key: cord-1011130-sny0h30h authors: Gohil, Nisarg; Bhattacharjee, Gargi; Singh, Vijai title: Synergistic bactericidal profiling of prodigiosin extracted from Serratia marcescens in combination with antibiotics against pathogenic bacteria date: 2020-09-18 journal: Microb Pathog DOI: 10.1016/j.micpath.2020.104508 sha: ce157087625ae643f5dc70bc6ea40eb9a5e5826a doc_id: 1011130 cord_uid: sny0h30h The emergence of multidrug resistance (MDR) bacteria is on the rise and the situation has been worsening on each successive day, which is evident from the outpouring number of reports about how more and more pathogens are becoming resistant to even the third and fourth generations of antibiotics. Lately, combination therapies or drug synergy have been giving promising results in curbing infections since it delineates its action on multiple aspects as compared to monotherapies. In this study, we used prodigiosin, a bacterial pigment endowed with magnificent biological properties, in combination with six antibiotics to study its effect on Pseudomonas aeruginosa, Staphylococcus aureus and Chromobacterium violaceum. Minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) of prodigiosin against the test organisms were determined and a checkerboard assay of prodigiosin with various antibiotics combinations was performed with an aim to abate antimicrobial resistance. MIC and MBC for prodigiosin were obtained in the range of 4–16 μg/mL, which was lower than that of most test antibiotics. Coupling of prodigiosin with other test antibiotics exhibited an excellent synergy profile against all test organisms and the effects were reported to be either synergistic or additive. In the case of S. aureus and C. violaceum, all combinations were found to be synergic, and remarkably for S. aureus, FBC index was reported to be as low as ≤0.25 with all tested antibiotics. Therefore, it is deduced that prodigiosin augments and intensifies the action of antibiotics, and results in a double-whammy against the emergence of MDR strains. The discovery of antibiotics has revolutionized the field of medicines and saved millions of lives [1] . However, the rapid and dramatic co-evolution of microbes against antibiotics has rusted the potency of these 'miracle drugs' and has ultimately rung the global alarm of antibiotic resistance. Even the World Health Organization (WHO) has confirmed this ever-increasing menace and announced the threat by stating "The world is running out of antibiotics" [2] . According to the report 'Tackling Drug-Resistant Infections Globally' published in May 2016, it is predicted that if proper actions are not taken by 2050, the multidrug-resistant (MDR) or extremely drugresistant (XDR) pathogens could take lives of 10 million people every year [3] . To address these consequences, it is imperative to devise novel approaches or develop a new antibiotics or biocidal compounds at the earliest for controlling of pathogens [4] . In the last couple of decades, combinational therapy has emerged as a promising strategy for treating several diseases such as cancer [5, 6] , diabetes [7] , AIDS [8] , hypertension [9] , autoimmune diseases [6] , tuberculosis [10] , infectious diseases [11, 12] and even against the recent COVID-19 pandemic [13] . In context to drug-resistance, 'drug cocktails' could be a game-changer as they come with several benefits such as (1) a broadened antimicrobial spectrum, (2) the likelihood of developing drug resistance would reduce substantially as the chances of acquiring resistance towards a novel combination would be much lower, (3) it could be more efficacious against polymicrobial infections, (4) if the exercised drugs act synergistically, i.e., the combined effect of antimicrobial agents used is greater than the sum of their individual effects. It can provide even better pathogen clearance than the single drug alone and that too at a very low concentration and (5) the lower concentration of antimicrobial agents could minimize the drug toxicity in the host [11, 14] . Most clinically used antibiotics, to date, are either microbial natural metabolites or semisynthetic derivatives of these molecules [15] . However, the conventional antibiotics are lately failing in their efficacy towards the MDR pathogens. Therefore, scientists and pharmaceutical companies are diving deep into the bacterial sources, particularly in novel potent bacteria to discover a unique backbone of antibiotics that can outperform in the race between pathogens and drug discovery. Amongst many such candidates, a crimson red bacterial pigment called Prodigiosin, is J o u r n a l P r e -p r o o f one such alkaloid secondary metabolite produced by numerous microorganisms including Serratia marcescens, S. rubidaea, Pseudomonas magneslorubra, Pseudovibrio denitrificans, Pseudoalteromonas rubra, Vibrio psychroerythrous, V. gazogenes, Streptomyces lividans, Nocardia spp., etc. [16] . It contains three pyrrole rings in its molecular structure. True to its name, this natural pigment is prodigious and manifests multifaceted characteristics such as antimicrobial, anticancer, antimalarial, antifungal, immunosuppressive, and many more [16, 17] . This phenomenal potential of prodigiosin has caught the attention of many researchers, nutraceutical, pharmaceutical, cosmetic and other industries [18] . The aim of this study was to evaluate the action of prodigiosin as an antimicrobial agent against infectious microorganisms. Most importantly, we investigated the synergic efficacy of prodigiosin with antibiotics, the amalgamation of which could intensify the potency of conventional antibiotics against the pathogenic microorganisms as a novel resolution that could be used to address drug-resistance issues. Microorganisms used in experiments were P. aeruginosa MG, P. aeruginosa PG30, were maintained and preserved in 20-25% glycerol at -80 °C for further use. Overnight grown activated culture of S. marcescens was spread on LB agar (HiMedia, India) and incubated at 28 °C for 96 hr. The cells were scraped, washed twice with sterile distilled water and collected in 20 mL acidified methanol (4 mL of 1 N HCl in 96 mL of methanol) and the suspension was vortexed for 5 min. This was followed by centrifugation (Eppendorf 5810R, Germany) at the agitation speed of 7500 rpm for 15 min at 4 °C. The methanol extract was left to evaporate to dryness at 28 °C, following which the residue was re-suspended in 1 mL acidified methanol and stored at 0-4 °C for further use [19] . The concentration of prodigiosin was determined by the standard curve equation based on absorbance at 492 nm [20] . The extracted prodigiosin was characterized through ultravioletvisible spectroscopy (Shimadzu UV-1900, Japan) and a spectrum was generated in the range of 300-700 nm. The purity of prodigiosin was also determined by HPLC (Shimadzu LC-20AT, Japan) using Kinetex C18 column (250 × 4.6 mm, 5 μm, Phenomenex, USA). The separation was achieved using methanol and water (HPLC grade, Merck, Germany) as the mobile phase at a flow rate of 1 mL/min. The MIC of prodigiosin and the antibiotics used in this study were determined through microbroth dilution assay in 96-well flat-bottom sterile microplates (SPL Life Sciences, Korea) [17, 21] . 100 µL of 2X Mueller-Hinton (MH) broth (HiMedia, India), 10% v/v (20 µL) J o u r n a l P r e -p r o o f standardized microbial inoculum and 80 µL of varying concentrations of prodigiosin (1 to 128 µg/mL) or antibiotics (1 to 2048 µg/mL) were added into each well. Controls such as sterility control and growth control were also considered for observation. The desired microbial inoculum was prepared using overnight grown culture which was standardized as per 0.5 McFarland turbidity standard at an optical density of 600 nm (OD 600 ) (Systronics Visiscan Spectrophotometer 167, India) and adjusted with sterile normal saline solution (0.85% NaCl). Antibiotics used in the experiments were ampicillin (stock solution -100 mg/mL), chloramphenicol (34 mg/mL), spectinomycin (50 mg/mL), tetracycline (10 mg/mL), kanamycin (50 mg/mL) and nalidixic acid (10 mg/mL). All the antibiotics were purchased from HiMedia, India. The microtiter plates were incubated at 37 °C for 24 hr. The lowest concentration that inhibited the visible growth of the organism was considered to be the MIC, while the minimum concentration at which no viable cells were found after 24 hr incubation at 37 °C when streaked on an MH agar plate in the absence of antibiotic was taken as the MBC. All experiments were performed in triplicates. Synergistic antimicrobial profiling of prodigiosin with different antibiotics against the pathogenic bacterial strains was constructed using one of the authenticated procedure used for determining the synergistic effect as suggested by American Society for Microbiology (ASM): the checkerboard titration assay [22, 23] . In this study, different concentrations of prodigiosin and antibiotics were combined in a 2-dimensional fashion with the test organisms in 96-well flatbottom sterile microtiter plates. Thus, each well contained a unique combination of prodigiosin and antibiotic concentration against tested pathogens. The inoculum for the test organisms was standardized as described earlier. Controls were set as stated earlier. The plates were incubated at 37°C for 24 hr and MBC was determined. For assessment of results, fractional bactericidal concentration (FBC) index [24, 25] was determined by calculating the sum of FBC of prodigiosin and the antibiotic (i.e., ∑ FBC index = FBC prodigiosin + FBC antibiotic ). FBC can be defined as the MBC of drug used in combination divided by the MBC of the drug when used alone. The interaction is considered as synergistic when the FBC index is found to be ≤0.5; additive when J o u r n a l P r e -p r o o f the FBC index is >0.5 to ≤1.0; indifferent when the FBC index is >1.0 to ≤2.0 and antagonistic when FBC index is >2.0 [22] . where, FBCI = FBC index, MBC = minimum bactericidal concentration, PRO = prodigiosin, ATB = antibiotic As per Darshan and Manonmani [19] , the prodigiosin gave absorption spectrum maxima at 535 nm. A single peak at 535 nm was obtained in the UV-visible absorption spectrum of prodigiosin confirming its purity (Fig. 1) . The purity of the prodigiosin extracted from S. marcescens was achieved up to 95% as determined by HPLC. The HPLC profile of prodigiosin pigment is shown The MIC as well as MBC of prodigiosin and antibiotics against test pathogens are given in Table 1 . Prodigiosin showed excellent antimicrobial and bactericidal activity against all tested pathogenic strains. In all assays, MIC and MBC of prodigiosin ranged from 4 to 16 µg/mL. P. aeruginosa MG and C. violaceum were found to be resistant to ampicillin. In majority (75%, n = 18 out of 24 cases; Note: two resistant cases were included in total cases) of the cases, prodigiosin alone as an antimicrobial agent exhibited much lower MBC as compared to the test antibiotics. Apart from that, in a few cases (37.50%, n = 9/24), the MBC of prodigiosin was even ≤128-fold lower than the MBC of test antibiotics. In a way, it could also be claimed that against J o u r n a l P r e -p r o o f all the test organisms, prodigiosin was found to be more or equally effective than ampicillin, chloramphenicol, spectinomycin and nalidixic acid as the obtained MBC of prodigiosin was lower than or equal to the said antibiotics. Interestingly, in both P. aeruginosa strains, the MBC concerning prodigiosin was lower than all the tested antibiotics. The combination effect of prodigiosin with commercially available antibiotics against pathogenic microorganisms is given in Table 2 and depicted in Fig. 3 A serious shortfall of new antibiotics has dragged the world towards global health emergencies [2, 26] . In response to defeat this growing threat, a pressing need has arisen to discover novel biocidal substances or to find an alternative approach to eradicate MDR issues [4] . Combination antimicrobial therapy (CAT) is a potent approach that can be used to treat many infectious J o u r n a l P r e -p r o o f diseases [12] . Many studies have shown that CAT has an edge over the monotherapy of an antibiotic against infections [11, 12, 21, 22] . In our attempt to prove so, prodigiosin, an exemplary bacterial pigment known for its innumerable features was used. The amphiphilic nature of prodigiosin endows it to easily incorporate within the bacterial cell membrane and affect its integrity [27, 28] . Thereafter, it inhibits the action of DNA gyrase and topoisomerase, the enzymes that are obligatory for DNA replication [16, 29] . It is proven that microorganisms communicate with each other via transferring of signalling molecules or auto-inducers through a phenomenon which is commonly known as quorumsensing that plays a pivotal role in expressing pathogenicity, motility, biofilm formation, production of antibiotics and pigments, spore formation and many others [26] . In clinical infections, pathogenic microorganisms cause threats majorly by forming a biofilm. By doing so, the capability of delineating resistance towards antibiotics in pathogens could increase up to 1,000-1,500 times [30] . Amongst the biofilm producers, P. aeruginosa is renowned for its ability to produce very strong biofilm, which has made this superbug recognized as a 'critical priority' pathogen in the list of "antibiotic-resistant priority pathogens" published by WHO [31] . In this present study, P. aeruginosa (as well as C. violaceum) showed resistance to ampicillin, a βlactam antibiotic that is a potent cell wall synthesis inhibitor. According to the data shown by Darshan and Manonmani [19] , the obtained MBC for P. aeruginosa, Escherichia coli, S. aureus and Bacillus cereus were 16, 20, 9 and 12 µg/ml, respectively. These results are in agreement with the data sets obtained by the present study. In synergistic profiling of both P. aeruginosa, we obtained additive effect with most antibiotics which is predicted to be due to the formation of strong biofilm by these strains that might have hindered the permeability of antimicrobials to enter the cell [21, 32] . Similar to P. aeruginosa, S. aureus is also amongst the 'high priority' pathogens [31] and falls under the class of ESKAPE pathogens (six pathogens exhibiting MDR and virulence: Enterococcus faecium, S. aureus, Klebsiella pneumoniae, Acinetobacter baumannii, P. aeruginosa and Enterobacter spp.) [33] . It is also regarded as the most common human pathogen that causes a wide range of infections. In an extensive study of prodigiosin against twenty oxacillin-resistant clinical isolates of S. aureus, the MBC was manifested to range from 2 to 16 µg/ml [17] . These results are also consistent with the present study, where the MIC and MBC J o u r n a l P r e -p r o o f values against S. aureus were 8 and 16 µg/ml, respectively. Likewise, Suryavanshi et al. [34] have also reported extraordinary MIC value (2.5 µg/ml) against S. aureus. Notably, combinations of prodigiosin with all tested antibiotics against S. aureus gave an excellent FBC index of ≤0.25, which was half of the required value to be synergic (i.e., 0.50). In other words, S. aureus got even more sensitized to prodigiosin in the presence of antibiotics. Numerous studies have reported that gram-positive bacteria, especially S. aureus, are more susceptible to prodigiosin compared to gram-negative bacteria [16] [17] [18] 34] . Our synergistic profiling results of S. aureus are concurrent with the above-mentioned studies. Apart from that, opportunistic pathogen C. violaceum was also considered in our study, even though the infections caused by these microbes are rare, though are usually fatal [35] . The combined effect of prodigiosin with antibiotics was synergistic, suggesting that prodigiosin can potentiate the action of commercially available antibiotics against these pathogens. We predict that prodigiosin orchestrates the membrane integrity of the bacterial cell and promotes the entry of itself and other antibiotics into the cell by altering the membrane permeability. Recently, prodigiosin was also tested for a synergy effect against Mycobacterium smegmatis with 9 cell wall synthesis inhibiting antibiotics [25] . Prodigiosin presented a considerable MIC value of 2.8 against M. smegmatis, which was higher than all of the used antibiotics, except vancomycin. In their results, prodigiosin was found to be either synergistic (66.67%, n = 6/9) or indifferent (33.33%, n = 3/9), thus suggesting prodigiosin as a promising candidate to be used as a supplement to potentiate the cell wall synthesis inhibitors. They were relatively lesser preferred drugs as compared to other drug targets in the treatment of infections caused by the Mycobacterium [25] . Besides antibiotics, the synergism of prodigiosin with biosurfactants [36, 37] and with chitinolytic enzymes [38] has already been reported for antibacterial and antifungal activities, respectively. From these studies, it was deduced that prodigiosin intensifies the efficacy of the amalgamated agents against pathogenic microorganisms. Similar to prodigiosin, a purple-hued bacterial pigment violacein produced by C. violaceum, Janthinobacterium lividum, Alteromonas luteoviolacea, Dugenella sp. etc. has also exhibited tremendous synergistic antimicrobial efficiency against S. epidermidis [21] , Salmonella typhi, J o u r n a l P r e -p r o o f Vibrio cholerae, P. aeruginosa, K. pneumoniae and S. aureus [22] and connoted its potential to be used in combination with antibiotics. We are experiencing a serious shortfall in its available antibiotic arsenal. With the emergence of some highly resistant superbugs in tandem with very few novel antibiotics in the pipeline, it is time to look for alternative approaches besides developing next-generation antibiotics. Combining the proficiency of novel molecules such as prodigiosin with commercial antibiotics may prove to be a plausible substitute to subjugate the surging morbidity and mortality. Prodigiosin can be a treasured chemical of inestimable value owing to the range of function it is involved in, naming from antimicrobial, anti-tumorigenic, antimalarial, immunosuppressive and even as a food colourant. Though it is predicted to have different mechanisms that it uses to execute its aforementioned functions on different hosts, the exact molecular target still remains an enigma. With regard to its antibacterial properties, it is proposed to function as a bacteriolytic and bacteriostatic agent during the exponential and stationary phases of the growth cycle respectively [39] . In our study, it is evident that in most cases, prodigiosin when coupled with antibiotics having different modes of action was able to display synergism and wipe out pathogens at minimal possible concentrations. In some cases, these combinations depicted additive responses but in absolutely no case did we get antagonistic or insignificant interaction. Therefore, in a nutshell, fusing prodigiosin with antibiotics may increase the efficacy of the drugs by several folds, which in the future, might lead to its emergence as a compelling substitute providing the same effects as antibiotics alone, that too at a much lower concentration. The authors declare they have no competing interests. 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The bacterial strains P. aeruginosa MG, C. violaceum, S. marcescens and S. aureus were kindly gifted by Dr Vijay Kothari from Institute of Science, Nirma University, Ahmedabad, India.