Absence of a functional erm gene in isolates of Mycobacterium immunogenum and the Mycobacterium mucogenicum group, based on in vitro clarithromycin susceptibility.

Macrolide resistance has been linked to the presence of a functional erythromycin ribosomal methylase (erm) gene in most species of pathogenic rapidly growing mycobacteria (RGM). For these Mycobacterium isolates, extended incubation in clarithromycin is necessary to determine macrolide susceptibility. In contrast, the absence of a detectable erm gene in isolates of M. chelonae, M. senegalense, and M. peregrinum and a nonfunctional erm gene in M. abscessus subsp. massiliense and 15% to 20% of M. abscessus subsp. abscessus isolates renders these species intrinsically macrolide susceptible. Not all RGM species have been screened for the presence of an erm gene, including the Mycobacterium mucogenicum group (M. mucogenicum, M. phocaicum, and M. aubagnense) and Mycobacterium immunogenum. A total of 356 isolates of these two pathogenic RGM taxa from two reference laboratories (A.R.U.P. Reference Laboratories and the Mycobacteria/Nocardia Laboratory at the University of Texas Health Science Center at Tyler) underwent clarithromycin susceptibility testing with readings at 3 to 5 days and 14 days. Only 13 of the 356 isolates had resistant clarithromycin MICs at initial extended MIC readings, and repeat values on all available isolates were ≤2 µg/ml. These studies suggest that these two additional RGM groups do not harbor functional erm genes and, like M. chelonae, do not require extended clarithromycin susceptibility testing. We propose to the Clinical Laboratory and Standards Institute that isolates belonging to these above-mentioned six rapidly growing mycobacterial groups based on molecular identification with no known functional erm genes undergo only 3 to 5 days of susceptibility testing (to exclude mutational resistance).

CAF-secreted annexin A1 induces prostate cancer cells to gain stem cell-like features.

UNLABELLED: Annexin A1 (AnxA1), a phospholipid-binding protein and regulator of glucocorticoid-induced inflammatory signaling, has implications in cancer. Here, a role for AnxA1 in prostate adenocarcinoma was determined using primary cultures and a tumor cell line (cE1), all derived from the conditional Pten deletion mouse model of prostate cancer. AnxA1 secretion by prostate-derived cancer-associated fibroblasts (CAF) was significantly higher than by normal prostate fibroblasts (NPF). Prostate tumor cells were sorted to enrich for epithelial subpopulations based on nonhematopoietic lineage, high SCA-1, and high or medium levels of CD49f. Compared with controls, AnxA1 enhanced stem cell-like properties in high- and medium-expression subpopulations of sorted cE1 and primary cells, in vitro, through formation of greater number of spheroids with increased complexity, and in vivo, through generation of more, larger, and histologically complex glandular structures, along with increased expression of p63, a basal/progenitor marker. The differentiated medium-expression subpopulations from cE1 and primary cells were most susceptible to gain stem cell-like properties as shown by increased spheroid and glandular formation. Further supporting this increased plasticity, AnxA1 was shown to regulate epithelial-to-mesenchymal transition in cE1 cells. These results suggest that CAF-secreted AnxA1 contributes to tumor stem cell dynamics via two separate but complementary pathways: induction of a dedifferentiation process leading to generation of stem-like cells from a subpopulation of cancer epithelial cells and stimulation of proliferation and differentiation of the cancer stem-like cells. IMPLICATIONS: AnxA1 participates in a paradigm in which malignant prostate epithelial cells that are not cancer stem cells are induced to gain cancer stem cell-like properties.

Antimicrobial susceptibility testing, drug resistance mechanisms, and therapy of infections with nontuberculous mycobacteria.

Within the past 10 years, treatment and diagnostic guidelines for nontuberculous mycobacteria have been recommended by the American Thoracic Society (ATS) and the Infectious Diseases Society of America (IDSA). Moreover, the Clinical and Laboratory Standards Institute (CLSI) has published and recently (in 2011) updated recommendations including suggested antimicrobial and susceptibility breakpoints. The CLSI has also recommended the broth microdilution method as the gold standard for laboratories performing antimicrobial susceptibility testing of nontuberculous mycobacteria. This article reviews the laboratory, diagnostic, and treatment guidelines together with established and probable drug resistance mechanisms of the nontuberculous mycobacteria.

Expression of tmRNA in mycobacteria is increased by antimicrobial agents that target the ribosome.

The specialized RNA, tmRNA, is a central component of prokaryote trans-translation; a process that salvages stalled translational complexes. Evidence from other bacteria suggested that exposure to ribosome inhibitors elevated tmRNA levels, although it was unclear whether such changes resulted from increased tmRNA synthesis. Consequently, this study was initiated to determine the effect of ribosome inhibitors on the expression of tmRNA in mycobacteria. Exposure of Mycobacterium smegmatis to ribosome-targeting antimicrobial agents was associated with increased levels of the tmRNA precursor, pre-tmRNA, and mature tmRNA. For example, exposure to 16 µg mL⁻¹ erythromycin for 3 h increased pre-tmRNA and tmRNA by 18- and 6-fold, respectively. Equivalent results were found following exposure of Mycobacterium bovis BCG to streptomycin. Exposure to antimicrobial agents with nonribosome targets did not affect tmRNA levels. The increased tmRNA levels were associated with increased output from the ssrA promoter, which controls tmRNA transcription, without evidence of a change in tmRNA degradation. These results suggest that the upregulation of tmRNA expression was an important response of bacteria to exposure to ribosome-inhibiting antimicrobial agents.

Prospective monitoring for invasive aspergillosis using galactomannan and polymerase chain reaction in high risk pediatric patients.

BACKGROUND: The diagnosis of invasive aspergillus remains a challenge in the care of high-risk patients. Outcomes are improved when invasive aspergillus is diagnosed early, prompting the initiation of appropriate antifungal therapy. We evaluated the utility of prospective monitoring for invasive aspergillosis (IA) using biomarkers such as serum galactomannan (GM) and/or blood polymerase chain reaction (PCR) in high-risk pediatric patients. METHODS: Patients with high-risk leukemia (HRL) or allogenic hematopoietic cell transplant (HCT) recipients were prospectively monitored twice weekly for IA using GM and PCR for Aspergillus species. RESULTS: Sixty-eight patients had collected >or=2 specimens. The 1086 specimens were collected; 627 from HRL (58%) and 459 (42%) from HCT recipients. Median specimens/patient was 11.0 (2 to 58), and median follow-up/patient was 98.5 days (14 to 437). Fifty-six percent of samples were obtained from patients receiving mold-active agents; 32% HRL and 89% HCT. There were no proven, 3 probable, and 20 possible episodes of IA. Thirteen specimens (1.2%) from 4 patients (5%) were GM+. None were positive by PCR. CONCLUSIONS: The prospective use of GM and PCR in this high-risk pediatric population did not identify cases of proven IA. A high false positive rate was not detected. It is speculated that changes in clinical practice, such as early use of empiric and/or prophylactic mold-active agent and frequent imaging studies have impacted the epidemiology of IA. In a population with low incidence of IA, the use of these assays as a screening device on blood may not further enhance current outcomes.

A novel gene, erm(41), confers inducible macrolide resistance to clinical isolates of Mycobacterium abscessus but is absent from Mycobacterium chelonae.

Mycobacterium abscessus infections tend to respond poorly to macrolide-based chemotherapy, even though the organisms appear to be susceptible to clarithromycin. Circumstantial evidence suggested that at least some M. abscessus isolates might be inducibly resistant to macrolides. Thus, the purpose of this study was to investigate the macrolide phenotype of M. abscessus clinical isolates. Inducible resistance to clarithromycin (MIC > 32 microg/ml) was found for 7 of 10 clinical isolates of M. abscessus previously considered susceptible; the remaining 3 isolates were deemed to be susceptible (MIC

Intrinsic macrolide resistance in rapidly growing mycobacteria.

This study reports the discovery of erm genes in seven species of rapidly growing mycobacteria (RGM): Mycobacterium boenickei, M. goodii, M. houstonense, M. mageritense, M. neworleansense, M. porcinum, and M. wolinskyi. This study further substantiates the role of erm genes in intrinsic macrolide resistance in RGM.

Intrinsic macrolide resistance of the Mycobacterium tuberculosis complex is inducible.

Mycobacterium tuberculosis is intrinsically resistant to macrolides, a characteristic associated with expression of the erm(37) gene. This intrinsic resistance was found to be inducible with clarithromycin and the ketolide HMR3004. Furthermore, underlying the phenotypic induction was an increase in erm(37) mRNA levels.

Molecular basis of intrinsic macrolide resistance in clinical isolates of Mycobacterium fortuitum.

OBJECTIVES: Some clinical isolates of Mycobacterium fortuitum are naturally resistant to macrolides, e.g. clarithromycin. Thus, the aim of this study was to identify the gene(s) conferring this resistance. METHODS: M. fortuitum ATCC 6841T DNA libraries were screened for plasmids that complemented the macrolide-susceptible phenotype of Mycobacterium smegmatis variant ermKO4 [erm(38)-negative]. Macrolide-resistant M. smegmatis transformants were selected on agar containing 128 mg/L erythromycin. RESULTS: Genetic complementation identified an M. fortuitum rRNA methylase gene, termed erm(39), 69% identical to erm(38) of M. smegmatis. In addition, erm(39) was found to be in the same chromosomal location as erm(38) in their respective hosts. Like erm(38), erm(39) conferred resistance (MIC >128 mg/L) to macrolide-lincosamide (ML) agents, but not to streptogramin B. Analysis of erm gene expression in M. fortuitum showed that ML agents increased erm(39) RNA levels, reaching a steady state level approximately 20-fold higher than baseline. Screening of 32 M. fortuitum clinical isolates by PCR showed that all were positive for erm(39), irrespective of clarithromycin susceptibility. A majority of clarithromycin-susceptible (MIC < or = 2 mg/L) isolates were postulated to carry a disabled erm(39) gene as they had a GTG-->CTG mutation in the putative initiation codon of the erm(39) gene. CONCLUSIONS: The similarity of the erm genes of M. smegmatis and M. fortuitum suggests that they were inherited from a common ancestor. Although the clinical impact of erm(39) on the therapeutic utility of clarithromycin is unclear, induction of this gene is consistent with the trailing end-points commonly seen during susceptibility testing of M. fortuitum isolates against macrolides.

Differential virulence of Mycobacterium avium strains isolated from HIV-infected patients with disseminated M. avium complex disease.

The role that colonization with Mycobacterium avium plays in the development of disseminated disease is unclear. In this study, we determined whether all M. avium strains isolated from the portals of M. avium infection are capable of crossing the mucosal border and causing infection. The patients in this study were enrolled in AIDS Clinical Trial Group protocol 341. The patients were divided into 3 groups; 2 groups differed in their immunological and clinical risk for M. avium disease. A third group (n=22 patients) had culture-documented disseminated M. avium complex disease at the time of entry in the study. Eight of 22 patients had M. avium isolated from both a colonized site and blood or bone marrow specimens. All 8 patients had distinct M. avium strains; 2 patients had a polyclonal infection. The virulence properties of 13 strains were determined, including invasion of gastrointestinal cells and replication in macrophages. There were significant differences in the virulence properties, and these differences may provide insight into the interplay between microbial pathogenesis and host defense.

Intrinsic macrolide resistance in Mycobacterium smegmatis is conferred by a novel erm gene, erm(38).

High-level, acquired macrolide resistance in mycobacteria is conferred by mutation within the 23S rRNA gene. However, several mycobacteria are naturally resistant to macrolides, including the Mycobacterium smegmatis group and Mycobacterium tuberculosis complex. Thus, the aim of this study was to characterize this resistance. Intrinsic macrolide resistance in M. smegmatis was inducible and showed cross-resistance to lincosamides but not to streptogramin B (i.e., ML resistance). A similar phenotype was found with Mycobacterium microti and macrolide-resistant Mycobacterium fortuitum. A search of the DNA sequence data for M. smegmatis strain mc(2)155 identified a novel erm gene, erm(38), and expression analysis showed that erm(38) RNA levels increased >10-fold after a 2-h incubation with macrolide. Inducible ML resistance was not expressed by an erm(38) knockout mutant, and complementation of this mutant with intact erm(38) in trans resulted in high-level ML resistance (e.g., clarithromycin MIC of >512 micro g/ml). Thus, the results indicate that erm(38) confers the intrinsic ML resistance of M. smegmatis. Southern blot analysis with an erm(38)-specific probe indicated that a similar gene may be present in macrolide-resistant M. fortuitum. This finding, with the presence of the erm(37) gene (Rv1988) in the M. tuberculosis complex, suggests that such genes are widespread in mycobacteria with intrinsic macrolide resistance.