Hall, Leslie

ABSTRACT Mycobacterium tuberculosis is an important human pathogen that is routinely cultured in clinical and research laboratories. M. tuberculosis can contaminate surfaces and is highly resistant to disinfection. We investigated whether hydrogen peroxide vapor (HPV) is effective for the deactivation of M. tuberculosis on experimentally contaminated surfaces in a biological safety cabinet (BSC) and a room. Biological indicators (BIs) consisting of an ∼3-log 10 inoculum of M. tuberculosis on stainless steel discs and a 6-log 10 inoculum of Geobacillus stearothermophilus were exposed to HPV in BSC time course experiments and at 10 locations during room experiments. In three separate BSC experiments, M. tuberculosis BIs were transferred to growth media at 15-min intervals during a 180-min HPV exposure period. No M. tuberculosis BIs grew following 30 min of HPV exposure. In three separate room experiments, M. tuberculosis and G. stearothermophilus BIs were exposed to HPV for 90, 120, and 150 min, respectively. BIs for both microorganisms were deactivated in all 10 locations following 90 min of HPV exposure. HPV provides an alternative to traditional decontamination methods, such as formaldehyde fumigation, for laboratories and other areas contaminated with M. tuberculosis .

ABSTRACT Mycobacterium peregrinum consists of two taxa: types I and II. We evaluated 43 clinical type II strains from throughout the United States. They were responsible for soft-tissue and bone infections, catheter-related infections, and possible pneumonitis. By carbohydrate utilization, they were indistinguishable from type I strains, being d -mannitol and trehalose positive. However, they had a distinct susceptibility pattern that included intermediate ciprofloxacin MICs but low clarithromycin and doxycycline MICs of ≤1 μg/ml. These features were also shared by reference isolates of Mycobacterium senegalense from African bovine cases of “farcy.” By 16S rRNA gene sequencing, the type II isolates shared 100% sequence identity with M. senegalense . Partial sequencing of the type II hsp65 gene (441 bp) revealed four sequevars showing ≥98.4% identity with each other and ≥98.6% identity with the sequence of five bovine strains of M. senegalense . There was ≤97.1% identity with M. peregrinum type I isolates and other Mycobacterium fortuitum group species. Sequencing of additional gene targets including the 16S-23S rDNA internal transcribed spacer region and the rpoB gene (partial sequence) revealed a similar phylogenetic grouping. DNA-DNA hybridization showed 76 to 99% relatedness between the bovine and human strains. These studies demonstrate that type II isolates are not isolates of M. peregrinum but represent human strains of M. senegalense . This study is the first to demonstrate this species as a human pathogen. Representative human M. senegalense strains include ATCC 35755 and newly submitted strains ATCC BAA-849, ATCC BAA-850, and ATCC BAA-851.

ABSTRACT Recent molecular studies have shown Mycobacterium porcinum , recovered from cases of lymphadenitis in swine, to have complete 16S rDNA sequence identity and >70% DNA-DNA homology with human isolates within the M. fortuitum third biovariant complex. We identified 67 clinical and two environmental isolates of the M. fortuitum third biovariant sorbitol-negative group, of which 48 (70%) had the same PCR restriction enzyme analysis (PRA) profile as the hsp65 gene of M. porcinum (ATCC 33776 T ) and were studied in more detail. Most U.S. patient isolates were from Texas (44%), Florida (19%), or other southern coastal states (15%). Clinical infections included wound infections (62%), central catheter infections and/or bacteremia (16%), and possible pneumonitis (18%). Sequencing of the 16S rRNA gene (1,463 bp) showed 100% identity with M. porcinum ATCC 33776 T . Sequencing of 441 bp of the hsp65 gene showed four sequevars that differed by 2 to 3 bp from the porcine strains. Clinical isolates were positive for arylsulfatase activity at 3 days, nitrate, iron uptake, d -mannitol, i- myo -inositol, and catalase at 68°C. They were negative for l -rhamnose and d -glucitol (sorbitol). Clinical isolates were susceptible to ciprofloxacin, sulfamethoxazole, and linezolid and susceptible or intermediate to cefoxitin, clarithromycin, imipenem, and amikacin. M. porcinum ATCC 33776 T gave similar results except for being nitrate negative. These studies showed almost complete phenotypic and molecular identity between clinical isolates of the M. fortuitum third biovariant d -sorbitol-negative group and porcine strains of M. porcinum and confirmed that they belong to the same species. Identification of M. porcinum presently requires hsp65 gene PRA or 16S rRNA or hsp65 gene sequencing.

The isolation and identification of a novel, slow-growing, scotochromogenic, mycobacterial species is reported. A strain, designated MUP 1182T, was isolated from a cervical lymph node of a 3-year-old child. MUP 1182T is alcohol- and acid-fast, with a lipid pattern that is consistent with those of species that belong to the genus Mycobacterium. It grows slowly at 25–37 °C, but does not grow at 42 °C. The isolate was revealed to be biochemically distinct from previously described mycobacterial species: it has urease and Tween hydrolysis activities and lacks nitrate reductase, 3-day arylsulfatase and β-glucosidase activities. Comparative 16S rDNA sequencing showed that isolate MUP 1182T represents a novel, slow-growing species that is related closely to Mycobacterium lentiflavum and Mycobacterium simiae. On the basis of these findings, the name Mycobacterium parmense sp. nov. is proposed, with MUP 1182T (=CIP 107385T=DSM 44553T) as the type strain.

ABSTRACT Described herein is our experience with the MicroSeq D2 large-subunit rDNA sequencing kit for the identification of filamentous fungi encountered in the mycology laboratory at the Mayo Clinic. A total of 234 filamentous fungi recovered from clinical specimens were used in the evaluation. All were identified by using phenotypic characteristics as observed macroscopically and microscopically on any medium or a combination of media, which included Sabouraud's dextrose, inhibitory mold, cornmeal, Czapek-Dox, potato dextrose, and V8 juice agars; all isolates were sequenced using the MicroSeq D2 large-subunit rDNA sequencing kit. Of the of 234 isolates, 158 were correctly identified to the appropriate genus or genus and species by using nucleic acid sequencing. Sequences for 70 (29.9%) of the isolates (27 genera) were not included in the MicroSeq library. Of the 80 dematiaceous and 154 hyaline fungi sequenced, 65 and 51.2%, respectively, gave results concordant with those determined by phenotypic identification. Nucleic acid sequencing using the MicroSeq D2 large-subunit rDNA sequencing kit offers promise of being an accurate identification system; however, the associated library needs to include more of the clinically important genera and species.

ABSTRACT Experience with a MicroSeq D2 large-subunit (LSU) ribosomal DNA (rDNA) sequencing kit for identification of yeast species commonly encountered in the mycology laboratory at Mayo Clinic is described here. A total of 131 isolates of yeasts recovered from clinical specimens were included in the study. Phenotypic methods used for initial identification included germ tube formation, urease production, microscopic morphological features on cornmeal agar, and an API 20C AUX system; all isolates were sequenced using a MicroSeq D2 LSU rDNA sequencing kit. Nucleic acid sequencing identified 93.9% of the isolates to the correct genus and species. A total of 100 of the isolates (representing 19 species of Candida ) were sequenced, and 98% gave results concordant with identifications made by the API 20C AUX system; distance scores ranged from 0 to 1.88%, with an average value of 0.23%. Candida dubliniensis was not included in the MicroSeq database and was identified as Candida albicans . A total of 32 isolates representing 9 other genera (including Cryptococcus , Filobasidium , Kloeckera , Malassezia , Pichia , Sporidiobolus , Rhodotorula , Zygosaccharomyces , and Trichosporon ) were included, and 81.3% showed concordant results when phenotypic and sequencing results were compared. Most discrepancies were attributed to the lack of inclusion of the species in the MicroSeq or API 20C AUX database. The MicroSeq D2 LSU rDNA sequencing kit appears to be accurate and useful for the identification of yeasts that might be seen in a clinical laboratory.

ABSTRACT An evaluation of the MicroSeq 500 microbial identification system by nucleic acid sequencing and the Mayo Clinic experience with its integration into a routine clinical laboratory setting are described. Evaluation of the MicroSeq 500 microbial identification system was accomplished with 59 American Type Culture Collection (ATCC) strains and 328 clinical isolates of mycobacteria identified by conventional and 16S ribosomal DNA sequencing by using the MicroSeq 500 microbial identification system. Nucleic acid sequencing identified 58 of 59 (98.3%) ATCC strains to the species level or to the correct group or complex level. The identification results for 219 of 243 clinical isolates (90.1%) with a distance score of <1% were concordant with the identifications made by phenotypic methods. The remaining 85 isolates had distance scores of >1%; 35 (41.1%) were identified to the appropriate species level or group or complex level; 13 (15.3%) were identified to the species level. All 85 isolates were determined to be mycobacterial species, either novel species or species that exhibited significant genotypic divergence from an organism in the database with the closest match. Integration of nucleic acid sequencing into the routine mycobacteriology laboratory and use of the MicroSeq 500 microbial identification system and Mayo Clinic databases containing additional genotypes of common species and added species significantly reduced the number of organisms that could not be identified by phenotypic methods. The turnaround time was shortened to 24 h, and results were reported much earlier. A limited number of species could not be differentiated from one another by 16S ribosomal DNA sequencing; however, the method provides for the identification of unusual species and more accurate identifications and offers the promise of being the most accurate method available.

ABSTRACT A 45-year-old man from Nepal with a 13-year history of polycystic kidney disease was diagnosed as suffering from chronic renal failure with end-stage renal disease. After receiving empirical antituberculosis treatment, he was treated with broad-spectrum antibiotics. A left nephrectomy was performed, and after 4 months, he received a kidney transplant. The left kidney was grossly enlarged, with multiple cystic spaces filled with blackish material. Histologic examination of the excised left kidney tissue stained with hematoxylin and eosin and Gomori's methenamine silver stains showed numerous hyaline, septate, fungal hyphae of various lengths, many broken into rectangular arthroconidia in the cystic spaces. Culture of the kidney tissue yielded white, glabrous, yeast-like colonies. Based on its micromorphology, growth at 42°C, and ribosomal DNA (rDNA) sequence analysis, and also sequence analysis of the internal-transcribed-spacer and D1/D2 rDNA regions, the yeast was identified as Trichosporon loubieri. Postsurgically, the patient was treated with amphotericin B and oral itraconazole, followed by maintenance therapy with fluconazole. He remained afebrile and asymptomatic. At the final follow-up, all parameters were found normal and the patient was doing well, with normal renal function reports. This paper presents the first known case of human infection caused by T. loubieri.

ABSTRACT Six clinical isolates of the nonpigmented, rapidly growing species Mycobacterium mageritense were recovered from sputum, bronchial wash, blood, sinus drainage, and two surgical wound infections from separate patients in Texas, New York, Louisiana, and Florida. The isolates matched the ATCC type strain by PCR restriction enzyme analysis of the 65-kDa hsp gene sequence of Telenti, high-performance liquid chromatography, biochemical reactions, and partial 16S rRNA gene sequencing. These are the first isolates of this species to be described in the United States and the first isolates to be associated with clinical disease. Susceptibility testing of all known isolates of the species revealed all isolates to be susceptible or intermediate to amikacin, cefoxitin, imipenem, and the fluoroquinolones and sulfonamides but resistant to clarithromycin. Because of their phenotypic and clinical similarity to isolates of the Mycobacterium fortuitum third biovariant complex (sorbitol positive), isolates of M. mageritense are likely to go undetected unless selected carbohydrate utilization or molecular identification methods are used.