Targeting Antibiotic Resistant Salmonella enterica: Bio-matrix Based Selection and Bioactivity Prediction of Potential Nutraceuticals

Natural Plant Products (NPPs) with medicinal potential, derived from either traditional or modern medicine, serves as a source of approx. 5000 compounds per species to fight against multifarious diseases. Such tremendous source of secondary plant metabolites employs to defend themselves against bacteria, fungi or viruses and can be used in almost the same way in medicine to treat microbial or viral infections [1]. The effective management of re-emerging pathogens with enhanced scope of attack and attributable resistance require intensive efforts in this direction. NPPs have become an ideal alternative to be explored for their therapeutic utilities by the virtue of their holistic broad spectrum activity; limited or no side effects; better acceptability and ease of local availability [2]. However, the Indian subcontinent has only 2% global market share of herbal medicine despite having 7000 Ayurvedic, 700 Unani, 600 Siddha medicines and 30 modern medicines. This clearly indicates the lack of directed research in instigating the search for newer antimicrobials based on either reverse pharmacology or classical quest of lead moiety. The expected market for NPPs is $5 Trillion by 2050; if focused research is triggered so as to yield therapeutic leads [3]. Such focused research demands an in depth knowledge of pathophysiology of disease causing microorganisms and rationale based selection of holistic agents having bio-protective potential.


Introduction
Natural Plant Products (NPPs) with medicinal potential, derived from either traditional or modern medicine, serves as a source of approx. 5000 compounds per species to fight against multifarious diseases. Such tremendous source of secondary plant metabolites employs to defend themselves against bacteria, fungi or viruses and can be used in almost the same way in medicine to treat microbial or viral infections [1]. The effective management of re-emerging pathogens with enhanced scope of attack and attributable resistance require intensive efforts in this direction. NPPs have become an ideal alternative to be explored for their therapeutic utilities by the virtue of their holistic broad spectrum activity; limited or no side effects; better acceptability and ease of local availability [2]. However, the Indian subcontinent has only 2% global market share of herbal medicine despite having 7000 Ayurvedic, 700 Unani, 600 Siddha medicines and 30 modern medicines. This clearly indicates the lack of directed research in instigating the search for newer antimicrobials based on either reverse pharmacology or classical quest of lead moiety. The expected market for NPPs is $5 Trillion by 2050; if focused research is triggered so as to yield therapeutic leads [3]. Such focused research demands an in depth knowledge of pathophysiology of disease causing microorganisms and rationale based selection of holistic agents having bio-protective potential.
in the ecosystem and limited / no treatment options. The outbreaks of Multi-Drug Resistant (MDR) strains of Salmonella enterica serovar typhimurium in the Indian subcontinent, Southeast Asia, and Africa showed approximately 16 million cases and 60,0000 deaths each year [4]. These infections which were earlier contained within hospitals, have now higher penetration even at community level [5]. Salmonella is also associated with reactive arthritis having global population expressivity in a range of 6 to 30%, out of which 0.2%-7.3% cases were of joint inflammation alone. Incubation period of reactive arthritis is ambiguous spanning a time period of 2-4 weeks after the onset of enteric infection [6]. Reactive arthritis is also reported to be associated with other food borne illness like Campylobacter and Shigella with reported infection rate of 1 and 4% in adults respectively [7]. Such findings implied that such infections are burdening the economic Page 2 of 9 setup of healthcare management attributed towards a longer duration of treatment with expensive antibiotics. Resistance in multidrug resistant pathogens is accredited to chromosomal mutations and selective pressure of several synthetic drugs used for the treatment [8]. Also, the resistance profile of Salmonella enterica serovar typhimurium indicates a shift from multidrug resistance to extensive drug resistance with resistance against tetracycline, streptomycin, sulfamethoxazole, ampicillin and ceftriaxone [9]. Emergence of strains resistant to ciprofloxacin further broadened its risk spectra. The mitigation of such risk at early stages is necessary so as to cease the consequent cascade of its use with biothreat intent. New therapeutic modalities with multitargeted mode of action are needed to be investigated. The holistic mode of physiological action of natural plant products with reduced or nil adverse drug reactions might serve as the probable candidate(s) to be explored [10].
The present study utilized in silico bioprospection herbal informatics tool to filter the potential herbals targeted against virulence factors of MDR Salmonella enterica serovar typhimurium. It included web based classical data search, matrix linked data mining followed by fuzzy logic based optimization/ validation to identify potent herbals with probable therapeutic efficacy [11]. Furthermore, the selected herbals were screened for their biochemical activity at in silico level using 'Mol inspiration', a chemiinformatics tool [12]. This study hence also illustrated the active leads for ascertaining desired biological activity, which further warrants antimicrobial efficacy testing at both in vitro and in vivo levels.

Classical literature surge model
Antibiotic resistant Salmonella enterica serovar typhimurium was selected as the target pathogen. A classical literature surge model was utilized and ascertained Enterochelin/enterobactin (ENT) Inhibition, Type III secretory system (TSSIII) Inhibition, Lipopolysaccharides (LPS) Inhibition, Superoxide dismutase (SOD) Inhibition and Symptomatic relief provision as virulence factors [13]. Similarly, the database set of herbals was selected with attributable factors as a) Ethnopharmacological importance of plant; b) Relevance of Herb in traditional medicine; c) Availability factor or cultural acceptability in localized regions; d) Any vedic literature supporting its use; e) Investigations/ prior experience on potential of the herb; f) Indirect indications, if any.

Relevance factor linked binary matrix analysis
PubMed was used as the assigned search engine for keyword hits analysis. A combination keyword converging 'anti-microbial activity and virulence factor' was entered in the query search box of PubMed. Observation based analysis of first 20 hits was done for linking the relevance of each virulence factor with its direct significance in triggering the disease [14]. Percentage relevance of each virulence factor was evaluated using the following formula;

No of relevant hits based on observational analysis Relevance avg X Total No of Hits screened =
The scrutinization of primary database set of NPPs was done with respect to presence or absence of a given bioactivity parameter in the NPP, as reported in either traditional databases or PubMed search engine.

Relevance factor linked weightage matrix analysis
On the basis of average percentage relevance, each bioactivity parameter was assigned with a weightage score. Bioactivity parameter having maximum percentage relevance was assigned with a score of 05 and other succeeding parameters were provided with relative weightage scores based on unitary method evaluation. Evaluation of overall weightage of plants having a binary score >3 was done by multiplying their binary score with weightage score of existing bioactivity parameter. This step definitely out-ruled the possibility of any 'uncertainty factor', thereby reducing investigator's biasness. Plants with scores ≥3 from previous step were only utilized to identify potent leads [15].

Fuzzy set membership analysis and optimization
The following mathematical relationship was used to ascertain relative relevance within an identified set of herbals; Where, μS represents the desirability values of selected NPPs of the fuzzy set S. Min(S) and Max(S) are minimum and maximum scores, respectively, in the fuzzy set S [16]. The estimated μS were converted into a leveled score by using a scaled magnitude as optimization of identified potential set of NPPs.

In silico biochemical and molecular activity prediction using 'Mol Inspiration' Preparation of ligand dataset
The 2D structures and the SMILES (Simplified Molecular-Input Line-Entry System) notation of predominant phyto-constituents of scrutinized herbals (~ Fuzzy Set Score of 1) were obtained from Pubchem [17].

Biological activity prediction
Canonical SMILES of each of the selected biologically active phytoconstituent was considered as the test set. Based on the analysis of large training set consisting of tens of thousands of the known biologically active compounds, computer program PASS analyzed the presence or absence of specific substructures in the test set in relation to the training set. Each substructure fragment contributed towards a bioactivity score for each listed activity i.e., GPCR Ligand, Ion Channel modulator, Kinase inhibitor, Nuclear receptor ligand and Protease inhibitor. This provided a total molecule activity score (a number, typically between -3 and 3). Molecules with the highest activity score had the highest probability to be active [12].

Classical literature surge model
Classical Bioprospection was done utilizing Vedic literature, ethnopharmacological databases, PubMed and direct/indirect scientific evidences as the foundation for rationale based selection of herbals targeting MDR S. enterica. Also, pathophysiology of MDR S. enterica was reviewed so as to select the crucial 05 virulence factors as drug targets (Bioactivity parameters). These bioactivity parameters were selected on the basis of certain important characteristics including a) category of microorganism as lethal, sub-lethal, incapacitating agent; b) unavailability of a treatment regime/vaccine; c) re-emerging virulent forms from past; d) intended use as a bioweapon. Similarly, classical bioprospection of 50 herbals was done on the basis of their direct significance in a) symptomatic relief provision; b) cell wall synthesis inhibition; c) bacterial enzyme inhibition; and/or d) antibiotic resistance modification.

Relevance factor linked binary matrix analysis
On the basis of keyword hits scoring matrix, average percentage relevance was ascertained for each virulence factor of Salmonella enterica subvar typhimurium. Rationale for selection of each of these parameters is elucidated in Table 1. Highest percentage relevance was obtained for Lipopolysaccharide inhibition (~ 65%), followed by other parameters like Symptomatic relief provision, Type III secretory system (TSSIII) inhibition, Superoxide dismutase (SOD) inhibition and Enterochelin/ enterobactin inhibition. Consequently weightage was given to selected parameters in the range of 0.05-5, based on statistical unitary approach, with highest weightage i.e. 5, given to Lipopolysaccharide inhibition, followed by other parameters in decreasing order, as explicated in Table  2. The binary matrix analysis of presence / absence of these virulence factors in 50 herbals revealed that 28 herbal plants showed score ≥3 i.e., the median cut off value (Table 3).

Weightage matrix based analysis and optimization
The weightage score matrix analysis of 28 scrutinized plants revealed that 10 plants showed highest weightage score ≥ 14.98, thereby indicating their probable therapeutic utility against one or more of the targeted virulence factors. The final selection included Abrus precatorius, Azadirachta indica, Camellia sinensis, Holarrhena antidysenterica, Andrographis paniculata, Adhatoda vasica, Euphorbia hirta, Ocimum sanctum, Terminalia arjuna and Terminalia belerica with an optimized fuzzy score of 1.0 (Table 4).

S.No.
Bioactivity parameter Rationale for selection (Based on Classical Approach)

Enterochelin/enterobactin(ENT) Inhibition [23]
• Enterochelin is a cyclic polyester consisting of three residues of 2,3-dihydroxy-N-benzoylserine. Enterobactin, a physiologically active iron sequestering agent, is excreted into the medium by S. typhimurium under low iron conditions and is indispensable for growth.

•
Enterochelin regulates the capacity of S. typhimurium to grow both in vivo and in vitro.

•
Crude extracts of several plants like Allium sativum, Acacia catechu, A. nilotica etc have been found to be bacteriostatic against multidrug resistant (MDR) strains of S. typhimurium.

2.
Type III secretory system (TSSIII) Inhibition (Injectisome or Injectosome) [24] • Contains proteins that form a channel in the inner and outer bacterial membranes, as well as an extracellular needle that is used for transporting and injecting effector proteins into a host cell. that exhibited significant effects on type III secretory system.

Lipopolysaccharide Inhibition (LPS) [25]
• LPS, key component of the cell wall and is required for the cellular integrity.

•
Toxicity is due to an outer membrane consisting largely of lipo-polysaccharides (LPS) which protect the bacteria from the environment. It is made up of an O-antigen (responsible for the host immune response), and lipid A (made up of two phosphorylated glucosamine which are attached to fatty acids. Phosphorylation of this leads to the toxicity, which connects it to the outer membrane. • Several plant extracts have exhibited synergistic activity like Zingiber officinalis, Semecarpus anacardium, and Terminalia belerica against gram negative microorganisms and inhibits inflammation.

Superoxide dismutase (SOD) Inhibition [26]
• Enzymes that catalyze the dismutation of superoxide (O 2 − ) into oxygen and hydrogen peroxide. Human WBC's generate superoxide and other reactive oxygen species to kill bacteria.
• During infection, some bacteria therefore produce superoxide dismutase (Specially sodA and sodB) to protect themselves from being killed, which might be helpful for the survival of strain by acting against the phagocytic mechanism of the host.

•
Plants extracts that have shown synergistic effects include Terminalia belerica + Zingiber officinalis + Embelia ribes to inhibit the SOD antioxidant activity of S. typhimurium.

5.
Symptomatic relief [27] • S. typhimurium is known to cause bacteremia, with or without gastrointestinal involvement; typhoid or enteric fever; localized infections (e.g, bones, joints, and meninges) • One of the possible disease management strategies is to alleviate the symptoms by using herbals like Vitis vinifera, Piper longum, Allium sativum etc.

•
Active ingredient such as alkaloids, flavones, glycosides, etc of medicinal herbals have been known to show such symptomatic relief providing effects.

Discussion
Antibiotics, the defense resort against the sturdy pathogens of commensal as well as nosocomial origin, are now losing the battle against multi-drug resistant re-emerging pathogens e.g., Salmonella enterica serovar typhimurium (diversified food borne pathogen) [18]. Such conflict has arisen due to the irrational usage of antibiotics, thereby transforming commensals into extensively drug resistant forms. Natural selection and sudden mutations might be conferring resistance in these pathogens [8]. The present day synthetic drugs are not competent enough to overcome the irreversible effects of such resistance patterns. Limitations of chemotherapeutic moieties i.e., systemic toxicity and adverse drug reactions, hence require to be outruled by instigating the search for newer antimicrobials of plant origin having holistic potential [1].
The present study utilizes an ethnopharmacological approach amalgamated with informatics for rationale based selection of herbals. Random search model approach was devised using PubMed as the search engine. 05 bioactivity parameters were selected on the basis of their direct/ indirect role in a) symptomatic relief provision, b) antibiotic resistance modification, c) bacterial enzyme modification, d) cell wall synthesis inhibition (Table 1). This bioprospection study was found to be in consonance with a similar study of the evaluation of sleep wake cycle in healthy individuals conducted by Elizabeth S. Jenuwine et al. [19]. The importance of herbal informatics model can further be viewed from the fact that herbals have been used as the main reservoir for a variety of natural plant products having bio-protection abilities e.g., Taxol (anticancerous, Taxus baccata), Silymarin (radioprotector, Silybum mariannum), Vincristine (anticancerous, Rosmarinus officinalis), Vinblastin (anticancerous, Rosmarinus officinalis) etc [20].
Binary coefficient matrix analysis, the first screening step of herbal informatics, was used to identify herbals on the basis of all or none principle, thereby eliminating the outliers which have scored less than the median cut off value i.e., >3. After the first screening step, 28 plants were filtered from a pool of 50 plants, thus reducing the total time of execution. Furthermore, weightage matrix based analysis was used to scrutinize the selection process in case of 02 or more herbals with identical binary score. Final decision of selection of herbals was based on fuzzy set membership analysis, so as to provide a universally acceptable optimized score [16].
The final categorized set of plants included Abrus precatorius (Rati), Azadirachta indica (Neem), Camellia sinensis (Green tea), Holarrhena antidysenterica (Indrajava), Andrographis paniculata (Kalmegh), Adhatoda vasica (Adosa), Euphorbia hirta (Dudhi), Ocimum sanctum (Tulsi), Terminalia arjuna (Arjuna) and Terminalia belerica (Behada). All these above mentioned plants have been reported to be contain a variety of secondary metabolites like conessine (alkaloid, Holarrhena antidysenterica); andrographolide (alkaloid, Andrographis paniculata) and epicatechin gallate (flavonoid, Camellia sinensis) etc [1,20]. 16 Punica   Furthermore, the chemiinformatics based biochemical activity prediction of predominant phytoconstituents of these NPPs gave an insight of their mechanistic mode of action. Phytoconstituents which were exhibiting bioactivity with respect to either GPCR or Nuclear receptor ligand (i.e., Conessine, Andrographolide, Amyrin, Arjunolic acid) might have a direct or indirect role in regulation of immune system activity and inflammation. Both G protein-coupled receptor signaling and Nuclear Receptor binding lead to mast cell degranulation which subsequently induces localized inflammation and bacterial antigen clearance [21]. Moreover secondary metabolites with bioactivity linked to protease inhibition (e.g., Conessine, Andrographolide, Amyrin, Arjunolic acid) is directly contemplating the antimicrobial activity of these phytoconstituents as protease is known to be a key enzyme of bacteria required for evasion of host immune defenses, nutrients acquisition for growth and proliferation and tissue necrosis [22]. These secondary metabolites of NPPs might therefore, be responsible for their holistic antibacterial action, which can further be tested and validated at in vitro and in vivo levels.

Conclusion
The study has provided 06 NPPs i.e., Holarrhena antidysenterica, Andrographis paniculata, Euphorbia hirta, Terminalia arjuna, Abrus precatorius and Camellia sinensis with significant therapeutic potential that need to be explored at in vitro and pre-clinical level to manage MDR Salmonella enterica serovar typhimurium and other related infections.