Recognition of a Nocardia transvalensis complex by resistance to aminoglycosides, including amikacin, and PCR-restriction fragment length polymorphism analysis.

Amikacin resistance, rare among nocardiae, was observed in 58 clinical isolates of nocardiae. All of these isolates hydrolyzed hypoxanthine, and 75 to 100% utilized citrate, D-galactose, and D-trehalose as sole carbon sources. Based on utilization of I-erythritol, D-glucitol, i-myo-inositol, D-mannitol, and ribitol and susceptibility to amoxicillin-clavulanic acid, the 58 isolates were separable into four groups. One group was negative for I-erythritol and ribitol and included all the isolates belonging to Nocardia asteroides complex antibiogram type IV. The remaining three groups were positive for I-erythritol and ribitol and were grouped within Nocardia transvalensis. The group that included the type strain was designated N. transvalensis sensu stricto, and the other two groups were designated new taxons 1 and 2. PCR-restriction fragment length polymorphism (RFLP) analysis of a 439-bp segment of the 65-kDa heat shock protein gene with XhoI and HinfI produced identical patterns for 53 (91%) and 58 (100%) isolates, respectively, and differentiated them from all other Nocardia taxa. NarI- and HaeIII-derived RFLP patterns clearly differentiated each of the four biochemically defined taxa. These four groups were also distinguishable by using the chromogenic substrates in Dade MicroScan test panels. By high-performance liquid chromatography, these isolates exhibited the same unique mycolic acid-ester elution patterns that differed from those of all other clinically significant nocardiae. Gas-liquid chromatographic analysis of fatty acids also produced similar patterns for all isolates that distinguished them from all other Nocardia taxa, but did not differentiate the four taxa within the complex. We propose the designation N. transvalensis complex for these four groups of nocardiae, pending further genetic evaluation.

Rapid identification of clinically significant species and taxa of aerobic actinomycetes, including Actinomadura, Gordona, Nocardia, Rhodococcus, Streptomyces, and Tsukamurella isolates, by DNA amplification and restriction endonuclease analysis.

A previously described PCR-restriction fragment length polymorphism (RFLP) identification schema for Nocardia that used an amplified 439-bp segment (amplicon) of the 65-kDa heat shock protein gene was evaluated for potential use with isolates of all clinically significant aerobic actinomycetes. The study included 28 reference (American Type Culture Collection) strains and 198 clinical isolates belonging to 20 taxonomic groups. Of these 198 isolates, 188 could be differentiated by this PCR-RFLP method. Amplicons from all aerobic actinomycete isolates lacked BstEII recognition sites, thereby distinguishing them from those of mycobacteria that contain one or more such sites. Of 29 restriction endonucleases, MspI plus HinfI produced RFLP patterns that differentiated 16 of the 20 taxa. A single RFLP pattern was observed for 15 of 20 taxa that included 65% of phenotypically clustered isolates. Multiple patterns were seen with Gordona bronchialis, Nocardia asteroides complex type VI, Nocardia otitidiscaviarum, Nocardia transvalensis, and Streptomyces spp. Streptomyces RFLP patterns were the most heterogeneous (five patterns among 19 isolates), but exhibited a unique HinfI fragment of > 320 bp. RFLP patterns that matched those from type strains of Streptomyces albus, Streptomyces griseus, or Streptomyces somaliensis were obtained from 14 of 19 Streptomyces isolates. Only 10 of 28 isolates of N. otitidiscaviarum failed to yield satisfactory amplicons, while only 6 of 188 (3.2%) clinical isolates exhibited patterns that failed to match one of the 21 defined RFLP patterns. These studies extended the feasibility of using PCR-RFLP analysis as a rapid method for the identification of all clinically significant species and taxa of aerobic actinomycetes.

DNA amplification and restriction endonuclease analysis for differentiation of 12 species and taxa of Nocardia, including recognition of four new taxa within the Nocardia asteroides complex.

Nineteen reference and 156 clinical strains of the genus Nocardia belonging to 12 taxonomic groups were studied for restriction fragment length polymorphism (RFLP) by using an amplified 439-bp segment of the 65-kDa heat shock protein gene. Of 30 restriction endonucleases, digestion with MspI and then digestion with BsaHI produced RFLP band patterns which separated all 12 groups except N. asteroides type IV from 6 of 12 N. transvalensis isolates and N. carnea from the N. asteroides type VI isolates. Commonly encountered species such as N. nova, N. farcinica, N. brasiliensis sensu stricto, and N. otitidiscaviarum were easily separated. Each taxon resulted in a single RFLP band pattern that included > or = 96% of all biochemically grouped isolates for 9 of 12 taxa with MspI and for 8 of 12 taxa with BsaHI. With the use of both patterns, only 6 of 175 (3.4%) isolates failed to fit the biochemically defined group patterns. These studies provide the first evidence for the separate identities of four antibiogram-defined (but currently unnamed) groups within the N. asteroides complex (types I, II, IV, and VI) and the presence of two subgroups within N. transvalensis. They also provide genotypic evidence for the separate identities of N. nova and N. farcinica. The lack of BstEII recognition sites in amplicons obtained from nocardiae provides a simple and rapid method for the differentiation of nocardiae from mycobacteria. DNA amplification with RFLP analysis is the first rapid method that distinguishes all clinically significant taxa and recognized species within the genus Nocardia.

New Nocardia taxon among isolates of Nocardia brasiliensis associated with invasive disease.

Nocardia brasiliensis, the second most frequently isolated aerobic actinomycete in the clinical laboratory, is usually associated with localized cutaneous infections. However, 22% of 238 N. brasiliensis isolates from the United States and 12% of 66 isolates from Queensland, Australia, which had been collected over a 17-year period, were associated with extracutaneous and/or disseminated diseases. Of the 62 invasive isolates, 37 (60%) were susceptible to ciprofloxacin and/or were susceptible to clarithromycin and resistant to minocycline, compared with only 6 (3%) of 242 localized cutaneous isolates. The 43 isolates with this susceptibility pattern appeared to define a new taxon. They were similar to Nocardia asteroides complex isolates clinically in proportions from persons with pulmonary (70%), central nervous system (23%), and/or disseminated diseases (37%) in the setting of corticosteroids (74%) or AIDS (14%). This putative new taxon differed from N. brasiliensis in the hydrolysis of adenine (92 versus 4%), beta-lactamase patterns on isoelectric focusing, and the presence of two early mycolic acid-ester peaks by high-performance liquid chromatography. Restriction analysis of a 439-bp fragment of the 65-kDa heat shock protein gene revealed that N. brasiliensis and the new taxon had different restriction patterns with 8 of the 11 enzymes tested. Screening of invasive isolates of N. brasiliensis for susceptibility to ciprofloxacin will identify most isolates of the new taxon, which likely represents a new Nocardia species.

Mycobacterium asiaticum as the probable causative agent in a case of olecranon bursitis.

Mycobacterium asiaticum was isolated from fluid aspirated from an olecranon bursa that had become inflamed following a superficial injury. Other possible causes of the inflammation were excluded. No specific antimycobacterial therapy was given. The infection responded to drainage, regular dressing, and immobilization. Our experience suggests that M. asiaticum is a potential cause of infection of the joints and surrounding tissues.

Mycobacterium marinum infection: epidemiology and presentation in Queensland 1971-1990.

OBJECTIVE: Evaluation of the clinical and epidemiological features of Mycobacterium marinum infection in Queensland. DESIGN: Laboratory identification and in-vitro susceptibility tests of 29 isolates from the Queensland Health Department Tuberculosis Reference Laboratory were retrospectively gathered and followed up by contacting referring practitioners and obtaining clinical details of patients involved. SUBJECTS: 29 patients from whom M. marinum was isolated, with a male:female ratio of 3.1:1, and a mean age of 47.4 years. RESULTS: Of 26 patients for whom adequate information was available, 12 had evidence of involvement of deep tissues (including two cases of arthritis) and five suffered sporotrichoid spread of infection. The delay between onset of symptoms and consultation with a medical practitioner was five months (range, two weeks to two years), with a further mean delay to definitive diagnosis of 4.4 weeks. Cure was apparent in 22 of 23 cases. Chemotherapy alone was adequate in 11 cases, as was surgical intervention in three, while a combination approach was successful in eight cases. Trimethoprim/sulfamethoxazole was successful in seven of nine cases and combination rifampicin and ethambutol in six of seven. Tetracyclines were employed as single-agent therapy in nine patients and were effective in seven. CONCLUSIONS: Synovitis was a common presenting feature of M. marinum infection in Queensland patients. Occupational and recreational exposure to salt or fresh water was common, and although this history was available to practitioners a mean delay to definitive diagnosis of 4.4 weeks still occurred. The data suggest that chemotherapy alone is often adequate, even with deep tissue involvement. Combinations of conventional antimycobacterial drugs may be the therapy of choice, especially for serious infections, although success was recorded with trimethoprim/sulfamethoxazole alone.

Infections due to Nocardia transvalensis: clinical spectrum and antimicrobial therapy.

Nocardia transvalensis, a rare Nocardia species, has previously been recognized as a cause of actinomycotic mycetoma. In a retrospective review of N. transvalensis isolates referred to the Centers for Disease Control (Atlanta) during the period January 1981 through January 1990, we identified 15 patient isolates. Four N. transvalensis isolates originated from one Australian reference laboratory; one patient's isolate that was identified by the Australian laboratory but that was not received at the Centers for Disease Control was also included in our study. A review of the cases of these 16 patients found that N. transvalensis caused infection in 10 patients and colonization in two patients. Six (75%) of eight patients with primary pulmonary or disseminated N. transvalensis infections had an underlying immunologic disorder or were receiving immunosuppressive therapy; three patients with disseminated infection died. All nine infected patients for whom specific antimicrobial therapy was prescribed received trimethoprim-sulfamethoxazole. Results of in vitro antimicrobial susceptibility tests of 11 N. transvalensis isolates revealed increased antimicrobial resistance to amikacin and other drugs when compared with that of other Nocardia species. Severely immunocompromised patients are predisposed to N. transvalensis pneumonia or disseminated infection, and the lung may be the portal of entry.

Infection with Nocardia species in Queensland. A review of 102 clinical isolates.

OBJECTIVE: To review the species distribution, pathologic significance and disease associations of clinical isolates of Nocardia and related bacteria in Queensland, and to examine the characteristics, treatment and outcome of patients infected with these organisms. DESIGN AND SETTING: A retrospective review of Queensland State Health Laboratory records provided microbiological data for Nocardia isolates referred from other laboratories during the period January 1983 to December 1988. Clinical information was extracted from hospital case notes, or obtained from detailed questionnaires completed by attending physicians. Nocardia isolates were classified as "significant" if specific treatment for nocardiosis was given, or on the basis of autopsy findings. PATIENTS: One hundred and two patients had a Nocardia species or a related organism isolated from clinical specimens during the study period. RESULTS: The 102 isolates included Nocardia asteroides (45), N, brasiliensis (35), N. caviae (5) and N. transvalensis (5). Clinical results were available for 93 patients, of whom 74 (80%) had a significant isolate recovered. Primary pulmonary or disseminated disease occurred in 35 patients, and was caused mainly by N. asteroides. Significant infections of skin and soft tissues, primarily due to N. brasiliensis, were found in 39 patients. Preexisting lung disease and treatment with steroids and immunosuppression were risk factors for pulmonary and disseminated nocardiosis. A history of inoculation in an outdoor setting was frequent in patients with cutaneous disease. Antibiotic regimens that included trimethoprim-sulfamethoxazole or another sulfonamide agent were used to treat the majority of patients with significant infection. Deaths were confined to those with pulmonary and disseminated disease, with a case fatality rate of 40% in that group. CONCLUSION: Infection with Nocardia species appears to be more common than is generally appreciated. The local species distribution and disease spectrum are similar to those described elsewhere. A high index of suspicion for nocardiosis should be maintained in susceptible hosts with pulmonary infiltrates, particularly when there is evidence for metastatic infection, and in patients with superficial infections and a history of outdoor injury.

Fourth report of the cooperative, open-ended study of slowly growing mycobacteria by the International Working Group on Mycobacterial Taxonomy.

The open-ended study of the International Working Group on Mycobacterial Taxonomy is an ongoing project to characterize slowly growing strains of mycobacteria that do not belong to well-established or thoroughly characterized species. In this fourth report we describe two numerical taxonomic clusters that represent subspecies or biovars of Mycobacterium simiae, one cluster that encompasses the erstwhile type strain of the presently invalid species "Mycobacterium paraffinicum," one cluster that is phenotypically very similar to Mycobacterium avium and Mycobacterium intracellulare but may be a separate genospecies, one cluster that appears to be phenotypically distinct from M. avium but reacts with a nucleic acid probe specific for M. avium, and three tentatively defined clusters in proximity to a cluster that encompasses the type strain of Mycobacterium malmoense. Of special practical interest is the fact that one of the latter three clusters is composed of clinically significant scotochromogenic bacteria that can be misidentified as the nonpathogenic organism Mycobacterium gordonae if insufficient biochemical tests are performed.

Tuberculosis in Australia: an analysis of cases identified in reference laboratories in 1986-88.

The Special Interest Group in Mycobacteria within the Australian Society for Microbiology has carried out a collaborative study of cases of tuberculosis diagnosed in Australian reference laboratories in the years 1986, 1987 and 1988. Annual totals of 574, 584 and 613 respectively, suggest that the incidence of bacteriologically-positive tuberculosis is continuing at 3-4 cases per 100,000 population. The highest rates were detected in males over 50 and females over 65 years of age. Three-quarters of the total cases relate to pulmonary disease. Resistance to at least 1 anti-tuberculosis drug was detected in 78 (12.7%) of isolates tested in 1988. The negligible decline in incidence of tuberculosis in Australia, the high prevalence in S.E. Asian countries, and the fact that HIV-infection is an important risk factor, make it imperative that Australia's diagnostic and management programmes be maintained.

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.

Bacteremia caused by Mycobacterium neoaurum.

An immunocompromised patient with an indwelling Hickman catheter developed Mycobacterium neoaurum bacteremia. This rapidly growing mycobacterium was previously isolated from soil, dust, and water but has not been described as a human pathogen. The infection responded to therapy with cefoxitin and gentamicin. It was not necessary to remove the Hickman catheter.

Cutaneous infection with Mycobacterium gordonae.

A case of cutaneous infection with Mycobacterium gordonae and other reports of extrapulmonary infection due to this organism are reviewed. This case confirms the pathogenic potential of M. gordonae which must now be included among the scotochromogens capable of causing cutaneous disease. Isolates of this organism should be tested against a full range of antimicrobial agents since traditional antituberculous therapy may be of limited efficacy. Pending the results of in vitro susceptibility testing, amikacin, ethambutol, rifampicin and co-trimoxazole are suggested as empirical therapy for infections caused by this organism.

Mycobacterium asiaticum as a potential pulmonary pathogen for humans. A clinical and bacteriologic review of five cases.

Mycobacterium asiaticum was isolated from pulmonary material from 5 persons residing in Queensland, Australia. All patients had repeated positive specimens, but the organism was considered responsible for pulmonary mycobacteriosis in only 2 of them. This is the first report of disease caused by M. asiaticum. Clinical, bacteriologic, and epidemiologic details are presented.

Mycobacterial cross-contamination of bronchoscopy specimens.

Bronchoscopy specimens from 3 patients investigated during 1 wk at a large teaching hospital were culture positive for the same serovar of Mycobacterium intracellulare. Review of laboratory records and patient data suggested that only the first patient examined was a genuine excretor of mycobacteria; the isolates from the other 2 patients were false positives caused by inadequate disinfection of part of the collection apparatus between use. The investigations confirm the suitability of glutaraldehyde as a rapid, highly effective antimycobacterial disinfectant and illustrate the value of serotyping when dealing with mycobacterial isolates of questionable significance.

Differential identification of mycobacteria in smears of sputum.

As a test of their ability to differentiate mycobacteria in Ziehl-Neelsen stained smears of sputum, 4 readers examined an assortment of 200 smears, of which half were known to contain Mycobacterium tuberculosis and half were known to contain atypical mycobacteria. In 2 separate trials, readers made a prediction of either 'typical' or 'atypical' for each smear. Reader consistency between trials ranged from 69.5% to 86.5%, indicating that readers were basing their predictions on definite criteria. Overall accuracy ranged from 62.5% to 80%, with experienced readers being the most accurate. Reliability of readers' predictions was influenced by smear positivity, but certain smears exhibited uncharacteristic features which led to incorrect predictions. When considered along with other available data, smear-based presumptive diagnosis can assist the clinician in his management of the patient with suspected tuberculosis in the period before culture results are known.

Misleading results from bone marrows cultured for mycobacteria.

Re-evaluation of a previous study dealing with atypical mycobacteria grown from specimens of bone marrow provided strong evidence that the isolates were of no clinical significance. Bone marrow culture should not be undertaken as a diagnostic test for pulmonary atypical mycobacteriosis.

Mycobacterium haemophilum causing lymphadenitis in an otherwise healthy child.

Suppurating submandibular lymphadenitis in an otherwise healthy infant was caused by Mycobacterium haemophilum, A fastidious organism which requires media supplemented with ferric ammonium citrate or haemoglobin for its growth. It is recommended that material from superficial lesions and lymph nodes collected for mycobacteriological investigations be cultured on media known to support growth of M. haemophilum.

Atypical mycobacteria causing non-pulmonary disease in Queensland.

During the period 1971--7, the Tuberculosis Reference Laboratory in Queensland dealt with 52 isolates of atypical mycobacteria made from non-pulmonary sites under circumstances suggesting complicity in disease. Twenty-four isolates belonging to the MAIS complex were associated with lymph node infections in children. Twelve isolates belonged to the M. fortuitum-chelonei complex; most were from superficial abscesses. Five cases of M. marinum infection and 8 of M. ulcerans disease were detected.

Mycobacterium avium associated with chronic pulmonary disease in a Queensland resident.

Bacteriological investigation over an 11-year period has implicated Mycobacterium avium ser I as the most probable cause of chronic pulmonary disease in a resident of south-eastern Queensland. Although the patient had contact with domestic fowls and a pet budgerigar, the source of the organism is uncertain.