Biomedical subjects

S Sreevatsan

Publications and source records attributed to S Sreevatsan.

18 recordsLinked to original sources

PubMed2004

An overview of transmissible spongiform encephalopathies.

Transmissible spongiform encephalopathies (TSEs) are fatal neurodegenerative disorders of humans and animals associated with an accumulation of abnormal isoforms of prion protein (PrP) in nerve cells. The pathogenesis of TSEs involves conformational conversions of normal cellular PrP (PrP(c)) to abnormal isoforms of PrP (PrP(Sc)). While the protein-only hypothesis has been widely accepted as a causal mechanism of prion diseases, evidence from more recent research suggests a possible involvement of other cellular component(s) or as yet undefined infectious agent(s) in PrP pathogenesis. Although the underlying mechanisms of PrP strain variation and the determinants of interspecies transmissibility have not been fully elucidated, biochemical and molecular findings indicate that bovine spongiform encephalopathy in cattle and new-variant Creutzfeldt-Jakob disease in humans are caused by indistinguishable etiological agent(s). Cumulative evidence suggests that there may be risks of humans acquiring TSEs via a variety of exposures to infected material. The development of highly precise ligands is warranted to detect and differentiate strains, allelic variants and infectious isoforms of these PrPs. This article describes the general features of TSEs and PrP, the current understanding of their pathogenesis, recent advances in prion disease diagnostics, and PrP inactivation.

Animals↗

PubMed2000

A multiplex approach to molecular detection of Brucella abortus and/or Mycobacterium bovis infection in cattle.

A multiplex amplification and detection platform for the diagnosis of Mycobacterium bovis and Brucella abortus infection simultaneously in bovine milk and nasal secretions was developed. This system (designated the bovine pathogen detection assay [BPDA]-PCR) consists of duplex amplification of species-specific targets (a region of the BCSP31K gene of B. abortus and a repeat-sequence region in the hsp65 gene of M. bovis, respectively). This is followed by a solid-phase probe capture hybridization of amplicons for detection. On the basis of spiking experiments with normal milk, the analytical sensitivity of the assay was 800 CFU equivalents/ml of milk for B. abortus and as low as 4 CFU equivalents per ml of milk for M. bovis. BPDA-PCR was validated with 45 liver samples from lemmings experimentally infected with B. abortus. The assay sensitivity, based on culture status as a "gold standard," was 93.9%. In this experiment, BPDA-PCR also identified five culture-negative liver samples as positive (41.7%). Field studies for the evaluation of BPDA-PCR were performed with samples from dairy animals from geographically distinct regions (India, Mexico, and Argentina). A high prevalence of shedding of B. abortus (samples from India) and M. bovis (samples from Mexico) was identified by BPDA-PCR. In samples from India, B. abortus shedding was identified in 86% of milk ring test-positive animals (n = 15) and 80% of milk ring test-negative cows (n = 5). In samples from Mexico, M. bovis was identified by PCR in 32.6% of pools (n = 46) of milk that each contained milk from 10 animals and in 56.2% of nasal swabs (n = 121) from cattle from tuberculin test-positive herds. In contrast, the Argentine cattle (n = 70) had a modest prevalence of M. bovis shedding in nasal swabs (2.9%) and milk (1.4%) and of B. abortus in milk (11.4%). On the basis of these analyses, we identify BPDA-PCR as an optimal tool for both screening of herds and testing of individual animals in a disease eradication program. A combination of the duplex assay, screening of milk samples in pools, and the proposed algorithm provides a highly sensitive, cost-effective, and economically viable alternative to serological testing.

Animals↗

PubMed1998

Characterization of the phylogenetic distribution and chromosomal insertion sites of five IS6110 elements in Mycobacterium tuberculosis: non-random integration in the dnaA-dnaN region.

SETTING: Five IS6110 chromosomal insertion sites were characterized in the multidrug resistant Mycobacterium tuberculosis 'W' strain. OBJECTIVE: To use insertion site probes to study the phylogenetic distribution of IS6110 in the M. tuberculosis genome. DESIGN: A total of 722 M. tuberculosis isolates, previously genotyped using the standard IS6110 Southern blot hybridization methodology, were re-hybridized with the Region A insertion site probe and representative strains were further hybridized with the Region B and C probes. Strains were grouped on the basis of having IS6110 insertions in these different regions and their relatedness was further compared by sequencing the IS6110 insertion sites. RESULTS: The insertion site probes revealed that the collection of Chinese isolates previously grouped as the Beijing strain family shared IS6110 insertions in common with the W and other genotypic group 1 strains. Unexpectedly, we found that IS6110 integrated at least 10 independent times between the dnaA and dnaN genes encoding deoxyribonucleic acid replication proteins. CONCLUSIONS: IS6110 insertion site mapping is able to identify genetic relatedness among a collection of M. tuberculosis clinical strains representing the breadth of species diversity. The mapping data indicate that IS6110 insertion sites are not always random.

Blotting, Southern↗

PubMed1998

Algorithmic approach to high-throughput molecular screening for alpha interferon-resistant genotypes in hepatitis C patients.

This study was designed to analyze the feasibility and validity of using Cleavase Fragment Length Polymorphism (CFLP) analysis as an alternative to DNA sequencing for high-throughput screening of hepatitis C virus (HCV) genotypes in a high-volume molecular pathology laboratory setting. By using a 244-bp amplicon from the 5' untranslated region of the HCV genome, 61 clinical samples received for HCV reverse transcription-PCR (RT-PCR) were genotyped by this method. The genotype frequencies assigned by the CFLP method were 44.3% for type 1a, 26.2% for 1b, 13.1% for type 2b, and 5% type 3a. The results obtained by nucleotide sequence analysis provided 100% concordance with those obtained by CFLP analysis at the major genotype level, with resolvable differences as to subtype designations for five samples. CFLP analysis-derived HCV genotype frequencies also concurred with the national estimates (N. N. Zein et al., Ann. Intern. Med. 125:634-639, 1996). Reanalysis of 42 of these samples in parallel in a different research laboratory reproduced the CFLP fingerprints for 100% of the samples. Similarly, the major subtype designations for 19 samples subjected to different incubation temperature-time conditions were also 100% reproducible. Comparative cost analysis for genotyping of HCV by line probe assay, CFLP analysis, and automated DNA sequencing indicated that the average cost per amplicon was lowest for CFLP analysis, at $20 (direct costs). On the basis of these findings we propose that CFLP analysis is a robust, sensitive, specific, and an economical method for large-scale screening of HCV-infected patients for alpha interferon-resistant HCV genotypes. The paper describes an algorithm that uses as a reflex test the RT-PCR-based qualitative screening of samples for HCV detection and also addresses genotypes that are ambiguous.

Algorithms↗

PubMed1998

Comparative Evaluation of Cleavase Fragment Length Polymorphism With PCR-SSCP and PCR-RFLP to Detect Antimicrobial Agent Resistance in Mycobacterium tuberculosis.

Background: Several molecular methods potentially useful in the detection of Mycobacterium tuberculosis mutations, specifically in rpoB and katG, were compared. Methods and Results: DNA from 24 M. tuberculosis clinical isolates, with mutations associated with resistance to rifampin and/or isoniazid, was analyzed. A 128 bp amplicon, spanning the 81 bp rpoB region containing most mutations leading to rifampin resistance, was analyzed by polymerase chain reaction-single-strand conformation polymorphism (PCR-SSCP) and a recently introduced mutation scanning method, cleavase fragment length polymorphism (CFLP) analysis. Also, a 350 bp amplicon encompassing that region was analyzed by the CFLP method. CFLP analysis of the 350 bp amplicon (23 isolates) identified 14 of 17 mutants; however, CFLP analysis of the 128 bp amplicon accurately identified all mutants as did PCR-SSCP with interpretative difficulty for two codon 513 mutations. CFLP and PCR-restriction fragment length polymorphism (RFLP) analyses of a 623 bp amplicon encompassing katG codons 315 and 463 showed that the CFLP method identified single and dinucleotide codon 315 substitutions with or without codon 463 (CGG-->CTG) changes, whereas PCR-RFLP (MspI) missed one codon 315 polymorphism (AGC-->ACA) in three isolates. Conclusion: Both PCR-SSCP and CFLP analyses were sensitive in identifying all mutations on short sequences in the rpoB mutants. CFLP appears to be more efficient than SSCP and RFLP for the detection of mutations in large amplicons.

Journal Article↗

PubMed1997

Restricted structural gene polymorphism in the Mycobacterium tuberculosis complex indicates evolutionarily recent global dissemination.

One-third of humans are infected with Mycobacterium tuberculosis, the causative agent of tuberculosis. Sequence analysis of two megabases in 26 structural genes or loci in strains recovered globally discovered a striking reduction of silent nucleotide substitutions compared with other human bacterial pathogens. The lack of neutral mutations in structural genes indicates that M. tuberculosis is evolutionarily young and has recently spread globally. Species diversity is largely caused by rapidly evolving insertion sequences, which means that mobile element movement is a fundamental process generating genomic variation in this pathogen. Three genetic groups of M. tuberculosis were identified based on two polymorphisms that occur at high frequency in the genes encoding catalase-peroxidase and the A subunit of gyrase. Group 1 organisms are evolutionarily old and allied with M. bovis, the cause of bovine tuberculosis. A subset of several distinct insertion sequence IS6110 subtypes of this genetic group have IS6110 integrated at the identical chromosomal insertion site, located between dnaA and dnaN in the region containing the origin of replication. Remarkably, study of approximately 6,000 isolates from patients in Houston and the New York City area discovered that 47 of 48 relatively large case clusters were caused by genotypic group 1 and 2 but not group 3 organisms. The observation that the newly emergent group 3 organisms are associated with sporadic rather than clustered cases suggests that the pathogen is evolving toward a state of reduced transmissability or virulence.

Alleles↗

PubMed1997

Inactivation of Streptococcus pyogenes extracellular cysteine protease significantly decreases mouse lethality of serotype M3 and M49 strains.

Cysteine proteases have been implicated as important virulence factors in a wide range of prokaryotic and eukaryotic pathogens, but little direct evidence has been presented to support this notion. Virtually all strains of the human bacterial pathogen Streptococcus pyogenes express a highly conserved extracellular cysteine protease known as streptococcal pyrogenic exotoxin B (SpeB). Two sets of isogenic strains deficient in SpeB cysteine protease activity were constructed by integrational mutagenesis using nonreplicating recombinant plasmids containing a truncated segment of the speB gene. Immunoblot analyses and enzyme assays confirmed that the mutant derivatives were deficient in expression of enzymatically active SpeB cysteine protease. To test the hypothesis that the cysteine protease participates in host mortality, we assessed the ability of serotype M3 and M49 wild-type strains and isogenic protease-negative mutants to cause death in outbred mice after intraperitoneal inoculation. Compared to wild-type parental organisms, the serotype M3 speB mutant lost virtually all ability to cause mouse death (P < 0.00001), and similarly, the virulence of the M49 mutant was detrimentally altered (P < 0.005). The data unambiguously demonstrate that the streptococcal enzyme is a virulence factor, and thereby provide additional evidence that microbial cysteine proteases are critical in host-pathogen interactions.

Animals↗

PubMed1997

MMAS-1, the branch point between cis- and trans-cyclopropane-containing oxygenated mycolates in Mycobacterium tuberculosis.

The proportion of mycolic acid containing trans-substituents at the proximal position of the meromycolate chain is an important determinant of fluidity of the mycobacterial cell wall and is directly related to the sensitivity of mycobacterial species to hydrophobic antibiotics. MMAS-1, an enzyme encoded in the gene cluster responsible for the biosynthesis of methoxymycolates, was overexpressed in Mycobacterium tuberculosis and shown to result in the overproduction of trans-cyclopropane and trans-olefin-containing oxygenated mycolic acids. MMAS-1 converted a cis-olefin into a trans-olefin with concomitant introduction of an allylic methyl branch in a precursor to both the methoxy and ketone-containing mycolic acids. In addition to an increase in the amount of trans-mycolate, MMAS-1 expression resulted in a substantial increase in the amount of ketomycolate produced relative to methoxymycolate. Thus MMAS-1 may act at a complex branch point where expression of this enzyme directly affects the cis- to trans-ratio and indirectly affects the keto to methoxy ratio. Overexpression of MMAS-1 resulted in a substantially slower growth rate at moderately elevated temperature, decreased thermal stability of the cell wall as measured by differential scanning calorimetry, and an increased permeability to chenodeoxycholate. These results provide experimental evidence for the intermediacy of trans-olefinic mycolate precursors in trans-cyclopropane formation and suggest that increasing the proportion of the polar ketomycolate subclass may exert a significant fluidizing effect on the cell wall.

Bacterial Proteins↗

PubMed1997

The emb operon, a gene cluster of Mycobacterium tuberculosis involved in resistance to ethambutol.

Ethambutol (EMB), a frontline antituberculous drug, targets the mycobacterial cell wall, a unique structure among prokaryotes which consists of an outer layer of mycolic acids covalently bound to peptidoglycan via the arabinogalactan. EMB inhibits the polymerization of cell wall arabinan, and results in the accumulation of the lipid carrier decaprenol phosphoarabinose, which suggests that the drug interferes with the transfer of arabinose to the cell wall acceptor. Unfortunately, resistance to EMB has been described in up to 4% of clinical isolates of Mycobacterium tuberculosis and is prevalent among isolates from patients with multidrug-resistant tuberculosis. We used resistance to EMB as a tool to identify genes participating in the biosynthesis of the mycobacterial cell wall. This approach led to the identification of the embCAB gene cluster, recently proposed to encode for mycobacterial arabinosyl transferases. Resistance to EMB results from an accumulation of genetic events determining overexpression of the Emb protein(s), structural mutation in EmbB, or both. Further characterization of these proteins might provide information on targets for new chemotherapeutic agents and might help development of diagnostic strategies for the detection of resistant M. tuberculosis.

Amino Acid Sequence↗

PubMed1997

Analysis of the oxyR-ahpC region in isoniazid-resistant and -susceptible Mycobacterium tuberculosis complex organisms recovered from diseased humans and animals in diverse localities.

Automated DNA sequencing was used to analyze the oxyR-ahpC region in 229 Mycobacterium tuberculosis complex isolates recently recovered from diseased humans and animals. The entire 1,221-bp region was studied in 118 isolates, and 111 other isolates were sequenced for oxyR, ahpC, or the 105-bp oxyR-ahpC intergenic region. The sample included isoniazid (INH)-susceptible and -resistant organisms in which the katG gene and inhA locus had previously been sequenced in their entirety to identify polymorphisms. A total of 16 polymorphic sites was identified, including 5 located in oxyR, 2 in ahpC, and 9 in the 105-bp intergenic region. All polymorphic sites located in the intergenic region, and the two missense substitutions identified in ahpC, occurred in INH-resistant organisms. In contrast, there was no preferential association of polymorphisms in oxyR, a pseudogene, with INH-resistant organisms. Surprisingly, most INH-resistant strains with KatG codon 315 substitutions that substantially reduce catalase-peroxidase activity and confer high MICs of INH lacked alterations in the ahpC gene or oxyR-ahpC intervening region. Taken together, the data are consistent with the hypothesis that some polymorphisms located in the ahpC-oxyR intergenic region are selected for after reduction in catalase or peroxidase activity attributable to katG alterations arising with INH therapy. These mutations are uncommon in recently recovered clinically significant organisms, and hence, there is no strict association with INH-resistant patient isolates. The ahpC compensatory mutations are apparently uncommon because strains with a KatG null phenotype are relatively rare among epidemiologically independent INH-resistant organisms.

Animals↗

PubMed1997

Mutations associated with pyrazinamide resistance in pncA of Mycobacterium tuberculosis complex organisms.

A gene (pncA) with mutations associated with pyrazinamide resistance in Mycobacterium tuberculosis complex members was characterized in 67 pyrazinamide-resistant and 51 pyrazinamide-susceptible isolates recovered from diverse geographic localities and anatomic sites and typed by IS6110 profiling. All pyrazinamide-susceptible organisms had identical pncA alleles. In striking contrast, 72% of the 67 resistant organisms had pncA mutations that altered the primary amino acid sequence of pyrazinamidase. A total of 17 previously undescribed mutations were found, including upstream mutations, missense changes, nucleotide insertions and deletions, and termination mutations. The mutations were arrayed along virtually the entire length of the gene. These data are further evidence that most drug resistance in M. tuberculosis is due to simple mutations occurring in chromosomally encoded genes rather than to acquisition of resistance genes by horizontal transfer events.

Amidohydrolases↗

PubMed1997

Ethambutol resistance in Mycobacterium tuberculosis: critical role of embB mutations.

Ethambutol [(S,S')-2,2'-(ethylenediimino)di-1-butanol; EMB], is a first-line drug used to treat tuberculosis. To gain insight into the molecular basis of EMB resistance, we characterized the 10-kb embCAB locus in 16 EMB-resistant and 3 EMB-susceptible genetically distinct Mycobacterium tuberculosis strains from diverse localities by automated DNA sequencing and single-stranded conformation polymorphism analysis. All 19 organisms had virtually identical sequences for the entire 10-kb region. Eight EMB-resistant organisms had mutations located in codon 306 of embB that resulted in the replacement of the wild-type Met residue with Ile or Val. Automated sequence analysis of the 5' region (1,892 bp) of embB in an additional 69 EMB-resistant and 30 EMB-susceptible M. tuberculosis isolates from diverse geographic localities and representing 70 distinct IS6110 fingerprints confirmed the unique association of substitutions in amino acid residue 306 of EmbB with EMB resistance. Six other embB nucleotide substitutions resulting in four amino acid replacements were uniquely found in resistant strains. Sixty-nine percent of epidemiologically unassociated EMB-resistant organisms had an amino acid substitution not found in susceptible strains, and most (89%) replacements occurred at amino acid residue 306 of EmbB. For strains with the Met306Leu or Met306Val replacements EMB MICs were generally higher (40 microg/ml) than those for organisms with Met306Ile substitutions (20 microg/ml). The data are consistent with the idea that amino acid substitutions in EmbB alter the drug-protein interaction and thereby cause EMB resistance.

Antitubercular Agents↗

PubMed1997

Animal by-products contaminated with Salmonella in the diets of lactating dairy cows.

As part of a total mixed ration, two rumen-fistulated dairy cows were fed meat and bone meal that had been artificially contaminated with Salmonella spp. Samples from the rumen, feces, and milk were taken 3 d/wk and cultured for salmonella. Rectal temperatures and rumen pH were also measured at the time of sample collection. Over the 2-mo study, salmonella were intermittently recovered from rumen contents, from feces, and from necropsy specimens of rumen contents, cecal contents, and mesenteric lymph nodes. No excretion of salmonella in milk was detected. An elevated rumen pH was associated with increased isolation of salmonella. No clinical illness was observed for either cow. Meat and bone meal that has been contaminated with low concentrations of salmonella is unlikely to result in clinical illness in healthy adult lactating cows. However, dairy producers should continue to be concerned about feed biosecurity and water contamination of animal by-products to prevent and control contamination by salmonella.

Animal Feed↗

PubMed1996

Evaluation of three experimental subunit vaccines against pneumonic pasteurellosis in cattle.

The objective of this study was to evaluate the efficacy of three Pasteurella haemolytica A1 derived experimental subunit vaccines against pneumonic pasteurellosis in cattle. The three vaccines were: (a) culture supernatant (CS) containing leukotoxin (Lkt), lipopolysaccharide (LPS) and capsular polysaccharide (CP); (b) sodium salicylate extract (SSE) containing iron regulated outer membrane proteins (IROMPs), LPS and CP; (c) and a combination of the above two. Vaccine efficacy was defined in terms of reduction in clinical and pneumonic lesion scores after intrapulmonic challenge with live P. haemolytica. The results indicate that the CS vaccine elicited antibodies against both Lkt and CP, while the SSE vaccine elicited antibodies against IROMPs and CP. Animals inoculated with the combination vaccine showed increased levels of antibodies against IROMPs, Lkt and CP. There was significant correlation between lung and serum antibodies against Lkt, CP and IROMPs. Animals that received the combination vaccine had significantly lower mean pneumonic lung score as compared to SSE and control groups. The animals which received CS vaccine had mean pneumonic lung score significantly lower than that of control group. A strong negative correlation existed between serum antibody levels against Lkt, IROMPs, CP and pneumonic lung scores. The results from this study demonstrate the usefulness of CS vaccine alone or in combination with SSE vaccine in bringing about optimal protection in vaccinated calves, against experimental pneumonic pasteurellosis.

Animals↗

PubMed1996

Fluoroquinolone resistance associated with specific gyrase mutations in clinical isolates of multidrug-resistant Mycobacterium tuberculosis.

Fluoroquinolones are potent antibacterial agents being used clinically against multidrug-resistant tuberculosis. Treatment failure is thought to arise from acquisition of fluoroquinolone resistance by Mycobacterium tuberculosis. A collection of 13 resistant clinical isolates of M. tuberculosis was examined for ciprofloxacin sensitivity relative to controls exhibiting the same IS6110 DNA type. Specific alleles were associated with distinct levels of drug susceptibility for 11 isolates that contained nucleotide changes expected to alter the amino acid sequence of the A subunit of DNA gyrase. Five different gyrA (ciprofloxacin resistance) alleles were present among 7 isolates having the W DNA subtype. These isolates, which are representative of an outbreak strain, constitute a panel of organisms that can be used to evaluate contributions of gyrase and DNA topoisomerase IV to resistance.

Anti-Infective Agents↗

PubMed1996

Characterization of rpsL and rrs mutations in streptomycin-resistant Mycobacterium tuberculosis isolates from diverse geographic localities.

Two genes (rpsL and rrs) with mutations associated with streptomycin resistance in Mycobacterium tuberculosis were characterized in 78 streptomycin-resistant and 61 streptomycin-susceptible isolates recovered from patients living in the United States, South America, Europe, Africa, and Asia. Fifty-four percent of the 78 resistant organisms had missense mutations in codon 43 of rpsL resulting in a K-43-->R substitution. Mutations in codon 88 of rpsL were also identified in four Asian isolates.

Antibiotics, Antitubercular↗

PubMed1996

Identification of a polymorphic nucleotide in oxyR specific for Mycobacterium bovis.

Automated sequence analysis of a 410-bp region of the axyR gene in 105 Mycobacterium tuberculosis complex isolates identified a polymorphic nucleotide that differentiated Mycobacterium bovis isolates from other complex members. All 29 M. bovis isolates sequenced had an adenine residue at nucleotide 285, whereas all 76 other complex isolates had a guanine residue. PCR-restriction fragment length polymorphism analysis of oxyR with restriction endonuclease AluI in an additional 255 complex isolates from widespread intercontinental sources confirmed and extended the unique association of adenine at position 285 with M. bovis isolates.

Adenosine↗

PubMed1996

A dot immunobinding assay (dot-ELISA) for the rapid serodiagnosis of Salmonella enteritidis infection in chickens.

A dot immunobinding assay (DIA) was developed for the detection of antibodies to Salmonella enteritidis. Western blot analysis of outer membrane proteins from SE identified 2 polypeptides of molecular masses 43 and 46 kD that were specific for S. enteritidis. These 2 polypeptides were utilized as antigens in the DIA. The DIA was tested on sera from chickens experimentally infected with S. enteritidis. Results of the DIA were compared with that of conventional microagglutination and serum plate tests. The DIA was a highly specific and sensitive test that can be useful for screening birds to determine if they are infected with S. enteritidis. Its simplicity, reliability, reproducibility, and speed in interpreting the assay results makes it a useful screening test for flock monitoring.

Animals↗