β-lactamase (ESBL)-producing EAEC strains have been reported worldwide. To characterize EAEC strains in Japan, we investigated the population structure of EAEC. A total of 167 aggR-positive strains isolated from stool specimens from diarrheal patients in Kagoshima (139 strains) and Osaka (28 strains), Japan, between 1992 and 2010 were examined for the prevalence of EAEC virulence markers, the bla CTX-M gene, and the capacity to form biofilms. Multilocus sequence typing was also conducted. EAEC strains were widely distributed across four major E. coli phylogroups. Strains of O111:H21/clonal group 40 (CG40) (30 strains), O126:H27/CG200 (13 strains), and O86a:H27/CG3570 (11 strains) in phylogroup B1 are the historical EAEC clones in Japan, and they exhibited strong biofilm formation. Twenty-nine strains of EAEC O25:H4/CG131 were identified in phylogroup B2, 79% of which produced CTX-M-14. This clone has emerged since 2003. The clone harbored plasmid-encoded EAEC virulence genes but not chromosomal virulence genes and had lower biofilm-forming capacity than historical EAEC strains. This clone most likely emerged from a pandemic uropathogenic O25:H4/sequence type 131 clone by acquiring an EAEC virulence plasmid from canonical EAEC. Surveillance of the horizontal transfer of both virulence and ESBL genes among E. coli strains is important for preventing a worldwide increase in antimicrobial drug resistance.

The induction of intercellular adhesion molecule-1 on human umbilical vein endothelial cells by a heat-stable component of Porphyromonas gingivalis

Live Porphyromonas gingivalis enhanced the expression of intercellular adhesion molecule-1 (ICAM-1) on the surface of human umbilical vein endothelial cells (HUVECs) in a bacterial dose-dependent manner. Inactivation of P. gingivalis by ultraviolet (UV), heat (56°C, 30 min), or sonication did not alter its stimulatory activity. ICAM-1 expression began to increase at 4 h after stimulation, reached a maximum at 12 h, and remained at the maximum for at least the next 8 h. This time course was similar to that of expression by Escherichia coli LPS. Furthermore, the effect of UV-inactivated P. gingivalis was not inhibited by boiling or polymyxin B treatment. In addition, the effect of P. gingivalis strain W83 on ICAM-1 expression was stronger than that of strain ATCC 33277. Our results suggested that some unidentified, heat-stable proteins, polysaccharides, or lipids may be the stimulatory factor(s), although the participation of LPS could not be completely ruled out. The ability of P. gingivalis to stimulate ICAM-1 expression on endothelial cells may play an important role in the pathogenesis of periodontal disease.