IDENTIFICATION OF MYCOBACTERIUM GENAVENSE IN A DIANA MONKEY (CERCOPITHECUS DIANA) BY POLYMERASE CHAIN REACTION AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY.

A 25-yr-old Diana monkey (Cercopithecus diana) with a 1.5-yr history of chronic colitis and diarrhea was found to have disseminated granulomatous disease with intralesional acid fast bacilli. Bacilli were identified as Mycobacterium genavense by polymerase chain reaction, sequencing of the 16S-23S ribosomal RNA intergenic spacer (ITS) gene, and mycolic acid analysis by high-performance liquid chromatography. Mycobacterium genavense is a common cause of mycobacteriosis in free-ranging and captive birds. In addition, recognition of opportunistic infection in human immunodeficiency virus-positive patients is increasing. Disease manifestations of M. genavense are similar to Mycobacterium avium complex (MAC) and include fever, wasting, and diarrhea with disseminated disease. Similar clinical signs and lesions were observed in this monkey. Mycobacterium genavense should be considered as a differential for disseminated mycobacterial disease in nonhuman primates as this agent can mimic MAC and related mycobacteria.

Treatment of experimental bacterial keratitis with topical trovafloxacin.

OBJECTIVE: To investigate the therapeutic role of trovafloxacin mesylate, a newer-generation fluoroquinolone with an expanded spectrum of activity, in the treatment of experimental bacterial keratitis. METHODS: Susceptibility studies were performed on various strains of ocular isolates to determine the minimum inhibitory concentration (MIC) of trovafloxacin compared with ciprofloxacin and ofloxacin, using the E-test method. Pharmacokinetic studies were performed by a single topical administration of trovafloxacin to rabbit eyes with either an intact or denuded corneal epithelium. Aqueous humor, vitreous, and corneal concentrations of trovafloxacin were determined at different time points. Experimental bacterial keratitis studies were performed in rabbit eyes. Three identical studies were conducted using Staphylococcus aureus, Streptococcus pneumoniae, or Pseudomonas aeruginosa. Therapy groups included 0.5% trovafloxacin, 0.3% ciprofloxacin, 0.3% ofloxacin, and isotonic sodium chloride solution. After 12 hours of drops administration, corneas were excised, homogenized, and serially plated. The main outcome measure was quantitative bacteriologic analysis for residual colony-forming units. RESULTS: In vitro susceptibility study findings indicated that the MIC of trovafloxacin was significantly lower than the MIC of ciprofloxacin and ofloxacin for S. aureus, S. pneumoniae, and Haemophilus influenzae, lower than the MIC of ciprofloxacin and ofloxacin for Staphylococcus epidermidis, and intermediate between ciprofloxacin and ofloxacin for P. aeruginosa. Pharmacokinetic studies showed a significant concentration of trovafloxacin in the treated corneas, especially in eyes with a denuded epithelium. All serum samples had undetectable trovafloxacin concentrations. Experimental keratitis studies showed a statistically significant decrease of colony-forming units in trovafloxacin-treated eyes in the S. aureus model and a similar decrease in the S pneumoniae and P aeruginosa models. CONCLUSIONS: Topical 0.5% trovafloxacin proved to be an effective ocular medication for the therapy of gram-positive and gram-negative keratitis. Clinical Relevance Trovafloxacin may provide an excellent therapeutic alternative in bacterial keratitis.

Use of the "RAM" susceptibility testing method for rapid detection of clarithromycin resistance in the Mycobacterium avium complex.

Standard susceptibility testing of the Mycobacterium avium complex (MAC) can require 7 to 14 days from initial isolation. We evaluated a high-performance liquid chromatography-based susceptibility test for rapid determination of clarithromycin (CLR) resistance in MAC. This method can be completed in 72 h of incubation. A total of 110 MAC strains were tested using the following concentrations of CLR: 4, 16, and 64 microg/mL, for a total of 330 tests. Microbroth dilution was used as the reference method. Rapid analysis of mycolic acid ("RAM") concordance with the reference method for CLR susceptibility was 98% (254/258) and 100% for CLR resistance (72/72). The 4 discordant results occurred with 2 strains, which demonstrated intermediate resistance with an MIC of 16 microg/mL. This study demonstrates that "RAM"-based susceptibility testing for determination of CLR resistance in MAC is both rapid and accurate, providing a significant reduction in turn-around-time from 7 to 14 days to 72 h of incubation.

Rapid, standardized method for determination of Mycobacterium tuberculosis drug susceptibility by use of mycolic acid analysis.

Multidrug-resistant (MDR) Mycobacterium tuberculosis and extrensively drug-resistant (XDR) M. tuberculosis are emerging public health threats whose threats are compounded by the fact that current techniques for testing the susceptibility of M. tuberculosis require several days to weeks to complete. We investigated the use of high-performance liquid chromatography (HPLC)-based quantitation of mycolic acids as a means of rapidly determining drug resistance and susceptibility in M. tuberculosis. Standard susceptibility testing and determination of the MICs of drug-susceptible (n = 26) and drug-resistant M. tuberculosis strains, including MDR M. tuberculosis strains (n = 34), were performed by using the Bactec radiometric growth system as the reference method. The HPLC-based susceptibilities of the current first-line drugs, isoniazid (INH), rifampin (RIF), ethambutol (EMB), and pyrazinamide (PZA), were determined. The vials were incubated for 72 h, and aliquots were removed for HPLC analysis by using the Sherlock mycobacterial identification system. HPLC quantitation of total mycolic acid peaks (TMAPs) was performed with treated and untreated cultures. At 72 h, the levels of agreement of the HPLC method with the reference method were 99.5% for INH, EMB, and PZA and 98.7% for RIF. The inter- and intra-assay reproducibilities varied by drug, with an average precision of 13.4%. In summary, quantitation of TMAPs is a rapid, sensitive, and accurate method for antibiotic susceptibility testing of all first-line drugs currently used against M. tuberculosis and offers the potential of providing susceptibility testing results within hours, rather than days or weeks, for clinical M. tuberculosis isolates.

Mycobacterium pseudoshottsii sp. nov., a slowly growing chromogenic species isolated from Chesapeake Bay striped bass (Morone saxatilis).

A group of slowly growing photochromogenic mycobacteria was isolated from Chesapeake Bay striped bass (Morone saxatilis) during an epizootic of mycobacteriosis. Growth characteristics, acid-fastness and 16S rRNA gene sequencing results were consistent with those of the genus Mycobacterium. Biochemical reactions, growth characteristics and mycolic acid profiles (HPLC) resembled those of Mycobacterium shottsii, a non-pigmented mycobacterium also isolated during the same epizootic. Sequencing of the 16S rRNA genes, the gene encoding the exported repeated protein (erp) and the gene encoding the 65 kDa heat-shock protein (hsp65) and restriction enzyme analysis of the hsp65 gene demonstrated that this group of isolates is unique. Insertion sequences associated with Mycobacterium ulcerans, IS2404 and IS2606, were detected by PCR. These isolates could be differentiated from other slowly growing pigmented mycobacteria by their inability to grow at 37 degrees C, production of niacin and urease, absence of nitrate reductase, negative Tween 80 hydrolysis and resistance to isoniazid (1 mug ml(-1)), p-nitrobenzoic acid, thiacetazone and thiophene-2-carboxylic hydrazide. On the basis of this polyphasic study, it is proposed that these isolates represent a novel species, Mycobacterium pseudoshottsii sp. nov. The type strain, L15(T), has been deposited in the American Type Culture Collection as ATCC BAA-883(T) and the National Collection of Type Cultures (UK) as NCTC 13318(T).

Characterization of Mycobacterium montefiorense sp. nov., a novel pathogenic Mycobacterium from moray eels that is related to Mycobacterium triplex.

The characterization of a novel Mycobacterium sp. isolated from granulomatous skin lesions of moray eels is reported. Analysis of the hsp65 gene, small-subunit rRNA gene, rRNA spacer region, and phenotypic characteristics demonstrate that this organism is distinct from its closest genetic match, Mycobacterium triplex, and it has been named M. montefiorense sp. nov.