Biomedical subjects

B Swaminathan

Publications and source records attributed to B Swaminathan.

At least 37 records · Page 2Linked to original sources

PubMed1999

Identification of Bartonella species directly in clinical specimens by PCR-restriction fragment length polymorphism analysis of a 16S rRNA gene fragment.

It is now established that two species of Bartonella, namely, Bartonella henselae and B. quintana, cause bacillary angiomatosis in human immunodeficiency virus-infected patients. In addition, B. henselae causes cat scratch disease and B. quintana, B. henselae, and B. elizabethae can cause bacteremia and endocarditis in immunocompetent persons. We have developed a PCR-restriction fragment length polymorphism-based assay for direct detection and identification to species level of Bartonella in clinical specimens. This is accomplished by PCR amplification of Bartonella DNA using primers derived from conserved regions of the gene carrying the 16S ribosomal DNA, followed by restriction analysis using DdeI and MseI restriction endonucleases. We amplified a Bartonella genus-specific 296-bp fragment from 25 clinical samples obtained from 25 different individuals. Restriction analysis of amplicons showed that identical patterns were seen from digestion of B. henselae and B. quintana amplicons with DdeI, whereas a different unique pattern was seen by using the same enzyme with B. vinsonii and B. elizabethae. With MseI digestion, B. henselae and B. vinsonii gave nearly identical patterns while B. quintana and B. elizabethae gave a different pattern. By combining the restriction analysis data generated with MseI and DdeI, unique "signature" restriction patterns characteristic for each species were obtained. These patterns were useful in identifying the Bartonella species associated with each tissue specimen.

Angiomatosis, Bacillary↗

PubMed1999

Molecular subtyping of Clostridium perfringens by pulsed-field gel electrophoresis to facilitate food-borne-disease outbreak investigations.

Clostridium perfringens is a common cause of food-borne illness. The illness is characterized by profuse diarrhea and acute abdominal pain. Since the illness is usually self-limiting, many cases are undiagnosed and/or not reported. Investigations are often pursued after an outbreak involving large numbers of people in institutions, at restaurants, or at catered meals. Serotyping has been used in the past to assist epidemiologic investigations of C. perfringens outbreaks. However, serotyping reagents are not widely available, and many isolates are often untypeable with existing reagents. We developed a pulsed-field gel electrophoresis (PFGE) method for molecular subtyping of C. perfringens isolates to aid in epidemiologic investigations of food-borne outbreaks. Six restriction endonucleases (SmaI, ApaI, FspI, MluI, KspI, and XbaI) were evaluated with a select panel of C. perfringens strains. SmaI was chosen for further studies because it produced 11 to 13 well-distributed bands of 40 to approximately 1,100 kb which provided good discrimination between isolates. Seventeen distinct patterns were obtained with 62 isolates from seven outbreak investigations or control strains. In general, multiple isolates from a single individual had indistinguishable PFGE patterns. Epidemiologically unrelated isolates (outbreak or control strains) had unique patterns; isolates from different individuals within an outbreak had similar, if not identical, patterns. PFGE identifies clonal relationships of isolates which will assist epidemiologic investigations of food-borne-disease outbreaks caused by C. perfringens.

Centers for Disease Control and Prevention, U.S.↗

PubMed1998

Identification and characterization of nucleotide sequence differences in three virulence-associated genes of listeria monocytogenes strains representing clinically important serotypes.

Listeria monocytogenes is a Gram-positive, facultative intracellular bacterium that causes invasive, often fatal, disease in susceptible hosts. As a foodborne pathogen, the bacterium has emerged as a significant public health problem and has caused several epidemics in the United States and Europe. Three serotypes (1/2a, 1/2b, 4b) of L. monocytogenes are responsible for nearly 95% of all reported cases of human listeriosis. L. monocytogenes serotype 4b has caused all well-characterized foodborne epidemic outbreaks in North America and Europe between 1981 and 1993. However, most of the genetic studies to characterize virulence factors of L. monocytogenes have been done by using serotypes 1/2a and 1/2c. In this investigation, we examined three virulence-associated genes (hly encoding listeriolysin, plcA encoding phosphotidylinositol-specific phospholipase C, and inlA encoding internalin) of two serotype 4b and two serotype 1/2b strains. We chose these virulence-associated genes on the basis of published sequence differences among strains from Listeria subgroups containing serotypes 1/2a and 1/2c versus 4b, respectively. They correspond to sequence homologies that include very highly conserved (hlyA), highly conserved (plcA) and mostly conserved (inlA). We found by using nucleotide sequence analysis of the hly, plcA, and inlA genes, the two L. monocytogenes strains (including a strain associated with a foodborne disease outbreak in California in 1985) in this study, two serotype 1/2b strains from a study that we recently reported, and other similar published data for serotypes 1/2a, 1/2c, and 4b, had a high degree of sequence conservation at the gene and protein levels for all three genes. However, the sequences for the hly gene of L. monocytogenes strains of serotypes 1/2b and 4b were more closely related to each other and showed significant divergence from serotypes 1/2a and 1/2c. A unique nonsynonymous mutation was found in the hly gene of L. monocytogenes isolates that were associated with the 1985 California outbreak and were the epidemic phage type. When 158 L. monocytogenes isolates from the collection at the Centers for Disease Control and Prevention were screened, the mutation was found only in one other strain that had been isolated in California 3 years before the epidemic. Although the California epidemic clone was lactose negative, other L. monocytogenes serotype 4b isolates that were lactose negative did not possess the unique mutation observed in that epidemic clone.

Bacterial Proteins↗

PubMed1998

Computerized analysis of restriction fragment length polymorphism patterns: comparative evaluation of two commercial software packages.

Two computerized restriction fragment length polymorphism pattern analysis systems, the BioImage system and the GelCompar system (Molecular Analyst Fingerprinting Plus in the United States), were compared. The two systems use different approaches to compare patterns from different gels. In GelCompar, a standard reference pattern in one gel is used to normalize subsequent gels containing lanes with the same reference pattern. In BioImage, the molecular sizes of the fragments are calculated from size standards present in each gel. The molecular size estimates obtained with the two systems for 12 restriction fragments of phage lambda were between 97 and 101% of their actual sizes, with a standard deviation of less than 1% of the average estimated size for most fragments. At the window sizes used for analysis, the GelCompar system performed somewhat better than BioImage in identifying visually identical patterns generated by electrophoretic separation of HhaI-restricted DNA of Listeria monocytogenes. Both systems require the user to make critical decisions in the analysis. It is very important to visually verify that the systems are finding all bands in each lane and that no artifacts are being detected; both systems allow manual editing. It is also important to verify results obtained in the pattern matching or clustering portions of the analysis.

Bacteriophage lambda↗

PubMed1997

An outbreak of gastroenteritis and fever due to Listeria monocytogenes in milk.

BACKGROUND: After an outbreak of gastroenteritis and fever among persons who attended a picnic in Illinois, chocolate milk served at the picnic was found to be contaminated with Listeria monocytogenes. METHODS: In investigating this outbreak, we interviewed the people who attended the picnic about what they ate and their symptoms. Surveillance for invasive listeriosis was initiated in the states that receive milk from the implicated dairy. Stool and milk samples were cultured for L. monocytogenes. Serum samples were tested for IgG antibody to listeriolysin O. RESULTS: Forty-five persons had symptoms that met the case definition for illness due to L. monocytogenes, and cultures of stool from 11 persons yielded the organism. Illness in the week after the picnic was associated with the consumption of chocolate milk. The most common symptoms were diarrhea (present in 79 percent of the cases) and fever (72 percent). Four persons were hospitalized. The median incubation period for infection was 20 hours (range, 9 to 32), and persons who became ill had elevated levels of antibody to listeriolysin O. Isolates from stool specimens from patients who became ill after the picnic, from sterile sites in three additional patients identified by surveillance, from the implicated chocolate milk, and from a tank drain at the dairy were all serotype 1/2b and were indistinguishable on multilocus enzyme electrophoresis, ribotyping, and DNA macrorestriction analysis. CONCLUSIONS: L. monocytogenes is a cause of gastroenteritis with fever, and sporadic cases of invasive listeriosis may be due to unrecognized outbreaks caused by contaminated food.

Animals↗

PubMed1997

Nucleotide sequence analysis of two virulence-associated genes in Listeria monocytogenes serotype 1/2b and comparison with the same genes in other serotypes important in human disease.

The nucleotide sequences of the hly and plcA genes (encoding listeriolysin, a thiol-activated cytolysin and a non-specific phospholipase C, respectively) were determined for two strains of Listeria monocytogenes serotype 1/2b. The deduced amino acid sequences for listeriolysin were identical for the two strains and identical to that of listeriolysin from serotype 4b. The two serotype 1/2b strains differed in one amino acid (histidine vs asparagine) in the deduced amino acid sequence for phosopholipase C.

Cloning, Molecular↗

PubMed1997

Molecular characterization of the gene encoding H antigen in Escherichia coli and development of a PCR-restriction fragment length polymorphism test for identification of E. coli O157:H7 and O157:NM.

Recent outbreaks of disease caused by Escherichia coli O157:H7 have focused much attention on this newly emerged pathogen. Identification of the H7 flagellar antigen is critical for the confirmation of E. coli O157:H7; however, clinical isolates are frequently nonmotile and do not produce detectable H antigen. To further characterize nonmotile isolates (designated NM), we developed a PCR-restriction fragment length polymorphism (PCR-RFLP) test to identify and characterize the gene encoding the H antigen (fliC) in E. coli. The entire coding sequence of fliC was amplified by PCR, the amplicon was restricted with RsaI, and the restriction fragment pattern was examined after gel electrophoresis. Two hundred eighty E. coli isolates representing serotypes O157:H7 and O157:NM, flagellar antigen H7 groups associated with other O serogroups, and all other flagellar antigen groups were analyzed. A single restriction pattern (pattern A) was identified for O157:H7 isolates, O157:NM isolates that produced Shiga toxin (formerly Shiga-like toxin or verotoxin), and 16 of 18 O55:H7 isolates. Flagellar antigen group H7 isolates of non-O157 serotypes had one of three banding patterns distinct from pattern A. A wide variety of patterns were found among isolates of the other 52 flagellar antigen groups; however, none was identical to the O157:H7 pattern. Thirteen of 15 nonmotile strains that did not produce the A pattern had patterns that matched those of other known H groups. The PCR-RFLP in conjunction with O serogroup determination will be useful in identifying E. coli O157:H7 and related strains that do not express immunoreactive H antigen and could be expanded to include other clinically important E. coli strains.

Antigens, Bacterial↗

PubMed1996

WHO-sponsored international collaborative study to evaluate methods for subtyping Listeria monocytogenes: restriction fragment length polymorphism (RFLP) analysis using ribotyping and Southern hybridization with two probes derived from L. monocytogenes chromosome.

Seven laboratories participated in a WHO-sponsored international collaborative study, to evaluate methods for subtyping Listeria monocytogenes, by performing restriction fragment length polymorph sm (RFLP) analysis-based subtyping of an international study set of 80 strains of L. monocytogenes that included 22 epidemiologically related groups. The RFLP analysis was done by Southern hybridization with one of two types of probes found in multiple copies on the chromosome of L. monocytogenes. Six laboratories performed ribotyping. These laboratories used EcoRI enzyme to restrict the L. monocytogenes DNA and ribosomal RNA or DNA as the probe for Southern hybridizations. The seventh laboratory used Ncil to restrict the DNA, and two probes, one randomly cloned and the other containing repeat sequences cloned from L. monocytogenes DNA. The overall discriminating power of ribotyping, as estimated by calculation of Simpson's index of diversity, ranged from 0.83 to 0.88 for the six laboratories. The discriminating power of the combination of two probes used by Laboratory 7 was 0.91. Ribotyping and the cloned probes used by Laboratory 7 discriminated poorly between serotype 4b strains. Neither method identified three atypical strains (identified by other subtyping methods) included in three apparently epidemiologically related groups. Ribotyping did not discriminate between strains of serotypes 4b and 4b(X) in one epidemiologically related group of strains; one cloned probe used by Laboratory 7 discriminated between these strains. Intra-laboratory reproducibilities for the seven laboratories ranged from 80.0 to 100%. as determined by their abilities to correctly identify 11 pairs of duplicate strains included in the study set. Inter-laboratory reproducibilities were generally very good considering that no attempt was made to standardize protocols used by the participants.

Bacterial Typing Techniques↗

PubMed1996

A foodborne outbreak of gastroenteritis involving Listeria monocytogenes.

An outbreak of gastroenteritis occurred in Italy among 39 persons who had attended a private supper. All guests were previously healthy, young, non-pregnant adults; 18 (46%) had symptoms, mostly gastrointestinal (78%), with a short incubation period. Four were hospitalized with acute febrile gastroenteritis, two of whom had blood cultures positive for Listeria monocytogenes. No other microorganisms were recovered from the hospitalized patients' specimens. Epidemiological investigation identified rice salad as the most likely vehicle of the food-borne outbreak. L. monocytogenes was isolated from three leftover foods, the kitchen freezer and blender. Isolates from the patients, the foods and the freezer were indistinguishable: serotype 1/2b, same phage type and multilocus enzyme electrophoretic type. Eight (36%) of 22 guests tested were found to have antibodies against L. monocytogenes, compared with none of 11 controls from the general population. This point source outbreak was probably caused by infection with L. monocytogenes. Unusual features included the high attack rate among immunocompetent adults and the predominance of gastrointestinal symptoms.

Adolescent↗

PubMed1996

Bartonella vinsonii subsp. berkhoffii subsp. nov., isolated from dogs; Bartonella vinsonii subsp. vinsonii; and emended description of Bartonella vinsonii.

Two bacterial strains, one isolated from the blood of a dog with valvular endocarditis and one isolated from the blood of a healthy dog, were similar to Bartonella species, as determined by a number of phenotypic criteria, including growth characteristics, biochemical reactions, and cell wall fatty acid composition. The results of 16S rRNA gene sequence similarity studies confirmed that these strains are closely related and belong in the genus Bartonella and that Bartonella vinsonii is their closest relative (the 16S rRNA of isolate 93-C01T [T = type strain] was 99.37% identical to the 16S rRNA of the type strain of B. vinsonii, the 16S rRNA of isolate G7464 was 99.61% identical to the 16S rRNA of the type strain, and the 16S rRNAs of the dog isolates were 99.77% identical to each other). The 16S rRNAs of both strains contained a 12-base insertion that was not present in the 16S rRNA of the type strain of any Bartonella species. DNA relatedness tests revealed that these strains were related at the species level to the type strain of B. vinsonii. They were, however, significantly more closely related to each other than to B. vinsonii. On the basis of their unique 16S rRNA sequence insertion, their preferentially high level of relatedness, and their similar origins (dogs), we believe that strains 93-C01(T) and G7464 should be placed in a separate subspecies of B. vinsonii, for which we propose the name B. vinsonii subsp. berkhoffii subsp. nov. The type strain of B. vinsonii subsp. berkhoffii is strain 93-C01 (= ATCC 51672). The description of B. vinsonii is emended to accommodate the new subspecies, and B. vinsonii subsp. vinsonii is described.

Animals↗

PubMed1996

Rapid identification of Campylobacter species by restriction fragment length polymorphism analysis of a PCR-amplified fragment of the gene coding for 16S rRNA.

Restriction fragment length polymorphism analysis of a PCR-amplified DNA fragment of the gene coding for 16S rRNA was performed on 148 previously characterized strains of Campylobacter, Helicobacter, Arcobacter, and Wolinella succinogenes and 13 Campylobacter-like isolates. These strains included clinical, animal, and environmental isolates. PCR amplification generated a 283-bp fragment from all species. The amplicon from each strain was digested with six restriction endonucleases (AccI, AvaI, DdeI, HaeIII, HpaII, XhoI). DdeI was useful for the initial grouping of the strains. Additional discrimination within the different DdeI groups was obtained with AccI, HaeIII, HpaII, and XhoI digestions. The PCR-restriction fragment length polymorphism analysis allowed for the discrimination of members of the genus Campylobacter from members of closely related genera and discrimination between Campylobacter species. The proposed method is simple and rapid and can be useful for the routine identification of Campylobacter-like organisms in clinical or epidemiologic studies.

Animals↗

PubMed1996

Characterization of nonmotile variants of Escherichia coli O157 and other serotypes by using an antiflagellin monoclonal antibody.

An antiflagellin monoclonal antibody (15D8) was used to detect the presence of flagella in nonmotile variants of several pathogenic Escherichia coli serotypes. Of the 48 isolates examined, 15 reacted with monoclonal antibody 15D8 and were culturally confirmed to be motile. Of the 38 clinical strains designated O157:NM or O157:H-, 7 were antibody reactive and motile and agglutinated with anti-H7 sera.

Antibodies, Monoclonal↗

PubMed1996

Molecular subtyping of Neisseria meningitidis serogroup B: comparison of five methods.

In order to compare methods for subtyping Neisseria meningitidis serogroup B isolates, 96 isolates obtained from various locations in the United States and northwestern Europe were subtyped by five methods: monoclonal antibody (MAb)-based serotyping and serosubtyping, DNA macrorestriction analysis by pulsed-field gel electrophoresis (PFGE), multilocus enzyme electrophoresis (MEE), ribotyping, and PCR-restriction fragment length polymorphism of the internally transcribed spacer region of the rRNA operon (ITS PCR-RFLP). All N. meningitidis serogroup B isolates were typeable by PFGE, MEE, ribotyping, and ITS PCR-RFLP. Only 44.8% of the isolates were completely typeable (both serotype and serosubtype determination) by MAb-based serotyping and serosubtyping. 60.4% of the isolates could be serotyped but not serosubtyped, and 90.6% of the isolates could be either serotyped or serosubtyped. Simpson's discrimination indices of diversity for the methods were as follows: PFGE, 99.7%; MEE, 99.4%; ribotyping, 98.8%; MAb serotyping, 75.8%; MAb serotyping and/or serosubtyping 97.5%; and ITS PCR-RFLP, 84.2%. The high degree of diversity observed by PFGE, MEE, and ribotyping can be explained by the fact that isolates were collected from different geographic locations at various times. PFGE, MEE, and ribotyping showed greater discriminatory abilities than MAb-based serotyping and serosubtyping or ITS PCR-RFLP.

Antibodies, Monoclonal↗

PubMed1995

Bartonella (Rochalimaea) quintana bacteremia in inner-city patients with chronic alcoholism.

BACKGROUND: Bartonella (Rochalimaea) quintana is a fastidious gram-negative bacterium known to cause trench fever, cutaneous bacillary angiomatosis, and endocarditis. Between January and June 1993 in Seattle, we isolated B. quintana from 34 blood cultures obtained from 10 patients not known to be infected with the human immunodeficiency virus (HIV). METHODS: After identifying the isolates as B. quintana by direct immunofluorescence and DNA-hybridization studies, we determined strain hybridization with studies of restriction-fragment-length polymorphisms (RFLPs) of the intergenic spacer (noncoding) region of ribosomal DNA amplified by the polymerase chain reaction (PCR). To characterize the epidemiologic and clinical features of bartonella infections in these patients, we performed a retrospective case-control study using as controls 20 patients with blood cultures obtained at approximately the same time as those obtained from the index patients. RESULTS: B. quintana isolates from the 10 patients were indistinguishable by PCR-RFLP typing. All 10 patients had chronic alcoholism, and 8 were homeless (P = 0.001 for both comparisons with controls). The six patients who underwent HIV testing were seronegative. At the time of their initial presentation, seven patients had temperatures of at least 38.5 degrees C. Six patients had three or more blood cultures that were positive for B. quintana, and in four of these patients B. quintana was isolated from blood cultures obtained 10 or more days apart. Subacute endocarditis developed in two patients and required surgical removal of the infected aortic valve in one of them. Nine patients recovered; one died of sepsis from Streptococcus pneumoniae infection. CONCLUSIONS: B. quintana is a cause of fever, bacteremia, and endocarditis in HIV-seronegative, homeless, inner-city patients with chronic alcoholism.

Adult↗

PubMed1995

A rapid dot immunoassay for detecting the Brazilian purpuric fever clone of Haemophilus influenzae biogroup aegyptius with a "flow through" device.

Brazilian purpuric fever (BPF) is a highly fatal pediatric disease that may follow an episode of purulent conjunctivitis caused by a virulent clone of Haemophilus influenzae biogroup aegyptius (Hae). Oral rifampin prophylaxis, by eliminating carriage of the BPF clone in children with conjunctivitis, may prevent onset of the systemic disease. A test to detect the BPF clone directly from eye swabs could identify those in need of prophylaxis. This is a preliminary report of a rapid dot immunoassay performed on a "flow-through" cartridge that was developed for use under field conditions. The test is based upon recognition of a unique epitope of the 25-kDa pilin protein on the surface of BPF clone cells by a monoclonal antibody. With 36 laboratory-maintained cultures of Hae (15 clone isolates and 21 others), sensitivity of the assay was 67% and specificity was 95%. When fimbrial-enriched (25-kDa+) phenotypes of five false-negative clone strains were prepared for use as test antigens, sensitivity rose to 100%. Evaluation of the immunoassay under field conditions is necessary to prove its efficacy.

Conjunctivitis, Bacterial↗

PubMed1995

Bartonella (Rochalimaea) species as a cause of apparent "culture-negative" endocarditis.

Bartonella quintana (formerly Rochalimaea quintana) is a recently recognized cause of apparent "culture-negative" endocarditis. We describe a 39-year-old, homeless man who developed aortic valve endocarditis caused by B. quintana. He had a history of alcoholism and was seronegative for the human immunodeficiency virus. We established that B. quintana was the cause of the endocarditis on the basis of the isolation of B. quintana from blood cultures, the compatibility of histochemical stains of cardiac valve tissue, the reactivity of the polymerase chain reaction specific for B. quintana on cardiac valve tissue, and the failure to isolate an alternative causative organism despite extensive efforts. This is the second report of endocarditis caused by B. quintana and the fourth report of endocarditis caused by a Bartonella species. On the basis of the findings of this report and those of other recent reports, further study is warranted to determine the overall role of Bartonella species in apparent culture-negative endocarditis.

Adult↗

PubMed1995

Isolation and characterization of a beta-D-glucuronidase-producing strain of Escherichia coli serotype O157:H7 in the United States.

A phenotypic variant of Escherichia coli serotype O157:H7 (G5101) was isolated from a patient with bloody diarrhea. Strain G5101 does not ferment sorbitol but is beta-D-glucuronidase and urease positive. Serotyping and colony hybridization using a serotype-specific DNA probe confirmed that the isolate was O157:H7. G5101 produces Shiga-like toxins I and II and contains an eae gene that is highly conserved in the O157:H7 serotype. This strain would have been missed by laboratories that screen for the sorbitol-negative, beta-D-glucuronidase-negative phenotype in isolating E. coli O157:H7 from clinical and food specimens.

DNA Probes↗

PubMed1995

Identification of Campylobacter fetus by PCR-DNA probe method.

A PCR method for rapid identification of Campylobacter fetus subsp. fetus was evaluated. A fragment of the gene coding for 16S rRNA was amplified from crude cell lysates of 18 C. fetus strains and 30 strains representing other Campylobacter species and subspecies. The amplicons were probed by dot blot hybridization with a digoxigenin-labeled C. fetus-specific oligonucleotide probe. The probe reacted only with C. fetus subsp. fetus and C. fetus subsp. venerealis and may be useful for rapid identification in clinical laboratories.

Bacteriological Techniques↗