Genotypic Characterization and Antimicrobial Susceptibility Testing of Actinomycetes from Sputum of Tuberculosis Smear Negative Patients from Selected Referral Facilities, Kenya

Abstract

Actinomycetes are organisms belonging to the order Actinomycetale. Most of them are opportunistic pathogens causing infections in immunocompromised patients. In most cases, respiratory infections caused by Actinomycetes display symptoms that mimic tuberculosis. This can lead to misdiagnosis resulting to delayed or inappropriate treatment. Therefore, this study aimed at isolating, characterizing and determining drug susceptibility of Actinomycetes from the sputum of TB smear negative patients from selected referral facilities in Kenya. This employed a cross-sectional study design where a total of three hundred and eight-five (385) sputum samples from TB smear negative patients were collected and screened for the presence of Actinomycetes. Phenotypic identification was done by Gram staining, culture and biochemical test. Genotypic identification was done using rDNA primers to amplify 16S rRNA gene as well as Phylogenetic analysis for confirmation. Antimicrobial susceptibility testing was done by minimum inhibition concentration method (MIC) using 9 antimicrobial agents; amikacin (64 μg/ml), amoxicillin-clavulanate (64/32 μg/ml), ceftriaxone (256 μg/ml), ciprofloxacin (64 μg/ml), clarithromycin (64 μg/ml), gentamycin (64 μg/ml), co-trimoxazole (trimethoprim-sulfamethoxazole) (8/152 μg/ml), doxycycline (64 μg/ml) and linezolid (64 μg/ml). Data was analyzed by statistical package, R Windows using the Kruskal-Wallis test, a P value of less than 0.05 was considered significant. Fifty two (13.5%) of 385, were identified as Actinomycetes using the phenotypic methods. Of the 52 Actinomycetes isolates, 32 (8.3%) were amplified for16S rRNA gene. Nine of the amplified genes were sequenced and compared with those in National Center for Biotechnology Information (NCBI). Phylogenetic analysis confirmed the 9 sequenced genes were members of Actinomycetes. Eight of them were members of the genus Streptomyces while only one was a Nocardiopsis species. Antimicrobial susceptibility testing was done on the 52 isolates. The isolates showed highest susceptibility to Gentamycin and Ciprofloxacin at 100% and 98% respectively. Highest resistance was observed in Ceftriazone and Clarithromycin at 11.5% and 26.9% respectively. Multidrug resistance was observed in four isolates. Staphylococcus aureus ATCC 25923 was used for quality control. There is Actinomycetes infections in TB smear negative patients hence it is important to investigate suspected pulmonary pathologies for potential Actinomycetes infection for proper diagnosis and interventions.