In vitro ACTIVITY OF PRODIGIOSIN ISOLATED FROM Serratia marcescens IN COMBINATION WITH TWO GROUPS OF ANTIBIOTICS AGAINST GRAM-POSITIVE MICROORGANISMS

The work was aimed to study the synergy of the antimicrobial activity of the prodigiosin pigment with antibiotics against bacteria of the genera Bacillus , Staphylococcus and Streptococcus . The serial dilution method was used to evaluate antimicrobial compositions, which included inhibitors of cell wall synthesis: ampicillin, benzylpenicillin, vancomycin, cefazolin, and metronidazole (nitroimidazole derivatives) in combination with the pigment prodigiosin isolated from Serratia marcescens . Each combination was tested against the studied strains. The fractional inhibitory concentration index (FICI) for each combination was calculated to determine synergy, and the results were interpreted as follows: FICI  0.5 — synergism; FICI> 4.0 — antagonism; and FICI> 0.5–4 — neutralism. It was shown that the ethanol extract of prodigiosin in combination with benzylpenicillin, vancomycin, cefazolin, and metronidazole interacted differently synergistically depending on the type of microorganism. The combinations of prodigiosin and metronidazole showed a synergistic effect against Bacillus subtilis , vancomycin and cefazolin against Staphylococcus aureus and benzylpenicillin against Streptococcus pyogenes . Other combinations of prodigiosin and antibiotics showed a neutral effect, and in the case of cefazolin against Str. pyogenes , even an antagonistic effect. Thus, the study showed the synergism of prodigiosin with antibiotics depending on the type of microorganism, contributed to a several-fold decrease in the minimum inhibitory and bactericidal concentrations of each component separately, and the results indicated that prodigiosin acted separately more efficiently against gram-positive non-spore-forming bacteria. This synergistic combination of antimicrobial В(+A) ampicillin, benzylpenicillin, vancomycin, cephazolin, metronidazole (+ prodigiosin); ˆ the absence of inhibitory effect when making the maximum test concentration; P < 0.05.

The work was aimed to study the synergy of the antimicrobial activity of the prodigiosin pigment with antibiotics against bacteria of the genera Bacillus, Staphylococcus and Streptococcus. The serial dilution method was used to evaluate antimicrobial compositions, which included inhibitors of cell wall synthesis: ampicillin, benzylpenicillin, vancomycin, cefazolin, and metronidazole (nitroimidazole derivatives) in combination with the pigment prodigiosin isolated from Serratia marcescens. Each combination was tested against the studied strains. The fractional inhibitory concentration index (FICI) for each combination was calculated to determine synergy, and the results were interpreted as follows: FICI 0.5 -synergism; FICI> 4.0 -antagonism; and FICI> 0.5-4 -neutralism.
It was shown that the ethanol extract of prodigiosin in combination with benzylpenicillin, vancomycin, cefazolin, and metronidazole interacted differently synergistically depending on the type of microorganism. The combinations of prodigiosin and metronidazole showed a synergistic effect against Bacillus subtilis, vancomycin and cefazolin against Staphylococcus aureus and benzylpenicillin against Streptococcus pyogenes. Other combinations of prodigiosin and antibiotics showed a neutral effect, and in the case of cefazolin against Str. pyogenes, even an antagonistic effect.
Thus, the study showed the synergism of prodigiosin with antibiotics depending on the type of microorganism, contributed to a several-fold decrease in the minimum inhibitory and bactericidal concentrations of each component separately, and the results indicated that prodigiosin acted separately more efficiently against gram-positive non-spore-forming bacteria. This synergistic combination of antimicrobial agents had great potency to prevent bacterial resistance. greater depth for its use in anticancer and immunosuppressive therapy, than as an agent to fight infectious agents [1,4,7], but antimicrobial resistance threatens a resurgence of life-threatening bacterial infections and the potential demise of many aspects of modern medicine. Despite intensive drug discovery efforts, no new classes of antibiotics have been developed into new medicines for decades, in large part owing to the stringent chemical, biological and pharmacological requisites for effective antibiotic drugs. A new option for combating such pathogens is combination therapy. Combinations of antibiotics and antibiotics with non-antibiotic activityenhancing compounds offer a productive strategy to address the widespread emergence of antibiotic-resistant strains [8,9]. The purpose of the study was to investigate the synergism of antimicrobial activity of the prodigiosin pigment in combination with antibiotics against gram-positive test strains of bacteria.

Materials and Methods
Isolation and identification of pigmentproducing strains of bacteria. As a pigment producer, we used the species S. marcescens, namely the pigment-forming strain, which were isolated in the laboratories of the Department of Microbiology, virology, and immunology of Bogomolets National Medical University from the bentonite clays of Kurtsivskyi deposit (Crimea, Ukraine). Red color pigment-producing bacteria with different morphology and individual colonies were picked up separately and purified by quadrant streaking in nutrient agar plates for the isolation of bacterium S. marcescens. The pigmented colonies of bacteria were selectively isolated and transferred by the method of loop inoculum on nutrient agar surface of the following composition: peptone -10 (g/l), glycerol -10 (ml/l), K 2 SO 4 -10 (g/l), yeast extract -2 (g/l), MgCl 2 -1.4 (g/l), agar 15 (g/l), pH 6.5-7.0 ( Fig.). Then Petri dishes with inoculated strains of S. marcescens were incubated in a thermostat at +28 C for 24-72 h in an inverted position for the screening of pigment-producing strains. These obtained isolates were taken and identified by morphological and biochemical characterization using Bergey's manual of systematic bacteriology [10,11].
The method of obtaining purified prodigiosin. The extraction of prodigiosin pigment from biomass of bacteria was carried out by double proсessing of biomass with 96% ethanol. The resulting preparation dried in air and reextracted. The procedure was repeated several times before the release of insoluble admixtures. The resulting homogeneous solution was designated as a crude pigment complex or ethanol extract. The ethanol extract was evaporated dry in a drying oven at a temperature of +45-50 C and the residue dissolved in chloroform (10 ml/l of precipitate). The resulting solution was mixed with an equal volume of a water-ethanol mixture (4:1) and emulsified on a magnetic stirrer for 1 hour at room temperature. A water-ethanol mixture containing watersoluble admixture separated by a separating funnel. The procedure was repeated by increasing the volume content of ethanol by half. The drug was then redried in a vacuum oven and redissolved in ethanol (10 ml/g precipitate) [3].
Quantification of prodigiosin. The purity of prodigiosin isolated from the pigmented strain was determined by high-performance Serratia marcescens isolated from the bentonite clays on nutrient agar surface liquid chromatography (HPLC-MS) on the Agilent 1200 device (Agilent Technologies, USA) with diode-matrix and mass-selective detectors. Detection was performed using a diode-matrix detector with 315 and 535 nm signal recording. The molecular weight of the compounds determined on a massive detector with ionization in positive and negative APCI mode. Determination of the absorption spectrals of the isolated pigment determined by UV/VIS spectrophotometry method. Absorption spectra of the extract were tested by Portlab 512 spectrophotometer in the range 400-700 nm. Absorption of bacterial cells before extraction noted at each stage. The concentration of pigment was calculated using the following equation [12]: where OD -optical density; OD 534represent pigment absorption; OD 620represent bacterial cells absorption; 1.381constant.
Evaluation of antibacterial activity. To evaluate the antimicrobial properties of prodigiosin pigment in combination with antibiotics, we used the next reference strains of microorganisms: Bacillus subtilis ATCC 6633, Staphylococcus aureus ATCC 25923 and Streptococcus pyogenes АТСС 21059. The strains obtained from Gromashevsky Institute of Epidemiology and Infectious Diseases of the National Academy of Medical Sciences of Ukraine and Zabolotny Institute of Microbiology and Virology of the National Academy of Sciences of Ukraine.
Antimicrobial activities and synergistic interaction with antibiotics of the obtained purified prodigiosin pigment were determined at the Department of Microbiology, Virology, and Immunology of Bogomolets National Medical University. The minimum inhibitory and minimum bactericidal concentrations (MICs and MBCs) were determined by the micro broth dilution method. The broth microdilution format is traditionally set up as 2-fold dilutions of the compounds in sterile polystyrene plates at a lower volume of the drugs and nutrient medium used in the study. Each tube of a 96-well plate (except control) in a volume of 150 μl was injected with a suspension of microorganism cells in a liquid nutrient medium in the amount of 110 6 CFU/ml. The compounds were added in the same amount to the first tube, followed by 2-fold dilutions, and then incubated at + 37 C for 24 h. The MIC and MBC were determined after 24 hours. The MBC was determined by adding 50 μl of the suspensions from the wells, which did not show any growth after incubation during MIC assays, to 150 μl of fresh broth. These suspensions were reincubated at +37 C from 72 to 96 h. The MBC was determined as the lowest concentration of extract which inhibited 100% growth of microorganisms [13].
To evaluate the effect of the combination, the fractional inhibitory concentration index (FICI) calculated for each antibiotic combination [14] by computing the ratio of the MIC of the combination divided by the MIC of the antimicrobial alone for each agent and then adding those two ratios together (Equation A). Briefly, FICI was calculated as follows: where All studies performed in triplicate and the statistical processing of the obtained results carried out by using the specialized software Statistica 9.0 (StatSoft Inc., USA). A value of P < 0.05 was considered as statistically significant [15].

Results and Discussion
Combinatorial compounds sensitivity assays showed that metronidazole, vancomycin/cefazolin, and benzylpenicillin with prodigiosin (in ratio 1:1) presented remarkably synergistic activities against B. subtilis, St. aureus, and Str. pyogenes, which were selected for the study based on differences in the structure of their cell walls and differents sensitivity to antibiotics, and FICI values were ranging from 0.279 to 0.498. The MICs of five antimicrobials and FICI combinations of prodigiosin against test strains of microorganisms are shown in Table 1.

MIC MIC Equation A : FICI MIC MIC
These results reflect a > 4-fold decrease in MIC for metronidazole and vancomycin, > 8-fold decrease for cefazolin and more than 250-fold for benzylpenicillin, and a greater than 4-fold decrease in prodigiosin (synergistic MIC) compared to the MIC of each compound. Other combinations of antibiotics with prodigiosin did not show synergistic activity.
Ampicillin, benzylpenicillin, vanco my cin, cefazolin, and metronidazole in combination with prodigiosin were tested in vitro to determine whether they were bacteriostatic or bactericidal against the test strains of microorganisms. The MBC of five antimicrobial compounds and FICI combinations of them with prodigiosin against test strains of microorganisms are shown in Table 2.
In determining the MBC/MIC ratio for individual compounds, four different combinations of antibiotics with prodigiosin had a bactericidal action of 0.05 to 31.25 μg/ ml. Interestingly, when prodigiosin was assayed in a combination with a different fixed concentration of antibiotics, the MBC of prodigiosin was in the range 0.1 to 5.62 μg/ml, probably because the bactericidal nature of prodigiosin was not dominant in the combination, due to the selective effect on the target structure different in the bacterial cell.
The first area of research is the combined use of prodigiosin with antibacterial drugs, presented in this publication, reproduced by the simultaneous effect of the pigment of S. marcescens and inhibitors of cell wall Table 1

. Value of MIC (range) of combined effect of antibiotics and prodigiosin relative to test strains of microorganisms
Hereinafter: МІC А -prodigiosin pigment; МІC В -ampicillin, benzylpenicillin, vancomycin, cephazolin, metronidazole; МІC А(+B) -prodigiosin (+ ampicillin, benzylpenicillin, vancomycin, cephazolin, metronidazole); МІC В(+A) -ampicillin, benzylpenicillin, vancomycin, cephazolin, metronidazole (+ prodigiosin); ˆ -the absence of inhibitory effect when making the maximum test concentration; * -P < 0.05.  The second area of research, presented in the paper, was on evaluate in the susceptibility of spore-forming bacteria to antimicrobial preparations, due to the impact of prodigiosin and nitroimidazole derivatives on B. subtilis. The combinations of prodigiosin and metronidazole had synergistic effects on B. subtilis, and the mechanism of action combination can associate with inhibiting nucleic acid synthesis by disrupting the DNA of microbial cells.
The study of the interactions of the compounds showed that prodigiosin may be competing for the same cellular target as antibiotics, leading to a neutral or antagonistic effect. Thus, the results of studies indicate that prodigiosin by peptidoglycan hydrolysis, which is predominant in the cell walls of grampositive bacteria and/or accumulation inside the bacterial cell by mediating redox reactions, leads to impaired membrane permeability and/ or disruption of DNA structure. We have demonstrated that prodigiosin can enhance the activity of individual antibiotics depending on the type of microorganism and possibly other non-clinically effective antibiotics against pathogenic bacteria while providing lower FICI values, which is an important finding of our study. The results indicate that prodigiosin acts more effectively against gram-positive non-spore-forming bacteria, and synergistic combinations of antimicrobial agents have Summarizing, a set of five antibiotics with different structures was analyzed in the presence of prodigiosin and found that in total only four combinations had synergistic activity against test strains of gram-positive bacteria. The study has indicated that synergistic combinations of antimicrobial agents being susceptible to pathogenic bacteria had a great potency to prevent resistance. The resultant synergy in the combination of prodigiosin and inhibitors cell wall synthesis is a novel concept, as such combinations will have identical or different mechanisms of action, which may lead to new choices of therapeutic agents for the treatment, especially infections caused by multidrug-resistant microorganisms having no effective therapy available. Combinations of inhibitors cell wall synthesis or nitroimidazole derivatives with prodigiosin may warrant further clinical investigation for treating the diseases associated with pathogenic grampositive microorganisms.

Conclusions
The work was performed at the Department of Microbiology, virology, and immunology of Bogomolets National Medical University within the framework of the initiative-search topic "Biological activity of prodigiosin pigment isolated from Serratia marcescens".