Over the years, the rate of discovery of new drugs has declined significantly, leading to an urge to explore alternative environmental sources apart from the soil, for novel antibiotics. In this study, water samples were collected from different places around Bath and they were screened for antibacterial activity against six test bacteria. A total of 106 isolates were isolated and tested against methicillin-sensitive Staphylococcus aureus (MSSA) to confirm their antagonistic activity. 34 isolates showed promising inhibition and were further tested against bacteria which included MSSA, methicillin-resistant Staphylococcus aureus (MRSA), Enterococcus faecium (E. faecium), Escherichia coli (E. coli), Pseudomonas aeruginosa (P. aeruginosa) and Klebsiella pneumoniae (K. pneumoniae) through perpendicular streak test. The supernatant of the isolates was also extracted by centrifugation and assayed for its antibacterial activity. For E. coli-inhibiting isolates, further tests against strains of E. coli with different antibiotic resistance were performed to identify the types of antibiotics produced. 11 active isolates were effective against both Gram-positive and Gram-negative bacteria in the perpendicular streak test. The supernatant of the isolates exhibited minimal antibacterial activity. Following the results of 16S rRNA sequencing, two active isolates belonged to Pseudomonas species while the other three isolates were classified as Bacillus species. Isolate 107 was identified as Bacillus pumilus and it demonstrated the strongest inhibition against MSSA, MRSA, E. faecium and E. coli with a zone of inhibition of 20.8mm, 24.3mm, 16.2mm and 13.6mm respectively. Moreover, isolates 18 and 107 both had strong activity against all strains of E. coli tested, while isolate 71 was only active against four strains of E. coli tested, suggesting that one of the antibiotics produced may be kanamycin. Our findings indicate that aquatic environment is a potent source for the isolation of bioactive microorganism potential for the production of antibacterial compounds.
1. Introduction
Microbes play an essential role in the production of antibiotics, antifungal as well as antiviral infections and this role is expanding each day (1). Owing to their ability to produce useful secondary metabolites, microbes have contributed greatly in the development of pharmaceutical industry and the control of many medical conditions as they are now widely used as antitumour drugs, immunosuppressants, enzyme inhibitors, and in many other applications (1). Back in 1928, Alexander Fleming found a compound produced by a mould, which was later identified as Penicillium notatum, had the capability of killing the bacterium Staphylococcus aureus in his laboratory. The active compound was known as penicillin, a beta-lactam antibiotic, and it was used massively as a potent antimicrobial drug during World War II (1). This discovery has marked the beginning of the microbial drug era as many useful antibiotics have since been isolated from soil bacteria, for instance, streptomycin, chloramphenicol, and tetracycline (2). These antibiotics produced have had remarkable biological activities to human beings, to illustrate, streptomycin was the first active drug against tuberculosis whereas chloramphenicol was the drug of choice for typhoid fever (3-5).
The soil is incontestably a rich reservoir that allows the screening of drug compounds as it can harbour an enormous number of soil inhabitants, such as bacteria, fungi, algae, protozoa, insects, and other more complex living organisms (6). Many soil organisms have the ability to produce secondary metabolites which enable them to inhibit the growth of other microorganisms in the same niche in order to compete for survival (7). Over the past decades, a significant amount of bioactive compounds have been discovered from the terrestrial environment (3). The majority of the useful soil microorganisms belong to the genera Penicillium, Streptomyces, Cephalosporium, Micromonospora and Bacillus, and they are still being studied continuously (8). Bacillus is found abundantly in the soil environment and it is known to produce antibiotic like bacitracin, pumulin and gramicidin which are active against Gram-positive bacteria, and polymyxin, colistin and circulin which are effective against Gram-negative bacteria, demonstrating a vast range of antimicrobial activity (9; 10). Furthermore, more than 60% of antimicrobial agents used in human and animals were originated from the genus Streptomyces, some of which are chloramphenicol, erythromycin, and tetracycline, which have a broad spectrum of activity (5; 9).
In the recent years, the search for antibiotics has plateaued whereby limited new antibacterial drugs are being introduced to the market, posing a challenge to the healthcare sector. The rate of discovery of new compounds has declined and the scientists are facing a bottleneck where the same known antimicrobial compounds have been isolated over the past few years (11). Nowadays, antibiotics are widely used therapeutically and prophylactically in the healthcare setting for human, animals and agricultural purposes. They are commercially exploited and the overconsumption of these compounds has brought about a rapid evolution in microorganisms where resistance to the antibiotics is spreading dramatically. Furthermore, the crisis of multidrug resistance is expanding uncontrollably, leading to a consistent demand for more effective and useful antibacterial medicines. Staphylococcus aureus, Pseudomonas sp., Klebsiella sp., and Enterococcus sp. are examples of common nosocomial bacteria and they often cause a serious problem to hospitalised patients (1). The development of resistance of these pathogens to currently available antibiotics, both natural and synthetic classes of antibiotics, has rendered many of the standard drugs ineffective (12).
In view of the scarcity of new antimicrobial medications being found from terrestrial environment, there is an urge to explore different environmental sources. The marine environment consists of a wide diversity of microorganisms, and thus the discovery of many medically useful compounds. The antivirals acyclovir and cytarabine used for herpes virus and non-Hodgkin’s lymphoma respectively were originally isolated from marine sponges, showing the potential of marine life as a novel source of medicines (1). As reported in a study conducted in South East coast of India, organisms originated from the genera Vibrio, Pseudomonas, Marinobacter and Bacillus have been isolated and proven to exhibit antimicrobial activity, where the isolate belonged to Bacillus species had the highest activity against Bacillus subtilis, E. coli and C. albicans (13). Besides that, aquatic actinomycetes have also been reported to produce bioactive compounds with potential clinical uses such as salinosporamide-A (from Salinispora sp.), marinopyrroles (from Streptomyces sp.) and marinobactin (from Marinobacter sp.) (14-16). Antibacterial actinomycetes have been successfully isolated from Lake Tana, Ethiopia, of which 13 isolates showed antibacterial ability against at least one of the tested bacteria, such as K. pneumoniae, S. aureus, P. aeruginosa and E. coli (17). Similarly, a study conducted in Ghana has isolated 27 antibiotic-producing microorganisms from marine and freshwater sources and it has been found that one of the active isolates produced metabolites with a broad antibacterial activity against both tested Gram-positive and Gram-negative bacteria (18).
Although it is apparent that the ocean is a boundless source for novel antibacterial compounds, the water sources remain underexplored and unexploited. The aim of this study was to isolate antibiotic-producing microorganisms from the aquatic environment around Bath area and to determine their antibacterial activity against six test bacteria, three of which belonged to Gram-positive bacteria and the other three were Gram-negative bacteria.
2. Methods and Materials
2.1 Sample collection
Water samples with sediments were collected from River Avon, Bath City Farm, Rainbow Wood Farm and pond in the University of Bath.
2.2 Isolation of microorganisms
The water samples were serially diluted up to 10-4 in phosphate buffered saline (PBS) (Oxoid). Each diluted sample was inoculated into media by spread plate technique and incubated at 28°C. Three different media were used, namely minimal salt agar (Sigma), nutrient agar (Oxoid) and tryptic soy agar (TSA) (Oxoid).
2.3 Screening of isolates for antibacterial activity
Crowded plate technique was used to screen microorganisms with antagonistic activity. Inhibition activity can be demonstrated by the formation of clear zones surrounding the colonies, after being incubated for three days. Colonies of interest were selected and repeated streaking technique was used to purify the isolated colonies.
An overlay of soft agar with a concentration of 0.75% was performed using double-layer agar technique to confirm the antibacterial activity of the isolates. 10ml of soft agar maintained in a water bath at 42°C was mixed with 100l of methicillin-sensitive Staphylococcus aureus (MSSA) Newman (19) before pouring it onto the solid agar. Clear zones produced indicated the synthesis of compounds active against the test bacterium.
Perpendicular streak test was carried out where isolates were streaked as a single line along the diameter of TSA and incubated at 37°C and 28°C for one day each or 28°C for three days. The test organisms were then cross streaked at right angles to the original streak of isolates. The test organisms used included MSSA Newman, methicillin-resistant Staphylococcus aureus (MRSA) 252 (20), Enterococcus faecium (E. faecium) E1162 (21), Escherichia coli (E.coli) BW25113 (22), Pseudomonas aeruginosa (P. aeruginosa) PA01 (23), and Klebsiella pneumoniae (K. pneumoniae) (departmental strain collection). The plates were incubated at 37°C for 24 hours and the length of zones of inhibition was measured. Control plates without isolates of interest were simultaneously streaked with test organisms to study their normal growth.
For isolates that showed activity against E. coli BW25113, they were further tested with four other E. coli strains: E. coli ER2420 (pACYC184) (24), E. coli ER2420 (pACYC177) (24), E. coli SURE (pET-Amy) (25), and E. coli XL1Blue (pSG1164) (26), which displayed different antibiotics resistance. Perpendicular streak test was carried out to identify the antibiotic(s) produced.
2.4 Antibacterial activity of supernatant
Five isolates were selected and grown in the nutrient broth (NB) (Oxoid) for three days. The supernatant was obtained by centrifugation at 4 °C, 5000 g for 20 minutes and it was filter sterilised before use. The antibacterial activity of the supernatant of each isolate was investigated as described:
(i) 100l of the supernatant of each isolate was mixed with 100l of each test organism culture (106 cfu/ml) and plated in 96-well plates. Eight replicates were done for each isolate. 200l of NB served as a negative control, while a mixture of 100l of each test organism culture and 100l of NB served as a positive control. The plates were incubated at 37°C for 24 hours and the OD of the mixture was measured.
(ii) Agar well diffusion test was carried out where 100l of each test organism culture (106 cfu/ml) were plated on TSA and left for 30 minutes. Five wells of 9mm were punched in the TSA with a sterile cork borer and 100l of each supernatant sample was filled into the wells of each plate inoculated with different test organisms. The plates were incubated at 37°C for 24 hours and observed for growth inhibition zones.
2.5 Characterisation of colonies of interest
The strains of interest were characterised using gram staining to identify their classes, shapes and sizes under a light microscope.
2.6 Genomic DNA extraction
Genomic DNA of each isolate was extracted by alkaline lysis as described in (27; 28) with some modifications. In this experiment, a loopful of cells was emulsified in 20l lysis buffer (0.25% sodium dodecyl sulfate (SDS) (SIGMA), 0.05N NaOH (Fisherbrand)) and heated at 95°C for 15 minutes (28). The cells were then centrifuged at 13,000rpm for 30 seconds and the supernatant was diluted with 180l sterile water and stored at -20°C for future use.
2.7 PCR primers and conditions
Five isolates of interest were chosen for 16S rRNA sequencing. The 16S rRNA gene was amplified by PCR using forward primer 27F (5’- AGAGTTTGATCMTGGCTCAG-3’) (Sigma) and reverse primer 1492R (5’-TACGGYTACCTTGTTACGACTT-3’) (Sigma). PCR was conducted with One Taq @ Standard Reaction Buffer (5x) (New England Biolabs Inc.) using manufacturer’s instruction.
PCR protocol was as follow: at 94°C for 2 min followed by 30 cycles at 94°C for 30 s, 55°C for 60 s, and 68°C for 90 s, then 68°C for 5 min in a single step. The samples were then held at 4°C. The PCR products were washed using Monarch PCR & DNA Cleanup Kit (5g) (New England BioLabs Inc.) before performing agarose gel electrophoresis to evaluate their purity and quality.
2.8 Agarose gel electrophoresis
Visualisation of the PCR products was carried out on 1% agarose-TAE gel (Invitrogen, ThermoScientific) as described in (27) and Bioline Hyperladder I 100lanes was used as the ladder. Following the result of electrophoresis, Mix2Seq protocol was followed to prepare the samples for 16S rRNA sequencing.
2.9 Statistical analysis
Student t-test was used to calculate the significance of the difference between the experimental and the control samples. Differences were considered significant at p<0.05.
3. Results
3.1 Isolation and screening of microbial isolates
A total of 106 colonies were found to show inhibition of the growth of the surrounding microorganisms. The majority of the active isolates were collected from Bath City Farm while the least was collected from River Avon. It was also shown that most of the isolates of interest grew better on TSA compared to NA.
Following primary isolation, the isolates were overlaid with MSSA to confirm their antibacterial activity as shown in Figure 1. Out of the 106 colonies, only 34 of them inhibited the growth of MSSA and they were further investigated through perpendicular streak test with six bacteria. The zone of inhibition from perpendicular streaking of the isolates indicated antibiotic-producing activity as depicted in Figure 2. From the 34 isolates, only 11 isolates showed good outcomes, five independent repeats were done and the results are shown in Table 1. The average length of the inhibition zone produced by the isolates varied greatly, depending on the susceptibility of the test organisms to the antibiotics produced. In the perpendicular streak test, MSSA, which served as a control, was inhibited by all of the tested isolates. It was found that none of the isolates was active against P. aeruginosa, while MRSA was sensitive to every antibiotic-producing isolate. The number of isolates that inhibited the growth of E. coli and E. faecium was six and five respectively, and only
Figure 1. An overlay of MSSA was performed on TSA to determine the antibacterial activity of the isolates. A prominent zone of inhibition was shown by isolate 44, where the growth of MSSA was inhibited.
three of the isolates antagonised the growth of K. pneumoniae. Out o
f the eleven isolates, five isolates were active against all three Gram-positive test organisms, three of which were active only against Gram-positive bacteria. Nine isolates demonstrated inhibition on at least three of the test organisms, of which five isolates inhibited four test organisms in total, and only two isolates inhibited two test organisms. The isolate 107 showed the most potent antagonistic activity against the test organisms as the lengths of inhibition observed were the longest among all (20.8 6.4 mm, 24.3 5.6 mm, 16.2 7.6 mm and 13.6 4.7 mm against MSSA, MRSA, E. faecium and E. coli respectively), while the isolate 103 exhibited the least potent
Isolates Degree of inhibition
MSSA MRSA E. faecium E. coli P. aeruginosa K. pneumoniae
18 ++ ++ – + – ++
33 + + – + – +
36 + ++ – – – –
40 + + – – – –
71 ++ ++ – ++ – ++
99 ++ ++ ++ + – –
101 + + ++ – – –
102 + + + – – –
103 + + – + – –
104 + + ++ – – –
107 +++ +++ ++ ++ – –
Figure 2. Perpendicular streak method was used to study the activity of the isolates against six bacteria. Colony 104 demonstrated clear inhibition of the growth of MSSA, MRSA and E. faecium.
antagonistic ability with the weakest inhibition against the test organisms MSSA, MRSA and E. coli (4.4 2.2 mm, 4.0 1.2 mm and 6.0 1.4 mm). MSSA and MRSA were both the Gram-positive bacteria most commonly inhibited by the isolates while E. coli was the most commonly inhibited Gram-negative bacterium among all.
3.2 Antibacterial activity of the supernatant
Two tests were carried out to study the antibacterial activity of the supernatant of each isolate:
(i) In the agar well diffusion test, the diameter of the zone of inhibition was measured and recorded. A fairly weak zone of inhibition was shown by all isolates, ranging from 9.5mm to 12.5mm. Isolate 18 was active against all bacteria, also demonstrating the strongest inhibition among all, with a maximum zone of inhibition of 12.5 0.7 mm shown against E. faecium. Isolate 71 had a fairly low activity against the test organisms E. coli (10.2 0.7mm), P. aeruginosa (10 0mm) and K. pneumoniae (11.5 2.1mm), and showed no effect against any of the Gram-positive bacteria. The supernatant of isolates 102, 104 and 107 exhibited some antibacterial activity against both Gram-positive and Gram-negative bacteria, although the activity was weak (<2mm zone of inhibition).
(ii) In the 96-well assays, the OD of the isolates in test organisms’ broth was compared to the OD of the positive controls as shown in Figure 3. The reduction in OD indicated that there was antibacterial activity, whereas a similar reading of OD showed that there was no inhibition. It was shown that isolates 18, 71 and 102 demonstrated slight antagonistic activity against MSSA (only 102 showed significance with p=0.038), isolate 104 did not seem to have a great effect on MSSA while 107 had a mild promoting effect on the growth of MSSA (p=0.111).
All tested isolates antagonised MRSA and E. coli. For MRSA, the differences in the OD were significant, showing a significant inhibition effect from each isolate. The strongest inhibition against MRSA was shown by isolate 71 with a difference of 0.207 (90%) in the OD while isolate 18 showed the strongest activity against E. coli, giving a difference of 0.441 (97%) in the OD. Isolate 107 was less active against both MRSA and E. coli, giving the minimum zone of inhibition in both tests.
There was no big difference in the OD shown among the isolates tested with E. faecium, except that isolate 18 was shown to have promoted the growth of the test organism, with a difference of 0.031 in the OD but it was not significant (p= 0.122). Isolates 18, 104 and 107 demonstrated slight enhancing effect while the other two isolates had an inhibitory effect on P. aeruginosa. All p values except for 104 are <0.05. A prominent inhibitory effect has been shown by isolate 18 against K. pneumoniae (p=0.0009), and isolate 71 gave a reduction in the OD (p=0.086). Three other isolates demonstrated a slight promoting effect on the growth of K. pneumoniae (p values >0.05).
As a whole, the supernatant of isolate 71 displayed the strongest activity against all test bacteria, while isolate 18 gave the greatest degree of zone inhibition, especially against MSSA, E. coli and K. pneumoniae.
3.3 Determination of the types of antibiotics produced by the isolates of interest
Three isolates that displayed antagonistic effect against E. coli were further screened against four different strains of E. coli to determine the possible antibiotic(s) produced. Isolates 18 showed powerful inhibition against all strains of E. coli, with every zone of inhibition exceeding 10mm. Isolate 107 inhibited the growth of every strain tested, with a zone of inhibition ranging from 5mm to 15mm. The strongest inhibition was shown against E. coli SURE (pET-Amy) while the weakest inhibition was shown against E. coli BW 25113. This indicated that the antibiotics produced were not any of the antibiotics that the tested E. coli was resistant to. On the other hand, isolate 71 inhibited the growth of four E. coli strains, except for E. coli ER2420 (pACYC184), showing that one of the antibiotics produced may be kanamycin.
Table 2. E. coli strains and their respective antibiotic resistance
Strains of E. coli Antibiotic resistance
E. coli BW25113 None
E. coli ER2420 (pACYC184) Chloramphenicol, tetracycline
E. coli ER2420 (pACYC177) Ampicillin, kanamycin
E. coli XL1Blue (pSG1164) Ampicillin, tetracycline, chloramphenicol
E. coli SURE
(pET-Amy) Ampicillin, kanamycin, tetracycline
3.4 Gram stain
11 active isolates with promising antibacterial activity were gram stained. It was found that five isolates of interest belonged to Gram-negative bacteria, whereas the other six isolates belonged to Gram-positive bacteria. Among the five Gram-negative isolates, four of them were short rods, while isolate 36 had the shape of coccobacilli. On the other hand, all Gram-positive isolates were rod-shaped and half of the Gram-positive isolates were thin rods.
3.5 Visualisation of PCR products
Genomic DNA of the isolates of interest was amplified using PCR and the fragments of the PCR products were visualised under UV light, using electrophoresis as pictured in Figure 5.
It was shown that the molecular weight of the PCR products was approximately 1.5kb when compared to the molecular weight marker. The intensity of the marker was used to measure the amount required for the 16S rRNA sequencing.
3.6 16S rRNA sequencing result
The 16S rRNA sequences of five isolates of interest were identified and compared using the BLAST tool to determine their species.
Isolates 18 and 71 were classified as Pseudomonas species with isolate 18 having near 100% identity to P. donghuensis. Isolate 71 was identified as P. protegens as both the forward and reverse sequences were 99% identical to the one proposed. On the other hand, isolates 102, 104 and 107 were closely related to Bacillus sp., with the strain unknown. Isolates 102 and 104 were highly similar and both had high possibility to be B. subtilis or B. velezensis as they were 99% identical to these species. For isolate 107, it showed 99-100% similarity with B. pumilus. These results were compatible with the one obtained from gram stain test. Pseu
domonas species are Gram-negative and isolates 18 and 71 were shown to be Gram-negative in gram stain, while Bacillus species are Gram-positive, whereby 102, 104 and 107 stained purple in the gram stain test.
Isolate Type Shape
18 G-ve Short rods
33 G-ve Short rods
36 G-ve Coccobacilli
40 G-ve Short rods
71 G-ve Short rods
99 G+ve Rods
101 G+ve Rods
102 G+ve Rods
103 G+ve Rods
104 G+ve Rods
107 G+ve Rods
4. Discussion
Effective antibiotics are essential to maintaining the high standard of healthcare and the emergence of multidrug resistance has urged the process of research and development of novel antibacterial products. The search for new bioactive compounds is necessary to combat these multi-drug resistant pathogens and it might help to delay the progress of antibiotic resistance growth. Aquatic microorganisms have slowly emerged as a new source of active metabolites producing microorganisms and more investigations in this area should be carried out to prompt the success of new discoveries.
In this study, a number of active isolates have been found to exhibit antagonistic activity against a variety of test organisms, showing that there is a synthesis of antimicrobial active compounds in the aquatic life. Five out of eleven isolates were active against at least four test organisms, suggesting the production of wide spectrum antibacterial compounds. The supernatant of the active isolates was tested to determine the antibacterial activity of extracellular metabolites. The overall results from the agar well diffusion showed a generally broader spectrum of activity against the tested bacteria. However, the degree of inhibition was insufficient to make any promising conclusions. Surprisingly, P. aeruginosa was shown to be inhibited by all of the isolates’ supernatant, with a very minimal degree of inhibition. In the 96-well plate test, the OD shown by the samples with E. faecium was the lowest among all, this might be due to its slow-growing property as observed during our study. The increase in OD was detected and it may be caused by the promoting effect on the growth of test organisms or contamination in the samples. The promoting effect shown was significant only for isolates 18 and 107 against P. aeruginosa and this should be further studied. However, the lack of repetition of the OD test may affect the reliability and consistency of the results. Repeated streaking of the active isolates may also lead to the loss of their ability to produce antibiotics or the synthesis of new active compounds due to the variation of growth media (alteration in nutrients, temperature, osmotic conditions etc.)(29). In addition, TSA was used in perpendicular streak test while NB was used for the supernatant test. One of the factors causing limited antibacterial activity shown in the agar well diffusion may be the medium which was possibly less conducive to the production of active compounds.
As shown from the results, the isolated microorganisms showed more activity against Gram-positive bacteria than Gram-negative bacteria. Different sensitivity between the two groups of bacteria has been observed, this could be ascribed to morphological differences of their outer polysaccharide membranes. The outer membrane of Gram-negative bacteria consists of lipopolysaccharide which makes it impermeable to lipophilic solutes whereas the Gram-positive bacteria lack an outer membrane, making them more susceptible and receptive to antibiotics (17). MSSA and MRSA were the most susceptible bacteria to the antibiotics produced by the isolates. This also revealed that the isolates found may be potential to be used to extract the active metabolites that may be useful in treating various severe infections caused by MSSA and MRSA. As expected, P. aeruginosa showed the least susceptibility to the isolates as Pseudomonas species contain innate resistance to a vast array of antibiotics owing to its low outer membrane permeability which acts as a barrier to the uptake of antibiotics and substrate molecules, as well as its active efflux pump system that rapidly pumps the antibiotics out of the bacterial cell (30).
Isolates 18 and 71 were identified as Pseudomonas donghuensis and Pseudomonas protegens respectively. A marine isolate of Pseudomonas sp. has been proven to have the capability of synthesising secondary metabolites that can inhibit a broad spectrum of microorganisms, including K. pneumoniae, MSSA, MRSA, Shigella flexneri, P. aeruginosa and B. subtilis (31). P. donghuensis has demonstrated its ability to excrete a large number of siderophores, including fluorescent pyoverdine and non-fluorescent 7-hydroxytropolones, in numerous studies (32; 33). Under the iron-deficient condition, siderophores act as an iron chelator to increase iron uptake, limiting the amount of iron availability to phytopathogens. It has also demonstrated its antibacterial ability by inhibiting the growth of several virulent species of soft rot plant bacteria from the Dickeya and Pectobacterium genera (33; 34). Due to its iron-chelating properties, P. donghuensis is a potential candidate of biocontrol agent as it can restrict the proliferation and root colonisation by phytopathogens. Isolate 71 was classified as P. protegens, which had been reported that it had plant-protecting ability against a range of soil-borne phytopathogens (35). P. protegens produce secondary metabolites with broad-spectrum antibiotic activity, such as 2,4-diacetylphloroglucinol (DAPG), pyoluteorin, hydrogen cyanide and pyrrolnitrin, where DAPG has been known for its antibiotic and antifungal ability due to its toxicity against a wide range of bacteria, fungi, oomycetes and plants (35; 36). In a study, it was reported that a strain of P. protegens was active against E. coli, S. aureus as well as Bacillus cereus, whereas in another study, P. protegens isolated from treated wastewater had demonstrated multidrug-resistance against ceftazidime, cefepine, ticarcillin-clavulanic acid and aztreonam (37; 38). This shows that P. protegens possess antibacterial activity against numerous plant pathogens, although limited studies have been carried out in human pathogens.
Of all of the isolates, isolate 107 showed the strongest activity, especially against Gram-positive bacteria. Together with isolates 102 and 104, they were classified as part of the genus of Bacillus species. It is not surprising that three microorganisms isolated belong to the genus Bacillus as they have been known as producers of a wide range of biologically active molecules (39). Bacillus sp. are Gram-positive bacteria which are known to generate spores under adverse conditions to ensure their survival in adverse conditions, and both marine and terrestrial isolates of Bacillus have displayed capability of producing potent metabolites (13; 40). Peptide antibiotics produced by Bacillus sp. can be classified into two major groups, (i) non-ribosomally synthesised, such as bacitracin, gramicidin, surfactin, tyrotricidin, and (ii) ribosomally synthesised, such as bacteriocins (12; 41). In the present study, isolates 102 and 104 were identified as either Bacillus subtilis or Bacillus velezensis.
In a few studies, it has been shown that B. subtilis isolates have the ability to synthesise protein antibiotics, like subtilin, subtilosin and sublancin, which are mostly active against Gram-positive bacteria (42-44). This is consistent with our results in the perpendicular streak test where isolates 102 and 104 only inhibited the growth of Gram-positive test organisms. Subtilosin A is one of the antibiotics synthesised by B. subtilis, it has demonstrated to be active against a wide range of bacteria, including Listeria monocytogenes, E. coli, S. aureus, and K. pneumoniae, although its effect on human pathogenic
bacteria was moderate (45). Besides antibacterial activity, both B. subtilis and B. velezensis also produce antifungal lipopeptides, such as iturin, bacillomycin, and fengycin, where strains of B. subtilis have demonstrated strong antifungal properties against Aspergillus ochraceus, Penicillium roqueforti, and strains of B. velezensis were active against Alternaria panax and Magnaporthe oryzae. (12; 46; 47).
16S rRNA sequence of isolate 107 has suggested it to be B. pumilus. Limited studies have been conducted using B. pumilus, especially against human pathogens. However, it was found that one strain of B. pumilus produced pumilicin 4, a bacteriocin with good heat and pH stability, and has activity against a vast array of Gram-positive bacteria, including MRSA and VRE (48). In the present study, isolate 104 has demonstrated antibacterial action only against Gram-positive bacteria, which is in contrast to a few studies which have reported that B. pumilus showed inhibition against both Gram-positive (eg: MSSA) and Gram-negative bacteria (eg: E. coli) (49; 50). This shows that B. pumilus could potentially produce more than one type of bioactive compounds (eg: pumilicin, surfactin, iturin) which are active against different classes of bacteria (46; 50). The strain of B. pumilus isolated in our study also showed powerful inhibition against different strains of E. coli. Bacillus species is widely investigated for its effectiveness against pathogenic fungi and plant disease, but only a few studies have been done on human pathogenic bacteria which hinder us from making a constructive conclusion that can contribute to the drug research and development.
In our study, the concentration of the active compounds synthesised by the supernatant was fairly low, which may result in the loss of the antibacterial activity. The culture filtrates can be further purified using solvent extraction procedure in the future to enhance the antibiotic extraction (51). Furthermore, upcoming research should focus on the identification of the bioactive metabolites produced and the extraction of the active compounds synthesised. Scientists should also come up with methods to mass produce the useful products for pharmaceutical purposes. Plenty of research have been conducted on phytopathogens using the isolates that have been found in this study and an emphasis on their effects on human diseases or infections should be discussed in the future. All in all, a number of useful isolates have been found in the present study, showing that the aquatic environment is a potential source to be employed to isolate antibiotic-producing microorganisms.
5. Conclusion
Our findings in the present study have demonstrated that water can harbour microorganisms that are capable of producing antibiotics active against a variety of bacteria, including both Gram-positive and Gram-negative bacteria. Environmental sources apart from soil should be explored for the search of potential bioactive compounds to treat infections, especially those caused by multi-drug resistant pathogens. It is essential to improve on current standard drugs as well as to carry out incessant screening of useful microbial products for the development of new therapeutic agents to counteract drug resistance in the microbial population. In addition, more effective and user-friendly methods of screening and extracting useful metabolites should be invented to ease the process. However, the development of new classes of antibiotics cannot completely prevent the expansion of resistant strains of pathogenic bacteria and hence, antibiotic stewardship and patient education regarding the appropriate use of antibiotics come into play to slow down the progress of the spreading of antibiotic resistance.
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