Differentiation of Mycobacterium genavense and Mycobacterium simiae by automated mycolic acid analysis with high-performance liquid chromatography.

Mycobacterium genavense, a fastidious opportunist in patients with AIDS, cannot be identified by conventional biochemical methods. Computerized mycolic acid analysis by high-performance liquid chromatography offers an alternative that distinguishes the mycolic acid profile of M. genavense from those of all other organisms in the database developed at the Centers for Disease Control and Prevention.

Isolation and characterization of a unique group of slowly growing mycobacteria: description of Mycobacterium lentiflavum sp. nov.

A distinct group of slowly growing mycobacteria was identified on the basis of growth characteristics, biochemical and lipid profiles, and nucleic acid analyses. The isolates showed growth at 22 to 37 degrees C, yellow pigmentation, and negative tests for Tween 80 hydrolysis, nicotinic acid, nitrate reductase, and urease; tests for arylsulfatase, pyrazinamidase, and heat-stable catalase were variable. Analysis of cellular fatty acids by gas-liquid chromatography and mycolic acids by thin-layer chromatography and high-performance liquid chromatography indicated a distinctive pattern which was unlike those of other species. Determination of the 16S rRNA gene sequence showed a unique sequence closely related to Mycobacterium simiae and M. genavense. On the basis of DNA homology studies, we suggest that these organisms are representatives of a novel species, for which the name M. lentiflavum sp. nov. is proposed.

In vitro activities of U-100592 and U-100766, novel oxazolidinone antibacterial agents.

Oxazolidinones make up a relatively new class of antimicrobial agents which possess a unique mechanism of bacterial protein synthesis inhibition. U-100592 (S)-N-[[3-[3-fluoro-4-[4-(hydroxyacetyl)-1-piperazinyl]- phenyl]-2-oxo-5-oxazolidinyl]methyl]-acetamide and U-100766 (S)-N-[[3-[3-fluoro-4-(4-morpholinyl)phenyl]- 2-oxo-5-oxazolidinyl]methyl]-acetamide are novel oxazolidinone analogs from a directed chemical modification program. MICs were determined for a variety of bacterial clinical isolates; the respective MICs of U-100592 and U-100766 at which 90% of isolates are inhibited were as follows: methicillin-susceptible Staphylococcus aureus, 4 and 4 micrograms/ml; methicillin-resistant S. aureus, 4 and 4 micrograms/ml; methicillin-susceptible Staphylococcus epidermidis, 2 and 2 micrograms/ml; methicillin-resistant S. epidermidis, 1 and 2 micrograms/ml; Enterococcus faecalis, 2 and 4 micrograms/ml; Enterococcus faecium, 2 and 4 micrograms/ml; Streptococcus pyogenes, 1 and 2 micrograms/ml; Streptococcus pneumoniae, 0.50 and 1 microgram/ml; Corynebacterium spp., 0.50 and 0.50 micrograms/ml; Moraxella catarrhalis, 4 and 4 micrograms/ml; Listeria monocytogenes, 8 and 2 micrograms/ml; and Bacteroides fragilis, 16 and 4 micrograms/ml. Most strains of Mycobacterium tuberculosis and the gram-positive anaerobes were inhibited in the range of 0.50 to 2 micrograms/ml. Enterococcal strains resistant to vancomycin (VanA, VanB, and VanC resistance phenotypes), pneumococcal strains resistant to penicillin, and M. tuberculosis strains resistant to common antitubercular agents (isoniazid, streptomycin, rifampin, ethionamide, and ethambutol) were not cross-resistant to the oxazolidinones. The presence of 10, 20, and 40% pooled human serum did not affect the antibacterial activities of the oxazolidinones. Time-kill studies demonstrated a bacteriostatic effect of the analogs against staphylococci and enterococci but a bactericidal effect against streptococci. The spontaneous mutation frequencies of S. aureus ATCC 29213 were <3.8 x 10(-10) and <8 x 10(-11) for U-100592 and U-100766, respectively. Serial transfer of three staphylococcal and two enterococcal strains on drug gradient plates produced no evidence of rapid resistance development. Thus, these new oxazolidinone analogs demonstrated in vitro antibacterial activities against a variety of clinically important human pathogens.

Identification of a novel oxazolidinone (U-100480) with potent antimycobacterial activity.

During the course of our investigations in the oxazolidinone antibacterial agent area, we have identified a subclass with especially potent in vitro activity against mycobacteria. The salient structural feature of these oxazolidinone analogues, 6 (U-100480), 7 (U-101603), and 8 (U-101244), is their appended thiomorpholine moiety. The rational design, synthesis, and evaluation of the in vitro antimycobacterial activity of these analogues is described. Potent activity against a screening strain of Mycobacterium tuberculosis was demonstrated by 6 and 7 (minimum inhibitory concentrations or MIC's < or = 0.125 micrograms/mL). Oxazolidinones 6 and 8 exhibit MIC90 values of 0.50 micrograms/mL or less against a panel of organisms consisting of five drug-sensitive and five multidrug-resistant strains of M. tuberculosis, with 6 being the most active congener. Potent in vitro activity against other mycobacterial species was also demonstrated by 6. For example, 6 exhibited excellent in vitro activity against multiple clinical isolates of Mycobacterium avium complex (MIC's = 0.5-4 micrograms/mL). Orally administered 6 displays in vivo efficacy against M. tuberculosis and M. avium similar to that of clinical comparators isoniazid and azithromycin, respectively. Consideration of these factors, along with a favorable pharmaco-kinetic and chronic toxicity profile in rats, suggests that 6 (U-100480) is a promising antimycobacterial agent.

Multidrug-resistant tuberculosis in the New York State prison system, 1990-1991.

Three epidemiologically linked multidrug-resistant (MDR) tuberculosis (TB) outbreaks in 1990-1991 involving New York State (NYS) inmates suggested MDR-TB was widespread in NYS prisons. Inmate lists were linked to 1990-1992 TB registries, medical records were reviewed, and movement histories for inmates with MDR-TB were examined within and between prisons and hospitals. In 1990-1991, 171 inmates were diagnosed with TB. This rate (156.2/100,000) was significantly higher than the 1990-1991 US rate (10.4/100,000) and the 1986 rate among NYS inmates (105.5/100,000). Of 171 cases, 155 were cultured-confirmed; 37 (32%) of 116 with drug susceptibilities determined had MDR-TB. Two other inmates with TB before 1990 were diagnosed with MDR-TB in 1990-1991. Of 39 inmates with MDR-TB, 38 (97%) were infected with the human immunodeficiency virus and 34 (87%) have died. These 39 lived in 23 of the 68 NYS prisons while potentially infectious; 12 were transferred through 20 prisons while ill with MDR-TB. Policies of correctional systems on infection control and inmate transfers need to be reevaluated to prevent spread of TB.

Rapid identification of mycolic acid patterns of mycobacteria by high-performance liquid chromatography using pattern recognition software and a Mycobacterium library.

Current methods for identifying mycobacteria by high-performance liquid chromatography (HPLC) require a visual assessment of the generated chromatographic data, which often involves time-consuming hand calculations and the use of flow charts. Our laboratory has developed a personal computer-based file containing patterns of mycolic acids detected in 45 species of Mycobacterium, including both slowly and rapidly growing species, as well as Tsukamurella paurometabolum and members of the genera Corynebacterium, Nocardia, Rhodococcus, and Gordona. The library was designed to be used in conjunction with a commercially available pattern recognition software package, Pirouette (Infometrix, Seattle, Wash.). Pirouette uses the K-nearest neighbor algorithm, a similarity-based classification method, to categorize unknown samples on the basis of their multivariate proximities to samples of a preassigned category. Multivariate proximity is calculated from peak height data, while peak heights are named by retention time matching. The system was tested for accuracy by using 24 species of Mycobacterium. Of the 1,333 strains evaluated, > or = 97% were correctly identified. Identification of M. tuberculosis (n = 649) was 99.85% accurate, and identification of the M. avium complex (n = 211) was > or = 98% accurate; > or = 95% of strains of both double-cluster and single-cluster M. gordonae (n = 47) were correctly identified. This system provides a rapid, highly reliable assessment of HPLC-generated chromatographic data for the identification of mycobacteria.

Clinical and epidemiologic characteristics of Mycobacterium haemophilum, an emerging pathogen in immunocompromised patients.

OBJECTIVE: To describe 13 infections caused by Mycobacterium haemophilum. DESIGN: Identification of patients by microbiologic record review, followed by medical record review and a case-control study. SETTING: Seven metropolitan hospitals in New York. PATIENTS: All patients with M. haemophilum infections diagnosed between January 1989 and September 1991 and followed through September 1992. Surviving patients were enrolled in the case-control study. RESULTS: Infection with M. haemophilum causes disseminated cutaneous lesions, bacteremia, and diseases of the bones, joints, lymphatics, and the lungs. Improper culture techniques may delay laboratory diagnosis, and isolates may be identified incorrectly as other mycobacterial species. Persons with profound deficits in cell-mediated immunity have an increased risk for infection. These include persons with human immunodeficiency virus infection or lymphoma and those receiving medication to treat immunosuppression after organ transplant. Various antimycobacterial regimens have been used with apparent success to treat M. haemophilum infection. However, standards for defining antimicrobial susceptibility to the organism do not exist. CONCLUSIONS: Clinicians should consider this pathogen when evaluating an immunocompromised patient with cutaneous ulcerating lesions, joint effusions, or osteomyelitis. Microbiologists must be familiar with the fastidious growth requirements of this organism and screen appropriate specimens for mycobacteria using an acid-fast stain. If acid-fast bacilli are seen, M. haemophilum should be considered as the infecting organism as well as other mycobacteria, and appropriate media and incubation conditions should be used.

Clinical significance, biochemical features, and susceptibility patterns of sporadic isolates of the Mycobacterium chelonae-like organism.

Mycobacterium chelonae-like organisms are nonpigmented rapidly growing mycobacteria whose clinical significance is unknown. We evaluated 87 sporadic isolates encountered in a clinical laboratory. Most isolates (62%) were respiratory; only 2 of 54 (4%) (both from patients with AIDS) were clinically significant. Among 33 nonrespiratory isolates, 20 of 33 (or 61%) were clinically significant. Clinical diseases included posttraumatic wound infections and catheter-related sepsis. Routine biochemical features included growth inhibition by 5% NaCl (100%), a smooth colony morphology (94%), positive 3-day arylsulfatase reaction (84%), no color or a light tan color on iron uptake (100%), and variable nitrate reduction (45%). Additional characteristics that helped to separate this group from M. chelonae and Mycobacterium abscessus were susceptibility to cephalothin (90%) and ciprofloxacin (100%), utilization of mannitol (94%) and citrate (83%) as carbon sources, and unique patterns of mycolic acid esters by high-performance liquid chromatography. This group was quite drug susceptible, with 100% of isolates inhibited by amikacin, imipenem, cefoxitin, cefmetazole, and the newer quinolones ciprofloxacin and ofloxacin. Three examples of this group, including a proposed type strain, have been deposited in the American Type Culture Collection.

Transmission of multidrug-resistant Mycobacterium tuberculosis among persons with human immunodeficiency virus infection in an urban hospital: epidemiologic and restriction fragment length polymorphism analysis.

From January 1990 to December 1991, 16 patients with multidrug-resistant tuberculosis (MDR-TB) caused by Mycobacterium tuberculosis resistant to isoniazid, rifampin, and streptomycin were diagnosed at Elmhurst Hospital. Compared with other TB patients, MDR-TB patients were more likely to have human immunodeficiency virus (HIV) infection (14/16 vs. 21/204, P < .001) and a prior admission (10/16 vs. 3/204, P < .001). HIV-infected patients hospitalized for > 10 days within three rooms of an infectious MDR-TB patient had higher risk of acquiring MDR-TB than did HIV-infected patients with shorter hospitalizations or locations further from the MDR-TB patient(s) (6/28 vs. 2/90, P < .001). Isolates of 6 of 8 MDR-TB patients in a chain of transmission were identical by restriction fragment length polymorphism DNA typing. Ambulation on the wards of inadequately masked TB patients and lack of negative pressure in isolation rooms probably facilitated transmission. This report documents nosocomial transmission of MDR-TB and underscores the need for effective isolation practices and facilities in health care institutions.

Mycobacterium celatum sp. nov.

A new slowly growing nonphotochromogenic Mycobacterium species of clinical importance is described. The biochemical characteristics of this organism were similar to those of Mycobacterium xenopi and members of the Mycobacterium avium complex. However, none of the strains reacted with commercially available genetic probes for the M. avium complex. The strains were resistant to most antituberculosis drugs. Multilocus enzyme electrophoresis revealed two original electrophoretic types, which was suggestive of new species. The strains contained alpha-, keto-, and dicarboxylic mycolates, as determined by thin-layer chromatography. A mycolic acid analysis by high-performance liquid chromatography revealed a chromatographic pattern similar to that of M. xenopi, but distinct from the patterns of previously described Mycobacterium species. Hexadecanoic and tuberculostearic acids were identified as the major cell wall fatty acids by gas-liquid chromatographic analysis; hexacosanoic acid was the major mycolic acid cleavage product, and 2-eicosanol was the major alcohol. Evaluation of the 16S rRNA sequence confirmed the phylogenetic position of the organism among the slowly growing Mycobacterium species. Cultures representing this new species have been deposited in the American Type Culture Collection as strains ATCC 51130 and ATCC 51131T (T = type strain). The name Mycobacterium celatum is proposed.

Bacteriologic investigations of unusual mycobacteria isolated from immunocompromised patients.

Mycobacterial isolates from blood and other extrapulmonary sites of six patients with AIDS were investigated because the isolates grew only in liquid media and failed to grow on solid culture media even on subculturing. Our investigations indicated that these mycobacteria possess common, but unusual, characteristics and probably belong to an unrecognized species recently reported as "Mycobacterium genavense."

The emergence of drug-resistant tuberculosis in New York City.

BACKGROUND: In the past decade the incidence of tuberculosis has increased nationwide and more than doubled in New York City, where there have been recent nosocomial outbreaks of multidrug-resistant tuberculosis. METHODS: We collected information on every patient in New York City with a positive culture for Mycobacterium tuberculosis during April 1991. Drug-susceptibility testing was performed at the Centers for Disease Control and Prevention. RESULTS: Of the 518 patients with positive cultures, 466 (90 percent) had isolates available for testing. Overall, 33 percent of these patients had isolates resistant to one or more antituberculosis drugs, 26 percent had isolates resistant to at least isoniazid, and 19 percent had isolates resistant to both isoniazid and rifampin. Of the 239 patients who had received antituberculosis therapy, 44 percent had isolates resistant to one or more drugs and 30 percent had isolates resistant to both isoniazid and rifampin. Among the patients who had never been treated, the proportion with resistance to one or more drugs increased from 10 percent in 1982 through 1984 to 23 percent in 1991 (P = 0.003). Patients who had never been treated and who were infected with the human immunodeficiency virus (HIV) or reported injection-drug use were more likely to have resistant isolates. Among patients with the acquired immunodeficiency syndrome, those with resistant isolates were more likely to die during follow-up through January 1992 (80 percent vs. 47 percent, P = 0.02). A history of antituberculosis therapy was the strongest predictor of the presence of resistant organisms (odds ratio, 2.7; P < 0.001). CONCLUSIONS: There has been a marked increase in drug-resistant tuberculosis in New York City. Previously treated patients, those infected with HIV, and injection-drug users are at increased risk for drug resistance. Measures to control and prevent drug-resistant tuberculosis are urgently needed.

Laboratory aspects of "Mycobacterium genavense," a proposed species isolated from AIDS patients.

"Mycobacterium genavense" is a proposed new species recently reported to cause disseminated infections in 18 patients with AIDS in Europe. We have recovered "M. genavense" as slowly growing fastidious mycobacteria in blood cultures of seven patients with AIDS. In the original studies of "M. genavense," the fastidious organism grew only in BACTEC 13A vials. The Seattle, Washington, isolates of "M. genavense" also failed to grow when subcultured from 13A vials to routine solid media, but dysgonic colonies were produced on Middlebrook 7H11 agar supplemented with mycobactin J. The mycolic acid pattern of patients' isolates closely resembled that of the type strain of Mycobacterium simiae when analyzed by one- and two-dimensional thin-layer chromatography and by high-performance liquid chromatography. Whole-cell fatty acid analyses by gas-liquid chromatography distinguished the isolates from M. simiae but misidentified them as Mycobacterium fortuitum. Sequence determinations of the hypervariable regions of the 16S rRNA gene indicate that these organisms belong to the recently proposed new species "M. genavense." Growth from Middlebrook 7H11 agar supplemented with mycobactin J consistently yielded positive tests for catalase (semiquantitative and at 68 degrees C), pyrazinamidase, and urease which enable mycobacteriology laboratories to presumptively identify "M. genavense" without nucleic acid analyses. The failure of "M. genavense" to grow on conventional mycobacterial solid media suggests that mycobacterial blood cultures should include a broth medium incubated for at least 8 weeks.

HIV infection and primary resistance to antituberculosis drugs in Abidjan, Côte d'Ivoire.

OBJECTIVE: To determine the prevalence of Mycobacterium tuberculosis resistance to antituberculosis drugs, and to relate this resistance to HIV serologic status. DESIGN: Cross-sectional prevalence study. SETTING: The two major outpatient tuberculosis clinics in Abidjan, Côte d'Ivoire, West Africa. PATIENTS: Sixty individuals with newly diagnosed pulmonary tuberculosis and sputum smears positive for acid-fast bacilli. MAIN OUTCOME MEASURES: HIV serologic status and in vitro testing for susceptibility of M. tuberculosis isolates to antituberculosis drugs. RESULTS: M. tuberculosis was isolated from 82% (49 out of 60) of sputum specimens. Thirty-five per cent (17 out of 49) were obtained from HIV-seropositive and 65% (32 out of 49) from HIV-seronegative patients. There was no statistically significant difference in the proportion of resistant isolates from HIV-seropositive versus HIV-seronegative patients, although the relatively small sample size limited power. Of the total number of isolates, 17% were resistant to isoniazid; resistance was less to streptomycin (7%), rifampin (2%), pyrazinamide (0%), and ethambutol (0%). Eighteen and 21% of mycobacterial isolates from HIV-seropositive and HIV-seronegative individuals, respectively, were resistant to one or more of these drugs. CONCLUSIONS: Surveys of this type are useful in planning and evaluating tuberculosis preventive therapy in individuals with dual infection.

Identification of Mycobacterium avium complex strains and some similar species by high-performance liquid chromatography.

Strains of Mycobacterium avium, Mycobacterium intracellulare, Mycobacterium scrofulaceum, Mycobacterium xenopi, and Mycobacterium gordonae were identified by high-performance liquid chromatography (HPLC) analysis of mycolic acids as bromophenacyl esters. HPLC criteria were used to develop a flow chart identification scheme, which was evaluated in our laboratory with a set of 234 strains representing five species and a hitherto undescribed species. Correct identifications of M. gordonae and M. xenopi were easily made. Flow chart differentiation of M. avium, M. intracellulare, and M. scrofulaceum was done with 97.9, 97.5, and 89.2% accuracies, respectively. Independent evaluation of the flow chart at a separate laboratory demonstrated an overall identification accuracy of 97% for M. avium complex. Strains that have been described biochemically as being intermediate between M. avium-M. intracellulare and M. scrofulaceum were identified as one or the other of these known species. Strains which were negative with the species-specific radioactive probe for M. avium complex but which were positive with the nonradioactive SNAP X probe were usually identified as M. intracellulare and M. scrofulaceum but rarely as M. avium.

An outbreak of multidrug-resistant tuberculosis among hospitalized patients with the acquired immunodeficiency syndrome.

BACKGROUND: Since 1990 several clusters of multidrug-resistant tuberculosis have been identified among hospitalized patients with the acquired immunodeficiency syndrome (AIDS). We investigated one such cluster in a voluntary hospital in New York. METHODS: We compared exposures among 18 patients with AIDS in whom tuberculosis resistant to isoniazid and streptomycin was diagnosed from January 1989 through April 1990 (the case patients) with exposures among 30 control patients who had AIDS and tuberculosis susceptible to isoniazid, streptomycin, or both. We also compared exposures among the 14 case patients hospitalized during the six months before the diagnosis of tuberculosis (the exposure period) with those among 44 control patients with AIDS matched for duration of hospitalization. Mycobacterium tuberculosis isolates were typed with analysis of restriction-fragment-length polymorphism (RFLP). RESULTS: Case patients with drug-resistant tuberculosis were significantly more likely than controls with drug-susceptible tuberculosis to have been hospitalized during their exposure periods (14 of 18 vs. 10 of 30) (odds ratio, 7.0; 95 percent confidence interval, 1.6 to 36; P = 0.006). Case patients hospitalized during their exposure periods were significantly more likely to have been hospitalized on the same ward as a patient with infectious drug-resistant tuberculosis than were either controls with drug-susceptible tuberculosis hospitalized during their exposure periods or controls matched for duration of hospitalization (13 of 14 vs. 2 of 10 and 23 of 44) (odds ratio, 52; 95 percent confidence interval, 3.1 to 2474; P less than 0.001; and odds ratio, infinity; 95 percent confidence interval, 2.4 to infinity; P = 0.005, respectively). Among those hospitalized on the same ward, the rooms of case patients were closer to that of the nearest patient with infectious tuberculosis than were the rooms of controls matched for duration of hospitalization. M. tuberculosis isolates from 15 of 16 case patients had identical patterns on RFLP analysis. Of 16 patients' rooms tested with air-flow studies, only 1 had the recommended negative-pressure ventilation. CONCLUSIONS: Multidrug-resistant tuberculosis is readily transmitted among hospitalized patients with AIDS. Physicians must be alert to this danger and must enforce adherence to the measures recommended to prevent nosocomial transmission of tuberculosis.

Separation of Mycobacterium bovis BCG from Mycobacterium tuberculosis and Mycobacterium bovis by using high-performance liquid chromatography of mycolic acids.

Profile analysis of mycolic acid ester patterns of Mycobacterium tuberculosis, Mycobacterium bovis, and Mycobacterium bovis bacillus Calmette-Gúerin (BCG) using high-performance liquid chromatography indicated that separation of BCG from M. tuberculosis and M. bovis by elution and relative retention times is possible. Mycolic acid patterns of BCG eluted from the column 0.5 min before M. tuberculosis or M. bovis, resulting in relative retention times for two peaks not seen in the pattern of M. tuberculosis or M. bovis. Identification was confirmed by phage typing, which has been the standard procedure for confirmation of BCG strains. These results showed that high-performance liquid chromatographic analysis of mycolic acid esters can be used in the mycobacterial reference laboratory for separation of BCG from M. tuberculosis and M. bovis.

Identification of mycobacteria by high-performance liquid chromatography.

Mycolic acids extracted from saponified mycobacterial cells were examined as p-bromophenacyl esters by high-performance liquid chromatography (HPLC). Standard HPLC patterns were developed for species of Mycobacterium by examination of strains from culture collections and other well-characterized isolates. Relative retention times of peaks and peak height comparisons were used to develop a differentiation scheme that was 98% accurate for the species examined. A rapid, cost-effective HPLC method which offers an alternative approach to the identification of mycobacteria is described.

Chronic tenosynovitis of the hand due to Mycobacterium nonchromogenicum: use of high-performance liquid chromatography for identification of isolates.

Six cases of chronic tenosynovitis of the hand due to the Mycobacterium terrae complex were identified. All isolates from the six cases were identified as Mycobacterium nonchromogenicum by high-performance liquid chromatography and by testing for susceptibility to ofloxacin and to 5% NaCl. Ethambutol, sulfonamides (or trimethoprim-sulfamethoxazole), erythromycin, and streptomycin are the drugs most active against isolates of the M. terrae complex, and therapy with some combination of these agents plus surgical debridement offers the best current treatment of this disease. This study supports the contention arising from previous case reports of pulmonary disease that M. nonchromogenicum is the pathogenic member of the M. terrae complex.