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1.  Anatomy research under the knife of medical ethics 
Journal of Pharmacy & Bioallied Sciences  2015;7(Suppl 1):S46-S48.
There is increased awareness and anxiety in conducting research for publication and at the same time ignorance about getting Ethical Committee clearance at least in Anatomy Departments among Basic Medical Sciences. While people are actively presenting papers, collect data, Indian Council for Medical Research guidelines does not cover aspects pertaining to Anatomy oriented research activities. This review article is an eye opener for fraternity in the medical field, especially in anatomy.
PMCID: PMC4439706  PMID: 26015746
Body donation; cadaver; fetus
2.  Detection of the aerolysin gene in Aeromonas hydrophila by the polymerase chain reaction. 
Journal of Clinical Microbiology  1990;28(11):2477-2481.
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.
PMCID: PMC268209  PMID: 2254423
3.  Differentiation of Genes Coding for Escherichia coli Verotoxin 2 and the Verotoxin Associated with Porcine Edema Disease (VTe) by the Polymerase Chain Reaction 
Journal of Clinical Microbiology  1990;28(10):2351-2353.
Two sets of synthetic oligonucleotide primers were used in a polymerase chain reaction adaptation to distinguish the closely related genes for type 2 verotoxin (VT2 or Shiga-like toxin [SLT-II]) and the verotoxin associated with porcine edema disease (VTe or SLT-II variant [SLT-IIv]) in Escherichia coli.
PMCID: PMC268176  PMID: 2229362
4.  Virulence properties of enterotoxigenic Escherichia coli O8: KX105 strains isolated from diarrheic piglets. 
Infection and Immunity  1988;56(1):241-246.
Twenty-two enterotoxigenic Escherichia coli (ETEC) O8:KX105 strains isolated from 1- to 7-week-old diarrheic piglets were examined for virulence properties. Thirteen strains caused acute watery diarrhea in orally infected, colostrum-deprived newborn piglets, whereas the remaining nine did not. The enteropathogenic strains colonized the small intestine, albeit with lower intensity than classical porcine ETEC. They produced the heat-stable STb and heat-labile LT enterotoxins, whereas the nonenteropathogenic strains produced the STb enterotoxin alone. None of the E. coli O8:KX105 strains exhibited mannose-resistant hemagglutination with erythrocytes from 12 species. Ten of the enteropathogenic and two of the nonenteropathogenic strains were positive for mannose-sensitive hemagglutination. These strains produced rodlike fimbriae 3 to 5 nm in diameter, whereas no fimbriae were detected on the other strains. None of the 22 strains produced the fimbrial antigens F4, F5, F41, F2, F3, FY(Att 25), and F165. Of the 13 enteropathogenic strains, 10 expressed the F6 antigen in the intestines of infected piglets but not in cultures. The other three enteropathogenic strains apparently lacked all of the known fimbrial antigens from porcine ETEC.
PMCID: PMC259263  PMID: 2891618
5.  Discriminatory genomic fingerprinting of Legionella pneumophila by pulsed-field electrophoresis. 
Journal of Clinical Microbiology  1994;32(10):2620-2621.
Eight strains of Legionella pneumophila were used to optimize cleavage of DNA with BssHII, SalI, or SpeI and separation by pulsed-field electrophoresis. Isolates from a community outbreak involving a contaminated hot tub were genomically identical. Cleavage patterns were distinctly different for unrelated environmental and nosocomial strains from a single hospital.
PMCID: PMC264120  PMID: 7814513
6.  Rapid and specific detection of verotoxin genes in Escherichia coli by the polymerase chain reaction. 
Journal of Clinical Microbiology  1990;28(3):540-545.
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.
PMCID: PMC269659  PMID: 2182671
7.  Cytotoxic and cytotonic factors produced by Campylobacter jejuni, Campylobacter coli, and Campylobacter laridis. 
Journal of Clinical Microbiology  1986;24(2):275-281.
Complete toxigenicity studies were performed on 341 strains of Campylobacter spp., including 23 nonhuman isolates. Toxin profiles based on both cytotonic and cytotoxic factors were determined after analyzing responses in Vero, HeLa, CHO and Y-1 cells. Suckling mouse assays were consistently negative for all culture filtrates tested. Toxin-producing strains were frequently encountered among both the human and nonhuman strains of Campylobacter jejuni, C. coli, and C. laridis investigated. Strains isolated from outbreaks demonstrated parallels in serotype, biotype, and toxigenicity profile, although no clear association could be demonstrated. Biphasic culture conditions conducive to the production of both toxic factors were delineated for the propagation of test Campylobacter strains. Cytotonic effects of Campylobacter culture filtrates were determined in Vero and CHO cells, and cyclic AMP accumulation in cells exposed to these culture filtrates was compared with that in cells exposed to reference toxigenic strains of Vibrio cholerae and Escherichia coli. Partial neutralization of C. jejuni enterotoxin was demonstrated by using antitoxins to cholera toxin and E. coli heat-labile enterotoxin. No neutralization of C. jejuni cytotoxin could be achieved by using antitoxins to either Clostridium difficile cytotoxin or E. coli Verotoxin (0157:H7).
PMCID: PMC268889  PMID: 3018039
8.  Natural infection with an attaching and effacing Escherichia coli in a diarrheic puppy. 
Enteric infection with an attaching and effacing Escherichia coli was diagnosed in a puppy with protracted diarrhea. Extensive colonization of the small intestinal mucosa was observed by light and scanning electron microscopy and characteristic lesions of bacterial attachment of the brush border of the enterocytes were demonstrated by transmission electron microscopy. The E. coli strain isolated from the small intestine belonged to serotype O49:H10, did not produce any known E. coli enterotoxin or cytotoxin, was not invasive, and was negative for the known fimbrial colonization factors produced by animal and human enterotoxigenic E. coli. A positive immunoperoxidase reaction was obtained on the bacteria attached to the enterocytes with an anti-E. coli O49 antiserum.
PMCID: PMC1255441  PMID: 3285983
9.  Cytotoxic and enterotoxic activities of Campylobacter jejuni are not specified by tetracycline resistance plasmids pMAK175 and pUA466. 
Journal of Clinical Microbiology  1987;25(1):150-151.
The 45-kilobase tetracycline resistance plasmids pMAK175 and pUA466 from Campylobacter jejuni were examined using curing and mating experiments. However, these plasmids encoded neither cytotoxin production, as determined in Vero cells, nor enterotoxin activity, as determined in Chinese hamster ovary cells.
PMCID: PMC265844  PMID: 3793867
10.  Comparison of Molecular Methods for Typing Vibrio parahaemolyticus 
Journal of Clinical Microbiology  1999;37(8):2473-2478.
An outbreak of Vibrio parahaemolyticus gastroenteritis on Canada’s west coast in 1997 emphasized the need to develop molecular methods for differentiation and typing of these organisms. Isolates were analyzed by enterobacterial repetitive intergenic consensus sequence (ERIC) PCR, detection of restriction fragment length polymorphisms (RFLP) in rRNA genes (ribotyping), pulsed-field gel electrophoresis (PFGE), and RFLP analysis of the genetic locus encoding the polar flagellum (Fla locus RFLP analysis). ERIC PCR and ribotyping were the most informative typing methods, especially when used together, while Fla locus RFLP analysis was the least discriminatory. PFGE exhibited good discrimination but suffered from a high incidence of DNA degradation. ERIC PCR and ribotyping will be useful for the evaluation of genetic and epidemiological relationships among V. parahaemolyticus strains.
PMCID: PMC85259  PMID: 10405387
11.  Human salmonellosis associated with exotic pets. 
Journal of Clinical Microbiology  1997;35(11):2786-2790.
During the period from 1994 to 1996, an increase in the number of laboratory-confirmed cases of human salmonellosis associated with exposure to exotic pets including iguanas, pet turtles, sugar gliders, and hedgehogs was observed in Canada. Pet turtle-associated salmonellosis was recognized as a serious public health problem in the 1960s and 1970s, and in February 1975 legislation banning the importation of turtles into Canada was enacted by Agriculture Canada. Reptile-associated salmonellosis is once again being recognized as a resurgent disease. From 1993 to 1995, there were more than 20,000 laboratory-confirmed human cases of salmonellosis in Canada. The major source of Salmonella infection is food; however, an estimated 3 to 5% of all cases of salmonellosis in humans are associated with exposure to exotic pets. Among the isolates from these patients with salmonellosis, a variety of Salmonella serotypes were also associated with exotic pets and included the following: S. java, S. stanley, S. poona, S. jangwani, S. tilene, S. litchfield, S. manhattan, S. pomona, S. miami, S. rubislaw, S. marina subsp. IV, and S. wassenaar subsp. IV.
PMCID: PMC230062  PMID: 9350734
12.  Analysis of genetic polymorphism in the phospholipase region of Mycobacterium tuberculosis. 
Journal of Clinical Microbiology  1997;35(5):1190-1195.
mtp40 was originally identified as a short genomic region that was found in strains of Mycobacterium tuberculosis but not in Mycobacterium bovis. Subsequent studies have revealed that the sequence is part of the mpcA gene, which encodes a phospholipase C. To investigate further the distribution of the mtp40 sequence, we analyzed strains of the M. tuberculosis complex by PCR and were able to amplify the mtp40 sequence in 90 of 94 strains of M. tuberculosis and in 2 strains of Mycobacterium microti but not in M. bovis or M. bovis BCG. Based on this, we developed a dot blot assay using genomic DNA which allows M. bovis to be distinguished from the majority of M. tuberculosis strains. We also probed Southern blots of 140 clinical isolates of M. tuberculosis to determine the frequency of strains lacking mtp40. This revealed an unexpected polymorphism in the phospholipase region. Two fragments were detected in 57% of samples. The expected fragment of 0.75 kbp corresponds to the region of mpcA containing mtp40. A 2.1-kbp fragment was observed to belong to a recently discovered second phospholipase gene, mpcB. In addition, some strains appeared to lack both genes, while others showed only the presence of mpcA. A few strains had additional bands, suggesting the existence of other homologs to the two phospholipase genes. We also detected the insertion of IS6110 in the mpcA coding region of one strain. The absence of these genes in some clinical isolates raises questions about their function during infection and in the development of tuberculosis disease in humans.
PMCID: PMC232727  PMID: 9114405
13.  Genetic identification of Mycobacterium bovis BCG by restriction fragment length polymorphism analysis of the direct-repeat region. 
Journal of Clinical Microbiology  1997;35(4):965-968.
Restriction fragment length polymorphism (RFLP) analysis was performed on the direct repeat (DR) regions of 14 strains of Mycobacterium bovis BCG. With AluI-digested DNA, BCG Japanese, Russian, and Mexican had differing RFLP patterns but 11 strains, including Pasteur, Glaxo, and Tice, had an identical pattern not detected in over 60 strains of the M. tuberculosis complex. DR analysis can aid in confirming the identification of clinical BCG isolates.
PMCID: PMC229711  PMID: 9157163
15.  Characterization of cytotoxic, hemolytic Aeromonas caviae clinical isolates and their identification by determining presence of a unique hemolysin gene. 
Journal of Clinical Microbiology  1996;34(12):3203-3205.
Aeromonas caviae has recently been recognized as an important enteropathogen and its hemolysin is purported to be one of the virulence factors, In this study, a total of 80 clinical isolates of Aeromonas spp. were investigated by PCR with synthetic oligonucleotides targeting a cloned hemolysin-encoding sequence from an A. caviae isolate of clinical origin. Of the 35 clinical A. caviae isolates tested, only 6 contained the target sequence.
PMCID: PMC229483  PMID: 8940472
16.  Identification of IS1356, a new insertion sequence, and its association with IS402 in epidemic strains of Burkholderia cepacia infecting cystic fibrosis patients. 
Journal of Clinical Microbiology  1996;34(7):1610-1616.
Burkholderia cepacia is now recognized as an important opportunistic pathogen in cystic fibrosis (CF) and other compromised patients. Epidemicity among CF patients has been attributed to at least one particularly infectious strain (strain ET12), and both genetic evidence and anecdotal evidence suggest that this strain, currently endemic in Ontario, and those causing an epidemic in the United Kingdom, are indeed the same. Our study was conducted to determine whether there was any association between the presence of various insertion sequence (IS) elements, the cable pilin subunit gene (cblA), electrophoretic type (ET), and ribotype (RT) in a collection of 97 clinical and 2 environmental isolates of B. cepacia. No apparent linkage was found for IS elements IS401, IS402, IS406, IS407, and IS408 with ET or RT. The cblA target, said to be a marker for high infectivity, was detected in 100% (38 of 38) of strains of B. cepacia ET12 and in a single strain of ET13 that differed in a single enzyme allele. A new IS, IS1356, identified during the investigation, was present in 71.7% of all isolates, and 50.7% of these isolates harbored IS1356 as a hybrid IS element inserted into IS402. IS1356 is 1,353 bp in length, and when it is inserted into IS402 it results in a 10-bp duplication at the site of insertion. IS1356 contains one major open reading frame of 1,260 bp coding for a putative transposase which has significant homology to ISRm3 in Rhizobium meliloti (59%) and to an undesignated IS element in Corynebacterium diphtheriae (49%). The IS402-IS1356 element was found exclusively in the epidemic strains from Ontario and the United Kingdom, being detected in 94.7% (36 of 38 isolates) of B. cepacia ET12 isolates. Of the two ET12 isolates found to be devoid of the IS402-IS1356 element, both contained IS1356 unassociated with IS402, one was temporally unrelated to the epidemic, and the other was from a CF patient in a geographic area remote from Ontario and the United Kingdom. It is evident that the IS402-IS1356 hybrid element, the cblA pilin subunit gene, and the allelic suite represented by multilocus enzyme electrophoretic type ET12 may provide useful markers for the epidemic, highly transmissible transatlantic strain isolated in Ontario and the United Kingdom.
PMCID: PMC229080  PMID: 8784555
17.  Evaluation of ribotyping as epidemiologic tool for typing Escherichia coli serogroup O157 isolates. 
Journal of Clinical Microbiology  1996;34(3):720-723.
A total of 121 representative Escherichia coli O157:H7 and O157:NM (nonmotile) isolates were characterized by ribotype, phage type, verotoxin genotype, and genomic fingerprints generated by pulsed-field gel electrophoresis. Ribotyping was not able to discriminate between O157:H7 isolates, and phage typing and pulsed-field gel electrophoresis were the most valuable and discriminatory techniques.
PMCID: PMC228877  PMID: 8904445
18.  Oligonucleotide primers designed to differentiate pathogenic pseudomonads on the basis of the sequencing of genes coding for 16S-23S rRNA internal transcribed spacers. 
Universal primers targeting conserved sequences flanking the 3' end of the 16S and the 5' end of the 23S rRNA genes (rDNAs) were used to amplify the 16S-23S rDNA internal transcribed spacers (ITS) from eight species of pseudomonads which have been associated with human infections. Amplicons from reference strains of Pseudomonas aeruginosa, Pseudomonas cepacia, Pseudomonas gladioli, Pseudomonas mallei, Pseudomonas mendocina, Pseudomonas pickettii, Pseudomonas pseudomallei, and Xanthomonas maltophilia were cloned from each species, and sequence analysis revealed a total of 19 distinct ITS regions, each defining a unique sequevar with ITS sizes ranging from 394 (P. cepacia) to 641 (P. pseudomallei) bp. Five distinct ITS sequevars in P. cepacia, four in P. mendocina, three in P. aeruginosa, two each in P. gladioli and P. pseudomallei, and one each in P. mallei, P. pickettii, and X. maltophilia were identified. With the exception of one P. cepacia ITS, all ITS regions contained potential tRNA sequences for isoleucine and/or alanine. On the basis of these ITS sequence data, species-specific oligonucleotide primers were designed to differentiate P. aeruginosa, P. cepacia, and P. pickettii. The specificities of these primers were investigated by testing 220 clinical isolates, including 101 strains of P. aeruginosa, 103 strains of P. cepacia, and 16 strains of P. pickettii, in addition to 24 American Type Culture Collection (ATCC) Pseudomonas strains. The results showed that single primer pairs directed at particular ITSs were capable of specifically identifying the ATCC reference strains and all of the clinical isolates of P. aeruginosa and P. pickettii, but this was not the case with several ITS-based primer pairs tested for P. cepacia. This pathogen, on the other hand, could be specifically identified by primer pairs directed against the 23S rDNA.
PMCID: PMC170177  PMID: 7583922
19.  Linkage analysis of geographic and clinical clusters in Pseudomonas cepacia infections by multilocus enzyme electrophoresis and ribotyping. 
Journal of Clinical Microbiology  1994;32(4):924-930.
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.
PMCID: PMC263164  PMID: 7517953
20.  Genomic fingerprinting of Neisseria meningitidis associated with group C meningococcal disease in Canada. 
Journal of Clinical Microbiology  1993;31(9):2506-2508.
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.
PMCID: PMC265787  PMID: 8408577
21.  Streptococcal erythrogenic toxin genes: detection by polymerase chain reaction and association with disease in strains isolated in Canada from 1940 to 1991. 
Journal of Clinical Microbiology  1992;30(12):3127-3131.
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.
PMCID: PMC270600  PMID: 1452695
22.  Identification of verotoxin type 2 variant B subunit genes in Escherichia coli by the polymerase chain reaction and restriction fragment length polymorphism analysis. 
Journal of Clinical Microbiology  1991;29(7):1339-1343.
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.
PMCID: PMC270112  PMID: 1679436
23.  Detection of genes for enterotoxins, exfoliative toxins, and toxic shock syndrome toxin 1 in Staphylococcus aureus by the polymerase chain reaction. 
Journal of Clinical Microbiology  1991;29(3):426-430.
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.
PMCID: PMC269793  PMID: 2037659
24.  Purification of an Escherichia coli serogroup O157:H7 verotoxin and its detection in North American hemorrhagic colitis isolates. 
Journal of Clinical Microbiology  1989;27(9):1973-1978.
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.
PMCID: PMC267721  PMID: 2674193
25.  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)
PMCID: PMC1255543  PMID: 2469532

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