A meet-up of acetyl phosphate and c-di-GMP modulates BldD activity for development and antibiotic production.

Actinobacteria are ubiquitous bacteria undergoing complex developmental transitions coinciding with antibiotic production in response to stress or nutrient starvation. This transition is mainly controlled by the interaction between the second messenger c-di-GMP and the master repressor BldD. To date, the upstream factors and the global signal networks that regulate these intriguing cell biological processes remain unknown. In Saccharopolyspora erythraea, we found that acetyl phosphate (AcP) accumulation resulting from environmental nitrogen stress participated in the regulation of BldD activity through cooperation with c-di-GMP. AcP-induced acetylation of BldD at K11 caused the BldD dimer to fall apart and dissociate from the target DNA and disrupted the signal transduction of c-di-GMP, thus governing both developmental transition and antibiotic production. Additionally, practical mutation of BldDK11R bypassing acetylation regulation could enhance the positive effect of BldD on antibiotic production. The study of AcP-dependent acetylation is usually confined to the control of enzyme activity. Our finding represents an entirely different role of the covalent modification caused by AcP, which integrated with c-di-GMP signal in modulating the activity of BldD for development and antibiotic production, coping with environmental stress. This coherent regulatory network might be widespread across actinobacteria, thus has broad implications.

Efficient in planta production of amidated antimicrobial peptides that are active against drug-resistant ESKAPE pathogens.

Antimicrobial peptides (AMPs) are promising next-generation antibiotics that can be used to combat drug-resistant pathogens. However, the high cost involved in AMP synthesis and their short plasma half-life render their clinical translation a challenge. To address these shortcomings, we report efficient production of bioactive amidated AMPs by transient expression of glycine-extended AMPs in Nicotiana benthamiana line expressing the mammalian enzyme peptidylglycine α-amidating mono-oxygenase (PAM). Cationic AMPs accumulate to substantial levels in PAM transgenic plants compare to nontransgenic N. benthamiana. Moreover, AMPs purified from plants exhibit robust killing activity against six highly virulent and antibiotic resistant ESKAPE pathogens, prevent their biofilm formation, analogous to their synthetic counterparts and synergize with antibiotics. We also perform a base case techno-economic analysis of our platform, demonstrating the potential economic advantages and scalability for industrial use. Taken together, our experimental data and techno-economic analysis demonstrate the potential use of plant chassis for large-scale production of clinical-grade AMPs.

Curation, inference, and assessment of a globally reconstructed gene regulatory network for Streptomyces coelicolor.

Streptomyces coelicolor A3(2) is a model microorganism for the study of Streptomycetes, antibiotic production, and secondary metabolism in general. Even though S. coelicolor has an outstanding variety of regulators among bacteria, little effort to globally study its transcription has been made. We manually curated 29 years of literature and databases to assemble a meta-curated experimentally-validated gene regulatory network (GRN) with 5386 genes and 9707 regulatory interactions (~ 41% of the total expected interactions). This provides the most extensive and up-to-date reconstruction available for the regulatory circuitry of this organism. Only ~ 6% (534/9707) are supported by experiments confirming the binding of the transcription factor to the upstream region of the target gene, the so-called "strong" evidence. While for the remaining interactions there is no confirmation of direct binding. To tackle network incompleteness, we performed network inference using several methods (including two proposed here) for motif identification in DNA sequences and GRN inference from transcriptomics. Further, we contrasted the structural properties and functional architecture of the networks to assess the reliability of the predictions, finding the inference from DNA sequence data to be the most trustworthy approach. Finally, we show two applications of the inferred and the curated networks. The inference allowed us to propose novel transcription factors for the key Streptomyces antibiotic regulatory proteins (SARPs). The curated network allowed us to study the conservation of the system-level components between S. coelicolor and Corynebacterium glutamicum. There we identified the basal machinery as the common signature between the two organisms. The curated networks were deposited in Abasy Atlas ( https://abasy.ccg.unam.mx/ ) while the inferences are available as Supplementary Material.

Biosynthesis of C-nucleoside antibiotics in actinobacteria: recent advances and future developments.

Epidemic diseases and antibiotic resistance are urgent threats to global health, and human is confronted with an unprecedented dilemma to conquer them by expediting development of new natural product related drugs. C-nucleoside antibiotics, a remarkable group of microbial natural products with diverse biological activities, feature a heterocycle base linked with a ribosyl moiety via an unusual C-glycosidic bond, and have played significant roles in healthcare and for plant protection. Elucidating how nature biosynthesizes such a group of antibiotics has provided the basis for engineered biosynthesis as well as targeted genome mining of more C-nucleoside antibiotics towards improved properties. In this review, we mainly summarize the recent advances on the biosynthesis of C-nucleoside antibiotics, and we also tentatively discuss the future developments on rationally accessing C-nucleoside diversities in a more efficient and economical way via synthetic biology strategies.

Discovery of novel septacidin congeners from a high yield heterologous expression strain Streptomyces albus 1597.

Septacidin is an adenine nucleoside antibiotic with antifungal and antitumor activities. During the efforts to construct a better septacidin producer, we obtained a high yield strain S. albus 1597 by putting the biosynthetic gene cluster (BGC) of septacidin under the control of the constitutive strong promoter ermE*. S. albus 1597 could produce new septacidin congeners SEP-538 and SEP-552 with shorter fatty acyl chains. Moreover, SEP-624 with an unprecedented hydroxylated fatty acyl chain was also isolated from this titre improved strain, enriching the diversity of septacidins. SEP-552 showed moderate inhibitory effects against Epidermophyton floccosum 57312 with MIC value 62.5 µM, while SEP-538 and SEP-624 only exhibited weak antifungal activities. The structure-activity relationship investigation revealed that the antifungal activity of septacidins is significantly influenced by the length of and the decoration on their fatty acyl chains.

Bioactive Properties of a Novel Antibacterial Dye Obtained from Laccase-Mediated Oxidation of 8-Anilino-1-naphthalenesulfonic Acid.

Fungal laccase obtained from a Cerrena unicolor strain was used as an effective biocatalyst for the transformation of 8-anilino-1-naphthalenesulfonic acid into a green-coloured antibacterial compound, which can be considered as both an antimicrobial agent and a textile dye, simultaneously. The process of biosynthesis was performed in buffered solutions containing methanol as a co-solvent, allowing better solubilisation of substrate. The transformation process was optimised in terms of the buffer pH value, laccase activity, and concentrations of the substrate and co-solvent. The crude product obtained exhibited low cytotoxicity, antibacterial properties against Staphylococcus aureus and Staphylococcus epidermidis, and antioxidant properties. Moreover, the synthesised green-coloured compound proved non-allergenic and demonstrated a high efficiency of dyeing wool fibres.

Resolving the conflict between antibiotic production and rapid growth by recognition of peptidoglycan of susceptible competitors.

Microbial communities employ a variety of complex strategies to compete successfully against competitors sharing their niche, with antibiotic production being a common strategy of aggression. Here, by systematic evaluation of four non-ribosomal peptides/polyketide (NRPs/PKS) antibiotics produced by Bacillus subtilis clade, we revealed that they acted synergistically to effectively eliminate phylogenetically distinct competitors. The production of these antibiotics came with a fitness cost manifested in growth inhibition, rendering their synthesis uneconomical when growing in proximity to a phylogenetically close species, carrying resistance against the same antibiotics. To resolve this conflict and ease the fitness cost, antibiotic production was only induced by the presence of a peptidoglycan cue from a sensitive competitor, a response mediated by the global regulator of cellular competence, ComA. These results experimentally demonstrate a general ecological concept - closely related communities are favoured during competition, due to compatibility in attack and defence mechanisms.

Chlororesistoflavins A and B, Chlorinated Benzopyrene Antibiotics Produced by the Marine-Derived Actinomycete Streptomyces sp. Strain EG32.

As part of a collaborative biomedical investigation of actinomycete bacteria isolated from sediments collected along the northern coast of Egypt (Mediterranean Sea), we explored the antibacterial metabolites from a bacterium identified as a Streptomyces sp., strain EG32. HPLC analysis and antibacterial testing against methicillin-resistant Staphylococcus aureus (MRSA) resulted in the identification of six compounds related to the resistoflavin and resistomycin class. Two of these metabolites were the chlorine-containing analogues chlororesistoflavins A (1) and B (2). The absolute configurations of the lone stereogenic center (C-11b) in these metabolites were assigned by analysis of their ECD spectra. Interestingly, the ECD spectrum of chlororesistoflavin A (1) shows a Cotton effect of the n-π* transition antipodal to that of the parent natural product, a consequence of 1,3-allylic strain induced by the adjacent bulky chlorine atom that distorts the coplanarity of the carbonyl group with the π-system. The chiroptical analysis thus resolves the paradox and uniformly aligns the configuration of all analogues as identical to that reported for natural resistoflavin. Chlororesistoflavins A (1) and B (2) exhibited antibacterial activity against MRSA with a minimum inhibitory concentration of 0.25 and 2.0 µg/mL, respectively.

Rid7C, a Member of the YjgF/YER057c/UK114 (Rid) Protein Family, Is a Novel Endoribonuclease That Regulates the Expression of a Specialist RNA Polymerase Involved in Differentiation in Nonomuraea gerenzanensis.

The YjgF/YER057c/UK114 (Rid) is a protein family breadth conserved in all domains of life and includes the widely distributed archetypal RidA (YjgF) subfamily and seven other subfamilies (Rid1 to Rid7). Among these subfamilies, RidA is the only family to have been biochemically well characterized and is involved in the deamination of the reactive enamine/imine intermediates. In this study, we have characterized a protein of the Rid7 subfamily, named Rid7C, in Nonomuraea gerenzanensis, an actinomycete that is characterized by the presence of two types of RNA polymerases. This is due to the coexistence in its genome of two RNA polymerase (RNAP) ß chain-encoding genes, rpoB(S) (the wild-type rpoB gene) and rpoB(R) (a specialist, mutant-type rpoB gene) that controls A40926 antibiotic production and a wide range of metabolic adaptive behaviors. Here, we found that expression of rpoB(R) is regulated posttranscriptionally by RNA processing in the 5' untranslated region (UTR) of rpoB(R) mRNA and that the endoribonuclease activity of Rid7C is responsible for mRNA processing, thereby overseeing several tracts of morphological and biochemical differentiation. We also provide evidence that Rid7C may be associated with RNase P M1 RNA, although M1 RNA is not required for rpoB(R) mRNA processing in vitro, and that Rid7C endoribonuclease activity is inhibited by A40926, suggesting the existence of a negative feedback loop in A40926 production and a role of the endogenous synthesis of A40926 in the modulation of biochemical differentiation in this microorganism. IMPORTANCE The YjgF/YER057c/UK114 family includes many proteins with diverse functions involved in detoxification, RNA maturation, and control of mRNA translation. We found that Rid7C is an endoribonuclease that is involved in processing of rpoB(R) mRNA, coding for a specialized RNA polymerase beta subunit that oversees morphological differentiation and A40926 antibiotic production in Nonomuraea gerenzanensis. Rid7C-mediated processing promotes rpoB(R) mRNA translation and antibiotic production, while Rid7C endoribonuclease activity is inhibited by A40926, suggesting a role of the endogenous synthesis of A40926 in modulation of biochemical differentiation in this microorganism. Finally, we show that recombinant Rid7C copurified with M1 RNA (the RNA subunit of RNase P) from Escherichia coli extract, suggesting a functional interaction between Rid7C and M1 RNA activities.

Heterologous expression of a natural product biosynthetic gene cluster from Cordyceps militaris.

Cordyceps is a genus of ascomycete fungi widely used in old Chinese medicine, and many investigations have focus on uncovering their biological activities. Until now, only a few compounds have been identified from Cordyceps, mainly due to their poor yield. So as to make full use of Cordyceps, we used the strategy of genome mining and heterologous expression to discover natural products (NPs) from Cordyceps militaris. Analysis of the genome sequence of Cordyceps militaris CM01 showed the presence of a cryptic gene cluster encoding a highly-reducing polyketide synthetase (HR-PKS), enoyl-reductase (ER) and cytochrome P450. Heterologous expression in Aspergillus nidulans enabled the identification of two new polyketides, cordypyrone A and B. Their structures were determined by 1D and 2D NMR techniques. They showed only modest activities against pathogenic bacteria including methicillin-resistant Staphylococcus aureus (MRSA), Mycobacteria tuberculosis and Bacillus cereus.

Expression, Purification and Characterization of a Novel Hybrid Peptide CLP with Excellent Antibacterial Activity.

CLP is a novel hybrid peptide derived from CM4, LL37 and TP5, with significantly reduced hemolytic activity and increased antibacterial activity than parental antimicrobial peptides. To avoid host toxicity and obtain high-level bio-production of CLP, we established a His-tagged SUMO fusion expression system in Escherichia coli. The fusion protein can be purified using a Nickel column, cleaved by TEV protease, and further purified in flow-through of the Nickel column. As a result, the recombinant CLP with a yield of 27.56 mg/L and a purity of 93.6% was obtained. The purified CLP exhibits potent antimicrobial activity against gram+ and gram- bacteria. Furthermore, the result of propidium iodide staining and scanning electron microscopy (SEM) showed that CLP can induce the membrane permeabilization and cell death of Enterotoxigenic Escherichia coli (ETEC) K88. The analysis of thermal stability results showed that the antibacterial activity of CLP decreases slightly below 70 °C for 30 min. However, when the temperature was above 70 °C, the antibacterial activity was significantly decreased. In addition, the antibacterial activity of CLP was stable in the pH range from 4.0 to 9.0; however, when pH was below 4.0 and over 9.0, the activity of CLP decreased significantly. In the presence of various proteases, such as pepsin, papain, trypsin and proteinase K, the antibacterial activity of CLP remained above 46.2%. In summary, this study not only provides an effective strategy for high-level production of antimicrobial peptides and evaluates the interference factors that affect the biological activity of hybrid peptide CLP, but also paves the way for further exploration of the treatment of multidrug-resistant bacterial infections.

AdpA Positively Regulates Morphological Differentiation and Chloramphenicol Biosynthesis in Streptomyces venezuelae.

In members of genus Streptomyces, AdpA is a master transcriptional regulator that controls the expression of hundreds of genes involved in morphological differentiation, secondary metabolite biosynthesis, chromosome replication, etc. However, the function of AdpASv, an AdpA ortholog of Streptomyces venezuelae, is unknown. This bacterial species is a natural producer of chloramphenicol and has recently become a model organism for studies on Streptomyces. Here, we demonstrate that AdpASv is essential for differentiation and antibiotic biosynthesis in S. venezuelae and provide evidence suggesting that AdpASv positively regulates its own gene expression. We speculate that the different modes of AdpA-dependent transcriptional autoregulation observed in S. venezuelae and other Streptomyces species reflect the arrangement of AdpA binding sites in relation to the transcription start site. Lastly, we present preliminary data suggesting that AdpA may undergo a proteolytic processing and we speculate that this may potentially constitute a novel regulatory mechanism controlling cellular abundance of AdpA in Streptomyces. IMPORTANCEStreptomyces are well-known producers of valuable secondary metabolites which include a large variety of antibiotics and important model organisms for developmental studies in multicellular bacteria. The conserved transcriptional regulator AdpA of Streptomyces exerts a pleiotropic effect on cellular processes, including the morphological differentiation and biosynthesis of secondary metabolites. Despite extensive studies, the function of AdpA in these processes remains elusive. This work provides insights into the role of a yet unstudied AdpA ortholog of Streptomyces venezuelae, now considered a novel model organism. We found that AdpA plays essential role in morphological differentiation and biosynthesis of chloramphenicol, a broad-spectrum antibiotic. We also propose that AdpA may undergo a proteolytic processing that presumably constitutes a novel mechanism regulating cellular abundance of this master regulator.

Why Do Antibiotics Exist?

In the struggle with antibiotic resistance, we are losing. There is now a serious threat of moving into a postantibiotic world. High levels of resistance, in terms of both frequency and strength, have evolved against all clinically approved antibiotics worldwide. The usable life span of new clinically approved antibiotics is typically less than a decade before resistance reaches frequencies so high as to require only guarded usage. However, microbes have produced antibiotics for millennia without resistance becoming an existential issue. If resistance is the inevitable consequence of antibiotic usage, as has been the human experience, why has it not become an issue for microbes as well, especially since resistance genes are as prevalent in nature as the genes responsible for antibiotic production? Here, we ask how antibiotics can exist given the almost ubiquitous presence of resistance genes in the very microbes that have produced and used antibiotics since before humans walked the planet. We find that the context of both production and usage of antibiotics by microbes may be key to understanding how resistance is managed over time, with antibiotic synthesis and resistance existing in a paired relationship, much like a cipher and key, that impacts microbial community assembly. Finally, we put forward the cohesive, ecologically based "secret society" hypothesis to explain the longevity of antibiotics in nature.

Reductive inactivation of the hemiaminal pharmacophore for resistance against tetrahydroisoquinoline antibiotics.

Antibiotic resistance is becoming one of the major crises, among which hydrolysis reaction is widely employed by bacteria to destroy the reactive pharmacophore. Correspondingly, antibiotic producer has canonically co-evolved this approach with the biosynthetic capability for self-resistance. Here we discover a self-defense strategy featuring with reductive inactivation of hemiaminal pharmacophore by short-chain dehydrogenases/reductases (SDRs) NapW and homW, which are integrated with the naphthyridinomycin biosynthetic pathway. We determine the crystal structure of NapW·NADPH complex and propose a catalytic mechanism by molecular dynamics simulation analysis. Additionally, a similar detoxification strategy is identified in the biosynthesis of saframycin A, another member of tetrahydroisoquinoline (THIQ) antibiotics. Remarkably, similar SDRs are widely spread in bacteria and able to inactive other THIQ members including the clinical anticancer drug, ET-743. These findings not only fill in the missing intracellular events of temporal-spatial shielding mode for cryptic self-resistance during THIQs biosynthesis, but also exhibit a sophisticated damage-control in secondary metabolism and general immunity toward this family of antibiotics.

Identification and Verification of the Prodigiosin Biosynthetic Gene Cluster (BGC) in Pseudoalteromonas rubra S4059.

Pseudoalteromonas rubra S4059 produces the red pigment prodigiosin, which has pharmaceutical and industrial potential. Here, we targeted a putative prodigiosin-synthesizing transferase PigC, and a pigC in-frame deletion mutant did not produce prodigiosin. However, extractions of the pigC mutant cultures retained antibacterial activity, and bioassay-guided fractionation found antibacterial activity in two fractions of blue color. A precursor of prodigiosin, 4-methoxy-2,2'-bipyrrole-5-carbaldehyde (MBC), was the dominant compound in both the fractions and likely caused the antibacterial activity. Also, a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin, was identified from the two fractions. We also discovered antibacterial activity in the sterile filtered (nonextracted) culture supernatant of both wild type and mutant, and both contained a heat-sensitive compound between 30 and 100 kDa. Deletion of prodigiosin production did not affect growth rate or biofilm formation of P. rubra and did not change its fitness, as the mutant and wild type coexisted in equal levels in mixed cultures. In conclusion, a prodigiosin biosynthetic gene cluster (BGC) was identified and verified genetically and chemically in P. rubra S4059 and a stable blue pigment was isolated from the pigC mutant of S4059, suggesting that this strain may produce several prodigiosin-derived compounds of pharmaceutical and/or industrial potential. IMPORTANCE Pigmented Pseudoalteromonas strains are renowned for their production of secondary metabolites, and genome mining has revealed a high number of biosynthetic gene clusters (BGCs) for which the chemistry is unknown. Identification of those BGCs is a prerequisite for linking products to gene clusters and for further exploitation through heterologous expression. In this study, we identified the BGCs for the red, bioactive pigment prodigiosin using genomic, genetic, and metabolomic approaches. We also report here for the first time the production of a stable blue pigment, di-pyrrolyl-dipyrromethene prodigiosin (Dip-PDG), being produced by the pigC mutant of Pseudoalteromonas rubra S4059.

Gene editing enables rapid engineering of complex antibiotic assembly lines.

Re-engineering biosynthetic assembly lines, including nonribosomal peptide synthetases (NRPS) and related megasynthase enzymes, is a powerful route to new antibiotics and other bioactive natural products that are too complex for chemical synthesis. However, engineering megasynthases is very challenging using current methods. Here, we describe how CRISPR-Cas9 gene editing can be exploited to rapidly engineer one of the most complex megasynthase assembly lines in nature, the 2.0 MDa NRPS enzymes that deliver the lipopeptide antibiotic enduracidin. Gene editing was used to exchange subdomains within the NRPS, altering substrate selectivity, leading to ten new lipopeptide variants in good yields. In contrast, attempts to engineer the same NRPS using a conventional homologous recombination-mediated gene knockout and complementation approach resulted in only traces of new enduracidin variants. In addition to exchanging subdomains within the enduracidin NRPS, subdomains from a range of NRPS enzymes of diverse bacterial origins were also successfully utilized.

Biosynthesis of fluopsin C, a copper-containing antibiotic from Pseudomonas aeruginosa.

Metal-binding natural products contribute to metal acquisition and bacterial virulence, but their roles in metal stress response are underexplored. We show that a five-enzyme pathway in Pseudomonas aeruginosa synthesizes a small-molecule copper complex, fluopsin C, in response to elevated copper concentrations. Fluopsin C is a broad-spectrum antibiotic that contains a copper ion chelated by two minimal thiohydroxamates. Biosynthesis of the thiohydroxamate begins with cysteine and requires two lyases, two iron-dependent enzymes, and a methyltransferase. The iron-dependent enzymes remove the carboxyl group and the α carbon from cysteine through decarboxylation, N-hydroxylation, and methylene excision. Conservation of the pathway in P. aeruginosa and other bacteria suggests a common role for fluopsin C in the copper stress response, which involves fusing copper into an antibiotic against other microbes.

Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 are producers of the antibiotic nucleocidin and 4'-fluoroadenosine is identified as a co-product.

Genome homology and the presence of a putative biosynthetic gene cluster identified Streptomyces aureorectus DSM 41692 and Streptomyces virens DSM 41465 as candidate producers of the antibiotic nucleocidin 1. Indeed when these bacterial strains were cultured in a medium supplemented with fluoride (4 mM) they each produced nucleocidin 1 and the previously identified 4'-fluoro-3'-O-ß-glucosylated adenosine 2 and its sulfamylated derivative 3. In both of these cases 4'-fluoroadenosine 9 is also identified as a natural product although it has never been observed during fermentations of Streptomyces calvus, the original source of nucleocidin 1. The identity of 4'-fluoroadenosine 9 was confirmed by a total synthesis as well as by its in vitro enzymatic conversion to metabolite 2 using the glucosyl transferase enzyme, NucGT.

Study of Antibacterial and Anticancer Properties of bioAgNPs Synthesized Using Streptomyces sp. PBD-311B and the Application of bioAgNP-CNC/Alg as an Antibacterial Hydrogel Film against P. aeruginosa USM-AR2 and MRSA.

Here, we report the extracellular biosynthesis of silver nanoparticles (AgNPs) and determination of their antibacterial and anticancer properties. We also explore the efficacy of bioAgNPs incorporated in cellulose nanocrystals (CNCs) and alginate (Alg) for the formation of an antibacterial hydrogel film. Streptomyces sp. PBD-311B was used for the biosynthesis of AgNPs. The synthesized bioAgNPs were characterized using UV-Vis spectroscopy, TEM, XRD, and FTIR analysis. Then, the bioAgNPs' antibacterial and anticancer properties were determined using TEMA and cytotoxicity analysis. To form the antibacterial hydrogel film, bioAgNPs were mixed with a CNC and Alg solution and further characterized using FTIR analysis and a disc diffusion test. The average size of the synthesized bioAgNPs is around 69 ± 2 nm with a spherical shape. XRD analysis confirmed the formation of silver nanocrystals. FTIR analysis showed the presence of protein capping at the bioAgNP surface and could be attributed to the extracellular protein binding to bioAgNPs. The MIC value of bioAgNPs against P. aeruginosa USM-AR2 and MRSA was 6.25 mg/mL and 3.13 mg/mL, respectively. In addition, the bioAgNPs displayed cytotoxicity effects against cancer cells (DBTRG-0.5MG and MCF-7) and showed minimal effects against normal cells (SVG-p12 and MCF-10A), conferring selective toxicity. Interestingly, the bioAgNPs still exhibited inhibition activity when incorporated into CNC/Alg, which implies that the hydrogel film has antibacterial properties. It was also found that bioAgNP-CNC/Alg displayed a minimal or slow release of bioAgNPs owing to the intermolecular interaction and the hydrogel's properties. Overall, bioAgNP-CNC/Alg is a promising antibacterial hydrogel film that showed inhibition against the pathogenic bacteria P. aeruginosa and MRSA and its application can be further evaluated for the inhibition of cancer cells. It showed benefits for surgical resection of a tumor to avoid post-operative wound infection and tumor recurrence at the surgical site.