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Biomedical subjects

W M Johnson

Publications and source records attributed to W M Johnson.

At least 55 records · Page 3Linked to original sources
PubMed1994

Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping.

Multilocus enzyme electrophoresis and ribotyping were used to characterize 83 strains of Pseudomonas cepacia, mostly isolated from cystic fibrosis (CF) patients, although a number of isolates from non-CF nosocomial infections and reference environmental strains were represented. Twenty enzyme electrophoretic types (ETs) were determined; of these, one clone (ET12) was associated with six of nine ribotypes (RTs) said to be geographically representative of the United Kingdom and all of the Ontario (Canada) isolates from CF patients. This clone was not associated with nosocomial infections or environmental strains and was never found in CF isolates from British Columbia or Nova Scotia, Canada, or a center in the eastern United States. Individual isolate EcoRI RT signatures did not cluster geographically as did the ET signatures by clonal analysis. Frequently RTs occurred in more than a single ET. Known point source focal nosocomial outbreaks were typified by single ETs and stable RTs. Dendrographic analysis of the strains grouped those strains from CF patients, nosocomial outbreaks, and environmental sources into separate ET families, and diversity analysis indicated that, with the exception of ET17, CF isolates clustered in unique and closely related ETs different from those from nosocomial and environmental sources. This study has also shown the potential of multilocus enzyme electrophoresis to monitor the intercontinental spread of P. cepacia strains in CF patients, and this may have a significant impact on plans for CF patient summer camps and design of infection control practices. Whether the intercontinental ET12 clone, which predominates in the United Kingdom and the province of Ontario, linked by summer camp acquisition, has increased virulence for CF patients remains to be established.

Alleles↗
PubMed1993

Silicosis following employment in the manufacture of silica flour and industrial sand.

We describe silicosis in a patient with chronic obstructive pulmonary disease who worked as a maintenance mechanic for less than 5 years in a plant in South Carolina that manufactures silica flour and industrial sand. An open lung biopsy showed an early stage of silicosis manifested by perivascular and peribronchial collections of macrophages as well as early granuloma formation. An antinuclear antibody profile compatible with Sjogren's syndrome or systemic lupus erythematosus occurred with no symptoms of either disease. Excessive occupational exposure levels of respirable free silica were documented.

Biopsy↗
PubMed1993

Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada.

A single electrophoretic type (ET15) of Neisseria meningitidis has been associated with an increased incidence of group C meningococcal disease in Canada. Genomic fingerprinting through pulsed-field gel electrophoresis of chromosomal DNA was used to characterize the clonal relationship among meningococcal isolates of different electrophoretic types and among isolates within ET15. The genomic fingerprints of the ET15 isolates, while similar as a group, were sufficiently distinct to confirm linkage for four pairs of strains from focal outbreaks and differed markedly from those of the other common electrophoretic types, ET5, ET9, and ET21.

Canada↗
PubMed1992

Detection of genes coding for listeriolysin and Listeria monocytogenes antigen A (ImaA) in Listeria spp. by the polymerase chain reaction.

Two pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect targeted sequences in genes coding for listeriolysin O and Listeria monocytogenes antigen A (ImaA). Strains of Listeria spp. used in this study were isolated from clinical specimens, contaminated foods, and environmental sources. Primers were targeted to internal regions of the genes coding for listeriolysin (hlyA) and Listeria antigen (ImaA) and amplification fragments were detected after the PCR by agarose gel electrophoresis. PCR was performed using nucleic acids extracted from a collection of 74 strains of Listeria spp. including 18 reference strains, 41 L. monocytogenes, nine L. innocua, five L. seeligeri and one L. ivanovii, encompassing representative sources, serovars, and enzyme electrophoretic types. Although the listeriolysin gene was found exclusively in L. monocytogenes, some strains of serovar 4c were negative. Simultaneous presence of both genes was restricted to L. monocytogenes strains of serovars 1/2, 3, and 4. The ImaA gene was identified in five of 10 L. innocua strains and one L. ivanovii isolated from pork. Strains of L. seeligeri, L. welshimeri, and L. grayi were negative for both genes. The detection limits in the PCR were found to be 10 pg of nucleic acids for the hlyA gene and 1 pg for the ImaA gene.

Antigens, Bacterial↗
PubMed1992

Streptococcal erythrogenic toxin genes: detection by polymerase chain reaction and association with disease in strains isolated in Canada from 1940 to 1991.

The presence of genes encoding pyrogenic exotoxins type A (speA), B (speB), and C (speC) and streptolysin O (slo) was determined by the polymerase chain reaction (PCR) to target specific sequences in 152 strains of group A streptococci. These included reference strains, representative M and T type strains, and strains associated with scarlet fever and pharyngitis collected between 1940 to 1991 and included strains from patients with severe invasive streptococcal infections. PCR amplicons were detected by agarose gel electrophoresis, and specificity was established by restriction fragment analysis. The frequency of occurrence for each target gene among all strains tested was 33.6% for speA, 99.3% for speB, 28.9% for speC, and 100% for slo. Strains of non-group A streptococci, recognized toxigenic bacterial pathogens, and pneumolysin-producing Streptococcus pneumoniae strains were negative for all targeted gene sequences. Detection limits in the PCR were found to be 100 pg of total nucleic acids for the speB and speC genes and 1 ng for the speA and slo genes. Isolates associated with scarlet fever, pharyngitis, and severe invasive infections showed statistically significant differences in the presence of speA, with scarlet fever strains having the highest association (81.3%), severe infections the next highest association (42.9%), and pharyngitis the lowest association (18.4%). Although no significant differences were observed in speC frequencies in isolated associated with the three disease categories, a genotype of speB slo was significantly higher in isolates associated with pharyngitis (54.1%) than in strains associated with scarlet fever (18.8%) or severe invasive disease (23.8%). Streptolysin O targets were present in all the isolates tested, and only a single strain (T-11-M-11) was devoid of targeted speB sequences, thereby demonstrating that neither speB nor slo is associated with any particular clinical presentation.

Bacterial Proteins↗
PubMed1991

Amplification by the polymerase chain reaction of a specific target sequence in the gene coding for Escherichia coli verotoxin (VTe variant).

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to target a specific sequence in the gene coding for the A subunit of Escherichia coli verotoxin (VTe-variant, VTev). This PCR protocol permits the VTe-variant target sequence to be distinguished from closely related sequences in the same coding regions for type 1, type 2, and type 2 variant E. coli verotoxins. This procedure will be a valuable adjunct to other DNA amplification techniques currently being used for molecular epidemiological studies of verotoxigenic E. coli.

Bacterial Toxins↗
PubMed1991

Application of the polymerase chain reaction (PCR) to detection of the aerolysin gene in whole cell cultures of beta-hemolytic Aeromonas hydrophila.

Oligonucleotide primers were used in a polymerase chain reaction protocol to detect the aerolysin gene in Aeromonas hydrophila. Primers detected template DNA in hemolytic, cytotoxic and enterotoxic strains of A. hydrophila and no amplification was detected with hemolytic A. sobria, non-hemolytic A. hydrophila and A. caviae strains.

Aeromonas↗
PubMed1991

Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction.

Eight pairs of synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) protocol to detect genes for staphylococcal enterotoxins A to E, exfoliative toxins A and B, and toxic shock syndrome toxin 1 in Staphylococcus aureus strains isolated from clinical specimens and contaminated foods. Primers were targeted to internal regions of the toxin genes, and amplification fragments were detected after the PCR by agarose gel electrophoresis. Unequivocal discrimination of toxin genes was obtained by the PCR by using nucleic acids extracted from 88 strains of S. aureus whose toxigenicity was established biologically and immunologically. In immunological assays, two strains of S. aureus produced equivocal results for production of enterotoxin C or toxic shock syndrome toxin 1, giving an overall concordance between phenotypic and genotypic identification of 97.7%. Primer specificity was established in the PCR by using nucleic acids from known toxin-producing bacterial pathogens and from nontoxigenic S. aureus. Strains of Streptococcus spp., including some producers of pyrogenic exotoxin A carrying the speA gene, were negative by the PCR designed to detect staphylococcal toxins. The detection limits were established for all the staphylococcal toxin genes within their respective PCR protocols. The identification of staphylococcal toxin genes in strains of S. aureus by the PCR offers a very specific, sensitive, relatively rapid, and inexpensive alternative to traditional immunological assays which depend on adequate gene expression for reliability and sensitivity.

Bacterial Toxins↗
PubMed1991

Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis.

A set of synthetic oligonucleotide primers was designed for use in a polymerase chain reaction protocol to specifically detect the B subunit genes in vtx2ha and vtx2hb, which code for the production of the VT2 (Shiga-like toxin II) variant cytotoxins VT2v-a and VT2v-b, respectively. An additional set of primers amplified a fragment common to the B subunits of the VT2 and the VT2 variant genes. Subsequent restriction endonuclease digestion of this amplicon permitted prediction of specific VT2 and variant genotypes on the basis of predetermined restriction fragment length polymorphisms. Genotypes of 21 VT2-producing strains of Escherichia coli were determined using this polymerase chain reaction-restriction fragment length polymorphism procedure. Four strains contained B subunit target sequences only for VT2 genes, 9 strains contained sequences only for VT2v-a genes, and 3 strains contained sequences only for VT2v-b. For genes in combination, one strain contained B subunit genes for both VT2 and VT2v-a and two strains contained B subunit genes for VT2 and VT2v-b. Two strains of E. coli O91:H21 contained both VT2v-a and VT2v-b B subunit genes. The VT2 reference strain of E. coli, E32511, was found to contain the targeted sequences from both VT2 and VT2v-a genes, whereas the recombinant E. coli, pEB1, possessed only that of the VT2 gene. The specific activities of extracellular VT2 determined in HeLa cells ranged from 0.3 to 41.7 TCD50 per microgram of protein in strains carrying the VT2 gene target and from 0 to 50.0 TCD50 per microgram of protein in strains carrying only the VT2 variant target (TCD50 is the tissue culture dose by which 50% of the cells were affected), suggesting that phenotypic expression does not correlate with genotype.

Animals↗
PubMed1990

Differentiation of Shiga toxin and Vero cytotoxin type 1 genes by polymerase chain reaction.

Two sets of synthetic oligonucleotide primers were used in a polymerase chain reaction technique to distinguish genes for Shiga toxin in Shigella dysenteriae 1 and type 1 Vero cytotoxin (VT1) in Escherichia coli. VT1a and VT1b primers directed at a common 130-base-pair (bp) fragment of the stx and sltI genes detected template nucleic acid in both Shiga toxin-positive S. dysenteriae 1 and VT1-producing E. coli strains. VT1c and VT1d primers, targeting a 140-bp fragment of the promoter region of the sltIA gene, were negative in the polymerase chain reaction with S. dysenteriae 1 nucleic acid and positive with nucleic acids from all strains found to produce VT1 in toxin-specific neutralization tests. Primer specificity was determined in the polymerase chain reaction using nucleic acid extracted from 49 strains of representative enteric pathogens defined in terms of their toxigenicity.

Bacterial Toxins↗
PubMed1990

Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction.

Synthetic oligonucleotide primers were used in a polymerase chain reaction (PCR) technique to detect the gene for aerolysin in strains of Aeromonas hydrophila and to screen for identical genes in A. caviae, A. sobria, and A. veronii isolated from patients with diarrheal disease. Primers targeted a 209-bp fragment of the aer gene coding for the beta-hemolysin and detected template DNA only in the PCR using nucleic acid (NA) from hemolytic strains of A. hydrophila which were also cytotoxic to Vero and CHO cells and enterotoxic in suckling-mouse assays. PCR amplification of NA from hemolytic A. sobria or nonhemolytic A. hydrophila and A. caviae strains was consistently negative. Primer specificity was determined in the PCR by using NA extracted from 56 strains of bacteria, including hemolytic Escherichia coli and Listeria monocytogenes as well as several recognized enteric pathogens defined in terms of their toxigenicity. The detection limit for the aerolysin gene by PCR amplification was 1 ng of total NA. The PCR clearly identified aerolysin-producing strains of A. hydrophila and may have application as a species-specific virulence test because other hemolytic Aeromonas species tested were negative.

Aeromonas↗
PubMed1990

Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction.

A set of four synthetic oligonucleotide probes derived from sequences of the VT1 (Shiga-like toxin I [SLT-I]) and VT2 (SLT-II) genes were used in a polymerase chain reaction (PCR) amplification procedure to detect these genes in some enteric pathogens. A total of 40 verotoxin-producing Escherichia coli strains and 43 isolates of other recognized enteric pathogens were studied. PCR amplification products identifying the VT1 and VT2 gene sequences were observed only in nucleic acid extracted from strains found to be VT positive in traditional tissue culture assays. Template nucleic acid extracted from other gram-negative bacteria was found to be negative with the exception of five isolates of Shigella dysenteriae type 1 in which good amplification with the VT1 probe was observed. The oligonucleotide probes clearly distinguished VT1 and VT2 strains of E. coli and did not give specific amplification with nucleic acid from VTe (a SLT-II variant)-producing E. coli. VT1 or VT2 genes or both were not detected in E. coli K-12 strain C600 or HB101 or in strains known to express other virulence factors, such as enterotoxins, adhesins, hemolysins, or unrelated cytotoxins. The sensitivity of the PCR procedure for detection of both VT1 and VT2 genes was determined to be 1 ng of total nucleic acid. Furthermore, the VT1 gene was easily detected when only 100 pg of nucleic acid was used as the template in the PCR procedure.

Animals↗
PubMed1989

Purification of an Escherichia coli serogroup O157:H7 verotoxin and its detection in North American hemorrhagic colitis isolates.

Verotoxin (VT), which is immunologically unrelated to VT1 (Shiga-like toxin I), was purified from the culture filtrate of Escherichia coli hemorrhagic colitis serogroup O157:H7 strain 3657 by copper ion chelate affinity chromatography followed by anion-exchange chromatography. The isoelectric point by sucrose density gradient isoelectric focusing was 5.0, the molecular weight by gel filtration on Superose 12 was about 60,000, and the 50% cytopathic dose for Vero cells was about 1 pg. This toxin was found by immunological methods to be the predominant VT in E. coli O157 isolates associated with illness in North America, with 38 of 42 strains tested producing this toxin, 20 in combination with VT1. VT from strain 3657 is immunologically identical to the described Shiga-like toxin II (VT2) of E. coli strains (from the United States) K-12(pEB1) and C600(933W) but only partially related to VT of strain E32511 (from the United Kingdom), the first to be named VT2.

Animals↗
PubMed1989

Protein A gold identification of ureaplasmas on the bovine zona pellucida.

The object of this study was to develop a prefixation protein A gold labelling technique for Ureaplasma diversum and to apply this to bovine embryos. Sixteen hour cultures of Ureaplasma diversum strain 2312 were incubated with either specific antiserum or nonimmune serum, followed by exposure to protein A gold and negative staining. The ureaplasmas which were incubated with specific antiserum were labelled with gold particles while those ureaplasmas which were incubated with nonimmune serum were not labelled. Twenty-three unhatched, day 7 bovine embryos were then incubated in either embryo culture medium (ECM) alone, ECM with sterile ureaplasma broth added or ECM with 1.7 X 10(6) colony forming units of Ureaplasma diversum strain 2312 per embryo. After 16 hours, the embryos were washed twice and incubated with either specific antiserum or nonimmune serum. The embryos were then incubated with medium containing protein A gold and examined by electron microscopy. No ureaplasmas were identified on the zona pellucida of the control embryos. Ureaplasmas were identified on the outer surface of the zona pellucida of 13 of the 17 embryos which had been exposed to the organism. Of these, the embryos which were incubated with specific antiserum had labelled ureaplasmas while the embryos which were incubated with nonimmune serum had unlabelled ureaplasmas on the zona pellucida. It was concluded that the protein A gold method was a suitable technique for the identification of ureaplasmas in EM preparations. The presence of ureaplasmas on the outer surface of the bovine zona pellucida following in vitro exposure to the organism was confirmed.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

Melioidosis: a reminder.

Recrudescent pulmonary melioidosis developed in two patients 12 and 16 years after their last travels to an endemic area. In one, a clinically silent prostatic abscess may have been the focus; and in both, the diagnosis was difficult to make even when the laboratory was notified of the possibility of infection with Pseudomonas pseudomallei. Recrudescent melioidosis should be considered in febrile patients who have been in endemic areas regardless of the interval from last exposure to the development of disease.

Humans↗
PubMed1988

A new heat-labile cytolethal distending toxin (CLDT) produced by Escherichia coli isolates from clinical material.

A new heat-labile Escherichia coli toxin cytolethal to Vero, HeLa, HEp-2 and CHO cells and negative in Y-1 cells has been demonstrated in culture filtrates of 43 E. coli strains associated with diarrheal disease. This new toxin was termed a cytolethal distending toxin (CLDT) to reflect the progressive cell distention and cytotoxicity evidenced in all sensitive tissue cells. CLDT was distinct from the classic heat-labile (LT) and heat-stable enterotoxins, Verotoxins and hemolysins and was produced by some strains of the following E. coli serogroups (02, 07, 08, 018, 022, 039, 044, 055, 083, 086, 091, 0113, 0119, 0128 and 0167). Moderate cyclic AMP accumulation (75-fold) was observed in CHO cells exposed to E. coli CLDT for 24 h. In contrast to E. coli LT, cyclic AMP levels were optimal at 24 h and were observed to decrease over the following 72 h period in CHO cells exposed to E. coli CLDT. In addition to heat-lability at 70 degrees C for 15 min, E. coli CLDT demonstrated a molecular weight over 30,000, was nondialyzable and trypsin-sensitive. E. coli CLDT was negative in adult rabbit ligated ileal loop and suckling mouse assays and provoked only an erythematous response in rabbit skin. All CLDT-positive E. coli strains identified to date were non-hemolytic and non-invasive. Verotoxin and heat-labile enterotoxin were found in combination with CLDT in a few E. coli strains.

Animals↗