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J B Kaper

Publications and source records attributed to J B Kaper.

At least 181 records · Page 10Linked to original sources
PubMed1987

Role of plasmid-encoded adherence factors in adhesion of enteropathogenic Escherichia coli to HEp-2 cells.

Plasmid-encoded adherence factors have been shown to be important for the full expression of enteropathogenic Escherichia coli (EPEC) pathogenicity and for EPEC adhesion to cultured HEp-2 cells. EPEC strain E2348 (O127) shows localized HEp-2 cell adhesion and possesses a 60-megadalton plasmid, pMAR2. When E2348 is cured of pMAR2 it loses the ability to adhere to HEp-2 cells, while nonadherent E. coli K-12 strains P678-54 and HB101 acquire HEp-2 adhesiveness after they gain the plasmid. By electron microscopy, E2348 was seen to adhere to HEp-2 cells in a manner that closely resembled EPEC adhesion to intestinal mucosa; bacteria were intimately attached to projections of the apical HEp-2 cell membrane and caused localized destruction of microvilli. The plasmid-containing K-12 strains, on the other hand, did not show intimate attachment and there was no modification of cell surface architecture. It is concluded that plasmid pMAR2 codes for an adhesin, possibly fimbrial in nature, that promotes HEp-2 adhesion but that other chromosomally encoded factors are required for EPEC to achieve the characteristic mode of intimate cell attachment.

Bacterial Adhesion↗
PubMed1987

Characterization of plasmids encoding the adherence factor of enteropathogenic Escherichia coli.

Volunteer studies have shown that a 60-megadalton plasmid is required for full virulence of the human enteropathogenic Escherichia coli (EPEC) strain E2348/69 (O127:H6). The plasmid, designated pMAR2, encodes localized adherence to HEp-2 cells in tissue culture via the adhesin known as the EPEC adherence factor (EAF). Using a DNA probe for the EAF, we have previously shown that these genes are specific for EPEC and are usually encoded on plasmids ranging from 55 to 65 megadaltons. In this study, Southern blot analysis and S1 nuclease homology determination reveal a high degree of sequence conservation among these plasmids, despite some variation in restriction maps. Phenotypic characterization of the prototype EAF plasmid pMAR2 reveals that the plasmid belongs to the group IncFII and is negative for alpha-hemolysin, colicin, and aerobactin synthesis, as well as biochemical markers and antibiotic resistance. Regions encoding adherence to HEp-2 cells were localized by Tn801 insertion mutagenesis. Adherence genes were then cloned as two distinct plasmid regions which confer the adherence phenotype only when complementing each other in trans.

Adhesins, Escherichia coli↗
PubMed1987

Determinants of the immunogenicity of live virulent and mutant Vibrio cholerae O1 in rabbit intestine.

Determinants of the capacity of live Vibrio cholerae O1 isolates to evoke specific immune responses in intestinal mucosa were studied in rabbits, using mucosal immunoglobulin A (IgA) antitoxin as the measured immune response. Antitoxin responses were evoked mostly by the primary inoculation and were dose dependent; secondary-type responses were modest and occurred only when the booster inoculum was large, i.e., 10(10) CFU. The efficiency of mucosal immunization correlated closely with the mucosal colonizing capacity of the infecting strain and was otherwise independent of toxin genotype (A+ B+ or A- B+) or whether the strain was motile or nonmotile. Live bacteria evoked mucosal antitoxin more efficiently than did purified cholera toxin. Prior immunization with a nontoxinogenic (A- B-) V. cholera strain interfered significantly with the induction of mucosal antitoxin by subsequent immunization with its fully toxinogenic (A+ B+) parent. These results demonstrate the marked efficiency with which live V. cholerae stimulate a specific enteric mucosal secretory IgA response. They support the view that mucosal colonization aids efficient delivery of bacterial antigens to responsive subepithelial lymphoid tissue. This might occur by transfer of colonizing bacteria through M cells into Peyer patches or by efficient delivery of secreted toxin to M cells by mucosa-associated organisms. Preexisting antibacterial immunity interferes with colonization, which may prevent efficient antigenic stimulation and which may explain the relatively weak response to booster immunization. The same process may also limit the efficacy of hybrid enteric bacterial vaccines when there is preexisting mucosal immunity to the carrier organism due to either natural exposure or prior immunization with another vaccine that uses the same carrier.

Animals↗
PubMed1986

Molecular epidemiology of group JK Corynebacterium on a cancer ward: lack of evidence for patient-to-patient transmission.

Plasmid profiles of 27 clinical isolates of group JK Corynebacterium, mostly from one cancer ward, revealed that only two strains harbored a 20-kilobase plasmid. These plasmid-bearing isolates had the same antimicrobial resistance pattern as non-plasmid-containing isolates: All were resistant to penicillin G, methicillin, cephalothin, clindamycin, and gentamicin. Restriction endonuclease analysis of chromosomal DNA was done on 18 clinical isolates of group JK Corynebacterium. Identical restriction patterns were seen when multiple isolates were from the same patient over several months of apparent colonization; in contrast, restriction patterns of isolates from patients from two clusters were all heterogeneous and suggested that patient-to-patient transmission of group JK Corynebacterium did not occur. Restriction endonuclease analysis of chromosomal DNA, but not plasmid profiling, appears to be a very sensitive typing tool for group JK Corynebacterium.

Corynebacterium↗
PubMed1986

Demonstration of a plasmid-borne gene encoding a thermostable direct hemolysin in Vibrio cholerae non-O1 strains.

Of 15 Vibrio cholerae non-O1 strains, 2 produced a hemolysin termed NAG-rTDH, which is very similar to the thermostable direct hemolysin of V. parahaemolyticus. These two strains contained DNA sequences which are homologous to a DNA probe for the V. parahaemolyticus thermostable direct hemolysin gene. A probe-positive 9-kilobase HindIII fragment was cloned from a plasmid of a V. cholerae non-O1 strain into plasmid pBR322, and the resulting Escherichia coli clones produced intracellular NAG-rTDH.

DNA, Bacterial↗
PubMed1986

Cloning of a gene involved in regulation of exotoxin A expression in Pseudomonas aeruginosa.

We have cloned a gene from Pseudomonas aeruginosa that stimulates the expression of exotoxin A. A recombinant library of genomic DNA from strain PA103 constructed with a broad-host-range plasmid vector containing chromosomal insert fragments generated by Sau3A was used to transform the hypotoxigenic mutant strain PA103-29. A recombinant plasmid, pFHK6, was isolated from a PA103-29 transformant which displayed increased toxin production. From pFHK6, which contained a 20-kilobase-pair chromosomal insert, a 3-kilobase-pair XhoI fragment was isolated and subcloned into the plasmid cloning vector pVK101 to give pFHK10. In toxigenic P. aeruginosa strains containing pFHK10, toxin expression was increased 10-fold and high levels of iron in the culture medium only partially inhibited the overproduction. Expression studies suggested that pFHK10 did not contain the toxin structural gene. In addition, Southern analysis with the 3-kilobase-pair XhoI fragment suggested that the putative toxin regulatory gene is common among different strains of P. aeruginosa including previously reported nontoxigenic strains.

ADP Ribose Transferases↗
PubMed1986

Localization of a determinant for HEp-2 adherence by enteropathogenic Escherichia coli.

pMAR2, a 60-megadalton plasmid encoding localized HEp-2 adherence in enteropathogenic Escherichia coli, was mapped with BamHI, HindIII, and SalI. Deletion and insertion mutants were constructed and used to define a potential DNA probe. Preliminary results indicate that this probe is sensitive and specific for the genes encoding the enteropathogenic E. coli adherence factor.

Adhesiveness↗
PubMed1986

Hemolysin production and cloning of two hemolysin determinants from classical Vibrio cholerae.

The hemolytic activity of 20 classical and 3 El Tor strains of V. cholerae O1 was examined phenotypically and genetically. The El Tor strains lysed bovine, chicken, human, rabbit, and sheep erythrocytes (RBCs), while the classical strains lysed only chicken and rabbit RBCs. The assay was done with RBCs in Tris-NaCl buffer, since phosphate-buffered saline was found to inhibit hemolytic activity. Hemolytic activity in culture supernatants from El Tor strains was more sensitive to heat inactivation than that in supernatants from the classical strain 395. A gene library of strain 395 was examined for hemolytic activity, and two distinct hemolytic clones were identified. One clone appeared identical to the previously cloned hemolysin structural gene from El Tor V. cholerae, while the other did not hybridize to the El Tor hemolysin probe, had a unique restriction enzyme digestion pattern, and encoded a hemolysin whose activity differed from that of the El Tor hemolysin clones. We suggest that the hemolysin specified by the determinant originally cloned from an El Tor vibrio be designated hemolysin I and the second hemolysin, cloned from the classical vibrio, be designated hemolysin II.

Animals↗
PubMed1986

Persistence of cholera in the United States: isolation of Vibrio cholerae O1 from a patient with diarrhea in Maryland.

A case of cholera was identified in Baltimore County, Md., in October 1984. The Vibrio cholerae O1 isolate from the patient was hemolytic, biotype El Tor, serotype Inaba, and was toxigenic by the Y-1 adrenal cell assay; on Southern blot analysis, the strain had a unique HindIII restriction site in the cholera toxin gene identical to that of other U.S. V. cholerae O1 isolates. Two days before he became ill, the patient had eaten meat from crabs harvested along the Texas coast.

Aged↗
PubMed1986

Molecular epidemiology of non-O1 Vibrio cholerae and Vibrio mimicus in the U.S. Gulf Coast region.

Ten toxigenic Vibrio cholerae non-O1 and V. mimicus strains isolated from clinical and environmental sources in the U.S. Gulf Coast region were examined for genetic relatedness. Restriction digest patterns of chromosomal DNA and Southern blot analysis with a cholera toxin gene probe revealed that the strains exhibited greater genetic divergence than the highly conserved V. cholerae O1 strains isolated from clinical and sewage samples in this region.

Cholera Toxin↗
PubMed1986

Synthetic oligodeoxyribonucleotide probes to detect Kanagawa phenomenon-positive Vibrio parahaemolyticus.

Synthetic oligodeoxyribonucleotide probes were used in the colony hybridization test to examine the association between the Kanagawa phenomenon (KP) and the thermostable direct hemolysin gene (tdh) of Vibrio parahaemolyticus. Representative V. parahaemolyticus strains with a variety of KP reactions and 17 other Vibrio species were examined for homology with four synthetic oligodeoxyribonucleotide probes (19 to 21 bases long) representing different regions of the tdh structural gene. Under stringent conditions, two of the probes were capable of distinguishing KP-positive V. parahaemolyticus from KP-negative or KP weak-positive V. parahaemolyticus which possesses mutated tdh genes. Vibrio hollisae strains hybridized with all four probes under reduced stringency, suggesting that they have tdh-related genes which are homologous but not identical to the tdh gene in all the regions examined. The results suggest that the colony hybridization test with the synthetic oligonucleotide probes is more suitable for the definitive determination of KP-positive strains than the hybridization with the larger gene probe or immunological assays.

DNA, Bacterial↗
PubMed1986

Comparison of DNA probes and the Sereny test for identification of invasive Shigella and Escherichia coli strains.

Forty-two Shigella and 29 Escherichia coli strains were screened for invasiveness in the Sereny test and for hybridization with two recently described DNA probes for the invasiveness plasmid. Both probes produced identical results. All Sereny-positive strains hybridized with both DNA probes. Three Sereny-negative strains also hybridized with the probes, suggesting that there are strains containing the invasiveness plasmid that are not pathogenic in animal models.

Animals↗
PubMed1986

Blinded, two-laboratory comparative analysis of Escherichia coli heat-stable enterotoxin production by using monoclonal antibody enzyme-linked immunosorbent assay, radioimmunoassay, suckling mouse assay, and gene probes.

Heat-stable enterotoxin (ST)-producing enterotoxigenic Escherichia coli (ETEC) can be identified by a variety of assays, including the suckling mouse assay (SMA), radioimmunoassay (RIA), polyclonal or monoclonal antibody enzyme-linked immunosorbent assay (ELISA), and DNA hybridization with STh and STp gene probes. To compare the sensitivity and reliability of these assays, 100 coded ETEC and non-ETEC isolates were blindly tested in two independent laboratories. SMA, RIA, and monoclonal ELISA were performed in Cincinnati, Ohio, while gene probe analysis was performed in Baltimore, Md. The method of storage of organisms had a profound effect on the stability of plasmids in certain strains. Hybridization experiments to determine the presence or absence of the enterotoxin gene showed that strains stored on Dorset egg medium at room temperature better retained their plasmids than strains stored frozen in skim milk. Forty-four of the 100 organisms obtained from the skim milk stock were found to produce STa in liquid culture by the RIA, SMA, and monoclonal ELISA (100% agreement). However, 50 of 54 of the strains stored on Dorset egg medium which were originally classified as STa+ or ST+ LT+ (positive for both heat-stable and heat-labile [LT] enterotoxins) were found to produce STa and retain the plasmid by each of these assays. Three additional strains were found which harbored the plasmid but did not elaborate STa by any of the assays (3% discrepancy). The monoclonal antibody ELISA appears to be highly reliable for determination of STa production by ETEC and can be easily scored visually even by untrained personnel. Furthermore, when this STa assay is coupled with a polyclonal antibody assay, it is possible to predict the genotype of STh- and STp-producing organisms.

Animals↗
PubMed1985

The diarrheal response of humans to some classic serotypes of enteropathogenic Escherichia coli is dependent on a plasmid encoding an enteroadhesiveness factor.

Isolates of the most common O serogroups of enteropathogenic Escherichia coli (EPEC) associated with infant diarrhea (designated class I) adhere to Hep-2 cells; the genes for this adhesin, termed EPEC adherence factor (EAF), are located on plasmids 50-70 MDa in size. Volunteers ingested 10(10) organisms of an O127:H6 Hep-2-adhesive class I strain (E2348/69) or its plasmid-minus, nonadhesive derivative. Diarrhea occurred in nine of 10 volunteers who ingested the parent strain (mean, 1,178 ml) but in only two of nine who took the plasmid-minus variant (mean, 433 ml; P less than .006). All volunteers ill from strain E2348/69 mounted serum IgA and IgG responses to a 94-kDa plasmid-associated outer membrane protein of E2348/69; this protein was found in other class I EPEC but not in enterotoxigenic or meningitic strains. The 50-70-MDa EAF plasmid seems necessary for full expression of pathogenicity in EPEC that exhibit Hep-2 adhesiveness. EPEC isolates of certain other, less common, O serogroups (O44, O86, and O114) are rarely Hep-2 adhesive. These EPEC, designated class II, possess distinct 50-70 MDa plasmids lacking EAF genes. Diarrhea was caused by 10(8) or 10(10) organisms of an O114:H2 class II EPEC strain (mean, 1,156 ml) in six of 11 volunteers. This result confirmed that class II EPEC are pathogenic by a mechanism not involving Hep-2 adhesiveness.

Adhesins, Escherichia coli↗
PubMed1985

Detection of an adherence factor of enteropathogenic Escherichia coli with a DNA probe.

A DNA probe to detect genes conferring localized adherence of enteropathogenic Escherichia coli (EPEC) to Hep-2 cells was evaluated by using E. coli isolates from the stools of Peruvian infants with and without diarrhea. The probe was both sensitive and specific and revealed that Hep-2 adherence (because of the EPEC adherence factor [EAF] was more frequent in some O serogroups of EPEC than in others. Those serogroups in which EAF is almost always found have been designated class I EPEC; serogroups in which EAF is rarely found have been designated class II. Both class I (EAF-positive) and class II EPEC are associated with diarrheal disease.

Adhesins, Escherichia coli↗
PubMed1985

Plasmid-mediated factors conferring diffuse and localized adherence of enteropathogenic Escherichia coli.

Histopathological evidence suggests that the adherence of enteropathogenic Escherichia coli (EPEC) to the mucosa of the small bowel is an important step in pathogenesis. Several reports have shown that many EPEC isolates adhere to HEp-2 and HeLa cells in tissue cultures. In the HeLa cell assay, there are at least two distinct patterns of adherence: localized adherence, which is characterized by the formation of bacterial microcolonies, and diffuse adherence, in which bacteria cover the cell uniformly. We have found that these two patterns can be demonstrated in HEp-2 cells as well as in HeLa cells and that the results of the two assays are closely correlated. Using a DNA probe which is sensitive and specific for localized adherence to HEp-2 cells, we provide evidence that localized adherence and diffuse adherence by EPEC are due to at least two genetically distinct adhesions which confer phenotypic differences in both the morphology of HEp-2 cell adherence and in surface hydrophobicity. The two factors are each encoded on plasmids which vary in size from 55 to 70 megadaltons; one strain exhibiting localized adherence carried these genes on the chromosome.

Adhesins, Escherichia coli↗
PubMed1985

Detection of the thermostable direct hemolysin gene and related DNA sequences in Vibrio parahaemolyticus and other vibrio species by the DNA colony hybridization test.

A specific gene probe for the Vibrio parahaemolyticus thermostable direct hemolysin gene was constructed and used to examine the presence or absence of the thermostable direct hemolysin gene or related DNA sequences in V. parahaemolyticus and other vibrios by the DNA colony hybridization method. The gene probe consisted of a 406-base-pair, completely internal fragment covering 71% of the structural gene with PstI linkers added to the ends. Six copies of this 415-base-pair PstI fragment were cloned into plasmid pBR322, which yielded large amounts of the probe DNA. One hundred forty-one V. parahaemolyticus strains were tested with the gene probe, and the results were compared with those of phenotypic assays for the thermostable direct hemolysin. All Kanagawa phenomenon-positive strains were gene positive. However, 86% of the strains that exhibited weak Kanagawa phenomenon and 16% of Kanagawa phenomenon-negative strains also reacted with the gene probe. Immunological methods for the detection of the thermostable direct hemolysin (modified Elek test, enzyme-linked immunosorbent assay) showed better correlation with gene probe results. All gene-positive strains produced hemolysin detectable in the enzyme-linked immunosorbent assay, although occasional strains showed weak reaction. The modified Elek test was slightly less sensitive than the enzyme-linked immunosorbent assay. All gene-negative strains were also negative in these immunological assays. One hundred twenty-one strains of Vibrio spp. other than V. parahaemolyticus were tested with the gene probe; only Vibrio hollisae strains reacted with the probe under stringent conditions.

Base Sequence↗