Evaluation of the antibacterial and anticancer activities of marine Bacillus subtilis ESRAA3010 against different multidrug resistant Enterococci (MDRE) and cancer cell lines

Fifty nine isolates belonging to six species of Enterococci namely, Enterococcus faecalis, Enterococcus faecium, Enterococcus raffi nosus, Enterococcus durans, Enterococcus mundtii and Enterococcus avium (n = 35, 15, 4, 3, 1 and 1 isolates, respectively) were obtained from diff erent clinical specimens including urine, pus, blood, wound, sputum and synovial fl uid. The highest numbers of enterococci were recorded from the pus (20 isolates, 33.90%) followed by urine (12 isolates, 20.34%) while the lowest frequency was observed with synovial fl uid samples (2 isolates, 3.39%). These isolates showed diff erent multidrug resistant patterns with the lowest resistant for linezolid (n = 5, 8.48%), followed by teicoplanin (n = 14, 23.73%) and vancomycin (n = 20, 33.90%) while they exhibited the highest resistant against penicillin (n = 53, 89.83%), oxacillin (n = 50, 84.75%), erythromycin (n = 49, 83.05%) and streptomycin (n = 47, 79.66 %). On the other hand, a free living marine bacterium under isolation code ESRAA3010 was isolated from seawater samples obtained from the fi shing area Masturah, Red Sea, Jeddah, Saudi Arabia. The phenotypic, chemotaxonomic, 16S rRNA gene analyses and phylogenetic data proved that isolate ESRAA3010 is very close to Bacillus subtilis and then it was designated as Bacillus subtilis ESRAA3010. It gave the highest antagonistic activity against all clinical Enterococcus faecalis, Enterococcus faecium, Enterococcus raffi nosus, Enterococcus durans, Enterococcus mundtii and Enterococcus avium isolates under study with minimum inhibitory concentration (MIC) ranged from 4 to 56 μg/mL, 4 to 12 μg/mL, 4 to 8 μg/mL, 4 to 8 μg/mL, 8 μg/mL and 4 μg/ mL, respectively as well as minimum bactericidal concentration (MBC) (8 to 64 μg/mL, 4 to 16 μg/mL, 4 to 12 μg/mL, 4 to 16 μg/mL, 12 μg/mL and 8 μg/mL, respectively). Moreover it showed anti-proliferative activity against colon (HCT-116), liver (HepG-2), breast (MCF-7) and lung (A549) carcinomas with IC50 equal to 39, 50, 75 and 19 μg/mL, respectively which indicates its prospective usage in the upcoming decades.


Introduction
Enterococcus species are Gram-positive cocci usually present in the human and sometimes become severely infectious agents; particularly they are frequently ind novel mechanisms to evade the antibiotics treatments [1,2]. They developed multidrug resistance to different antibiotics in common use (MDR) with markedly increasing prevalence by contacting with contaminating surfaces and apparatus or infected persons [3]. They gain increasing concern due to their facility for withstanding the in luence of various antibacterial agents, accordingly limit the drug of choices and leads to higher mortality and morbidity [4]. Therefore, inding of alternate powerful, inexpensive and harmless natural agents against multidrug resistant bacteria can be potent way for solving this serious global problem [5][6][7]. Marine bacteria are promising reservoirs of diverse effective bioactive natural products and many of them are being used in chemotherapy to treatment human diseases especially with the continuing need for new potent compounds against drug-resistant pathogens and https://www.heighpubs.org/hjb 019 https://doi.org/10.29328/journal.abb.1001018 managing of distressing cancers with high selective activity and less toxicity [8,9]. Red sea host diverse and abundant free living microorganisms have the ability to produce promising bioactive marine natural products [10]. Marine Bacillus species produce multipurpose biologically active compounds including lipopeptide, polypeptide, macrolactone, fatty acid, polyketide and isocoumarin metabolites [11,12] with wide variety of antifungal, antibacterial, antioxidant and antiproliferative activities [13,14]. The capability of Bacillus species to biosynthesis different antibiotics with varied structures has been demonstrated by numerous genetic studies, and genetic analysis of Bacillus strains has shown that about 8% of the genome is dedicated to antibiotic synthesis [15,16]. This work was aimed to assess the incidences and distributions of Enterococci among patient admitted to El-Demerdash teaching hospital (Cairo, Egypt) along with determining their antibiotic sensitivity pro iles against a panel of antibiotics to select the multidrug resistant Enterococci (MDRE). Moreover, this study aimed to explore potent marine Bacillus species that able to produce biologically active substances against these clinical isolates and diverse malignant cell lines including colon, liver, breast and lung carcinomas.

Clinical specimens
Different samples including urine, pus, blood, wound, sputum and synovial luid (5 samples for each) collected from patients ranging from 1 to over 60 years old (

Marine samples and isolation of marine Bacillus species
Ten samples of seawater from the ishing area Masturah, Red Sea, Jeddah, Saudi Arabia (latitude: 23°5031.4600N/ longitude: 38°49017.5200E) were collected in August 2018 at different depths in sterile screw cap bottles under iced conditions. Collected samples were taken to the laboratory, gathered and processed instantly. The isolation medium and process were prepared and done following the method of Ivanova, et al. [28] by plating serial dilutions of water sample individually to Petri dishes of nutrient agar (NA) supplemented with 100 μg/mL nystatin and cycloheximide, incubated at 30 °C for 3 days and recognized bacterial single colonies were transferred periodically to NA at 30 °C for 48 h and included in this study. Bacterial isolates were preserved on NA at 4 °C till using.

Antibacterial activity of marine bacterial isolates against diff erent MDR-Enterococci
Muller Hinton agar (MH) plates were inoculated with the clinical MDR-Enterococci isolates, individually and paper assay discs loaded with 30 μL of marine bacterial isolates supernatants separately were plated on the top of inoculated medium, incubated at 37 °C for 24 h and then antimicrobial activity of bacterial isolates was determined against the MDRE isolates by using the routine diffusion plate technique via evaluating the inhibition zone diameters in mm [data determined as no antagonistic activity (-),weak antagonistic activity (˂10 mm, +), moderate antagonistic activity (10 -15 mm, ++) and excellent antagonistic activity (16 -˃20 mm, +++)] [11,29].

Phenotypic and chemotypic properties of marine bacterial isolate ESRAA3010
ESRAA3010 strain was speci ied by conventional taxonomic procedures by means of API 20E and API 50CH methods along with other phenotypic and chemotypic characters [29][30][31][32]. Bacillus subtilis ATCC 6051T used as standard strain.

Bacillus subtilis ESRAA3010 was inoculated into
Erlenmeyer lasks containing tryptic soy broth medium then incubated for 24 h at 30 °C and 100 rpm, after incubation period the fermented broth of ESRAA3010 strain (5 L) was collected and the supernatant was separated under reduced pressure and then extracted twice with ethyl acetate (1:1, pH 4.5 under overnight shaking). The EtOAc extract obtained evaporated to dryness giving light brownish oil (6.41 g).
Determination of minimum inhibitory (MIC) and minimum bactericidal concentrations (MBC) of B. Subtilis ESRAA3010 extract against Enterococci strains.
The MIC and MBC of the extracted secondary metabolites were estimated in μg/mL against MDRE isolates as described by Cappuccino and Sherman [39] and Lavermicocca, et al. [40].
Cell viability test was measured by the mitochondrial dependent reduction of yellow MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide) to purple formazan [41,42]. HCT-116, MCF-7, HepG-2 and A-549 were achieved from Cancer Center, Karolinska Institute, Stokholm, Sweden; DMEM medium, RPMI 1640 medium and 1% antibioticantimycotic (10,000 U/mL potassium penicillin; 10,000 μg/ mL streptomycin sulfate and 25 μg/mL amphotericin B) were achieved from Life Technologies/Gibco (Grand Island, NY, USA). The effect of diverse amounts of B. subtilis ESRA-3010 extract ranging between 25 and 300 μg/mL on the cytotoxicity and cell viability of these tumor cell lines was measured as stated by Mosmann [41] and Wilson [42] in 96well microplate at 37 °C under 5% CO 2 using a water jacketed CO 2 incubator (Sheldon, TC2323, Cornelius, OR, USA) for 48 h followed by aspirating medium, adding 40 μL MTT salt (2.5 μg/mL) to wells, incubating for 4 h at 37 °C as mentioned above, ending the reaction along with dissolve the resulted crystals by adding 200 μL of 10% sodium dodecyl sulfate to wells and thus incubating overnight at 37 °C. The absorbance was estimated at 595 nm by the microplate multi-well reader (Bio-Rad Laboratories Inc. model 3350, Hercules, California, USA). The IC 50 (the amount of the extract that decreased cell viability by 50%) compared to the control (wells contain only solvent without any extract) were determined using SPSS 11 program. Percentages (%) of cell viabilities were estimated based on the formula: [(Absorbance of treated cell lines with extract / Absorbance of negative control) -1] X 100.

Isolation, characterization and occurrence of clinical Enterococci isolates
A total of ifty nine isolates of Gram-positive Enterococci isolates including 16, 11, 8, 5, 10 and 9 isolates were isolated from the age groups 1-6, ˃6-12, ˃12-20, ˃20-40, ˃40-60 and over 60 years old ( Table 1). The largest number of MDRE (27.12%) was collected from ages ranging from 1 to 6 years. Furthermore, 33 isolates (55.93%) were obtained from male samples while 26 isolates (44.07%) were obtained from female participants (Table 1) from El-Demerdash teaching hospital. The presence of resistant Enterococci isolates in human societies acts as a source for hospital infections [1]. In line with our results, Karna, et al. [4] revealed that the predominance of Enterococci isolates were achieved from age group 0-10 (20.9%), after that age group 20-30 (19.8%) along with detection of the high incidence of infection in male participants. Enterococci are recognized as unique reasons of hospital infections in patients with weakened immune systems. On the other hand, Table 2 showed the different department from which specimens were collected, 17 (28.81%); 16 (27.12%); 8 (13.56%); 7 (11.86%); 5 (8.48%); 4 (6.78%); 1 (1.70%) and 1 (1.70%) isolates were obtained from I.C.U (intensive care unit), surgery, diabetic, skin and venereal disease, hematology, emergency, E.N.T (ear, nose and throat) and neurology departments, respectively which indicating that hospitalization in the intensive care unit is an important risk factor for MDRE colonization and occurrence. Similar distribution observation were reported previously in resistant Enterococci collated from different humanoid  In agreement with our results, Karna, et al. [4] analyzed ninety one isolates of Enterococcus obtained from numerous clinical samples, among them the highest Enterococci isolates incidence reported from urine then pus and blood (61.5%, 19.8% and 5.5%, respectively).

Antibiotic susceptibility pattern in the Enterococci isolates
In the current work, the susceptibility patterns in the infections [19], clinical samples in Kashmir; North India [43], health care setting [3], tertiary care center of Eastern Nepal [4] and Turkey [1].

Characteristics of clinical Enterococcus strains
Following the standard guideline of species speci ication [2,[17][18][19][20], six different species of Enterococci isolates were identi ied, namely E. faecalis, E. faecium, E. raf inosus, E. durans, E. mundtii and E. avium (n = 35, 15, 4, 3, 1 and 1, respectively, Table 3. All isolates were Gram-positive, non-motile, positive for Voges-Proskauer reaction, negative for catalase activity and β-glucoronidase, produce acid from sorbitol and lactose as well as grown at 45 °C and pH 9.6. Only E. mundtii was able to produce yellow pigment which is a key feature of this species in addition to produce acid from trehalose; E. durans was the only negative strain for acid production from rhamnose, melezitose, arabinose and mannitol and E. avium was the only strain failed to produce acid from amidon but gelatin hydrolysis ability was only recorded in E. faecalis strains ( Table 3). The other details of phenotypic characterizations and chemotypic features of these species obtained in the current study are presented in table 3. Interestingly E. faecalis and E. faecium (Figures 1 and 2) together constitute more than 84.74% of total isolates and their increased proportion in current work might be attributed to their capability to attain and developed different resistance patterns against multiple antibiotics. In the majority of previous reports, E. faecalis has been documented as the main Enterococci species, followed by E. faecium. For example Karna, et al. [4] reported that among seven different identi ied species of Enterococci the highest frequency of strains among total isolates was reported for E. faecalis followed by E. faecium and together, they made up over 90% of the total isolates. Nevertheless, this result is higher than other studies [44,45]. These differences in bacterial occurrence can be attributed to the differences in geographic site, sample, period of hospitalization, and drugs used [46].

Isolation and evaluation of antagonistic activity of diff erent marine Bacillus isolates against diff erent Enterococci strains
Twelve isolates of free living marine Bacillus species were isolated, cultivated, and then their antagonistic activity toward Enterococci strains under study was evaluated and tabulated in table 6. Data clearly indicated that the isolate under the isolation code ESRAA3010 was the hyperactive strain that showed inhibitory activity against all Enterococci strains under study with inhibition power ranged from good (++) to excellent (+++) followed by ESRAA3012 isolate showed inhibitory activity toward 93.22% of all isolates (   bioactivities including antibacterial, antifungal, antitumor and antioxidant properties. Consequently, there is a potential for using these marine Bacillus species metabolites as promising medicines and in other biological treatments.

Anti-proliferative activity of B. subtilis ESRAA3010 extract
In vitro anti-proliferative activity of EtOAc extract of B. subtilis ESRAA3010 strain on different tumor cell lines was evaluated by MTT cell viability assay and illustrated in igure 5. Data clearly indicated that the cells viability of A-549 carcinoma was completely inhibited after treatment with ESRAA3010 extract at a concentration of 50 μg/mL ( Figure 5). Furthermore, HCT-116 and HepG-2 carcinomas totally inhibited at 100 μg/mL but the growth of MCF-7 cell line was completely inhibited at a concentration of 200 μg/mL with IC 50 equal to 39, 50, 75 and 19 μg/mL against colorectal (HCT-116),  [7,12,14] reported the anticancer activities of Bacillus species extracts on a large number of carcinomas. Also, Zhao, et al. [16] reported that treating carcinomas with products from cultured Bacillus strains had signi icant inhibitory effects on ovarian and colorectal carcinomas proliferation in a dose dependent manner.

Conclusion
Marine ecosystems in Egypt have proven to be proli ic resource for various types of marine bacteria, especially Bacillus species that produce different stimulating biological metabolites against infectious agents which widely distributed as multidrug resistant Enterococci strains and cancer.