Isolation, Examination and Characterization of Actinomycetes as a Source of Antimicrobial Agents from Libyan Soil

The rapid increase of bacterial resistance strains to multiple antibiotics has become a global public health concern. In the present study, actinomycetes from different districts of Libyan soil were isolated and screened for their inhibitory activity against pathogenic bacteria and fungi. Three hundred soil samples were taken from 77 diverse ecosystems, including deserts, forests, pastureland, and cropland located in different climatic regions in Libya. A total of 164 actinomycetes were obtained. Of 164 isolates, 38 (23.2%) isolates were morphologically and microscopically characterized by spore chain and surface morphology, aerial and substrate mycelia and soluble pigments. The preliminary classification of the isolates illustrates that all isolates belong to the genus Streptomyces. These isolates were further examined for their antagonistic potential against nine pathogenic bacteria and fungi. Out of 38, 11 (28.9%) isolates showed their capability to produce inhibitory substances against at least two tested strains. Among bacterial strains, Staphylococcus aureus was susceptible to almost all eleven isolates (90.9%), while Streptococcus pyogenes was found to be resistant to most selected isolates (18.2%). The isolate, 063 (Wadan soil-desert zone) was the only isolate that exhibited broad spectrum antimicrobial activity against all tested pathogenic microbes, and hence was selected for further study. According to its cultural, morphological, physiological, and biochemical characteristics, the isolate 063 was identified as Streptomyces rochei. The results obtained indicate that the Libyan soil, particularly in extreme environments, could be a potent source of bioactive metabolites with antimicrobial potency against a wide variety of microbial pathogens. Thus, this investigation suggested that exploring new habitats in unexplored and untapped regions of Libya could provide a promising source of biologically active compounds for therapeutic applications.

Diversity and bioactivity of actinomycetes from Signy Is-land terrestrial soils,maritime Antarctic

The Antarctic represents a largely untapped source for isolation of new microorganisms with potential to produce bio- active natural products. Actinomycetes are of special interest among such microorganisms as they are known to produce a large number of natural products, many of which have clinical, pharmaceutical or agricultural applications. We isolated, characterized and classified actinomycetes from soil samples collected from different locations on Signy Island, South Orkney Islands, in the maritime Antarctic. A total of 95 putative actinomyeete strains were isolated from eight soil samples using eight types of selective isolation media. The strains were dereplicated into 16 groups based on morphology and Amplified Ribosomal DNA Restriction Analysis （ARDRA） patterns. Analysis of 16S rRNA gene sequences of representatives from each group showed that streptomy- cetes were the dominant actinomycetes isolated from these soils; however, there were also several strains belonging to diverse and rare genera in the class Actinobacteria, including Demetria, Glaciibacter, Kocuria, Marmoricola, Nakamurella and Tsukamurella. In addition, screening for antibacterial activity and non-ribosomal peptide synthetase genes showed that many of the actinomycete strains have the potential to produce antibacterial compounds.

Isolation and characterization of potential antibiotic producing actinomycetes from water and sediments of Lake Tana,Ethiopia

Objective:To isolate,evaluate and characterize potential antibiotic producing actinomycetes from water and sediments of Lake Tana,Ethiopia.Methods:A total of 31 strains of actinomycetes were isolated and tested against Gram positive and Gram negative bacterial strains by primary screening.In the primary screening.11 promising isolates were identified and subjected to solid state and submerged state fermentation methods to produce crude extracts.The fermented biomass was extracted by organic solvent extraction method and tested against bacterial strains by disc and agar well diffusion methods.The isolates were characterized by using morphological,physiological and biochemical methods.Results:The result obtained from agar well diffusion method was belter than disc diffusion method.The crude extract showed higher inhibition zone against Gram positive bacteria than Cram negative bacteria.One-way analysis of variance confirmed most of the crude extracts were statistically significant at 95%confidence interval.The minimum inhibitor)concentration and minimum bactericidal concentration of crude extracts were 1.65 mg/mL and 3.30 mg/mL against Staphylococcus aureus,and 1.84 mg/mL and 3.80 mg/mL against Escherichia coli respectively.The growth of aerial and substrate mycelium varied in different culture media used.Most of the isolates were able to hydrolysis starch and urea:able to survive at 5%concentration of sodium chloride:optimum temperature for their growth was 30°C.Conclusions:The results of the present study revealed that freshwater actinomycetes of Luke Tana appear to have immense potential as a source ol antibacterial compounds.

Isolation and phylogenetic assignation of actinomycetes in the marine sediments from the Arctic Ocean

Actinomycetes in five marine sediments collected from the Arctic Ocean atdepths of 43 to 3 050 m were cultivated using a variety of media. A total of 61 actinomycetecolonies with substrate mycelia only were observed, and no colonies with aerial mycelia wereobserved under aerobic conditions at 15℃. From these colonies, 28 were selected to representdifferent morphological types. Denaturing gradient gel electrophoresis (DGGE) was used to check thepurity of isolates and select representatives for subsequent sequencing. Phylogentic analyses basedon nearly full-length 16S ribosomal RNA gene (rDNA) sequences indicated that the actinomycetesisolated were accommodated within genus Rhodococcus of family Nocardiaceae, genus Dietzia of familyDietziaceae, genera Janibacter and Terrabacter of family Instrasporangiaceae and genera Kocuria andA nhrobacter of family Micrococcaceae. One of the strains (P27-24) from the deep-sea sediment atdepth of 3 050 m was found to be identical in 16S rDNA sequence(1474/1474) with theradiation-resistant Kocuria rosea ATCC 187~T isolated from air. More than half of the isolatesshowed the similarities ranging from 99.5% to 99.9% in 16S rDNA sequence to dibenzofran-degrading,butyl 2-ethylhexanoate-hydrolysising and nitrile-metabolizing actinomycetes. All the strainsisolated were psychrotolerant bacteria and grew better on the media prepared with natural seawaterthan on the media prepared with deionized water. Three of them (Dietzia sp. P27-10, Rhodococcus sp.S11-3 and Rhodococcus sp.P11-5) had an obligate growth requirement for salt, confirming that thesestrains are indigenous marine actinomycetes.

Clear felling and burning effects on soil nitrogen transforming bacteria and actinomycetes population in Chittagong University campus, Bangladesh

The effect of forests clear felling and associated burning on the population of soil nitrogen transforming bacteria and actinomycetes are reported at three pair sites of Chittagong University campus, Bangla- desh in monsoon tropical climate. Clear felled area or burnt site and 15-21 year mixed plantation of native and exotic species, situated side by side on low hill having Typic Dystrochrepts soil was represented at each pair site. At all the three pair sites, clear felled area or burnt site showed very significantly （p~_0.001） lower population of actinomycetes, Rhizobium, Nitrosomonas, Nitrobacter and ammonifying as well as deni- trifying bacteria compared to their adjacent mixed plantation. From en- vironmental consideration, this finding has implication in managing natural ecosystem.

Isolation and characterization of marine-derived actinomycetes with cytotoxic activity from the Red Sea coast

Objective: To isolate and evaluate the cytotoxic activity of different actinomycetes species isolated from the Red Sea coast in Sharm el-Sheikh, Egypt.Methods: Forty actinomycetes strains were isolated from different sediments and seawater samples collected from the Red Sea coast in Egypt. Actinomycetes were recognized by morphological and microscopic examinations. Cell viability and cytotoxicity induced by the crude extracts on breast cancer cell lines MDA-MB-231 were assessed using methylene blue assay. The strains with promising cytotoxic activity were identified by sequencing and amplifying the 16 S r RNA genes. The antibacterial activities of the crude extracts were performed using Kirby–Bauer disc diffusion method.Results: The results indicated that five ethyl acetate extracts exhibited cytotoxicity towards breast cancer cell lines MDA-MB-231. The highest cytotoxic activity was found for the ethyl acetate extracts of EGY2 and EGY39. The isolate EGY3 was identified as a new Streptomyces species, while the actinomycete EGY22 was found to be a member of the genus Nocardiopsis sp. The crude extract of the isolate EGY8 showed slightly high antimicrobial activity against different test microorganisms.Conclusions: The results of the present study reveal that marine sediments of the Red Sea are a potent source of novel species of actinomycetes. The isolates may be useful in discovery of novel bioactive compounds and an important step in the development of microbial natural product research.

Characterization of cytotoxic compound from marine sediment derived actinomycete Streptomyces avidinii strain SU4

Objective:To investigate the cytotoxic activity of actinomycete isolated from marine sediment.Methods:In the present study the DNA was isolated and the ITS region of 16s rRNA was amplified by polymerase chain reaction,using two universal bacterial primers,1492K(5'-GGTTACCTTG'TTAC GACTT-3')and Eubac27F(5'-AGAGTTTGATCCTGGCTC AG-3').The amplified products were purified using TIANgel mini purification kit,ligated to MD18-T simple vector(TaKaRa),and transformed into competent cells of Escherichia coli DH5α.16S rRNA gene fragment was sequenced using forward primer M13F(-47)and reverse primer M13R(-48).Blast search sequence similarity was found against the existing non-redundanl nucleotide sequence database thus,identified as Streptomyces sp SU,Streptomyces rubralavandulae strain SU1,Streptomyces cacaoi strain SU2,Streptomyces cavourensis strain SU3,Streptomyces avidinii strain SU4,Streptomyces globisporus strain SU5,Streptomyces variabilis strain SU6,Streptomyces coelicolor strain SU 7.Among the eight identified isolates,one actinomycete Streptomyces avidinii strain SU4 was selected for further study.Results:Crude extract of the actinomycete isolate exhibited IC_(50)in 64.5μg against Hep-2 cell line,250μg in VERO cell line.This value is very close to the criteria of cytotoxicity activity for the crude extracts,as established by the American National Cancer Institute(NCI)is in IC_(50)<30μg/mL.The CC MS analysis showed that the active principle might be 1,2-benzenedicarboxylic acid,bis(2-methylpropyl)ester(12.17%),isooctyl phthalate(15.29%)with the retention time 15.642 and 21.612,respectively.Conclusions:This study clearly proves that the marine sediment derived actinomycetes with bioactive metabolites can be expected to provide high quality biological material for high throughout biochemical and anticancer screening programs.These results help us to conclude that the potential of using metabolic engineering and post genomic approaches to isolate more bioactive compounds and make their possible commercial application is not far off.

A New Degraded Sesquiterpene from Marine Actinomycete Streptomyces sp. 0616208

A new degraded sesquiterpene was isolated from the marine actinomycete Streptomyces sp. 0616208. Its structure was elucidated as （1α; 4aα; 5α, 7β, 8aβ）-5, 8a-dimethyl-decahydrona- phthalene-1, 4a, 7-triol on the basis of spectroscopic data.

Distribution of actinomycetes in oil contaminated ultisols of the Niger Delta(Nigeria)

The distribution of actinomycetes in oil contaminated sandy loam ultisols of the Niger Delta region of Nigeria was studied to aid in understanding the effect of hydrocarbons on indigenous microbial population in tropical soils. The average total counts of actinomycetes in all the oil samples analysed was 10 3 cfu/g. Higher counts of actinomycetes were observed during the dry season than during the wet season. The counts of hydrocarbonoclastic actinomycetes correlated positively with the total count of actinomycetes. The actinomycetes were generally restricted to the top soil(0—10 cm soil depth) although a seemingly deeper(down to 40 cm soil depth) distribution was noticed in the dry season. The isolates included oil degrading species of Actinoplanes, Norcadia, Streptomyces and Streptosporangium. Their high oil utilization ability indicates their positive potential and role in the bioremediation of oil spilled soils.

Bioprospection of marine actinomycetes: recent advances,challenges and future perspectives

In exploring new sources for economically important products, marine environment draws particular attention because of its remarkable diversity and extreme conditions;it is known to produce metabolic products of great value. It represents untapped source for the discovery of novel secondary metabolites with varying potential such as antibiotic, anti-tumor, antifouling and cytotoxic properties. Marine actinomycetes distributed throughout the marine environment from shallow to deep sea sediments have proved to be a finest source for this discovery. Secondary metabolites derived from marine actinomycetes have proved their worth in industries based on the research on their properties and wide range applications. Spotlight of the review is range of marine based actinomycetes products and significant research in this field. This shows the capability of marine actinomycetes as bioactive metabolite producers. Additionally, the present review addresses some effective and novel approaches of procuring marine microbial compounds utilizing the latest screening strategies of drug discovery from which traditional resources such as marine actinobacteria has decreased due to declining yields. The aim is in the context of promoting fruitful and profitable results in the near future. The recent surfacing of new technologies for bioprospection of marine actinomycetes are very promising, resulting in high quality value added products, and will be de?ning a new era for bioactive compounds with medical and biotechnological applications.

Identification and preliminary characterization of non-polyene antibiotics secreted by new strain of actinomycete isolated from sebkha of Kenadsa, Algeria

Objective: To study the antimicrobial activity of actinomycetes isolated from sabkha of Kenadsa and identification of the isolate interesting. Methods: Eighteen strains were isolated, using four culture media from sebkha of Kenadsa(Bechar, Southwestern Algeria). Screening of antimicrobial activity consisted of two steps: in primary screening, antibacterial activity was determined by using the agar plug method against test strains; in secondary screening, better isolate which showed a good activity in the first screening was selected to extract antimicrobial substances. The antimicrobial activities of extracts were evaluated by using Kirby-Bauer disc diffusion method. Partial characterization of antimicrobial products was performed on the basis of chemical revelations, UV-vis spectrometry and infrared spectroscopy. The identification of isolate interesting was performed through morphological, chemical, biochemical and physiological characteristics. Results: All isolates showed antimicrobial activity against at least one microorganism test. One isolate, LAM143 cG 3, was selected for its broad spectrum and high antimicrobial activity. The isolate LAM143 c G3 was identified as Spirillospora sp. The comparison between the species of this genus(Spirillospora rubra and Spirillospora albida) and our isolate indicated the existence of several physiological and biochemical differences which led us to suppose that this was a new member of this genus. Primary characterization of antimicrobial substances produced by the isolate LAM143 c G3 indicated the presence of amines and phenols. The UV–vis spectrum suggested a non-polyenic nature of substances secreted by our isolate, while infrared confirmed the presence of amine groups.Conclusions: The result of the present study revealed that sebkha of Kenadsa was rich in rare actinomycetes, that secreted interesting antimicrobial substance.

Hyoscyamine-producing marine Actinomycetes from Lagos Lagoon sediment

Objective:To isolate and screen Actinoniycetes from Lagos Lagoon soil sediments for production of bioactive metabolites.Methods:Sediment samples were collected from four different locations of Lagos Lagoon and were dried for 2 weeks after which the Actinoniycetes were isolated by serial dilution using the spread plate method on starch casein and Kuster's agar supplemented with 80 ug/mL cycloheximide to prevent fungal growth.The plates were incubated at 28 C for 1-2 weeks.Isolates were selected based on their colonial characteristics as well as their Gram's reaction and subciiltured using the same media for isolation until pure cultures were obtained and incubated at 28 C for 3 d.Thereafter,they were inoculated into starch casein and Kuster's broth media and incubated for 8 d.The secondary metabolites were screened for antimicrobial activity against the following microorganisms:methicillin resistant Staphylococcus aureus.Staphylococcus aureus ATCC 29213.Escherichia coli ATCC 29522.Pseudomonas aeruginosa ATCC 27853.Candida albicans and Enterocolitis faecal is ATCC 29212.Coagulasenegative staphylococci isolated from HIV patients were also used(Staphylococcus warneri.Staphylococcus xylosus and Staphylococcus epidennidis).The antimicrobial metabolites of the Actinoniycetes isolates were identified using gas chromatography(GC).Results:Crude extracts of isolates showed antimicrobial activity against some of the test organisms.The GC data analysis showed the antibiotic profile of these isolates.Conclusions:Analysis of the crude extracts of the isolates using GC method,revealed the presence of antibiotics including an anticholinergic hyoscyamine among other conclusions.

Xylanilyticolides A-C,Three New Compounds from Cultures of the Actinomycete Promicromonospora xylanilytica YIM 61515

Three new lactones,xylanilyticolides A-C(1-3),were isolated from cultures of the actinomycete Promicromonospora xylanilytica YIM 61515.Their structures were elucidated by 1D and 2D NMR spectroscopic data in conjunction with HRESIMS analysis.Compound 1 exhibited potent cytotoxicities against five human cancer cell lines HL-60,A-549,SMMC-7721,MCF-7 and SW480 with the IC_(50) values of 3.9,15.2,11.2,5.9,and 4.7 pM,respectively.

Caerulomycin A—An Antifungal Compound Isolated from Marine Actinomycetes

Actinomycetes have been prolific sources of novel secondary metabolites with a range of biological activities that may ultimately find application as therapeutic compounds. Hence several drug discovery companies are engaged in isolation of novel bioactive metabolites from these microbial sources. Antibiotics form the major class of such bioactive metabolites and have been widely used for treating infectious diseases. One of the most critical problems in clinical practice is the increase of prevalence of drug resistant strains, especially azole resistance among fungi. Due to this, there is a constant need for development of new antifungal antibiotics having novel scaffolds and/or mechanism of action. In our in-house screening program in the quest of novel and superior antifungal compounds, an actinomycetes strain PM0525875 was isolated from a marine invertebrate. The extracts of this microbe showed potent in-vitro antifungal activity against drug resistant fungal strains. The antifungal active peak from the extract obtained by shake flask fermentation was identified by chromatographic and other analytical techniques during bioactivity guided isolation. Later the fermentation conditions were optimized in 30 L fermentor for the production of sufficient amount antifungal compound for complete structural characterization. Consequently the fermented broth extract was subjected to bioactivity-guided fractionation, to isolate the active principle using different preparative chromatographic techniques followed by its characterization. The active principle was characterized to be Caerulomycin A. Minimum inhibitory concentration (MIC) of the compound was found in the range of 0.39 - 1.56 μg/ml against pathogenic fungal test strains. The phylogenetic analysis of producer strain using 16S rRNA sequence showed closest match with Actinoalloateichus cyanogriseus. Herewith we report the isolation of Caerulomycin A from marine invertebrate-associated Actinoalloteichus sp. using optimized medium and fermentation conditions.

Application of Potent Actinomycete Strains for Bio-Degradation of Domestic Agro-Waste by Composting and Treatment of Pulp-Paper Mill Effluent

Actinomycetes are known to produce an extensive range of bioactive compounds as well as variety of enzymes having multiple biotechnological applications. They are an important source of lignocellulose hydrolyzing enzymes and constitute considerable proportion of the soil or aquatic micro-flora responsible for degradation of biomass in natural environment. Presently, most of the commercially exploited lignocellulases and commercial biodegradation processes rely mostly on fungal or bacterial micro-organisms. Actinomycetes are relatively less explored for biodegradation processes that utilize lignocellulases for solid agro-waste management and waste water treatment. There is also a need to search and explore novel actinomycete strains for various biodegradation applications. This study involved examining the possibility of using only potent actinomycetes strains for the composting process by creating the consortium of such strains that could produce thermo-tolerant and alkali-tolerant key enzymes necessary for the degradation of cellulose, hemi-cellulose and lignin. The newly developed actinomycete consortium was tested for the composting activity and the composting process was optimized. The analysis of the composted material generated under ideal condition, demonstrated desirable physical and chemical characteristics. Paper pulp effluent poses a hazard to waterways due to toxicity. The toxicity of this mill effluent can be attributed to a compound called lignin and its chlorinated derivatives that are let out during the treatment of lignocellulosic constituents. Current study also involves the use of a strain of actinomycete having ability to produce enzyme laccase, which is active under alkaline condition for the treatment of paper pulp effluent. Enzyme laccase is known for its ability to attack phenolic components of lignin and common hazardous component of effluent, polycyclic aromatic hydrocarbons (PAH). Aerobic treatment of effluent by actinomycete strain indicated 21% reduction in COD at pH 7.5 after 14 days under optimum condition. This strain was identified as Streptomyces rochei based on molecular fingerprinting and was reported to be the producer of laccase probably for the first time. LCMS analysis of the treated effluent sample showed the presence of degradation compounds forming after 7 and 14 days of treatment. These compounds showed the degradation of lignin components and other phenolic, non-phenolic components of the effluent by intra molecular re-arrangement, oxidation and ring opening reactions. Overall, potent thermo-tolerant and alkali-tolerant actinomycete strains were successfully isolated and applied for bio-degradation of domestic agro-waste by composting and treatment of pulp-paper mill effluent. The consortium of these strains may be further utilized for scale-up studies in order to assess its commercial feasibility for biodegradation processes.

Genetic transformation of marine Actinomycete sp. isolate M048 and expression of a recombinant plasmid carrying the apc gene

Optimal conditions for protoplasts formation of marine Actinomycete sp. isolate M048 were described, dense and disperse mycelia were cultured in SGGP medium, 0.5% glycine, lysozyme exposure （2 mg/cm^3, 37 ℃, 40 min）, and the concentration of sucrose in protoplast buffer was 0.4 mol/dm^3 for keeping the balance of osmotic pressure. Using PEG-mediated pmtoplasts transformarion, the transformation frequency was 89 transformants per microgramme of pIJ702. Meanwhile, an effective transformation procedure was established based on intergeneric conjugation from E. coli ET12567 （pUZS002） using shuttle vectors pPM801, pPM803 and a φC31-derived integration vector pIJ8600 containing onT and attP fragments. Transformation frequencies were 5.30 ×10^-4 ±0.26 ×10^-4 , 8.92 ×10^-4 ±0. 19 ×10^-4 and 6.38 ×10^-5 ±0.41×10^-5 respectively. Further, the heterologous expression of the allophycocyanin gene （apc） in the strain M048 was used to demonstrate this transformation system. SDS - PAGE and Western blot analysis confirmed the expression of recombinant APC （rAPC）.

Screening of Actinomycetes from Medicinal Plant Rhizosphere Soils for Industrial Enzymes and Antimicrobial Activity

This study aimed to investigate the production of some metabolites （i.e., antibiotics, amylases and cellulases） of terrestrial actinomycetes isolated from medicinal plant rhizosphere soils. Initially, the soil samples were collected from Camellia sinensis （L） Okuntze., Peuraria mirifca Airy Shaw Suvatabandhua., Ananus comosus Merr., Elephantopus scaber Linn., Orthosiphon grandiforus Bolding., Jatropha multifda Linn. and Senna siamea. To screen and isolate actinomycetes, the soil samples were pretreated by air-drying and subsequent heat incubation. The bacterial isolates exhibiting actinomycetes features were then randomly screened for their production of amylases, cellulases and antibiotics. It was found that 130 isolates （from 136） could produce amylases; 40 （from 107） produced cellulases; and seven （from 45） exhibited antimicrobial activity. The data of this study were preliminary, and yet demonstrated a rich diversity of rhizo-actinomycetes from medicinal plants. Besides, these organisms could be an untapped source for discovering of biotechnologically useful metabolites.

Three New Compounds from the Actinomycete Actinocorallia aurantiaca

Aurantiadioic acids A(1)and B(2),two new furan-containing polyketides,and aurantoic acid A(3),a new natural product,were isolated from the liquid fermentation of the sika deer dung-derived actinomycete Actinocorallia aurantiaca.The structures of the new compounds were established by extensive spectroscopic methods,including 1D&2D NMR,HRESIMS spectroscopic analysis.The absolute configuration of 3 was assigned by comparison of the specific optical rotations with the reported derivatives.Biological activity evaluations suggested that compounds 1-3 showed weak inhibition on NO production in the murine monocytic RAW 264.7 macrophages with IC_(50)values of 35.8,41.8,45.2μM,respectively.Compound 3 showed weak inhibition on influenza A virus(A/PuertoRico/8/1934,H1N1)with an EC_(50)value of 35.9μM,and a selective index higher than 13.3.

Screening and Purification of Natural Products from Actinomycetes that Induce a“Rounded”Morphological Phenotype in Fission Yeast

This study was designed to identify and investigate bioactive natural product compounds that alter the cellular shape of the fission yeast Schizosaccharomyces pombe and induce a“rounded”or“small”cellular morphological phenotype.Bioassays using a range of antifungal agents against a multidrug-sensitive fission yeast strain,SAK950 showed that many induced a“rounded”phenotype.We then investigated whether 46 of the actinomycete strains identified in our previous study as inducing a similar phenotype produced antifungal agents of similar classes.We show that five of the strains produced streptothricin and that 26 strains produced polyenes,including fungichromin,filipin and candicidin,the last of which was produced by 24 strains.A taxonomic study of the strains indicated that the majority of the candicidin only producers were Streptomyces hydrogenans and S.albidoflavus whilst those that additionally produced streptothricin were related to S.enissocaesilis.A follow-up study to investigate the natural products made by related strains indicated that they followed a similar pattern.The identification of several compounds from the actinomycete strains similar to the antifungal agents initially tested confirm the validity of an approach using the S.pombe morphological phenotype and actinomycete taxonomy as a predictive tool for natural product identification.

Characterization and Antimicrobial Susceptibility of Actinomycetes from TB Smear Negative and Retreatment Patients in Nairobi, Kenya

Actinomycetes are opportunistic pathogens in immunosuppressive patients. Pulmonary actinomycetes infections display symptoms that mimic Mycobacteria tuberculosis and can be misdiagnosed and treated as pulmonary TB. Actinomycetes can be co-infection with tuberculosis leading to delayed or inappropriate treatment. This study aimed to identify and determine antimicrobial susceptibility profiles of Actinomycetes from the sputum of TB smear negative and re-treatment patients referred to TB reference facilities in Kenya. Sputum specimens were collected and direct smears stained with Gram’s reagents. Culture was done on Mueller Hinton agar and incubated at 35°C for two weeks. Identification was done using phenotypic and biochemical procedures. Confirmation of the isolates was done using Polymerase Chain Reaction. A total of 52/385 (14%) Actinomycetes were isolated and subjected to antimicrobial susceptibility testing using broth microdilution method to determine the Minimum Inhibitory Concentration. Nine antibiotics were tested which included: Amikacin, Amoxicillin/Clavulanic acid, Ceftriaxone, Ciprofloxacin, Clarithromycin, Linezolid, Doxycycline, Trimethoprim-Sulfamethoxazole and Gentamycin. Staphylococcus aureus (ATCC 25923) was used as a control. Most of the isolates were susceptible to the test antibiotics. However, four isolates showed multidrug resistance to Ceftriaxone and Clarithromycin with resistance of 11.5% and 26.9% respectively. Gentamycin and Ciprofloxacin showed the highest susceptibility of 100% and 98.1% respectively. The findings of this study confirm that Actinomycetes are significant pathogens in TB smear-negative cases. Although most antibiotics were susceptible, resistance to few antibiotics was observed;hence, there is a need for proper screening of TB smear-negative cases to detect infections by Actinomycetes and also conduct the antimicrobial susceptibility test to determine which antibiotic is effective.