Genetic analysis of the embB gene revealed mutations in 17 (68%) of 25 ethambutol (EMB) resistant isolates (M306I, M306V, M306L, Q497R) but also in 4 (20%) of 20 EMB-susceptible isolates of Mycobacterium tuberculosis, namely, an ATG3ATM substitution resulting in M306I, G406N, and the novel alterations M423I and A659T. Ethambutol (EMB) [(S,SЈ)-2,2Ј-(ethylenediimino)di-1-butanol] is a first-line drug used for antituberculosis therapy. It is often used in combination with isoniazid, rifampin, pyrazinamide, and streptomycin. Membrane-associated arabinosyl transferases have been implicated as the targets for EMB (2,3,14,15). The Mycobacterium tuberculosis emb operon is a gene cluster of three contiguous genes, namely, embC, embA, and embB, which encode mycobacterial arabinosyl transferases (26). These enzymes are involved in the polymerization of the cell wall arabinan (4,6,9,24,25,32). Inhibition of arabinan synthesis by EMB results in the accumulation of mycolic acids, leading to cell death.Alterations at codon 306 of embB have been identified as being the most common alteration in EMB-resistant M. tuberculosis clinical isolates (8, 12, 17-20, 23, 29). Initial work on 51 EMB-resistant isolates had shown that 89% of these isolates had alterations at residue 306 of embB, but these alterations were not detected in 30 EMB-susceptible isolates (23). A subsequent study confirmed this high frequency of embB306 alterations, with 67% of 75 EMB-resistant isolates having mutations not found in EMB-susceptible strains (19). This led to several groups developing targeted strategies for the detection of embB306 alterations (7,16,21,30). Amino acids within the EMB resistance-determining region of EmbB proteins are well conserved among mycobacterial species, including those from M. tuberculosis, M. leprae, and M. smegmatis (2), and mutations within this region have been detected in EMB-resistant isolates of M. tuberculosis.The aim of this present work was to screen all regions of the embB gene with previously reported mutations in order to assess the contribution of mutations within this gene to EMB resistance in M. tuberculosis clinical isolates from Singapore.Drug susceptibility testing was done using the BACTEC 460 radiometric method (Becton Dickinson, Towson, Md.) (2.5 g/ml). Twenty-five consecutive M. tuberculosis isolates resistant to EMB and 20 EMB-susceptible isolates from Singapore were collected as previously described (5, 10).DNA extracted from the isolates was analyzed by amplifying four fragments, using the PCR primers shown in Table 1. The PCR products were purified (QIAquick PCR purification kit or QIAquick gel extraction kit; QIAGEN) and directly sequenced using the BigDye Terminator sequencing kit and the ABI PRISM 377 automated sequencer (PE Biosystems, Branchburg, N.J.). Confirmation of mutations was done by reamplification and resequencing.IS6110 profiling was done according to standard procedures to determine if the isolates were epidemiologically independent (28). All isolates with the same nucleotide substit...

A task force evaluated an in vitro antibody-mediated chlamydial neutralization assay for its utility as a method to assess functional correlates of antibody responses to Chlamydia trachomatis. Two monoclonal antibodies that recognize different major outer membrane protein (MOMP) epitopes for a C. trachomatis serovar B strain exhibit good in vitro neutralizing activity, with a maximum of 90% neutralization. Calculations based on the 50% neutralization point indicated that 100% neutralization could theoretically be achieved when only 10% of the MOMP molecules bound antibody. Monoclonal antibodies that recognized either a heterologous MOMP or the genus-specific chlamydial lipopolysaccharide did not produce neutralizing activity. The standardized assay will be useful to establish if in vitro neutralizing antibody responses are predictive of protective immunity and will aid in defining chlamydial antigens and epitopes that may be attractive vaccine candidates.

Screening of 127 isoniazid (INH)-resistantMycobacterium tuberculosis isolates from Singapore for mutations within the dfrA and inhA genes revealed mutations in 0 and 5 (3.9%) isolates respectively, implying that mutations in dfrA do not contribute to the detection of INH-resistant M. tuberculosis and that mutations within inhA are rare. Thirty-seven (29%) of the 127 isolates had no mutations in any of the genes implicated in INH resistance (katG, kasA, and ndh; inhA and ahpC promoters), suggesting that there are new INH targets yet to be discovered.

Immunoglobulin G in human serum neutralizes chlamydial infectivity in vitro. Complement-intact, C5-depleted, and C8-depleted human serum all have significantly more neutralizing activity than serum heated to inactivate early components of complement. Cobra venom factor, an analog of human C3b, enhances neutralization of antichlamydial immunoglobulin G in the absence of early complement components. Chiamydia trachomatis infects mucous membranes of human eyes and genital tracts and causes chronic inflammation with the following resultant clinical syndromes: trachoma (serovars A, B, Ba, and C); conjunctivitis, urethritis, cervicitis, and pelvic inflammatory disease (serovars D, E, F, G, H, I, J, and K); and lymphogranuloma venerum (serovars Li, L2, and L3) (22). The immunology and pathogenesis of these diseases currently are not well understood. Because C. trachomatis infection induces diverse inflammatory reactions, the concept that chlamydial infection manifests different immunopathologic features has emerged (9, 13). As a step toward examining immune interactions of C. trachomatis with the human host, we have begun to examine the interaction of human antibody and complement with this organism in vitro.

Restriction fragments containing the major outer-membrane protein (MOMP) gene from two nonhuman (rodent) strains of Chlamydia trachomatis, the mouse pneumonitis (MoPn) strain and the SFPD strain isolated from hamsters with transmissible proliferative ileitis, were cloned and sequenced. The MOMP genes of both MoPn and SFPD encode an identical 22-amino acid leader peptide and mature polypeptides of 365 and 382 amino acids, respectively. Alignment of the MOMP genes of the two rodent strains revealed 91% identity. By comparison with other known chlamydial MOMP gene sequences, there was 80%-83% identity with human biovars strains of C. trachomatis, and there was 69%-70% identity with C. psittaci and C. pneumoniae strains. The main differences in these sequences were clustered into four variable domains. A minimum-length evolutionary tree was constructed on the basis of the MOMP gene variable positions by using PIMA package software. The minimum mutation distances indicated that (i) the MOMP genes of all chlamydial strains may have evolved from a common ancestor; (ii) all the strains of C. trachomatis compose one of the subtrees, and strains of C. psittaci and C. pneumoniae compose the other subtree; and (iii) in the C. trachomatis subtree, the human and the rodent strains are divided into two clusters. The branching pattern of this evolutionary tree is generally consistent with current classification based on serological, morphological, and other biological characteristics.