Strain classification of Mycobacterium tuberculosis: congruence between large sequence polymorphisms and spoligotypes.

Spoligotyping is used in molecular epidemiological studies, and signature patterns have identified strain families. However, homoplasy occurs in the markers used for spoligotyping, which could lead to identical spoligotypes in phylogenetically unrelated strains. We determined the accuracy of strain classification based on spoligotyping using the six large sequence and single nucleotide polymorphisms-defined lineages as a gold standard. Of 919 Mycobacterium tuberculosis isolates, 870 (95%) were classified into a spoligotype family. Strains from a particular spoligotype family belonged to the same lineage. We did not find convergence to the same spoligotype. Spoligotype families appear to be sub-lineages within the main lineages.

Use of the Gen-Probe amplified mycobacterium tuberculosis direct test for early detection of Mycobacterium tuberculosis in BACTEC 12B medium.

To achieve better sensitivity than direct testing and better turnaround time than current culture and identification methods, the Gen-Probe Mycobacterium Tuberculosis Direct method was used to detect Mycobacterium tuberculosis in BACTEC 12B medium cultures when they first gave a growth index (GI) of at least 10 (MTD/BACTEC method). Of 179 acid-fast, smear-positive specimens that were culture positive for M. tuberculosis, all were positive by the MTD/BACTEC method (sensitivity, 100%). Positive results were obtained only with tuberculosis patients. For diagnostic specimens from untreated patients, the mean time to achieve a GI of 10 was 6 days.

Pyrazinamide-monoresistant Mycobacterium tuberculosis in the United States.

Mycobacterium bovis is naturally resistant to the antituberculosis drug pyrazinamide (PZA). To determine whether all Mycobacterium tuberculosis complex isolates demonstrating PZA monoresistance were truly M. bovis, we examined the phenotype and genotype of isolates reported as PZA monoresistant in five counties in California from January 1996 through June 1999. Isolates reported by local laboratories to be PZA monoresistant were sent to the state reference laboratory for repeat susceptibility testing using the BACTEC radiometric method and to the Centers for Disease Control and Prevention for pncA sequencing and PCR-restriction fragment length polymorphism (RFLP) analysis of the oxyR gene. Of 1,916 isolates, 14 were reported as PZA monoresistant and 11 were available for retesting. On repeat testing, 6 of the 11 isolates were identified as PZA-susceptible M. tuberculosis, 1 was identified as PZA-monoresistant M. bovis, and 1 was identified as M. bovis BCG. The three remaining isolates were identified as PZA-monoresistant M. tuberculosis. Sequencing of the pncA and oxyR genes genotypically confirmed the two M. bovis and the six susceptible M. tuberculosis species. Each of the three PZA-monoresistant M. tuberculosis isolates had different, previously unreported, pncA gene mutations: a 24-bp deletion in frame after codon 88, a base substitution at codon 104 (Ser104Cys), and a base substitution at codon 90 (Ile90Ser). This study demonstrates that PZA monoresistance is not an absolute marker of M. bovis species but may also occur in M. tuberculosis, associated with a number of different mutational events in the pncA gene. It is the first report of PZA-monoresistant M. tuberculosis in the United States.

Access to newer laboratory procedures: a call for action.

Healthy People 2010, an initiative from the federal government, calls for action from tuberculosis controllers and tuberculosis laboratories in the fight to eliminate tuberculosis. Many patients, such as immunocompromised patients and those infected with multidrug-resistant tuberculosis strains, pose a challenge for care and diagnosis. Fortunately, many changes have occurred in the last decade to facilitate more rapid and accurate testing to assist with the care of these patients. California, Florida, New York and Texas have almost 50% of the tuberculosis cases in the United States, and their public health laboratories utilize different approaches to meet the same goal of rapid and accurate testing of specimens. With the targets of Healthy People 2010 (e.g., to reduce the average time for a laboratory to confirm and report tuberculosis cases to 2 days for 75% of cases) already looming on the horizon, innovative methods for achieving these goals should be evaluated. Using these public health laboratories as models, rapid, gold-standard testing methods should be provided to all patients in the United States. Soon it will be the year 2010..., are you ready to swiftly move forward?

Characterization of an SAV organism and proposal of Mycobacterium triplex sp. nov.

Polyphasic taxonomic methods were employed to characterize a new species of slowly growing, nonpigmented mycobacteria. We propose the name Mycobacterium triplex sp. nov. for this new taxon. Conventional identification testing demonstrated a group of similar organisms that were geographically widespread in the United States. Commercially available nucleic-acid probes specific for the Mycobacterium avium complex were unreactive for these strains. High-performance liquid chromatography analysis of the mycolic acids revealed mycolate profiles that closely resembled Mycobacterium simiae. Comparative 16S rRNA sequence data confirmed the phylogenetic relationship of the strains with the slowly growing mycobacteria. Representative-type strains have been deposited in the American Type Culture Collection as strain ATCC 700071 [corrected].

Mouse pathogenicity studies of Nocardia asteroides complex species and clinical correlation with human isolates.

Nocardia asteroides complex organisms derived from human specimens between 1979 and 1992 were identified to the species level. Of 117 N. asteroides complex organisms, 34 (29%) were N. farcinica, 28 (24%) were N. nova, and 55 (47%) were N. asteroides sensu stricto. An analysis of the specimen sites from which the organisms were derived showed that isolates derived from blood, brain, or bone marrow were more likely to be N. farcinica than the other two species. A study of the virulence of ten strains of each species was undertaken, using a mouse model with intravenous inoculation. The 50% lethal doses (LD50) for N. farcinica were significantly lower than those of the other two species. LD50 values for N. nova and N. asteroides were not significantly different. The above data confirming the greater virulence of N. farcinica support the identification of species within the N. asteroides complex.

False-positive cultures of Mycobacterium tuberculosis.

During a single week in April 1982, cultures for Mycobacterium tuberculosis were reported positive from nine patients who did not appear clinically to have active infection. Each of the patients had only one positive culture out of multiple specimens cultured. At the time of investigation, five specimens were available and were found to be all of the same phage type which strongly suggested cross-contamination. Four patients received antituberculosis chemotherapy. In one year of follow-up of the five who did not receive chemotherapy, none developed clinical disease. The contamination was probably due to faulty laboratory technique, but the source of the contaminant is uncertain. This investigation suggests that patients without clinical evidence of active infection and with isolated positive cultures for Mycobacterium tuberculosis should be carefully evaluated before they are subjected to a prolonged, potentially toxic, and expensive course of chemotherapy.

Comparative studies and laboratory diagnosis of Vibrio vulnificus, an invasive Vibrio sp.

Vibrio vulnificus was isolated from a bacteremic patient. This strain, together with other isolates of V. vulnificus, was compared with V. alginolyticus, V. fluvialis, and V. parahaemolyticus with regard to growth characteristics on enteric agar media (enabling isolation and identification) and production of exoenzymes which could correlate with invasive potential. V. vulnificus grew well on MacConkey. Endo, xylose-lysine deoxycholate, and Hektoen enteric agar plates. Because V. vulnificus colonies resembled those of lactose-fermenting strains of the family Enterobacteriaceae, however, isolation of this vibrio from mixed specimens or stools may require the use of thiosulfate-citrate-bile salts-sucrose agar. V. vulnificus produced numerous exoenzymes (protease, DNase, lipase, and esterase) but not elastase or lecithinase. Although differences in exoenzyme production were observed among the four vibrio species, no single exoenzyme could be linked to the invasive potential of V. vulnificus.

Inhibition of Bacillus subtilis aminopeptidase D by beta-lactam antibiotics.

Penicillins and cephalosporins inhibit the hydrolysis of D-alanyl-beta-naphthylamide by aminopeptidase D (EC 3.4.11.-) from Bacillus subtilis. The inhibition is predominantly non-competitive, although the more effective inhibitors, i.e., those with the lower Ki values, seem to exhibit a tendency toward competitive kinetics, while penicillin V, the antibiotic with the largest Ki, exhibits a strong tendency toward uncompetitive kinetics. The removal of antibiotic from the enzyme by gel filtration on Sephadex G-25 restores 100% activity to the enzyme, and suggests that the inhibition does not derive from a covalent antibiotic-enzyme complex. The antibiotics which have been studied, with their respective Ki values (mM) and inhibition type are: methicillin (1.01 +/- 0.34, mixed non-competitive-competitive); cephaloridine (2.43 +/- 0.17, non-competitive); cloxacillin (3.19 +/- 1.29, mixed non-competitive-competitive); oxacillin (6.88 +/- 0.77, non-competitive); cephalothin (8.12 +/- 0.99, non-competitive); penicillin G (20.0 +/- 3.21, non-competitive) and penicillin V (32.1 +/- 4.90, mixed non-competitive-uncompetitive). An empirical correlation exists between the Ki value and the freedom of rotation about the bond between the phenyl ring and the atom alpha to the ring in the variable side chain portion of the penicillin molecule.

Correlation of proteolytic activity of Pseudomonas aeruginosa with site of isolation.

Pseudomonas aeruginosa isolates were evaluated for protease activity by use of a semiquantitative plates assay. Differences were noted with respect to site of isolation, origin, and colonial morphology.

Immunoperoxidase staining for detection of Colorado tick fever virus, and a study of congenital infection in the mouse.

Indirect immunoperoxidase (IP) staining was evaluated for sensitivity and specificity in detecting Colorado tick fever (CTF) virus antigen in infected cell cultures and infected mouse tissues, and then was applied to a study of congenital CTF infection in mice. The sensitivity of IP staining was comparable to that of immunofluorescence staining in detecting CTF antigen in infected cell cultures. Endogenous peroxidase activity of mouse tissues caused nonspecific reactivity in the IP system, but this could be abolished by treatment with sodium azide and hydrogen peroxide without destroying CTF antigen. Offspring of mice infected with CTF virus during the 2nd week of pregnancy showed a highly significant increase in the incidence of stillbirths and neonatal deaths as compared with offspring of uninfected controls. CTF antigen or virus was demonstrable in only a low proportion (7%) of embryos, ill newborns or stillborns examined, but a high proportion of mice examined at a time when maternal antibody would be lost (6 and 12 weeks) showed CTF antibody, indicating a higher incidence of infection. IP staining showed potential for use in studies of viral pathogenesis in the mouse model.

Aminopeptidases of Bacillus subtilis.

Three enzymes with L- and one enzyme with D-aminopeptidase (EC 3.4.11; alpha-aminoacyl peptide hydrolase) activity have been separated from each other and partially purified from Bacillus subtilis 168 W.T., distinguished with respect to their molecular weights and catalytic properties, and studied in relation to the physiology of this bacterium. One L-aminopeptidase, designated aminopeptidase I, has a molecular weight of 210,000 +/- 20,000, is produced early in growth, and hydrolyzes L-alanyl-beta-naphthylamide most rapidly. Another, designated aminopeptidase II, molecular weight 67,000 +/- 10,000, is also produced early in growth and hydrolyzes L-lysyl-beta-naphthylamide most rapidly. A third, aminopeptidase III, molecular weight 228,000 +/- 20,000, is produced predominantly in early stationary phase and most efficiently utilizes L-alpha-aspartyl-beta-naphthylamide as substrate. The synthesis of aminopeptidase III in early stationary phase suggests that selective catabolism of peptides occurs at this time, perhaps related to the cessation of growth or the onset of early sporulation-associated events. A D-aminopeptidase which hydrolyzes the carboxyl-blocked dipeptide D-alanyl-D-alanyl-beta-naphthylamide (as well as D-alanyl-beta-naphthylamide and D-alanyl-D-alanyl-D-alanine) has also been identified, separated from aminopeptidase II, and purified 170-fold. D-Aminopeptidase, molecular weight 220,000 +/- 20,000, is localized predominantly in the cell wall and periplasm of the organism. This evidence and the variation of the activity during the growth cycle suggest an important function in cell wall or peptide antibiotic metabolism.