Yasuko Honma

Oxalate-Degrading Enterococcus faecalis

An oxalate‐degrading Enterococcus faecalis was isolated from human stools under anaerobic conditions. The bacteria required a poor nutritional environment and repeated subculturing to maintain their oxalate‐degrading ability. The E. faecalis produced 3 proteins (65, 48, and 40 kDa) that were not produced by non‐oxalate‐degrading E. faecalis as examined by SDS‐PAGE. Antibodies against oxalyl‐coenzyme A decarboxylase (65 kDa) and formyl‐coenzyme A transferase (48 kDa) obtained from Oxalobacter formigenes (an oxalate‐degrading anaerobic bacterium in the human intestine) reacted with 2 of the proteins (65 and 48 kDa) from the E. faecalis as examined by Western blottings. This is the first report on the isolation of oxalate‐degrading facultative anaerobic bacteria from humans.

A novel filamentous phage, fs-2, of Vibrio cholerae O139.

A novel filamentous bacteriophage, fs-2, was isolated from Vibrio cholerae O139 strain MDO14. The fs-2 phage was a long filamentous particle 1200 nm long and 7 nm wide. The purified phage formed a turbid plaque when spotted on a lawn of the host organisms. The plaque-formation activity was stable following heating to 70 degrees C but was inhibited by treatment with chloroform. fs-2 had a single-stranded DNA genome and was converted to a double-stranded replicative form in the host cell. Almost all V. cholerae O139 and O1 El Tor biotype strains tested were sensitive to the phage, but most O1 classical strains and non-O1 non-O139 strains were resistant. The fs-2 genome comprised 8651 nucleotides containing nine open reading frames, five of which had predicted protein products partially homologous to the reported protein products of other filamentous phages. Although the extent of the homology was not particularly high, the genetic organization of other filamentous phages appears to be preserved in fs-2. The phage was not integrated into the chromosome of its host, but a 715 nucleotide fragment located in the large intergenic region of fs-2 was highly homologous to a part of region RS2 (repetitive sequence 2) of the V. cholerae CTX phi sequence which is speculated to be required for integration of the phage into the V. cholerae chromosome at a specific site.

Pili of an Aeromonas hydrophila Strain as a Possible Colonization Factor

Aeromonas hydrophila Ae6 had 2 morphologically distinctive kinds of pili. One appeared rigid and straight with a diameter of 9 nm (R‐pili). The other appeared wavy and flexible with a diameter of 7 nm (W‐pili). W‐pili were very few on the cell as compared with R‐pili. In this study, W‐pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 21 kDa as estimated by SDS‐PAGE. There was no immunological cross‐reaction between W‐pili and other cellular components. The strain Ae6 and its purified W‐pili adhered to human and rabbit intestine and agglutinated human and rabbit erythrocytes. Organisms pretreated with the Fab fraction of anti‐pilus antibody failed to adhere to the intestine. Pretreatment of intestine with purified W‐pili blocked adherence of the organisms to the intestine. These results suggest that the W‐pili are the colonization factor of A. hydrophila Ae6.

Characterization of Vibrio cholerae O139 Synonym Bengal Isolated from Patients with Cholera-Like Disease in Bangladesh

Vibrio cholerae O139 (synonym Bengal), a novel serovar of V. cholerae, is the causative agent of large outbreaks of cholera‐like illness currently sweeping India and Bangladesh. Eight randomly selected V. cholerae O139 isolates were studied for their biological properties, which were compared with those of V. cholerae O1 and other V. cholerae non‐O1. The V. cholerae O139 isolates were characterized by the production of large amount of cholera toxin, hemagglutination, weak hemolytic properties, resistance to polymyxin B, lysogeny with, and production of, kappa type phage (4/8 isolates only), and resistance to both classical and El Tor‐specific phages. Thus, V. cholerae O139 isolates had an overall similarity with V. cholerae O1 El Tor.

Relationship between O-Serogroup and Presence of Pathogenic Factor Genes in Escherichia coli

A total of 383 isolates of serogroup‐based enteropathogenic and enteroinvasive Escherichia coli (310 strains of EPEC and 73 strains of EIEC) were examined for the presence of corresponding pathogenic genes. The serogroup‐based EPEC consisted of 232 strains isolated from diarrhea patients and of 78 strains from healthy carriers. The gene encoding intimin, eaeA, was detected in 42 of the 232 EPEC strains from patients (18.1%) and 9 of the 78 strains from carriers (11.5%). The difference was not significant. The bfp gene on the EAF plasmid was detected in 7 of the 42 eaeA‐positive EPEC strains from patients but was not detected in the 9 strains from carriers. In serogroup‐based EIEC, a chromosomal ipaH gene encoding one of the invasive plasmid antigens was detected in 4 of the 60 strains from patients (6%) but not in the 13 strains from carriers. The 4 ipaH‐positive strains possessed the invasive plasmid. These results suggested that the serogroup‐based diagnosis of EPEC and EIEC is not sufficient for identifying strains carrying the eaeA or ipaH gene.

Pili of Aeromonas hydrophila: purification, characterization, and biological role.

Aeromonas hydrophila (Ae6) has 2 morphologically distinctive kinds of pili. One appeared rigid, channeled, and straight with a diameter of 9 nm (Ae6‐R pili). The other looked flexible, wavy, and having helical structure with a diameter of 7 nm (Ae6‐W pili). Ae6‐R pili were purified and characterized. The pili consisted of a subunit protein with a molecular weight of 18 kDa as estimated by SDS‐PAGE, and contained 42.3% hydrophobic amino acids and one cysteine residue. The pilus was solubilized to 18 kDa subunit protein by 2‐mercaptoethanol, dithiothreitol, hydrochloric acid, or heating at 120 C for 5 min. The organism Ae6 was strongly adhesive to rabbit intestines as well as human intestines, and agglutinated erythrocytes. Anti‐pili antibody (Fab fraction) did not block the adhesion. Purified Ae6‐R pili did not adhere to the intestine or to the erythrocytes. However, the anti‐pili Fab inhibited pellicle formation of the organisms cultured in broth, and also inhibited salt agglutination with ammonium sulfate. From these results, Ae6‐R pili are not likely a colonization factor but probably play a role in the autoaggregation of the organisms.

Adhesive Property of Toxin‐Coregulated Pilus of Vibrio cholerae O1

The adhesive property of toxin‐coregulated pilus (TCP) to the human intestine (jejunum), and whether or not TCP mediates the adhesion of Vibrio cholerae 395 organisms to the intestinal epithelium were investigated using visually proving methods. The purified TCP did not agglutinate human erythrocytes nor adhere to the surface of human intestinal epithelium. V. cholerae 395 adhered to the epithelium, but the adhesion was not inhibited by blocking the pili with the Fab fraction of anti‐TCP IgG. The organisms adhered to the intestine treated with purified TCP in advance, as well as to the intact intestine. These findings suggest that TCP is not involved in the adhesion of these organisms to the intestinal epithelium.

MOLECULAR ANALYSIS OF A FILAMENTOUS PHAGE (FSL) OF VIBRIO CHOLERAE O139

A filamentous bacteriophage from Vibrio cholerae O139 strain A1-4450 was isolated (fsl). The phage fsl had a ssDNA genome and dsDNA as a replicative form (RF) in lysogenic host cell. The DNA sequence of fsl RF was determined. It consisted of 6340 bp and had a G + C content of 43.5%. Fifteen possible ORFs were found in fsl. One of them, ORF384, was estimated to encode 384 amino acid residues (44.6 kDa) and had homologous regions with the zot gene of V. cholerae and gene I of the coliphage group. ORF104, located upstream of ORF384, was homologous to gene 93 protein of Pf3 (filamentous phage of Pseudomonas sp.) corresponding to gene VI of coliphage. Other than ORF384 and ORF104, the ORF81, ORF44, ORF29, and ORF193 were speculated to correspond to gene V, gene VII, gene IX, and gene III, respectively, in the order as reported on f1 phage.

Detection of EspB using reversed passive latex agglutination: application to determination of enteropathogenic Escherichia coli.

We developed a new practical method to identify enteropathogenic Escherichia coli (EPEC) by detecting the pathogenic factor, EspB. E. coli were cultured in Dulbeccos Modification of Eagles Medium (DMEM), and EspB was detected in the culture supernatant by reversed passive latex agglutination (RPLA). All 63 E. coli strains harboring the eaeA gene encoding intimin were positive for RPLA, and all 25 strains without the eaeA gene were negative. Among these 63 eaeA-positive strains, 38 Shiga toxin-producing E. coli (STEC) produced Shiga toxin (Stx) under the same culture conditions (DMEM). Subtypes of EspB alpha, beta and gamma were antigenically cross-reactive to each other as determined by RPLA and Western blotting. A kit for Stx detection (RPLA) is commercially available and therefore this RPLA for detection of EspB could be a practical method to define EPEC in both clinical laboratories and the field.

Cloning, Sequencing and Expression of the Gene Encoding the Extracellular Metalloprotease of Aeromonas caviae

A gene (apk) encoding the extracellular protease of Aeromonas caviae Ae6 has been cloned and sequenced. For cloning the gene, the DNA genomic library was screened using skim milk LB agar. One clone harboring plasmid pKK3 was selected for sequencing. Nucleotide sequencing of the 3.5 kb region of pKK3 revealed a single open reading frame (ORF) of 1,785 bp encoding 595 amino acids. The deduced polypeptide contained a putative 16‐amino acid signal peptide followed by a large propeptide. The N‐terminal amino acid sequence of purified recombinant protein (APK) was consistent with the DNA sequence. This result suggested a mature protein of 412 amino acids with a molecular mass of 44 kDa. However, the molecular mass of purified recombinant APK revealed 34 kDa by SDS‐PAGE, suggesting that further processing at the C‐terminal region took place. The 2 motifs of zinc binding sites deduced are highly conserved in the APK as well as in other zinc metalloproteases including Vibrio proteolyticus neutral protease, Emp V from Vibrio vulnificus, HA/P from Vibrio cholerae, and Pseudomonas aeruginosa elastase. Proteolytic activity was inhibited by EDTA, Zincov, 1,10‐phenanthroline and tetraethylenepentamine while unaffected by the other inhibitors tested. The protease showed maximum activity at pH 7.0 and was inactivated by heating at 80 C for 15 min. These results together suggest that APK belongs to the thermolysin family of metalloendopeptidases.