Evaluation of antimicrobial activity and in vitro safety of the methanolic extract of Streptomyces manipurensis soil isolate H21 for potential industrial applications

Streptomyces manipurensis isolate H21 was recovered from a soil sample in Cairo, Egypt. Cell-free culture supernatant of Streptomyces manipurensis isolate H21 previously showed antifungal and broad-spectrum antibacterial activity against some Gram-positive, Gram-negative, MDR, and ESBL producer pathogens as well as some reference strains. The present study aimed at investigating antimicrobial and cytotoxic activities of its crude methanolic extract. The antibacterial activity of the crude methanolic extract was determined using broth-dilution method against Staphylococcus aureus ATCC 43300, Klebsiella oxytoca ATCC 700324, Klebsiella pneumoniae ATCC 700603, Klebsiella pneumoniae ATCC BAA-1705 and 2 MDR uropathogens. The minimum inhibitory concentrations (MICs) and the minimum bactericidal concentrations (MBCs) of the crude extract against the tested bacteria were in the range of 5-10 mg/mL and 10 mg/mL, respectively. The results revealed that the CD50 value of the extract was 1.17 mg/mL, against Caco-2 cell line, indicating in vitro safety and low cytotoxicity. Accordingly, Streptomyces manipurensis isolate H21 would be an excellent source of relatively safe and potent antibacterial agent.


INTRODUCTION
Microbial infections, with the emergence of multiple drug resistant (MDR) microorganisms, remain the second leading cause of death worldwide [1]. To overcome this crisis, an attempt has been made by multiple researchers to improve the existing antibiotics and/or discover novel antibiotics with improved effectiveness [2].
Screening microorganisms for the production of potent antibiotics have been intensively undertaken for many years by researchers [3]. Among the screened microorganisms, Streptomyces which represent an outstanding source of many antibiotics (around 80% of total antibiotic production) and active secondary metabolites [4,5].
Our previous work demonstrated the richness of antibiotic-producing Streptomyces in the Egyptian soil [6]. Streptomyces manipurensis soil isolate H21 which was previously recovered from Cairo, Egypt proved to have potent anti-ESBL activity and broad-spectrum antimicrobial activity [6]. The present work aimed to evaluate the antifungal and antibacterial activities of crude methanolic extract of cell-free culture supernatant (CFCS) of Streptomyces manipurensis soil isolate H21 against several references and MDR microorganisms and to determine its in vitro safety on a specific cell line.

Other reference strains
The used reference strains from the American Type Culture Collection (ATCC) were kindly provided by Prof. Dr. Mahmoud Abd El-Megead Yassien, Professor of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University. Other reference strains were provided by the Regional Center for Mycology and Biotechnology, Al-Azhar University, Cairo, Egypt. The reference strains were preserved at -80 °C in glycerol stock vials (Table 1).

Clinical pathogenic isolates
Two multiple drug resistant (MDR) bacterial uropathogens from El-Demerdash hospital were kindly provided by Ann Elshamy, assistant lecturer of Microbiology and Immunology, Faculty of Pharmacy, Ain Shams University (Table 2).

Cell line
Colorectal adenocarcinoma derived from human colon (Caco-2 cell line) was used for determination of cytotoxic activity of methanolic extract of Streptomyces manipurensis isolate H21. It was obtained from VACSERA, Cairo, Egypt.

Preparation of cell-free culture supernatant
This was carried out as previously described [8] with some modifications as follows, one pure colony of Streptomyces manipurensis isolate H21, grown on starch nitrate agar for 7 days, was used to inoculate 10 mL of the seed medium contained in 100 mL flask. The flask was then incubated at 28 °C and 200 rpm for 72 h. The culture obtained was used to inoculate the main culture (inoculum size 5% v/v) then incubated at 28 °C and 200 rpm for another 72 h. After incubation, the obtained culture broth was centrifuged at 15938g for 15 min to separate mycelium from the supernatant, and the cell-free culture supernatant (CFCS) obtained was harvested, filtered through 0.22 μm pore size cellulose membrane filters (CHEM LAB, Barcelona, Spain) and used for further study.

Extraction of the bioactive metabolite from CFCS
About five hundred milliliters of CFCS were dried by lyophilization. Then the dry lyophilized CFCS was extracted with distilled methanol (HPLC grade) for 2 h to ensure complete extraction. Finally, the extract was completely evaporated in vacuo using R3000 Rotary evaporator (Heidolph instruments GmbH and Co. Schwabach, Schwabach, Germany) at 40 °C until complete dryness.

Screening the methanolic extract for its antibacterial activity 2.5.1. Agar well diffusion method
The methanolic extract of lyophilized CFCS was tested for its anti-ESBL activity against K. pneumoniae ATCC 700603 using agar well diffusion as described formerly [9,10] with minor modifications. Methanol was used as negative control. In this experiment, 150 μL of 10 mg/mL dried extract was tested and the obtained zones of inhibition (IZ) were measured in millimeter (mm).

Disc diffusion method
The antimicrobial activity of the crude extract was estimated via comparison with some standard commercial antibiotics using disc diffusion method. The crude extract (40 µL) was gradually loaded on 6 mm sterile disc (Becton Dickinson, NJ, USA) and air dried under sterile conditions at room temperature. Three standard commercial antibiotic discs (Oxoid ® , Cheshire, UK) were used for the comparative study as positive controls. The used antibiotic discs were meropenem (10 µg/disc), ampicillin/ sulbactam (20/10 µg/disc) and amoxiclav (30 µg/disc). The discs were placed on the surface of Mueller Hinton agar (MHA) plate which was surface inoculated with ESBL producer K. pneumoniae ATCC 700603 (0.5 McFarland). The plates were incubated for 24 h at 37 °C and the produced inhibition zone around each disc was measured (mm). A negative control disc was loaded with methanol. The assay was done in duplicate. The antibacterial activity was evaluated by measuring the diameters of zones of inhibition in mm [11].

Minimum inhibitory concentrations (MICs)
The methanolic extract of the lyophilized CFCS was further tested against MDR uropathogens and some reference strains to find its minimum inhibitory concentrations (MICs) and minimal bactericidal concentrations (MBCs). The used bacteria were listed in Table 3. According to the Clinical and Laboratory Standards Institute (CLSI) guidelines, the MICs of the extract were determined using the broth microdilution method. Each well was first filled with 100 μL Mueller-Hinton broth (Difco, NJ, USA). Then, 100 μL of the extract was added to the first well in the row and two-fold serially diluted. Each well contained 100 μL extract in the dilution series. Suspension of the test organism (0.5 McFarland) was prepared and 10 μL of the prepared suspension was added into each well, except for the negative control wells. Column 11 and 12 were used for solvent control and positive control, respectively. The microtiter plate was incubated at 37 °C for 18-20 h and the MIC was determined by selecting the lowest concentration showing no visible growth of the tested organism. All experiments were performed twice [12].

Minimum bactericidal concentrations (MBCs)
To determine the MBCs, samples (10 μL and 50 μL) from wells showing no visible growth in the MIC assays were subcultured onto nutrient agar plates. The plates were incubated at 37 °C for 24 h. After incubation, the concentration that caused 99.9% killing of the original count was considered as the minimum bactericidal concentration (MBC) according to CLSI, 2017 guideline. Both MIC and MBC for the test bacteria were determined in triplicate. This was done as described previously [12,13,14]. When the ratio of MBC/MIC is ≤ 4, the active extract was considered as bactericidal otherwise as bacteriostatic. If the ratio is ≥ 16, the extract was considered ineffective [15].

Screening the extract for its antifungal activity
Methanolic extract of lyophilized supernatant was tested for its anticandidal activity using disc diffusion method. Discs were impregnated with 40 µL extract. The concentration of the used extract was 25 mg/mL. Tested species of Candida were mentioned in Table 4. Antifungal activity was tested using agar well diffusion against some fungi mentioned in Table 5. Cups (6 mm diameter) were loaded with 100 µL of 10 mg/mL of extract.

Cytotoxic activity of extract against Caco-2 cell line using MTT assay
MTT assay was carried out as described by Saliba et al. [16].

Maintenance of the Caco-2 cell line
Caco-2 cell line was grown in 50 mL sterile tissue culture (TC) flasks and subcultured every 3 days as follows: The spent RPMI with fetal bovine serum was discarded and cells were washed twice with phosphate buffered saline. The cells were then detached by incubating with 2 ml of the trypsin solution at 37 °C for 5-10 min. After cell detachment, 20 mL of fresh maintenance TC medium were added to the flask and 10 mL aliquot of this cell suspension was then transferred into another new TC flask (ATCC manual). The two flasks were then incubated at 37 °C in the presence of 5% CO 2 and humid atmosphere for 48 h.

Preparation of tissue culture plates
Caco-2 cell line maintained in the tissue culture flasks were trypsinized as described previously and suspended in propagation tissue culture medium to a count of 10 5 living cells/mL. This count was determined by using a hemocytometer (Shanghai, China) and cell viability was confirmed by staining with trypan blue solution. After gentle pipetting of the cell suspension for even distribution, aliquots of 100 µL were transferred to the wells of 96 well microplates (flat bottom sterile tissue culture plate). The plate was then incubated at 37 °C in the presence of 5% CO 2 and a humid atmosphere for 24 h to form a confluent monolayer (approximately contains 5 x 10 4 cells/well).

MTT assay
Caco-2 monolayer was prepared, washed twice with PBS then two aliquots of 100 µL of tissue culture medium (RPMI: fetal bovine serum 9:1 ratio) and 100 µL of provided sample (dissolved in 5% DMSO and RPMI tissue culture medium) of the test isolate were added to each well to reach final concentration 10 mg/mL, followed by twofold serial dilution. 12 dilutions were used for the extract to calculate its CD 50 . Control wells contained two aliquots of 100 µL of tissue culture medium (RPMI: fetal bovine serum 9:1 ratio) and 100 µL sterile DMSO (5%) and RPMI tissue culture medium, followed by two-fold serial dilutions. Following the specified incubation period (24 h) at 37 °C, the wells were washed with PBS and the cells were incubated with MTT solution (2 mg/mL) at 100 µL per each well for 1 h at 37 °C. After incubation, solutions were removed by decantation and cells in each well were treated with 100 µL DMSO to dissolve the formed formazan crystals. Elutes of each test isolate in the 12 wells were then collected and their absorbance was measured at 540 nm using Platos R496 Microplate reader AMD diagnostics (Graz, Austria). Control wells were treated similarly. The percentage of cytotoxicity was calculated using the following formula [17].

Cytotoxicity%= 1-[A 540 of test culture/A 540 of control culture] ×100
CD 50 is defined as the concentration of compounds that resulted in a 50% inhibition of mammalian cell growth.

Extraction of the bioactive metabolite from CFCS
Whenever different methods of extraction were used, all extracts should be tested for their activity. The best suitable recovery method to be used depends on the extraction method cost, which adds to the final product price, the required level of purity of the final agent and the adaptability of the method in the industry [18]. Results of the present study revealed that lyophilization is a good step in the extraction of the required antimicrobial agent.

Screening the methanolic extract for its antibacterial activity 3.2.1. Agar well diffusion method
The results obtained showed that methanolic extract of lyophilized CFCS of Streptomyces manipurensis isolate H21 exhibited inhibitory activity against reference ESBL producer, K. pneumoniae ATCC 700603. Extract of 10 mg/mL produced IZ of 17 mm.

Disc diffusion method
The anti ESBL activity of Streptomyces manipurensis isolate H21 crude extract against K. pneumoniae ATCC 700603 was recorded in the form of inhibition zone diameters surrounding the loaded discs. An extract that produced an inhibition zone ≥ 15 mm against tested strain was recorded as active. The methanolic extract of lyophilized CFCS of Streptomyces manipurensis isolate H21 had demonstrated anti-ESBL activity. Extract of Streptomyces manipurensis isolate H21 showed 18 mm inhibition zone. These results were comparable with amoxiclav (11 mm) and ampicillin/sulbactam (18 mm) discs as shown in (Fig. 1). Therefore, the methanolic extract of the lyophilized supernatant was further studied.

Minimum inhibitory concentrations (MICs)
The data obtained, through the determination of MICs of the extract are shown in (Table 3 However, it is difficult to compare the data with other researchers because several variables influence the results, and the choice of the extraction method and antibacterial test. Moreover, there are no standard criteria for the evaluation of crude extract activity.

Minimum bactericidal concentrations (MBCs)
The MBCs were established by subculturing the samples with no visible growth in the MIC assays. Most of the MBC values of the crude extract were double the MIC values in case of Streptomyces manipurensis isolate H21. Identical values were shown against K. pneumoniae ATCC BAA-1705 (Table 3).
For bactericidal agents, the MBC values are usually ≤ 4 times the MIC values. On the other hand, the MBC values of bacteriostatic agents are > 4 times higher than the MIC values. Such properties of antimicrobial agents can be influenced by the infecting bacterium as the antimicrobial agent may be bactericidal to one organism but bacteriostatic to another [15]. Therefore, the antimicrobial agent produced by Streptomyces manipurensis isolate H21 revealed to be bactericidal in nature for all tested bacteria.

Screening the extract for its antifungal activity
Methanolic extract of lyophilized CFCS from Streptomyces manipurensis isolate H21 showed positive anticandidal activity against Candida lusitaniae ATCC 3449 (Table 4). However, no antagonistic antifungal activity of the tested extract was observed against other tested fungi ( Table 5).
The extract of Streptomyces manipurensis isolate H21 exhibited various degrees of activities against all tested Gram-positive and Gram-negative bacteria. In addition, it exhibited antagonistic activity against reference MRSA strain Staphylococcus aureus ATCC 43300. Similar results reported previously [19]. The tested extract lacks antifungal activity except for Candida lusitaniae ATCC 3449. Although the CFCS showed anticandidal activity, extraction of the bioactive compound(s) with methanol revealed limited anticandidal activity. These finding envisaged that the extraction method had a definite effect on the isolation of bioactive principles. Similar results were published previously [20].

Cytotoxic activity of extract against Caco-2 cell line using MTT assay
According to the screening program of American National Cancer Institute (NCI), a crude extract is generally considered to have in vitro cytotoxic activity if the CD 50 value is ≤ 30 μg/ml. Methanolic extract of lyophilized CFCS of Streptomyces manipurensis isolate H21 showed no in vitro cytotoxic activity (CD 50 = 1.17 mg/mL). Results indicated that lyophilized supernatant of Streptomyces manipurensis isolate H21 had no cytotoxic activity may be considered as relatively safe based on the in vitro assay.

CONCLUSION
Methanolic extract of Streptomyces manipurensis isolate H21 showed a broad spectrum of antibacterial activity. To the best of our knowledge, this is the first report that addressed the production of the antimicrobial agent by Streptomyces manipurensis isolate H21. The antibacterial activity was assigned to the presence of bactericidal compounds with activity against MRSA and ESBL producer. No cytotoxic activity was detected indicating in vitro safety of the bioactive extract. The promising features of this bioactive extract can pave the way towards profitable large-scale production of antimicrobial drugs from Streptomyces manipurensis isolate H21.