No abstract

This report describes the first case in South Australia, Australia, of Mycobacterium pinnipedii tuberculosis in a free-ranging Australian fur seal (Arctocephalus pusillus doriferus). Severe pyogranulomatous pleuropneumonia with intrahistocytic acid-fast beaded filamentous bacilli was seen on histology. M. pinnipedii was confirmed by full 24-loci mycobacterial interspersed repetitive-unit-variable-number tandem-repeat (MIRU-VNTR) typing. Spillover concerns for public health and cattle are discussed.

Legionella -like bacteria were isolated from the respiratory tract of two patients in California, USA, and South Australia, but were not thought to cause disease. These bacteria, strains F2632 and IMVS-3376T, were found to have identical Legionella macrophage infectivity potentiator (mip) gene sequences and were therefore further characterized to determine their genetic and phenotypic relatedness and properties. Both of these Gram-negative-staining bacterial strains grew on buffered charcoal yeast extract medium, were cysteine auxotrophs and made a characteristic diffusible bright yellow fluorescent pigment, with one strain making a late appearing colony-bound blue–white fluorescent pigment. The optimal in vitro growth temperature was 35 °C, with very poor growth at 37 °C in broth or on solid media. There was no growth in human A549 cells at either 35 or 37 °C, but excellent growth in Acanthamoeba castellani at 30 °C and poorer growth at 35 °C. Phylogenetic analysis of these bacteria was performed by sequence analysis of 16S rRNA, mip, ribonuclease P, ribosomal polymerase B and zinc metalloprotease genes. These studies confirmed that the new strains represented a single novel species of the genus Legionella for which the name Legionella steelei sp. nov. is proposed. The type strain is IMVS-3376T ( = IMVS 3113T = ATCC BAA-2169T).

f Culture-based detection of nontuberculous Mycobacteria (NTM) in respiratory samples is time consuming and can be subject to overgrowth by nonmycobacterial bacteria. We describe a single-reaction TaqMan quantitative PCR assay for the direct detection of NTM species in clinical samples that is specific, sensitive, and robust. While rates of infection caused by members of the Mycobacterium tuberculosis complex continue to fall in developed countries (1, 2), disease caused by nontuberculous mycobacteria (NTM) is an area of growing concern (3-6). Pulmonary infection represents more than 90% of NTM cases (7) and has been described in a range of clinical contexts (8-11). Appropriate management of suspected pulmonary NTM infection requires the timely detection and identification of the etiological agent. The current "gold standard" for detection of NTM in respiratory samples relies on protracted in vitro culture, potentially delaying targeted therapy. It also requires samples to undergo decontamination prior to culture to lower levels of commensal microbiota (12) and is associated with variable sensitivity (13). The ability to perform a rapid quantitative screen for the presence of any NTM species would provide an important early indication of mycobacterial involvement and would be informative in cases where samples are culture negative, despite clinical or radiological signs.To prevent false-positive results arising from the detection of closely related species (14, 15), existing molecular assays target narrow phylogenetic groups or specific pathogens (16-21), require prior mycobacterial isolation by culture (22-24), or are unable to provide accurate species-level NTM identification (25). We describe a TaqMan quantitative PCR (qPCR) assay, based on the single-copy hsp65 gene, for the direct detection of NTM species in respiratory clinical samples.The assay design was based on the full-length hsp65 gene sequences that are available for 116 of the 174 currently described NTM species, including all 56 NTM species reported in respiratory disease (see Fig. S1 in the supplemental material). The PCR primers (forward, HSP171 [5=-CGCCAAGGAGATCGAGCTGG-3=], and reverse, HSP563 [5=-GGACAAGGTCGGCAACGAGGG-3=]) generate a 348-bp hsp65 amplicon and are used in conjunction with a TaqMan probe (5=-FAM-AGAAGGCCGTCGAGAAG GTCA-BHQ-3= [FAM, 6-carboxyfluorescein; BHQ, black hole quencher]) at an annealing temperature of 60°C (Fig. 1). A detailed description of assay development and methods is provided in the supplemental material.In silico analysis indicated complete homology to the targeted hsp65 gene region for 77 Mycobacterium species. Fourteen species had Յ2 nucleotide mismatches within the primer binding region, with a corresponding reduction in annealing temperature of up to 4.5°C. However, in all such cases, the corresponding primer binding region showed 100% sequence homology (see Tables S1 and S2 in the supplemental material). Twenty-one mycobacterial species (including M. tuberculosis and Mycobacterium leprae) and 40 assessed non...

No abstract