When the crude extracts of each of the test actinomycetes were assessed against a total of 32 test bacteria (seven Gram-positive and 25 Gram-negative) at a concentration of 10 mg/mL, extract obtained from Saccharopolyspora (TR 046) was observed to be active against nine test bacteria with zones of inhibition ranging from 8–28 mm (Table 1). Extract obtained from Saccharopolyspora (TR 039) was active against eight of the test bacteria with zones of inhibition varying from 8–15 mm; while extract obtained from Actinosynnema (TR 024) was active against four test bacteria with zones of inhibition ranging from 10–13 mm. Extracts from Actinosynnema and Saccharopolyspora (TR 039) appeared to be active against only Gram-negative bacteria while extract from Saccharopolyspora (TR 046) had a broad spectrum antibacterial activity. Proteus vulgaris CSIR 0030 was most susceptible to the extract obtained from Saccharopolyspora (TR 046) while the environmental Staphylococcus epidermidis was least susceptible. However, with extract from Saccharopolyspora (TR 039), Enterobacter cloacae ATCC 13047 was least susceptible, while Proteus vulgaris ATCC 6830 was most susceptible. There does not appear to be significant dynamics in the susceptibilities of the bacteria for which the extract from Actinosynnema was active.

It was also observed that the referenced strains Pseudomonas aeruginosa ATCC 7700 and Proteus vulgaris CSIR 0030, and environmental strain Klebsiella pneumoniae were reactive to all three extracts. Results of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of the test actinomycetes antibiotics are shown in Table 2. The MIC of extract from Saccharopolyspora (TR 046) ranged from 0.078 mg/mL to 10 mg/mL while its MBC varied between 1.25 mg/mL and >10 mg/mL. For the extract from Saccharopolyspora (TR 039), MIC ranged between 5 and >10 mg/mL while its MBC was generally >10 mg/mL. The extract from Actinosynnema on the other hand had MICs ranging between 1.25 mg/mL and 5 mg/mL and an MBC of >10 mg/mL.

Time-kill studies revealed that the Saccharopolyspora (TR 046) extract had bacteriostatic effects on referenced bacterial strain Pseudomonas aeruginosa (ATCC19582) with no major changes in the bacterial load with time (Table 3). The extract was however, strongly bactericidal against another referenced bacterial strain (Bacillus pumilus ATCC 14884) at 4× MIC resulting in the killing of approximately 10⁴ cfu/mL in 6 to 12 h and 10² cfu/mL at 2× MIC after 6 h of exposure to the extract suspension. It was however, weakly bactericidal at normal strength MIC (0.078 mg/mL). The extract also showed good bactericidal activity against the referenced strain Proteus vulgaris CSIR 0030 achieving reductions of 0.9log₁₀ and 0.13log₁₀ cfu/mL at 4× MIC (2.5 mg/mL) and 2× MIC (1.25 mg/mL) respectively after 12 h of exposure. The extract showed limited bactericidal activity against both Klebsiella pneumoniae (environmental strain) and Staphylococcus epidermidis (environmental strain) at all MIC levels after 6 h of exposure but showed bacteriostatic effects at all MIC levels after 12 h of exposure.

When the antibacterial activity of the three freshwater actinomycetes extracts was observed against a series of bacterial isolates, Gram positive test bacteria showed limited susceptibility to the extracts while the Gram negative bacteria were more susceptible. Whilst our findings confirm antibiotic production in the Saccharopolyspora and Actinosynnema isolated from the freshwater habitat, nevertheless, the Saccharopolyspora (both TR 046 and TR 039) species appear to exhibit consistently greater antibacterial activity than Actinosynnema (TR 024). The limited antibacterial activity of Actinosynnema extract is in agreement with previous findings [13] which reported that actinomycetes belonging to the genus Actinosynnema mainly produce maytansinoid antibiotics (potent anti-tumor agents) although it is worth noting that they still do produce antibacterials, howbeit against a limited range of bacteria, whereas Saccharopolyspora is a well documented producer of antibiotics [18], with Saccharopolyspora erythraea well known for the industrial production of the antibiotic erythromycin.

Of the two Saccharopolyspora strains (TR 046 and TR 039), extract obtained from TR 046 produced consistently larger inhibition zone diameters than that of TR 039. This result suggests that the extract from TR 046 has greater efficacy compared to the extract from TR 039. This result is further proven by the MIC and MBC results which show that for the extract from TR 039 MIC values ranged from 5 mg/mL to >10 mg/mL while the MBC values were all >10 mg/mL. The observed differences in the antibiotic producing potentials of the two Saccharopolyspora strains, TR 046 and TR 039, can possibly be explained in terms of the ecological conditions from where they were isolated. Actinomycetes isolated from a habitat richer in microorganism (competing bacterial species) tend to produce more antagonistic substances as a competitive tool for survival compared to those isolated from places with low numbers of competing microorganisms [19]. In this regard, it can be suggested that Saccharopolyspora TR 046 was isolated from a habitat richer in competing species of microorganisms compared to Saccharopolyspora TR 039.

Extracts obtained from both Saccharopolyspora TR 046 and TR 039 were active against five referenced bacterial strains each and whereas the extract from TR 046 was active against three environmental strains and showed no activity against clinical isolates, the extract from TR 039 showed activity against two environmental strains and one clinical isolate, a result which emphasizes the similarities in the genotypic origin of the two organisms. Their limited activity against clinical isolates confirms that these clinical strains of bacteria may possess characteristics that differ or are absent from the non-pathogenic strains [20]. Pathogenic strains may possess specific virulence determinants (toxins and adhesions, etc.) encoded by monocistronic genes, plasmids, or pathogenicity islands as well as plasmids that code for drug resistance [21,22], which may partially account for the ineffectiveness of the extracts against clinical strains.

Our findings suggest limited activity against Gram-positive bacteria by all the actinomycetes extracts used although it is worth noting that seven Gram-positive bacteria were tested as compared to 25 Gram-negative test bacteria and it can therefore be deduced that it may not be necessarily correct to suggest that the extracts lacked broad spectrum activity. Besides, Gram-negative bacteria are inherently more resistant to antimicrobials than Gram-positive bacteria due to the combined exclusion of the antimicrobial compounds by the double membrane barrier and transmembrane efflux present in this group of organisms [23], hence sourcing actinomycetes that produce effective antimicrobials against Gram-negative bacteria is a step in the right direction for the war against antibiotic resistance.

The rate of kill of the test organisms by the extract from Saccharopolyspora TR 046 appears to be both concentration and time dependent. Results suggest that the extract was mostly bactericidal at 2× MIC and 4× MIC up to 6 h of exposure with the effect waning off after 6 h. This might suggest that dosing frequency needs to be increased to once after every 6 h to maintain the bactericidal effect of the extract at its optimum. Complete eradication of the test organisms was not achieved. In view of the knowledge that actinomycetes produce secondary metabolites, especially antibiotics, in order to overcome other competing microorganisms by killing them [19], this could be attributed to the fact that the sediments from which the actinomycetes isolates were obtained have a small bacterial population which competes with them and hence rely less on antibiotic production as a competitive tool against other bacteria.

However, the extract exhibited a strong bactericidal efficacy against Bacillus pumilus (ATCC 14884) achieving a 1.26log₁₀ reduction in counts of the test organism after 6 h of exposure at 0.312 mg/mL (4× MIC) and moderate bactericidal efficacy against Proteus vulgaris CSIR 0030 achieving a 0.9log₁₀ reduction in counts of the test organism after 12 h of exposure at 2.5 mg/mL (4× MIC). The extract exhibited bacteriostatic effects on two test bacteria (Klebsiella pneumoniae {KZN} and Staphylococcus epidermidis {KZN}) with no major changes on the bacterial load with time. Pseudomonas aeruginosa showed persistive response to the extract, with a marked delay in its growth at 2.5 mg/mL (4× MIC) after 6 h of exposure increasing by 0.01log₁₀ counts as compared to an increase of 1.02log₁₀ counts after 6 h of exposure at 0.625 mg/mL (MIC). A 3log₁₀ or 99.9% reduction in viable bacterial density in an 18–24 h period is the generally accepted definition of bactericidal activity in antibiotics [24].

Results were analyzed under Minitab Release 14.2 using a 2-sample T-test and One-way Analysis Of Variance (ANOVA) as the statistical packages. Mean zone diameters were compared for all the three actinomycetes extracts. Analysis was carried out at 95% confidence interval.

Three actinomycetes strains belonging to the genera Saccharopolyspora (TR 046 and TR 039) and Actinosynnema (TR 024) were obtained from the culture collections of the Applied & Environmental Microbiology Research Group (AEMREG), University of Fort Hare, Alice, South Africa. The strains were isolated from the Tyume River in the Eastern Cape Province of South Africa [12]. The organisms were maintained on agar slants and in 20% glycerol (at −80 °C).

The stock culture of the test actinomycetes used in this study were prepared by streaking the actinomycetes from the agar slants onto starch casein agar (SCA) which was prepared as follows (per liter of filtered freshwater): soluble starch, 10 g; potassium phosphate dibasic, 2 g; potassium nitrate, 2 g; sodium chloride, 2 g; casein, 0.3 g; magnesium sulfate.7H₂O, 0.05 g; calcium carbonate, 0.002 g; ferrous sulfate.7H₂O, 0.01 g and bacteriological agar, 16 g. Each of these compounds was added to the diluents and allowed to dissolve completely using a magnetic stirrer. The medium was then autoclaved at 121 °C and 15 mm Hg for 15 minutes and allowed to cool down to 50 °C before being poured into 90 mm Petri dishes. The test actinomycetes were streaked on the prepared medium and incubated at 28 °C for between 7 and 14 days under aerobic conditions. Actinomycetes inocula were then prepared by transferring several colonies into sterile normal saline (10 mL) and the suspensions vortexed for 20 seconds to ensure homogeneity.

The fermentation broth was prepared following a method outlined by Muiru et al. [25] as follows (per liter of distilled water): 10 g starch, 4 g yeast extract, 2 g peptone, 5 ml potassium bromide (20 g/L) and 5 mL iron (iii) sulfate tetrahydrate (4.76 g/L). The medium was divided into 500 mL aliquots into 1 L Erlenmeyer flasks and sterilized by autoclaving at 121 °C and 15 mm Hg for 15 minutes. After the medium cooled, 100 μL volumes of actinomycetes suspensions (standardized to McFarland 0.5) were used to inoculate the flasks. The flasks were then incubated at 27 °C on a shaker at 300 rpm for 10 days. For quality control, confirmation of purity was done by streaking the fermentation cultures onto nutrient agar (NA), potato dextrose agar (PDA) and starch casein agar (SCA) plates.

Crude antibiotic extracts were recovered from the broth culture filtrate by solvent extraction using ethyl acetate in accordance with the description of Liu et al. [26]. Ethyl acetate was added to the filtrate in the ratio 1:1 (v/v) and shaken vigorously for 1 h for complete extraction. The ethyl acetate phase that contained the antibiotic was separated from the aqueous phase and concentrated in vacuo at 60 °C using a rotary evaporator. The residue obtained was weighed and reconstituted in 50% methanol to make a working concentration of 10 mg/mL for the antibacterial assays.

The test bacteria used in this study were obtained from the culture collection of the Applied and Environmental Microbiology Research Group (AEMREG) laboratory at the University of Fort Hare, Alice, South Africa and included the following:

Referenced strains: Escherichia coli ATCC 8739, Pseudomonas aeruginosa ATCC 19582, Staphylococcus aureus ATCC 6538, Enterococcus faecalis ATCC 29212, Bacillus cereus ATCC 10702, Bacillus pumilus ATCC 14884, Pseudomonas aeruginosa ATCC 7700, Enterobacter cloacae ATCC 13047, Klebsiella pneumoniae ATCC 10031, Klebsiella pneumoniae ATCC 4352, Proteus vulgaris ATCC 6830, Proteus vulgaris CSIR 0030, Serratia marcescens ATCC 9986, Acinetobacter calcoaceticus, Acinetobacter calcoaceticus anitratus, Escherichia coli 25922.

The test bacteria were confirmed for purity by streaking onto nutrient agar plates. These pure bacterial isolates were then inoculated into nutrient broth and incubated at 37 °C for 24 h. The turbid broths were later centrifuged at 7000 rpm and the supernatant discarded. The pellets of cells were resuspended and double washed in sterile normal saline and standardized to OD_600nm 0.1. The washed and standardized cells were subsequently used for various experiments described below.

Antibacterial activities of the crude extracts were determined using agar well diffusion technique as described by Pandey et al. [27]. Test organism cultures were grown overnight (18 h) in nutrient broth and standardized to OD_600nm 0.1. Test organisms were then spread-plated onto Muller Hinton agar (MHA) plates using sterile cotton swabs. A flame sterilized cork borer with a diameter of 6 mm was used to bore wells into the agar and 100 μL of the extract (10 mg/mL) loaded into the wells. Control wells were loaded with 100 μL of 50% methanol. The extract was allowed to diffuse into the agar before the plates were incubated under aerobic conditions at 37 °C for 24 h. At the end of the incubation period, the plates were observed for zones of inhibition around the wells. Inhibition zone is defined as the area free of growth in a bacterial lawn which results from the effect of antibiotic that has diffused into the medium from its applied source [28].

The MICs were determined using test organisms that showed susceptibility to the crude extracts by the broth microdilution method as outlined by the EUCAST Discussion Document [29]. Sterile plastic, disposable microtiter plates with 96 flat-bottom wells were used. The medium used in the plates was prepared at double the final strength to allow for a 50% dilution once the inoculum and solvents or antimicrobial were added. A 100 μL volume of double strength Muller Hinton broth was introduced into all the 96 wells and varying concentrations of the antibiotic were added in decreasing order along the wells after which wells were loaded with 50 μL of the test organism suspension. The plates were then incubated at 37 °C for 18–24 h. Wells in column 12 were used as the growth controls and contained 50 μL of test organism and 50 μL of sterile distilled water. Results were read using a microtiter plate reader (BIO-RAD model 680) at 490 nm. Visual reading of results was also done by first adding resazurin dye into all the wells. Wells with no growth turned blue in color while those with growth turned pink, and this helped to give a clear visual demarcation of the MIC wells. The MIC was estimated as the lowest concentration of the extract that inhibited growth of the test organisms.

The MBC was determined from the MIC plate following a method outlined by the CLSI [30], and is defined as the lowest concentration of an antibiotic that under defined in vitro conditions reduces by 99.9% the number of organisms in a medium containing a defined inoculum of bacteria, within a defined period of time [28]. It was determined by inoculating the broths in the MIC range into drug-free nutrient agar medium. The MBC was determined as the antibiotic concentration at which no growth was observed after incubation for 48 h.

The rate of kill assay was done only for TR 046 extract, which appeared to exhibit more antibacterial potency than the other actinomycetes. This was done by monitoring bacterial cell death over time in accordance with the description of Okoli and Iroegbu [31]. Also, five test bacteria were selected for this assay based on their susceptibility and Gram’s reaction. The inocula were prepared following the described guidelines of the EUCAST Discussion Document [29]. The resultant cell suspension was diluted 1:100 with fresh sterile broth and used to inoculate 50 mL volume of nutrient broth incorporated with the extract at multiples of the MIC to a final cell density of 5 × 10⁵ cfu/mL [32,33]. The flasks were then incubated with shaking at 37 °C on an orbital shaker at 120 rpm and samples of 100 μL were then withdrawn at 6 h and 12 h intervals and diluted appropriately. Approximately 100 μL volumes of the diluted samples were then plated out in triplicate on nutrient agar. Plates were incubated at 37 °C for 24 h, after which the numbers of surviving cells were enumerated [33]. Controls consisted of extract free nutrient broth inoculated with test organism.