Mycobacterium kubicae sp. nov., a slowly growing, scotochromogenic Mycobacterium.

A previously uncharacterized, slowly growing, scotochromogenic Mycobacterium species was detected by HPLC analysis of the cell-wall-bound mycolic acids. The mycolic acid pattern standard was shown to be a late-eluting, contiguous peak cluster occurring at approximately 8-9 min. The mycolic acid pattern was noted to be most similar in number of peaks and range of elution to that reported previously for Mycobacterium asiaticum. However, the relative distribution of peaks within the elution range demonstrated a pattern with prominent peaks that started to emerge later than the characteristic M. asiaticum pattern. Standard biochemical identification test results were similar to those of the photochromogenic species M. asiaticum. Comparative 16S rRNA gene sequence analysis confirmed the genetic uniqueness of the strains and demonstrated the unclassified mycobacteria to be in a unique, intermediate position between slow and rapid growers in the phylogenetic tree of Mycobacterium. The name Mycobacterium kubicae sp. nov. is proposed for this taxon. The type strain is CDC 941078T (= ATCC 700732T = CIP 106428T).

Central line sepsis in a child due to a previously unidentified mycobacterium.

A rapidly growing mycobacterium similar to strains in the present Mycobacterium fortuitum complex (M. fortuitum, M. peregrinum, and M. fortuitum third biovariant complex [sorbitol positive and sorbitol negative]) was isolated from a surgically placed central venous catheter tip and three cultures of blood from a 2-year-old child diagnosed with metastatic hepatoblastoma. The organism's unique phenotypic profile and ribotype patterns differed from those of the type and reference strains of the M. fortuitum complex and indicate that this organism may represent a new pathogenic taxon.

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].

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.

A nosocomial pseudo-outbreak of Mycobacterium xenopi due to a contaminated potable water supply: lessons in prevention.

OBJECTIVES: To determine risk factors for Mycobacterium xenopi isolation in patients following a pseudo-outbreak of infection with the organism. DESIGN: Retrospective cohort analysis of mycobacteriology laboratory specimen records and frequency-matched case-control study of hospital patients. SETTING: General community hospital. PATIENTS: For the case-control study, 13 case patients and 39 randomly selected controls with mycobacterial cultures negative for M xenopi, frequency matched by specimen source, whose specimens were submitted from June 1990 through June 1991. RESULTS: Between June 1990 and June 1991, M xenopi was isolated from 13 clinical specimens processed at a midwestern hospital, including sputum (n = 6), bronchial washings (2), urine (4), and stool (1). None of the patients with M xenopi-positive specimens had apparent mycobacterial disease, although five received antituberculosis drug therapy for a range of one to six months. Specimens collected in a nonsterile manner were more likely to grow the organism than those collected aseptically (3.1% versus 0, relative risk = infinity, P = 0.003). M xenopi isolation was attributed to exposure of clinical specimens to tap water, including rinsing of bronchoscopes with tap water after disinfection, irrigation with tap water during colonoscopy, gargling with tap water before sputum collection, and collecting urine in recently rinsed bedpans. M xenopi was isolated from tap water in 20 of 24 patient rooms tested, the endoscopy suite, and the central hot water mixing tank, but not from water in the microbiology laboratory. The pseudo-outbreak occurred following a decrease in the hot water temperature from 130 degrees F to 120 degrees F in 1989. CONCLUSIONS: Maintenance of a higher water temperature and improved specimen collection protocols and instrument disinfection procedures probably would have prevented this pseudo-outbreak.

Evaluation of Syngene DNA-DNA probe assays for the identification of the Mycobacterium tuberculosis complex and the Mycobacterium avium complex.

Two hundred mycobacterial cultures were used to evaluate two alkaline-phosphatase-labeled DNA probe (SNAP) kits developed by Syngene (San Diego, CA) for identification of Mycobacterium tuberculosis complex and M. avium complex. The M. tuberculosis complex SNAP probe, when compared with standard biochemical identification tests, gave results that were in agreement at 100% sensitivity and 98.7% specificity. Ninety-nine M. avium complex strains that were previously tested by the Gen-Probe M. avium complex probe assays and mycolic acid analysis were included to evaluate the M. avium complex SNAP assay which contained three probes, A (avium), I (intracellulare), and X. Eight strains identified as members of the M. avium complex by biochemical tests did not react with the three SNAP probes. These strains were also negative by the Gen-Probe assays. However, 23 strains identified as M. avium complex by biochemical tests and mycolic acid analysis and negative with the Gen-Probe assays gave positive results with the X probe and negative results with the A and I probes of the SNAP assay.

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.

Differentiation of slowly growing Mycobacterium species, including Mycobacterium tuberculosis, by gene amplification and restriction fragment length polymorphism analysis.

A two-step assay combining a gene amplification step and a restriction fragment length polymorphism analysis was developed to differentiate the Mycobacterium species that account for greater than 90% of potentially pathogenic isolates and greater than 86% of all isolates in clinical laboratories in the United States. These species are M. tuberculosis, M. bovis, M. avium, M. intracellulare, M. kansasii, and M. gordonae. With lysates of pure cultures as the template, two oligonucleotide primers that amplified an approximately 1,380-bp portion of the hsp65 gene from all 139 strains of 19 Mycobacterium species tested, but not from the 19 non-Mycobacterium species tested, were identified. Digestion of the amplicons from 126 strains of the six most commonly isolated Mycobacterium species with the restriction enzymes BstNI and XhoI in separate reactions generated restriction fragment patterns that were distinctive for each of these species, except for those of M. tuberculosis and M. bovis, which were not distinguishable. By including size standards in each sample, the restriction fragment profiles could be normalized to a fixed distance and the similarities of patterns could be calculated by using a computer-aided comparison program. The availability of this data base should enable the identification of an unknown Mycobacterium strain to the species level by a comparison of the restriction fragment pattern of the unknown with the data base of known patterns.

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.

Clinical disease, drug susceptibility, and biochemical patterns of the unnamed third biovariant complex of Mycobacterium fortuitum.

Previous studies of Mycobacterium fortuitum identified isolates that did not fit its two recognized biovariants. Eighty-five clinical isolates of this group, the "third biovariant complex", were evaluated. They represented 16% of 410 isolates of M. fortuitum submitted to a Texas laboratory and 22% of 45 isolates in Queensland, Australia. Most infections (76%) involved skin, soft tissue, or bone and occurred after metal puncture wounds or open fractures. Isolates differed from biovar fortuitum in resistance to pipemidic acid and use of mannitol and inositol as carbon sources. Two subgroups were present, and examples were deposited in the American Type Culture Collection. Isolates were resistant to doxycycline and one-third were resistant to cefoxitin. All were susceptible to amikacin, ciprofloxacin, sulfamethoxazole, and imipenem. Surgical debridement combined with drug therapy based on in vitro susceptibilities resulted in cures of cutaneous disease or osteomyelitis. DNA homology studies are needed to determine the taxonomic status of these organisms.

Heterogeneity among isolates of rapidly growing mycobacteria responsible for infections following augmentation mammaplasty despite case clustering in Texas and other southern coastal states.

Thirty-seven cases of rapidly growing mycobacterial wound infections following augmentation mammaplasty were identified between 1979 and 1988. The infections were usually unilateral and had a narrow geographic distribution: almost 60% were from Texas and 92% from five southern coastal states. In Texas a seasonal incidence was observed; 45% of all previously reported and current patients had undergone mammaplasty in April, May, or June. Although these findings suggested a possible common source, analysis of 35 available isolates revealed 19 different phenotype patterns. Five different taxonomic groups were represented, although most isolates (70%) were Mycobacterium fortuitum biovar fortuitum. Plasmid bands were identified in 10 of 15 strains studied, with nine different profiles. An additional 11 cases of breast infection due to rapidly growing mycobacteria not associated with augmentation were also identified, of which nine came from the same states that contributed mammaplasty cases. Rapidly growing mycobacterial infections of the breast are endemic in Texas and other southern coastal states, and the heterogeneity of the isolates suggests that most cases are unrelated.

Diversity and sources of rapidly growing mycobacteria associated with infections following cardiac surgery.

Eighty-nine isolates of rapidly growing mycobacteria associated with cardiac bypass-related infections were characterized. Isolates from sporadic infections belonged to eight taxonomic groups and displayed numerous multilocus enzyme genotypes, plasmid profiles, and heavy metal and antibiotic resistance patterns. Compared with 449 noncardiac wound isolates, 45 sporadic cardiac isolates were more likely to be Mycobacterium fortuitum and M. smegmatis and less likely to be M. chelonae. About 80% of cardiac and noncardiac isolates were from southern coastal states. Eight outbreaks of cardiac bypass-related infections were identified. Strains from each outbreak were genotypically distinctive, and five outbreaks involved more than one strain. In two outbreaks, isolates from environmental sources and noncardiac infections were similar or identical to isolates from sternal wound infections. The heterogeneity of these isolates suggests that most isolates are unrelated and are derived from local environmental sources rather than from contaminated commercial surgical materials or devices.

Nosocomial Mycobacterium fortuitum colonization from a contaminated ice machine.

Between October 15 and November 18, 1985, 5 patients on a medical ward of the Albany VA Medical Center (Ward 8A) became colonized with Mycobacterium fortuitum. Because other patients in Ward 8A were at risk of developing disease with M. fortuitum, microbiologic surveillance to identify colonization in sputum was begun. By February 15, 1986, 30 colonized patients had been identified in this ward but none in another ward with a comparable patient population, which suggests a source unique to Ward 8A. Because water has been recognized as a source of opportunistic mycobacterial pathogens, we conducted a retrospective case-control study using a telephone survey questionnaire to examine a number of water exposures in 10 patients and 20 control subjects. Exposure to ice from the Ward 8A ice machine, but not to potable water, was associated with colonization with M. fortuitum. Large-volume water samples from a variety of sources were cultured for acid-fast bacilli. M. fortuitum was isolated only from the ice machine in Ward 8A. The ice machine was disconnected, and no additional patients became colonized. Although ice machines are infrequently implicated in nosocomial outbreaks, they represent a potential source for pathogens that survive or replicate in water.

Human disease due to Mycobacterium smegmatis.

Mycobacterium smegmatis is a rapidly growing environmental species not considered a human pathogen. We identified 22 human isolates of M. smegmatis from Australia and the southern United States: 19 were from skin or soft-tissue infections, and none were from urine or the male genital tract. These isolates closely resembled Mycobacterium fortuitum, except for a negative three-day arylsulfatase test; growth at 43-45 C; a low semiquantitative catalase test; and, in 50% of isolates, a late-developing, yellow-to-orange pigment. The isolates were biochemically identical to four reference strains and the type strain of M. smegmatis. Isolates were resistant to isoniazid and rifampin but susceptible to ethambutol, doxycycline, sulfamethoxazole, ciprofloxacin, imipenem, and amikacin. Eleven patients treated on the basis of in vitro susceptibility tests responded well to therapy. The similarity of M. smegmatis to M. fortuitum and the failure to recognize that the former is an environmental species may have contributed to previous failures to recognize it as a human pathogen.

Mycobacterium chelonae wound infections after plastic surgery employing contaminated gentian violet skin-marking solution.

From April 1 to October 31, 1985, postoperative surgical-wound infections due to rapidly growing mycobacteria developed in eight patients undergoing cosmetic plastic surgery performed by one surgeon. All infections followed either face-lift or augmentation-mammoplasty procedures performed in the surgeon's office; no infections occurred after surgical procedures performed at the hospital or after other surgical procedures performed at the office. An epidemiologic investigation implicated a gentian violet skin-marking solution as the source of the infections (P less than 0.001). Among patients exposed to the gentian violet, infection was significantly more likely to develop in those undergoing a face lift or augmentation mammoplasty than in those undergoing blepharoplasty (P less than 0.001). Additional risk factors for infection included the postoperative use of antibiotics and glucocorticoids. Mycobacterium chelonae, subspecies abscessus, was isolated from the gentian violet stock used by the surgeon and from five of the eight patients. Additional studies showed that the same organism was present in the gentian violet stock at the pharmacy that supplied the agent to the surgeon. After a sterile skin-marking agent was substituted for the contaminated agent, no further cases occurred.

Analysis of mycolic acid cleavage products and cellular fatty acids of Mycobacterium species by capillary gas chromatography.

After growth and experimental conditions were established, the mycolic acid cleavage products, constituent fatty acids, and alcohols of representative strains of Mycobacterium tuberculosis, M. smegmatis, M. fortuitum complex, M. kansasii, M. gordonae, and M. avium complex were determined by capillary gas chromatography. Reproducible cleavage of mycolic acid methyl esters to tetracosanoic (24:0) or hexacosanoic (26:0) acid methyl esters was achieved by heating the sample in a high-temperature muffle furnace. The major constituent fatty acids in all species were hexadecanoic (16:0) and octadecenoic (18:1 omega 9-c, oleic) acids. With the exception of M. gordonae, 10-methyloctadecanoic acid was found in all species; moreover, M. gordonae was the only species tested which contained 2-methyltetradecanoic acid. M. kansasii was characterized by the presence of 2,4-dimethyltetradecanoic acid, M. avium complex by 2-eicosanol, and M. tuberculosis by 26:0 mycolic acid cleavage product. The mycolic acid cleavage product in the other five species tested was 24:0. Although a limited number of strains and species were tested, preliminary results indicate that this gas chromatographic method can be used to characterize mycobacterial cultures by their mycolic acid cleavage products and constituent fatty acid and alcohol content.

Antimicrobial susceptibility of five subgroups of Mycobacterium fortuitum and Mycobacterium chelonae.

Broth microdilution MICs were determined for 258 clinical isolates of Mycobacterium fortuitum (3 biovariants) and M. chelonae (2 subspecies) with amikacin, tobramycin, cefoxitin, doxycycline, erythromycin, and sulfamethoxazole-trimethoprim and with several new beta-lactams and aminoglycosides and ciprofloxacin. Variations in susceptibility by and within species subgroups confirm the need for susceptibility testing against clinically important strains.

Isoelectric focusing of beta-lactamases in Mycobacterium fortuitum. Association of a single enzyme pattern with cefoxitin resistance.

The uninduced culture supernatants and cell extracts from 58 strains of the 3 biovariants (biovar) of Mycobacterium fortuitum were all positive for beta-lactamase with the chromogenic cephalosporin substrate. By analytical isoelectric focusing (IEF), 29 of 30 strains of biovar fortuitum exhibited an identical beta-lactamase pattern with 1 major band. In contrast, the beta-lactamases of biovar peregrinum and the unnamed third biovar were heterogeneous, with multiple bands and a variety of patterns. The pH range of isoelectric points for the beta-lactamases was relatively narrow, however, with most bands appearing between pH 4.3 and 5.2. Although additional genetic studies are required, these enzymes appear to be chromosomal, as they are present in all strains including some without detectable plasmids. Repeat isolates from the same patient obtained up to six months apart always had the same beta-lactamase pattern by IEF. Of the third biovar complex, 30% are cefoxitin resistant with minimal inhibitory concentrations greater than 32 micrograms/ml. All 9 cefoxitin-resistant isolates tested had the same unique beta-lactamase pattern by IEF, although this enzyme failed to hydrolyze cefoxitin while hydrolyzing cephalothin and benzylpenicillin. Thus, despite the association of cefoxitin-resistance with a single enzyme pattern, the role of this beta-lactamase in resistance is not known.