Current scenario of antibiotic resistance
MDR has created a viscous cycle with more and more virulence developed in resistant strains and transferred to others by horizontal gene transfer [14]. Additionally, traditional treatment regimens with obsolete antibiotics available at cheap prices over the counter demanded the discovery of game changers. Not only human pathogens are involved in MDR but also veterinary pathogens and zoonotic bacteria with more than 200,000 tons of annual antibiotic production dedicated to agriculture and veterinary medicine, [15].
According to the Center for Disease Control and Prevention (CDC), the immunocompromised people, organ transplantation or rheumatoid arthritis patients, those receiving chemotherapy or dialysis treatment due to renal failure are the most affected with the ferocity of MDR [3].
Antibiotic resistance is afflicting both developing and developed countries. While in USA and Europe around 35,000 deaths were reported, Africa and Southwest Asia infection percentage was around 6 and 18%, respectively [1]. India and many other third world countries encountered deep uncontrolled usage of antibiotics as well as pollution cases in food and air, which supported the escalation of resistant infections [16].
Endophytes as an untapped source of antibiotics
Endophytes are unique since they colonize plants’ inner tissues and protect them from diseases. A strategy that warrants more investigation and is until now far from being understood. More than any other natural sources, endophytes are the factory of a wide range of bioactive metabolites such as alkaloids, phenolic acids, steroids, quinones, terpenoids, saponins, and tannins. This chemical diversity meant higher potential to act as anti-inflammatory, immunomodulating, antimalarial, antiviral, antidiabetic, ant arthritis, and anti-tuberculosis [17]. Fungal non-microbicidal molecules are to be used in combination with antibiotics to strengthen their activity. Therefore, drug discovery based on fungi antimicrobial adjuvants would be an auspicious approach, which will promote a better antibiotic regimen in clinical use. Many compounds are screened but show weaker activity that does not lead them to further advancement in clinical pipelines. These need to be studied in combination regimens with obsolete antibiotics rather than being excluded. A strategy that will even limit further genetic mutations and resistance [18].
Antibiotic adjuvants are non-microbicidal compounds that will render bacteria weaker and enhance effectiveness of the antimicrobial agents, among which are molecules that inhibit microbial adaptive responses and resistance. An example of combination therapies is trimethoprim and sulfa drugs, cefuroxime and ticlopidine whose additive effect was superior against MRSA; moreover, β-lactamase inhibitors as clavulanate, sulbactam and tazobactam were potent in their activity when combined with amoxicillin, ampicillin and piperacillin, respectively [18]
Hutchings et al went to the fact that root associated endophytes are the best producers of antimicrobial agents because they colonized and co-evolved with plants growth 440 million years ago with their filamentous growth stretching to the soil to reach plant roots for better interactions and survival. The production of these bioactive NPs mediated and favored such colonization [19, 20]. In the same vein, endophytes inside plants tissues could have been the source of antimicrobial activity, mostly attributed to plant extracts with no isolated bioactive molecules. Focusing on endophytes, bacteria and fungi, whether colonizing inside plants or plant soil puts forward a roadmap for effective assignments of research fundings in the next few decades in order to come up with potent treatment for threatening pathogens with MDR.
Endophytic fungi
Following in-silico screening, the highly methylated polyketide emerione A isolated from Aspergillus sp. TJ23 restored the activity of the β-lactam antibiotic meropenem and carbapenem towards New Delhi metal-β-lactamase 1 (NDM-1) producing strains of family Enterobacteriaceae [30]. The structure activity study revealed binding to the zinc ion inside NDM-1 active center. Carbapenem-resistant Enterobacteriaceae (CRE) are among the most critical virulent resistance because of their broad spectrum and challenging treatment. Both K. pneumoniae ATCC BAA-2146 and NDM-1-producing E. coli strains were rendered susceptible after treatment of emerione A [31]. Nor-harmane demonstrated an antibiofilm effect against P. aeruginosa PA14, Klebsiella oxytoca, E. coli O157: H7, and P. aeruginosa PAO1 [32]. The same alkaloid was isolated from Irpex lacteus fungus and showed anti-QS and anti-biofilm effect towards S. marcescens [32; 33]. Not only were biofilm genes downregulated as manifested by the qPCR but also nor-harmane restored sensitivity to ofloxacin. Additionally, the FT-IR revealed reduction of various metabolites of the extracellular matrix.
The previously reported from soil fungi, Eurotium chevalieri KUFA 0006, was isolated from the mangrove Rhizophora mucronate Poir yielding diketopiperazine and polyhydroxy anthraquionones as well as anthranilic acid, isochromone and prenylated indole 3-carbaldehyde derivatives [34]. While emodin augmented oxacillin effect against the MRSA S. aureus 66/1, it was bactericidal towards Gram-positive (E. faecalis ATCC 29212 and S. aureus ATCC 25923) and Gram-negative (P. aeruginosa ATCC27853 and E. coli ATCC 25922). Emodin, physcion and the two new prenylated indole carbaldehydes inhibited biofilm formation of S. aureus ATCC 25923, and others inhibited biofilm formation of E. coli ATCC 25922. Moreover, all of them were synergistic with vancomycin against multidrug-resistant VRE E. faecalis B3/101 [35].
From the endophytic Daldinia sp., the wood inhabiting tropical fungi, 2,4-Di-Tert-Butylphenol was isolated and showed anti-biofilm formation and anti- QS activity towards P. aeruginosa. This was in accordance with previous reports that showed 2, 4-DBP role in biofilm reduction of S. pyogenes as well as in restricting surface hydrophobicity, EPS production and bacterial adhesion [36]. While in C. albicans 2,4-DBP both restricted the formation and disrupted the preformed biofilms, it altered polysaccharides, proteins, and EPS components in Serratia marcescens [37]. 1-(4-amino-2-hydroxyphenyl) ethanone (AHE) suppressed acyl-homoserine lactones and QS-related genes; moreover, it inhibited antioxidant enzymes and subsequently enhanced oxidative stress in P. aeroginosa PAO1 [38]. 1,4-Naphthoquinones are remarkable in their antibacterial, antifungal or antiparasitic activities even though the structural features might differ from one molecule to another [Li et al, 2022]. Among the susceptible pathogens from the Gram-positive group is Staphylococcus spp.-representative bacterium of the ESKAPE pathogens, and from the Gram-negative group are Pseudomonas spp. (~ 9%) and E. coli (~ 11%). The plausible mechanisms of action for 1,4-naphthoquiones is inhibition of ROS, suppression of Eps, and curing plasmids elimination [39]
From the endophytic fungus Fusarium tricinctum, the naphthoquinone shikonin was isolated in reasonable amounts to facilitate its introduction to the industrial scale[40].
Shikonin revealed a synergistic activity with seven conventional antibiotics, namely, oxacillin, chloramphenicol, gentamicin, ceftazidime, amikacin, amoxicillin, and cefoxitin using the Checkerboard method against MRSA. the postulated mechanisms involved S. aureus biofilm and virulence factors inhibition, interfering with the cell membrane integrity and expression of PBP2a [41].
Chitosan or the deacetylated chitin, the linear polysaccharide obtained from insects’ cuticle, algae and fungi, was isolated in a yield of 53.8% from the endophytic Aspergillus flavus through a biological method of bacterial demineralization and deproteination. Not only did chitosan decrease lasR and rhlR gene expression up to 32 and 8 folds, respectively, but also reduced biofilm formation in nosocomial pathogen Pseudomonas aeruginosa. Chitosan synergistically enhanced the antimicrobial potential of ceftazidime, which decreased its MIC values from 1024 and 512 µg/ml to 128 and 64 µg/ml against P. aeruginosa and S. aureus, respectively [42]. The substituted pyran derivatives ……..and ………..,with the former comprising an α,β-unsaturated δ-lactone were isolated from the fungus Lasiodiplodia venezuelensis colonizing the ancient tropical palm trees and manifested anti-QS effect by reductionof lasB and rhlA expression and impaired key virulence factors without harming. P. aeruginosa growth [11]. The pyrone scaffold was reported in several studies to possess an antimicrobial effect because of their role as Michael acceptors. For instance, phomopsolides from the endophytic Penicillium sp. displayed feeding deterrent effects for Scolytid beetles [43, 44].
The diterpenoid sphaeropsidin A was isolated from the terrestrial endophyte Diplodia corticola and its synergistic activity in combination with epi-epoformin was assessed revealing a reduction in MIC values down to ¼ MIC regarding sphaerospidin A and 1/16 MIC regarding epi-epoformin against Gram-negative bacteria. The same combination was evaluated towards Gram-positive bacteria and showed lower MIC values of sphaerospidin A (1/2 MIC) and epi-epoformin (1/32 MIC) [45]. This adjuvant role of sphaerospidin A was safer to the cells, with a dose of 3.12 µg/mL for each of them and conferred cellular viability of 60%. as evidently shown b After measuring the Fractional Inhibitory concentration (FIC), an additive effect was noted with Gram-positive bacteria (FIC > 0.5) and synergistic effect with Gram-negative (FIC < 0.5). Moreover, biofilm formation of P. aeruginosa clinical and reference strains was inhibited by 62% and 50%, and that of MRSA strains was inhibited by 53% and 60%, respectively [46, 47]. From a total of seventy-eight marine natural products screened from a chemical library, the γ-pyrone derivative, kojic acid showed promising results as anti-QS against Chromobacterium violaceum CV017. Upon using LuxR-based reporter E. coli pSB401, kojic acid demonstrated potent inhibition at different concentrations from 0.2 to 36µM. [48]. The afore-mentioned results indicated the possibility of its use to reduce microbial communities without causing toxicity (49; 25]. The phenolic derivatives, vulculic acid and curvulol showed nearly the same inhibition of biofilm formation against Staphylococcus aureus by 96.82% at 256 µg/mL while their MIC values were around 33.33 µg/mL towards Staphylococcus aureus DSM 346 and Bacillus subtilis DSM 10 [50; 47]. Hypoxylon fragiforme, the beech wood associated fungus was endowed with high chemical diversity based on its developmental stages and fermentation conditions, which was clearly manifested in the previously reported cytochalasins and azaphilones production [51]. Cytochalasins from Hypoxylon sp. were uncommonly reported for their antibiofilm activity against S. aureus with up to 85% inhibition. It seemed that biofilm inhibition provided a critical advantage to fungi in restricting bacterial growth and thus outcompeting them in the biological niche because even the change of media composition of Hypoxylon stimulated the biosynthesis of other biofilm inhibiting compounds, sclerin and sclerin diacid (52; 53). A pair of thio-diketopiperazines were isolated from Phoma sp. with about 10µM MIC value against Staphylococcus aureus and Streptococcus pyogenes. Both thio-derivatives enhanced streptomycin activity with an anti- biofilm effect; furthermore, they regulated the pathogenicity genes in at sub-lethal concentration in Staphylococcus aureus; thus, they were good potential candidates as antibiotic adjuvants.[54].
From the marine fungi Penicillium sp. SCS-KFD08, naptho-α-pyrones penicitor A were isolated and characterized using the reference semiviriditoxin. While penicitor A absolute stereochemistry opposed the reference as having R-configuration of C-3, penicitor B was identified as the 2-methyl-7-hydroxy-5-propy-4H-chromen-4-one. The nor-sesquiterpene aculene E configuration was assigned by using the Harada-Nakanishi non-empirical rule as 1R,5S,10S. When tested for their quorum sensing inhibitory effect against C. violaceum CV026 by measuring the violacein production using 300 lM of single compounds, promising results were reported at sub-inhibitory concentrations for the naptho-α-pyrones, dihydro-iso-coumarins and nor-sesquiterpenes-[55].
Three depsidones were obtained in decent amounts from the marine associated fungus Aspergillus unguis IB1 with MIC values less than 16µg/mL against MRSA. Produced by the expression of MecA gene, the Penicillin-binding protein (PBP2a) is responsible for the low binding affinity of the cross-linking peptidoglycans; thus, conferring the resistance known for the S. aureus resistant strains. 2-chlorounguinol, unguinol, and nidulin interacted virtually to the binding pocket of PBP2a through hydrogen bonding and hydrophobic interactions, which suggested their synergistic role with antibiotics (56]
The strategy of using antibiotic adjuvants is quite attractive because you can activate many antibiotics with a single molecule [57]. The unique biological niches tapped by endophytes highly propose promising antimicrobial bioactivities that facilitate life in this ecology. The natural product streptazolin was isolated from Streptomyces C3 and showed weak antimicrobial effect with MIC values in the mM range against, MRSA, S. Typhi, V. cholerae and C. albicans [8. Aspergillomarasmine A, the polycarboxylic amino compound first isolated from Aspergillus versicolor [58] was recorded to influence Zn+ 2 availability and undermine bacterial survival. Similarly, it inhibited NDM-1 receptor in order to halt antibiotic resistance [59]. Stigmasterol is a phytosterol of tetracyclic triterpenoidal structure widely biosynthesized in plants and was recorded to greatly enhance the β-lactam resistant bacterial strains when combined with ampicillin [60]. Conessine alkaloid is a plant based antimicrobial agent active against Pseudomonas aureginosa by inhibiting bacterial efflux pump; therefore, was able to reduce the MIC by 8-fold of all the following antibiotics: levofloxacin, rifampicin, cefotaxime, erythromycin, novobiocin, and tetracycline (61]. The polyphenolic compound epigallocatechin gallate (EGCG) was isolated from Camellia sinensis and several phenolic rich plants. Its role was significant in inhibiting the MexAB-OprM efflux pump of 22 multi resistant Pseudomonas species and MRSA; thus, improving their antibiotic susceptibility. Methoctramine, the M2 muscarinic antagonist, potentiated the effectiveness of the nonpolar antibiotics novobiocin and erythromycin.
Marine endophytes
Squalamine was extracted from the dogfish shark and proved efficacy as a membrane permeabilizing agent in gram negative bacteria. Squalamine reduced the MIC values through synergism with the following antibiotics chloramphenicol, tetracycline, and ciprofloxacin; particularly, in strains overexpressing efflux pumps [62] [60]. Ianthelliformisamine A, B and C [57] isolated from the marine sponge Suberea ianthelliformis were active as enhancers of doxycycline against the three strains P. aeruginosa PAO1, Enterobacter aerogenes EA289, and K. pneumoniae KPC2 ST258 via depolarization of the PAO1 membrane.
The polyketides kaneoheoic acids A-F manifested an antibiotic adjuvant effect and reduced the MIC values against Staphylococcus aureus and Bacillus subtilis when administered with the established antibiotic chloramphenicol or the antibiotic adjuvant disulfiram [63]..
The meroterpenoid alkaloid, equisetin, biosynthesized from the PKS-NRPS pathway in marine associated fungi showed promising effect as an adjuvant to colistin and other eight classes of antibiotics when tested against S. aureus ATCC 29213. These results supported the hypothesis that equisetin was effective against Gram-positive bacteria, MCR- Gram-negativebcteria with FIC < 0.5 and colistin resistant E. coli isolates.[65].
Fatty acids identified in the endophytic Arthrographis kalrae obtained from Coriandrum sativum were active against the virulence factors of Streptomcossus mutans, the major producer of biofilm and extracellular matrix. A mixture of unsaturated and saturated fatty acids was regarded effective in suppressing the EPS of S. mutans at a concentration of 31.3 µg/mL[27].