Pyrosequencing for rapid detection of tuberculosis resistance to Rifampicin and Isoniazid in Syrian and Lebanese clinical isolates.

BACKGROUND: Rapid and accurate techniques are always welcomed for the detection of resistant strains of Mycobacterium tuberculosis MTB. OBJECTIVES: The objective of this study is to evaluate the pyrosequencing technology for the detection of MTB resistance to Rifampicin (RIF) and Isoniazid (INH) in Syrian and Lebanese clinical strains; 66 strains resistant to INH, among them 56 resistant also to RIF, were tested. METHODS: Four pyrosequencing assays were optimized and applied to the following loci: rpoBrpoB RIF resistance-determining region, katG, the promoter regions of inhA and ahpC-oxyR intergenic region. RESULTS: The prevalence of mutations on codon 315 of the katG gene, inhA and ahpc-oxyR were 42.4%, 21.2% and 9.0%, respectively, which make an overall sensitivity of 72.6% for INH resistance. All RIF-resistant strains contained at least one non-synonymous codon change in the sequenced rpoB region (507-533) relative to the ATCC reference strain. The RIF drug resistance region (RRDR) sequencing identified 96 modified codons representing 34 different mutations. CONCLUSIONS: The high sensitivity and the short turnaround time combined with multilocus sequencing of several isolates in parallel make pyrosequencing an attractive method for drug resistance screening for MTB.

Mycobacterium tuberculosis spoligotypes circulating in the Lebanese population: a retrospective study.

Genotyping Mycobacterium tuberculosis in Lebanon on the national level may be beneficial for assessing patients and monitoring the therapeutic response to DOTS. This study aimed to characterize the spoligotypes of clinical isolates of M. tuberculosis patients collected between April 2004 and October 2005 from all Lebanese provinces. Isolates (n = 60) were cultured and identified by their biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. Thirteen (13) patterns of M. tuberculosis complex family strains were identified: 41.6% of the strains belonged to the T 1 family, 25.0% to LAM 9, 10.0% to Haarlem 3, 3.3% to each of CAS, LAM 8, BCG and Family 36 and 1.7% to each of Haarlem 1, LAM 10, S, M. africanum, X 1 and T 3 families. The noticeable absence of Beijing and East African Indian families was not consistent with the patterns reported in neighbouring countries. A more inclusive study of the Lebanese population is necessary to accurately identify most of the prevailing families in the country.

Mycobacterium tuberculosis spoligotypes circulating in the Lebanese population: a retrospective study

Genotyping Mycobacterium tuberculosis in Lebanon on the national level may be beneficial for assessing patients and monitoring the therapeutic response to DOTS. This study aimed to characterize the spoligotypes of clinical isolates of M tuberculosis patients collected between April 2004 and October 2005 from all Lebanese provinces. Isolates [n = 60] were cultured and identified by their biochemical characteristics. DNA extracts of these samples were amplified by PCR and genotyped by spoligotyping. Thirteen [13] patterns of M tuberculosis complex family strains were identified: 41.6% of the strains belonged to the T 1 family, 25.0% to LAM 9,10.0% to Haarlem 3, 3.3% to each of CAS, LAM 8, BCG and Family 36 and17% to each of Haarlem 1, LAM 10, S, M. africanum, X 1 and T 3 families. The noticeable absence of Beijing and East African Indian families was not,consistent with the patterns reported in neighbouring countries. A more inclusive study of the Lebanese population Is necessary to accurately identify most of the prevailing families in the country

Characterization of Mycobacterium tuberculosis of Lebanese patients by double-repetitive-element polymerase chain reaction.

Molecular studies have been successfully applied in evaluating epidemiological linkages in tuberculosis. A total of 87 isolates of Mycobacterium tuberculosis were collected from patients in all regions of Lebanon and characterized in terms of drug sensitivity. Double-repetitive-element polymerase chain reaction was used to differentiate between strains. Various correlations related to age, sex, region, sensitivity and genotype were examined. Several genotypes were more common in certain age ranges. Male patients appeared more likely either to be infected by or to develop multi-drug resistant strains. There was also evidence for a distribution of genotype groups indicating some level of geographical isolation and hence separate evolution of M. tuberculosis strains.

Characterization of Mycobacterium tuberculosis in Syrian patients by double-repetitive-element polymerase chain reaction.

The role of previous treatment in the dynamics of tuberculosis transmission has not been adequately investigated. Mycobacterium tuberculosis isolates from previously treated patients (n = 88) from all regions of Syrian Arab Republic were characterized in terms of antibiotic sensitivity and genotyping using double-repetitive-element polymerase chain reaction (DRE-PCR) method for the proximity of the repetitive DNA elements IS6110 (a mobile genetic element) and PGRS. The 88 isolates resulted in 59 different DRE-PCR patterns. Correlations related to age, sex, region, sensitivity and genotype were examined. All regions of the country showed high levels of genotype diversity, suggesting a low level of transmission of M. tuberculosis strains in previously treated patients.

Characterization of Mycobacterium tuberculosis in Syrian patients by double-repetitive-element polymerase chain reaction

The role of previous treatment in the dynamics of tuberculosis transmission has not been adequately investigated. Mycobacterium tuberculosis isolates from previously treated patients [n = 88] from all regions of Syrian Arab Republic were characterized in terms of antibiotic sensitivity and genotyping using double-repetitive-element polymerase chain reaction [DRE-PCR] method for the proximity of the repetitive DNA elements IS6110 [a mobile genetic element] and PGRS. The 88 isolates resulted in 59 different DRE-PCR patterns. Correlations related to age, sex, region, sensitivity and genotype were examined. All regions of the country showed high levels of genotype diversity, suggesting a low level of transmission of M. tuberculosis strains in previously treated patients

Characterization of Mycobacterium tuberculosis of Lebanese patients by double-repetitive-element polymerase chain reaction

Molecular studies have been successfully applied in evaluating epidemiological linkages in tuberculosis. A total of 87 isolates of Mycobacterium tuberculosis were collected from patients in all regions of Lebanon and characterized in terms of drug sensitivity. Double-repetitive-element polymerase chain reaction was used to differentiate between strains. Various correlations related to age, sex, region, sensitivity and genotype were examined. Several genotypes were more common in certain age ranges. Male patients appeared more likely either to be infected by or to develop multi-drug resistant strains. There was also evidence for a distribution of genotype groups indicating some level of geographical isolation and hence separate evolution of M. tuberculosis strains

Inhibition Effects in the Hydrolysis Reactions of Esters and Peptides Catalyzed by Carboxypeptidase A: An Example of Cooperative Binding Effects with a Monomeric Enzyme.

N-benzoyl-L-phenylalanyl-L-phenylalanine is an excellent peptide substrate for carboxy-peptidase A; at 30 degrees C and pH 7.5, K(m) is 2.6 x 10(-5) M while k(cat) is 177 s(-1) (k(cat)/K(m) = 6.8 x 10(6) M(-1) s(-1)). Indole-3-acetic acid is a noncompetitive or mixed inhibitor towards the peptide and toward hippuryl-L-phenylalanine; plots of E/V vs [Inhibitor] are linear. N-Benzoyl-L-phenylalanine is a competitive inhibitor of peptide hydrolysis, and plots of E/V vs [Inhibitor] are again linear. One molecule of inhibitor binds per active site, and these inhibitors bind in different sites. At constant peptide substrate concentration and a series of constant concentrations of indole-3-acetic acid, plots of E/V vs the concentration of N-benzoyl-L-phenylalanine are linear and intersect behind the E/V axis and above the [Inhibitor] axis. This shows that both inhibitors can bind simultaneously and that binding of one facilitates the binding of the other (beta = 0.18). Employing the ester substrate hippuryl-DL,beta-phenyllactate, the same type of behavior is observed in the reverse sense; N-benzoyl-L-phenylalanine is a linear noncompetitive inhibitor and indole-3-acetic acid is a linear competitive inhibitor. Again the two inhibitor plot is linear and intersects above the [Inhibitor] axis (beta = 0.12). Previous X-ray crystallographic studies have indicated that indole-3-acetic acid binds in the hydrophobic pocket of the S'(1) site, while N-benzoyl-L-phenylalanine binds in the S(1)-S(2) site. The product complex for hydrolysis of N-benzoyl-L-phenylalanyl-L-phenylalanine (phenylalanine + N-benzoyl-L-phenylalanine) occupies both of these sites. However, the present work shows that the peptide substrate does not bind to the enzyme at pH 7.5 so as to be competitive with indole-3-acetic acid. The binding sites may be formed via conformational changes induced or stabilized by substrate and product binding. Copyright 2000 Academic Press.

Substituent effects in the carboxypeptidase A catalyzed hydrolysis of substituted L,beta-phenyllactate esters.

The carboxypeptidase A catalyzed hydrolysis of an extensive series of substituted cinnamoyl-L,beta-phenyllactate esters has been investigated. Plots of kcat vs pH are sigmoidal in the pH range 5-9 with an average apparent pKaES of 6.6 +/- 0.1. The values of Km are pH independent in the range pH 5-8. Plots of log kcat/Km vs pH give pKaE values of 6.4 and 9.0 that do not vary significantly through the series. A plot of log kcat (pH 8) vs sigma, the Hammett substituent constant, is linear with a slope rho of 0.5, while log Km vs sigma has a slope of -0.4. The plot of log kcat/Km vs sigma is also linear with rho = 0.9. The Hammett plots are linear at both pH 6 and 8 with closely similar slopes, which indicates that the apparent pKaES near pH 6 does not reflect a change in the rate-determining step. The enzymatic reactions and the nonenzymatic OH- catalyzed hydrolysis reactions are affected alike by changes in the substituent groups; a plot of log kOH, the second-order rate constant for alkaline hydrolysis of the esters, vs log kcat/Km is linear with a slope of 0.9. There is little effect of changing the substituent group in the nonenzymatic pH-independent hydrolysis of the Zn(II) complex of corresponding 4-substituted cinnamic acid 6-carboxypicolinic acid anhydrides (rho < or = 0.1).(ABSTRACT TRUNCATED AT 250 WORDS)