1. Introduction
Antibacterial resistance is a major public health problem worldwide, and is responsible for the mortality of 1 million people annually [
1].
Staphylococcus aureus is a pathogen that can cause serious infection, and several antibacterial drugs have become resistant by this pathogenic bacteria, especially multidrug-resistant
S. aureus. It is recognized by World Health Organization (WHO) as high priority in terms of urgency in the search for new antibiotics required for clinical situations [
2,
3,
4]. Methicillin-resistant
S. aureus (MRSA) is a clinically important pathogen that can tolerate many effective agents such as cephalosporin and fluoroquinolone. The situation regarding antibiotic resistance is deteriorating as a result of antibiotic misuse in agriculture, veterinary, and human medication, and the acquisition of bacterial resistance genes [
5,
6]. Meanwhile, the number of new antibiotics that are being launched in the market is limited. Most antibiotic drugs are derived from microorganisms, and are produced as a defense mechanism against external stimuli. Novel classes of drugs have been reported from soil bacteria, which are promising sources for drug discovery. This raises interest in the need to create different strategies for resolving this problem, and the discovery of new antibiotics is one of the effective approaches.
Soil is the most advantageous source for antibiotic discovery because of the high abundance of microbial communities. A number of antibacterial agents are produced from bacteria, and they are secondary metabolites for defense against other microorganisms or cell signaling within and between microbial communities. Soil bacteria are a promising resource that accounts for about 70% of antibiotics [
7,
8]. A number of these substances predominantly originated from soil
Streptomyces sp.,
Bacillus sp., and
Brevibacillus sp., which contain many biosynthetic gene clusters for streptomycin, daptomycin, tetracycline, and polymyxin [
9,
10]. Generally, the production of antibacterial substances is limited by their gene clusters. There are several approaches to improve the amount and potency of antibacterial activity through physical (gamma rays or UV radiation) and chemical (nitrous acid or
N-methyl-
N′-nitro-
N-nitrosoguanidine) mutagenesis. UV radiation is a convenient and readily applied method for increasing the capabilities of bioactive compounds via random mutagenesis [
11,
12]. The present study aimed to isolate and search for bacteria with anti-MRSA activity from soil samples in Thailand. Potential strains were studied in terms of the kinetics of antibacterial substance production, and its stability was assessed under deleterious conditions. The candidate isolate was mutated by UV radiation, and the resulting activity against
S. aureus and its antibiotic-resistant strains (MRSA) was evaluated. Drug susceptibility and the strain stability of the mutant were also determined.
4. Discussion
Heat treatment was performed on the terrestrial soil for the reduction of heat-labile microbes. Heat-tolerant bacteria were isolated in the search for antibacterial activity. Antibacterial properties exist in several types of bacteria as a result of nutrient competition between species under environments inadequate for survival [
24]. Predominant antibacterial producers are of the genera
Streptomyces,
Bacilli, and
Brevibacillus, which are heat-stable microorganisms. In this study, three potential isolates (Pr-18, Pr-82, and SPR-20) that exhibited high activity against indicator pathogens (
S. aureus TISTR 517, MRSA isolate 142, 1096, and 2468) were assigned to the
Bacillus and
Brevibacillus genera. Several studies have reported that
Bacillus sp. and
Brevibacillus sp. can be isolated from soil environments, and most of them are non-pathogenic agents that produce active peptide substances against the human pathogenic bacteria (
S. aureus and MRSA) [
24]. Loloathins, laterocidin, and bogorols are non-ribosomal synthesized antibacterial peptides from
Brevibacillus laterosporus, whereas bac-GM100 is a ribosomal-synthesized antimicrobial peptide from
B. brevis that showed broad-spectrum activity against clinical pathogens of
S. aureus,
P. aeruginosa, and
Candida tropicalis [
25,
26,
27,
28,
29]. The most potential isolate in this study,
Brevibacillus sp. SPR-20, was shown to be a promising target for inhibiting
S. aureus and MRSA. It produced the maximum anti-MRSA activity after 24 h of incubation, which corresponded to the stationary phase of the growth curve. The culture supernatant had a broad spectrum of activity with comparable potency to vancomycin, whereas cefoxitin and oxacillin were ineffective in treating MRSA strains. These inhibitory effects were also demonstrated in other
Brevibacillus sp., such as crude extract and brevibacillin prepared from
B. laterosporus strain SA14 and
B. laterosporus strain OSY-I, respectively [
27,
28].
B. halotolerans strain LAM0312 is a new species in this genus, and it was shown to be highly similar to
Brevibacillus sp. SPR-20 based on its 16S rRNA sequence [
29]. However, there is no scientific literature on the activity of
B. halotolerans, whereas
B. laterosporus strain DSM25 was demonstrated to have a close phylogenetic relation to SPR-20 and can produce antimicrobial compounds with predicted biosynthetic gene clusters of brevibacillin, paenibacterin, and petrobactin [
30]. Although the active substances from SPR-20 have not been fully investigated, these results provide an opportunity to search for drug candidates to combat antibiotic-resistant pathogens.
Bacillus sp. Pr-18, found in this study, has a close relationship with
B. sonorensis strain NBRC 101234. Previous studies have presented the isolation of
B. sonorensis MT93 and identified an antimicrobial peptide named sonorensin with a molecular mass of 6.27 kDa. It was demonstrated to be a potent antibacterial agent against
S. aureus,
P. aeruginosa, and
Listeria monocytogenes [
31].
Brevibacillus sp. Pr-82 was adjacent to
B. brevis strain NBRC 15304, and several studies have demonstrated small antibacterial peptides from those strains. Tostadin from
B. brevis strain XDH was isolated as a novel substance with a broad spectrum against
S. aureus and
E. coli [
31,
32].
The anti-MRSA substances from
Brevibacillus sp. SPR-20 are antimicrobial peptides which the remaining activity was more than 90% under various conditions, such as involving temperature (60–121 °C), proteolytic enzymes (proteinase K, trypsin, and α-chymotrypsin), surfactants (SDS and Triton X-100), and a wide pH range (2–14). Conversely, the peptide antibiotic nisin was more sensitive to temperature, proteolytic enzymes, and a basic pH. This result is consistent with previous stability studies, demonstrating that nisin was stable at −18 °C but substantially degraded at temperatures above 25 °C [
33]. The antibacterial activity of nisin was also shown to be diminished when treated with proteases or a pH above 10 because of the presence of enzymatic and chemical cleavage sites in the peptide antibiotic [
34,
35]. The activity of SPR-20 and nisin was enhanced when combined with surfactants. However, the addition of SDS to nisin reduced antibacterial activity when compared to SDS alone. This might result from the charge incompatibility between cationic nisin and anionic SDS complexes [
36]. The stability profile of SPR-20 was consistent with other studies, showing that some bacteriocin-like inhibitory substances (bacillocin Bb, brevicin AF01, entomocin 9, paenibacterin, and pumilicin) were tolerant to those conditions [
37,
38,
39,
40,
41]. These results show that the strain SPR-20 could produce anti-MRSA substances with a stable and broad spectrum of antibacterial activity.
Generally, UV mutagenesis is used to enhance the production of bioactive substances through dimerization of thymine bases, forming crosslinks in DNA that might result in base transversion, deletion, frameshifts, or transitions from GC to AT [
12]. Tolerant bacterial cells would use repair mechanisms to relieve genetic abnormalities through potentially generating mutagenesis, leading to selection and cell survival. UV-mediated mutagenesis is a useful, classical approach which is advantageous when genetic information of the microbial strains or biosynthetic pathway is lacking. It is assumed that random genetic alteration can bypass regulatory controls and checkpoints, contributing to the overproduction of active substances [
42]. In this study, the application of UV mutagenesis led to an increase in the overall production of antibacterial activity. Mutagenesis might lead to enhanced production of the same active substances. Alternatively, it might also activate the cryptic biosynthetic gene cluster of other antibacterial compounds that are not expressed under normal conditions. The mutant strains might then produce multiple active compounds. These two possibilities result in an increased zone of inhibition on the agar growth medium. The drawback of this method is the random selection of strains, and beneficial mutations may depend on several factors such as radiation power, frequency, and exposure time of mutagen treatment, type of bacterial culture, or conditions of treatment and post-treatment. The UV-treated
Brevibacillus sp. SPR-20 showed that the number of surviving cells decreased after prolonged UV exposure time, and they were sterilized within 4 min. The cell morphology and growth rate between mutant and wild-type strains did not differ, and both spores appeared to have unique characteristics, with rough and fold envelopes on the surface.
Brevibacillus sp. SPR-20 was more sensitive to UV irradiation than other
Brevibacillus sp., such as
Brevibacillus sp. strain AS-S10-IIM201 and
Brevibacillus borstelensis R1, which were more tolerant to UV irradiation for several minutes [
43,
44]. The agar overlay method was used as the pre-selection technique to screen several mutated isolates with higher antibacterial activity. An agar diffusion assay was carried out to determine antagonist effects against indicator pathogens for the remaining desired strains. The mutant M201 exhibited significantly higher activity (109.88–120.22%) when compared to the wild-type strain. This emphasizes UV mutagenesis as a potential approach for strain improvement. Similar observations were also reported in a number of strain improvement studies:
Bacillus licheniformis was irradiated by UV, and could produce bacitracin with 1.85-fold higher activity [
45]. A previous study also presented that the UV-treated
B. brevis strain FJAT-0809-GLX yielded a positive mutant rate of 7.8%, and enhanced the production of ethylparaben with higher activity toward pathogens [
46].
Furthermore, the mutant M201 was a stable strain as judged by the consistent antibacterial activity throughout many generations. This mutated isolate was more susceptible to some antibiotics (ceftriaxone) than the wild-type strain. This implies that UV radiation activated adaptive or protective responses and altered the bacterial cell wall or membrane. This resulted in an increase in the secretion of active substances into the culture medium and a higher sensitivity to antibiotic drugs that target cell wall synthesis [
47]. However, further experiments are required to elucidate the susceptible consequences and mechanism of action of these anti-MRSA substances for drug development in the antibiotic-resistant era.