Harry W. Taber

Phenotypic and Molecular Characterization of Mycobacterium tuberculosis Isolates Resistant to both Isoniazid and Ethambutol

ABSTRACT In performing radiometric susceptibility testing on over 2,000 patient isolates of Mycobacterium tuberculosis during the past 6 years, we found that resistance to 7.5 μg/ml ethambutol (EMB) occurred only in isolates that are also resistant to 0.4 μg/ml isoniazid (INH). Using 157 selected isolates in the present study, we performed radiometric and agar proportion susceptibility tests and DNA sequencing of genetic regions associated with resistance to these two drugs. The goal was to study the occurrence of the common mutations associated with resistance to each drug and also to determine whether any particular INH-resistance-associated mutation occurred more often in combination with any particular EMB-resistance-associated mutation. In an analysis of 128 isolates resistant to 0.4 μg/ml INH, we found that a mutation at katG Ser315 was more common in isolates also resistant to 7.5 μg/ml EMB (61 of 67 = 91.0%) than in isolates either susceptible to EMB or resistant to 2.5 μg/ml EMB (39 of 60 = 65.0%). These observations suggest that INH-resistant strains with a mutation at katG Ser315 are more likely to acquire resistance to 7.5 μg/ml EMB than are isolates with INH-resistance-associated mutations at other sites. In addition, we found that 64 of 67 (95.5%) isolates resistant to 7.5 μg/ml EMB contained a mutation in either codon 306 or codon 406 of embB. Met306Val was the most common embB mutation, present in 52 (77.6%) of the 67 isolates. Most occurrences of this mutation (49 of 52 = 94.2%) were found in isolates that also contained the katG Ser315Thr mutation. Finally, sequencing this region of embB appears to be sufficiently sensitive for use as a rapid screening tool for detection of high-level resistance to EMB.

Sequence and genetic organization of a Bacillus subtilis operon encoding 2,3-dihydroxybenzoate biosynthetic enzymes

Under iron-limiting conditions, Bacillus subtilis (Bs) produces the siderophore 2,3-dihydroxybenzoate (DHB) to acquire extracellular iron. In Escherichia coli (Ec), DHB is a precursor of the siderophore enterobactin, which suggested that Bs may possess similar biosynthetic enzymes. The sequences of two overlapping Bs clones capable of complementing Ec enterobactin mutants [Grossman, T.H., Tuckman, M., Ellestad, S. and Osburne, M.S. (1993) Isolation and characterization of Bacillus subtilis genes involved in siderophore biosynthesis: Relationship between B. subtilis sfpo and Escherichia coli entD genes. J. Bacteriol. 175, 6203-6211] were analyzed and five open reading frames were identified. These genes are located near 291 degrees on the Bs chromosome and have been termed dhbA, dhbC, dhbE, dhbB and dhbF, based on similarities to Ec ent homologs. Amino-acid identities between gene product homologs are: EntA and DhbA, 41%; EntC and DhbC, 35%; EntE and DhbE, 48%; EntB and DhbB, 54%; and EntF and DhbF, 29%. DhbC is also 35% identical to the Bs menaquinone-specific isochorismate synthase, MenF, illustrating an example of gene duplication. Operon disruption studies suggested that the dhb genes comprise an operon of at least four genes.

Estimated Costs of False Laboratory Diagnoses of Tuberculosis in Three Patients

We estimated direct medical and nonmedical costs associated with a false diagnosis of tuberculosis (TB) caused by laboratory cross-contamination of Mycobacterium tuberculosis cultures in Massachusetts in 1998 and 1999. For three patients who received misdiagnoses of active TB disease on the basis of laboratory cross-contamination, the costs totaled U.S.

Spoligologos: A Bioinformatic Approach to Displaying and Analyzing Mycobacterium tuberculosis Data

32,618. Of the total, 97% was attributed to the public sector (local and state health departments, public health hospital and laboratory, and county and state correctional facilities); 3% to the private sector (physicians, hospitals, and laboratories); and <1% to the patient. Hospitalizations and inpatient tests, procedures, and TB medications accounted for 69% of costs, and outpatient TB medications accounted for 18%. The average cost per patient was

Impact of Genotyping of Mycobacterium Tuberculosis on Public Health Practice in Massachusetts

10,873 (range,

Structural organization of a Bacillus subtilis operon encoding menaquinone biosynthetic enzymes

1,033-

The Bacillus subtilis menCD promoter is responsive to extracellular pH

21,306). Reducing laboratory cross-contamination and quickly identifying patients with cross-contaminated cultures can prevent unnecessary and potentially dangerous treatment regimens and anguish for the patient and financial burden to the health-care system.

Genomic analysis of the Mycobacterium tuberculosis complex: applications to laboratory diagnosis and genotyping

Spacer oligonucleotide (spoligotyping) analysis is a rapid polymerase chain reaction–based method of DNA fingerprinting the Mycobacterium tuberculosis complex. We examined spoligotype data using a bioinformatic tool (sequence logo analysis) to elucidate undisclosed phylogenetic relationships and gain insights into the global dissemination of strains of tuberculosis. Logo analysis of spoligotyping data provides a simple way to describe a fingerprint signature and may be useful in categorizing unique spoligotypes patterns as they are discovered. Large databases of DNA fingerprint information, such as those from the U.S. National Tuberculosis Genotyping and Surveillance Network and the European Concerted Action on Tuberculosis, contain information on thousands of strains from diverse regions. The description of related spoligotypes has depended on exhaustive listings of the individual spoligotyping patterns. Logo analysis may become another useful graphic method of visualizing and presenting spoligotyping clusters from these databases.

Isolation and sequence of ctaA, a gene required for cytochrome aa3 biosynthesis and sporulation in Bacillus subtilis.

Massachusetts was one of seven sentinel surveillance sites in the National Tuberculosis Genotyping and Surveillance Network. From 1996 through 2000, isolates from new patients with tuberculosis (TB) underwent genotyping. We describe the impact that genotyping had on public health practice in Massachusetts and some limitations of the technique. Through genotyping, we explored the dynamics of TB outbreaks, investigated laboratory cross-contamination, and identified Mycobacterium tuberculosis strains, transmission sites, and accurate epidemiologic links. Genotyping should be used with epidemiologic follow-up to identify how resources can best be allocated to investigate genotypic findings.

Sequence organization and regulation of the Bacillus subtilis menBE operon.

Menaquinone (MK) is a non-protein component of the Bacillus subtilis (Bs) electron transport chain synthesized from chorismate through a series of MK-specific reactions. The genes encoding biosynthesis of the naphthoquinone ring of MK are clustered at 273 degrees on the Bs chromosome. A 3.9-kb region capable of rescuing men mutants blocked in the early stages of MK biosynthesis was sequenced and found to contain three major open reading frames (ORFs). The first ORF (menF) has a predicted size of 51.8 kDa and 34% amino-acid identity with the isochorismate synthases of Escherichia coli (EntC) and Aeromonas hydrophila (AmoA), ORF2 (menD) a predicted size of 60.2 kDa and 21% identity with MenD of E. coli. ORF3 has a predicted size of 21.4 kDa and 29% identity to triacylglycerol lipase of Psychrobacter immobilis. No sequence corresponding to menC was identified. Plasmid integrational studies of the men gene cluster had suggested the presence of promoters secondary to the previously identified p1 men promoter. Sequence analysis revealed a putative promoter region upstream from ORF3.