Aminoglycoside-modifying enzymes determine the innate susceptibility to aminoglycoside antibiotics in rapidly growing mycobacteria.

OBJECTIVES: Infections caused by the rapidly growing mycobacterium (RGM) Mycobacterium abscessus are notoriously difficult to treat due to the innate resistance of M. abscessus to most clinically available antimicrobials. Aminoglycoside antibiotics (AGA) are a cornerstone of antimicrobial chemotherapy against M. abscessus infections, although little is known about intrinsic drug resistance mechanisms. We investigated the role of chromosomally encoded putative aminoglycoside-modifying enzymes (AME) in AGA susceptibility in M. abscessus. METHODS: Clinical isolates of M. abscessus were tested for susceptibility to a series of AGA with different substituents at positions 2', 3' and 4' of ring 1 in MIC assays. Cell-free extracts of M. abscessus type strain ATCC 19977 and Mycobacterium smegmatis strains SZ380 [aac(2')-Id(+)], EP10 [aac(2')-Id(-)] and SZ461 [aac(2')-Id(+), rrs A1408G] were investigated for AGA acetylation activity using thin-layer chromatography (TLC). Cell-free ribosome translation assays were performed to directly study drug-target interaction. RESULTS: Cell-free translation assays demonstrated that ribosomes of M. abscessus and M. smegmatis show comparable susceptibility to all tested AGA. MIC assays for M. abscessus and M. smegmatis, however, consistently showed the lowest MIC values for 2'-hydroxy-AGA as compared with 2'-amino-AGA, indicating that an aminoglycoside-2'-acetyltransferase, Aac(2'), contributes to innate AGA susceptibility. TLC experiments confirmed enzymatic activity consistent with Aac(2'). Using M. smegmatis as a model for RGM, acetyltransferase activity was shown to be up-regulated in response to AGA-induced inhibition of protein synthesis. CONCLUSIONS: Our findings point to AME as important determinants of AGA susceptibility in M. abscessus.

Lack of antimicrobial bactericidal activity in Mycobacterium abscessus.

Antibiotic therapy of infections caused by the emerging pathogen Mycobacterium abscessus is challenging due to the organism's natural resistance toward most clinically available antimicrobials. We investigated the bactericidal activity of antibiotics commonly administered in M. abscessus infections in order to better understand the poor therapeutic outcome. Time-kill curves were generated for clinical M. abscessus isolates, Mycobacterium smegmatis, and Escherichia coli by using antibiotics commonly categorized as bactericidal (amikacin and moxifloxacin) or bacteriostatic (tigecycline and linezolid). In addition, the impact of aminoglycoside-modifying enzymes on the mode of action of substrate and nonsubstrate aminoglycosides was studied by using M. smegmatis as a model organism. While amikacin and moxifloxacin were bactericidal against E. coli, none of the tested compounds showed bactericidal activity against M. abscessus. Further mechanistic investigations of the mode of action of aminoglycosides in M. smegmatis revealed that the bactericidal activity of tobramycin and gentamicin was restored by disruption of the chromosomal aac(2') gene in the mycobacterial genome. The lack of bactericidal antibiotics in currently recommended treatment regimens provides a reasonable explanation for the poor therapeutic outcome in M. abscessus infection. Our findings suggest that chromosomally encoded drug-modifying enzymes play an important role in the lack of aminoglycoside bactericidal activity against rapidly growing mycobacteria.

Tintelnotia destructans Keratitis: A Clinicopathological Report and Review of the Literature.

PURPOSE: To present the first clinicopathological report of Tintelnotia destructans keratitis, a filamentous fungus and as of yet uncommon virulent ocular pathogen. METHODS: A 70-year-old man presented with an infectious keratitis featuring a stromal infiltrate with feathery borders and a viscous hypopyon. Despite initial improvement under a combined therapy with natamycin and voriconazole, a perforation in the further course required a penetrating keratoplasty. Cultures and the corneal lenticule were available for microscopic examination and antifungal susceptibility testing. The limited literature on the subject was reviewed. RESULTS: Microscopic examination of cultures revealed hyphae and conidia being produced in globose fruiting bodies, a common characteristic of Tintelnotia sp. Histopathology showed short-branched hyphae that grew across the cornea regardless of the orientation of the collagen lamellae. Molecular methods identified the species T. destructans. The pattern of antifungal susceptibility included amphotericin B, ciclopirox, natamycin, posaconazole, voriconazole, and terbinafine. The postoperative clinical course was without complications. CONCLUSIONS: Although the clinical signs corresponded to the classic features of fungal keratitis, microscopic analysis revealed morphological characteristics of a fungal class that has shown little ophthalmological appearance so far. Data on T. destructans keratitis are highly limited in the literature, but all identified species shared sensitivity to terbinafine.

Detection of NDM-19, a novel variant of the New Delhi metallo-ß-lactamase with increased carbapenemase activity under zinc-limited conditions, in Switzerland.

A novel New Delhi metallo-ß-lactamase (NDM) variant, NDM-19, was identified in a carbapenem-resistant E. coli strain isolated from a subcutaneous infection of a laparotomy scar from an Egyptian patient in a Swiss hospital. NDM-19 is a derivative of NDM-7, from which it differs by a single amino acid substitution (Ala233Val). Under zinc-limiting growth conditions, E. coli DH5α transformants producing NDM-19 displayed reduced susceptibility towards expanded-spectrum cephalosporins and carbapenems as compared to transformants producing NDM-1 or NDM-7.