Kevin A. Nash

A Novel Gene, erm(41), Confers Inducible Macrolide Resistance to Clinical Isolates of Mycobacterium abscessus but Is Absent from Mycobacterium chelonae

ABSTRACT 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 μg/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 ≤ 0.5 μg/ml). Inducible resistance was conferred by a novel erm gene, erm(41), which was present in all 10 isolates and in an isolate of Mycobacterium bolletii (M. abscessus type II). However, the erm(41) alleles were nonfunctional in the three susceptible M. abscessus isolates. No evidence of erm(41) was found in Mycobacterium chelonae, and an isolate of Mycobacterium massiliense appeared to be an erm(41) deletion mutant. Expression of erm(41) in M. abscessus conferred resistance to clarithromycin and erythromycin and the ketolide HMR3004. However, this species was found to be intrinsically resistant, independent of erm(41), to clindamycin, quinupristin (streptogramin B), and telithromycin. The ability to confer resistance to clindamycin and telithromycin, but not quinupristin, was demonstrated by expressing erm(41) in Maycobacterium smegmatis. Exposure of M. abscessus to the macrolide-lincosamide-streptogramin B-ketolide agents increased the levels of erm(41) mRNA 23- to 250-fold within 24 h. The inducible macrolide resistance phenotype of some M. abscessus isolates may explain the lack of efficacy of macrolide-based chemotherapy against this organism.

Antimicrobial Susceptibility Testing, Drug Resistance Mechanisms, and Therapy of Infections with Nontuberculous Mycobacteria

SUMMARY 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.

Intrinsic Macrolide Resistance in Rapidly Growing Mycobacteria

ABSTRACT 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 in Mycobacterium smegmatis Is Conferred by a Novel erm Gene, erm(38)

ABSTRACT 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 mc2155 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 μg/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.

Detection of Rifampin Resistance in Mycobacterium tuberculosis by Use of a Rapid, Simple, and Specific RNA/RNA Mismatch Assay

An adaption of an RNA/RNA duplex, base pair-mismatch assay is capable of detecting rifampin resistance in Mycobacterium tuberculosis. The specificity and sensitivity of the mismatch assay in detecting rifampin resistance were 100% and 96%, respectively, when tested against 46 rifampin-resistant and rifampin-susceptible strains of M. tuberculosis. By use of a range of mycobacterial and nonmycobacterial prokaryote pathogens, the mismatch assay was shown to be specific for M. tuberculosis and Mycobacterium bovis. The assay is cost-effective compared with DNA sequencing and other molecular methods and is simple to perform and interpret. Furthermore, the assay can return a result within 24 h after receipt of an isolated organism and potentially can be used directly with smear-positive specimens.

Utility of Sequencing the erm(41) Gene in Isolates of Mycobacterium abscessus subsp. abscessus with Low and Intermediate Clarithromycin MICs

ABSTRACT The erm(41) gene confers inducible macrolide resistance in Mycobacterium abscessus subsp. abscessus, calling into question the usefulness of macrolides for treating M. abscessus subsp. abscessus infections. With an extended incubation (14 days), isolates with MICs of ≥8 μg/ml are considered macrolide resistant by current CLSI guidelines. Our goals were to determine the incidence of macrolide susceptibility in U.S. isolates, the validity of currently accepted MIC breakpoints, and the erm(41) sequences associated with susceptibility. Of 349 isolates (excluding those with 23S rRNA gene mutations), 85 (24%) had clarithromycin MICs of ≤8 μg/ml. Sequencing of the erm(41) genes from these isolates, as well as from isolates with MICs of ≥16 μg/ml, including ATCC 19977T, revealed 10 sequevars. The sequence in ATCC 19977T was designated sequevar (type) 1; most macrolide-resistant isolates were of this type. Seven sequevars contained isolates with MICs of >16 μg/ml. The T28C substitution in erm(41), previously associated with macrolide susceptibility, was identified in 62 isolates (18%) comprising three sequevars, with MICs of ≤2 (80%), 4 (10%), and 8 (10%) μg/ml. No other nucleotide substitution was associated with macrolide susceptibility. We recommend that clarithromycin susceptibility breakpoints for M. abscessus subsp. abscessus be changed from ≤2 to ≤4 μg/ml and that isolates with an MIC of 8 μg/ml have repeat MIC testing or erm sequencing performed. Our studies suggest that macrolides are useful for treating approximately 20% of U.S. isolates of M. abscessus subsp. abscessus. Sequencing of the erm gene of M. abscessus subsp. abscessus will predict inducible macrolide susceptibility.

Inducible Macrolide Resistance in Corynebacterium jeikeium

ABSTRACT Corynebacterium jeikeium is an opportunistic pathogen primarily of immunocompromised (neutropenic) patients. Broad-spectrum resistance to antimicrobial agents is a common feature of C. jeikeium clinical isolates. We studied the profiles of susceptibility of 20 clinical strains of C. jeikeium to a range of antimicrobial agents. The strains were separated into two groups depending on the susceptibility to erythromycin (ERY), with one group (17 strains) representing resistant organisms (MIC > 128 μg/ml) and the second group (3 strains) representing susceptible organisms (MIC ≤ 0.25 μg/ml). The ERY resistance crossed to other members of the macrolide-lincosamide-streptogramin B (MLSb) group. Furthermore, this resistance was inducible with MLSb agents but not non-MLSb agents. Expression of ERY resistance was linked to the presence of an allele of the class X erm genes,erm(X)cj, with >93% identity to other ermgenes of this class. Our evidence indicates that erm(X)cj is integrated within the chromosome, which contrasts with previous reports for the plasmid-associated erm(X) genes found inC. diphtheriae and C. xerosis. In 40% ofC. jeikeium strains, erm(X)cj is present within the transposon, Tn5432. However, in the remaining strains, the components of Tn5432 (i.e., the erm and transposase genes) have separated within the chromosome. The rearrangement of Tn5432 leads to the possibility that the other drug resistance genes have become included in a new composite transposon bound by the IS1249 elements.

Intrinsic Macrolide Resistance of the Mycobacterium tuberculosis Complex Is Inducible

ABSTRACT 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.

Rapid detection of mutations associated with macrolide resistance in Mycobacterium avium complex.

Macrolide resistance in Mycobacterium avium can be detected with an adaption of a commercially available RNA/RNA duplex mismatch assay (Ambion, Austin, Tex.). The sensitivity and specificity values for the assay were 100% when evaluated against 41 macrolide-resistant and -susceptible strains of M. avium. Resistant subpopulations of approximately 20% could be readily detected. The assay is simple to perform and interpret, inexpensive, and rapid (< 24-h turnaround).

CAF-Secreted Annexin A1 Induces Prostate Cancer Cells to Gain Stem Cell-like Features

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. Mol Cancer Res; 12(4); 607–21. ©2014 AACR.