Evaluation of Phenotyping and Genotyping Characterization of Serratia marcescens after Biofield Treatment

Serratia marcescens (S. marcescens) is Gram-negative bacterium, associated with hospital-acquired infections (HAIs), especially urinary tract and wound infections. The present study was aimed to evaluate the impact of biofield treatment on phenotyping and genotyping characteristics such as antimicrobial susceptibility, biochemical reactions, biotype, DNA polymorphism, and phylogenetic relationship of S. marcescens (ATCC 13880). The lyophilized cells of S. marcescens were divided into three groups (G1, G2, and G3). Control group (G1) and treated groups (G2 and G3) of S. marcescens cells assessed with respect to antimicrobial susceptibility, and biochemical reactions. In addition to that, samples from different groups of S. marcescens were evaluated for DNA polymorphism by Random Amplified Polymorphic DNA (RAPD), and 16S rDNA sequencing in order to establish the phylogenetic relationship of S. marcescens with different bacterial species. The treated cells of S. marcescens showed an alteration of 10.34% and 34.48% antimicrobials in G2 and G3 on 10th day, respectively as compared to control. The significant changes of biochemical reactions were also observed in treated groups of S. marcescens. The RAPD data showed an average range of 16-49.2% of polymorphism in treated samples as compared to control. Based on nucleotide homology sequences and phylogenetic analysis, the nearest homolog genus-species was found to be Pseudomonas fluorescence. These findings suggest that biofield treatment can prevent the emergence of absolute resistance to the useful antimicrobials against S. marcescens.


Introduction
Currently, many microorganisms have been acquired the resistance to number of antibiotics and other antimicrobial agents, which were effectively used earlier to cure a microbial infections. The antimicrobial resistant microbes (including bacteria, viruses, fungi, and parasites) can survive in antimicrobial drugs therapy. Therefore, regular treatments are ineffective. The frequent and improper use or misuse of antimicrobial medicines accelerates the emergence of drug-resistant microorganism, which was further spread by meagre infection control and poor sanitary conditions [1]. Serratia marcescens (S. marcescens) is a rod-shaped Gram-negative bacteria, belongs to family Enterobacteriaceae. It is a facultative anaerobic bacterium that can grow in presence and absence of oxygen at temperatures 30°C to 37°C. S. marcescens become an opportunist pathogen causing nosocomial infections and commonly involved in hospital-acquired infections (HAIs); specially urinary tract infections (UTIs), pneumonia, septicemia, meningitis and wound infections. Recently, S. marcescens drastically acquired the resistance to several existing antimicrobials like penicillin by decreasing the permeability and by β-lactamase to cleave the β-lactam ring of penicillin; fluoroquinolones (nalidixic acid, ciprofloxacin, ofloxacin, and norfloxacin), by proton dependent multidrug resistance (MDR) efflux pumps [2,3]. Therefore, development of effective antimicrobial therapy against S. marcescens is very needful for human health. Recently, biofield treatment came in focus that can cure the microbial infection by changing the microbial susceptibility against the antimicrobial drugs.
The relation between mass-energy was described by Friedrich, then after Einstein gave the well-known equation E=mc 2 for light and mass [4,5]. The mass (solid matter) is consist of energy and once this energy vibrates at a certain frequency, it gives physical, atomic and structural properties like shape, size, texture, crystal structure, and atomic weight to the matter. Similarly, human body also consists of vibratory energy particles like neutrons, protons, and electrons. Due to the vibration of these particles in the nucleus, an electrical impulse is generated [6]. Consequently, as per Ampere-Maxwell-Law, varying of these electrical impulses with time generates magnetic field, which cumulatively form electromagnetic field [7,8]. Thus, human has the ability to harness the energy from environment or universe and can transmit into any living or nonliving object(s) around the Globe. The objects always receive the energy and responding into useful way that is called biofield energy and the process is known as biofield treatment. Mr. Mahendra Trivedi's biofield treatment (The Trivedi Effect®) has been applied to transform the structural, physical, and chemical properties of materials in several fields like material science [9][10][11][12][13][14][15][16], agriculture [17][18][19], and biotechnology [20,21]. The biofield treatment has considerably altered the genotype of the microbes and thereby changed in susceptibility to antimicrobials [22][23][24].
After consideration of clinical significance of S. marcescens and significant impact of biofield treatment on microbes, we felt a detailed investigation was required to evaluate the effect of biofield treatment on S. marcescens. After that, the organism was assessed in relation to antimicrobials susceptibility and biotyping based on various Molecular and Genetic Medicine biochemical reactions. We also explored the genotyping of this organism using polymerase chain reaction (PCR) based methodologies of randomly amplified polymorphic DNA (RAPD) and 16S rDNA sequencing techniques. To the best of our knowledge, this is the first report that explores the impact of biofield treatment on S. marcescens.

Materials and Methods
Two vials of S. marcescens [American Type Culture Collection (ATCC) 13880] were procured from MicroBioLogics, Inc., USA, in sealed packs, and stored as per the recommended storage conditions until further use. The anti-microbial susceptibility, biochemical reactions, and biotype number were evaluated on MicroScan Walk-Away® (Dade Behring Inc., West Sacramento, CA) using Negative Breakpoint Combo 30 (NBPC30). DNA Fingerprinting by RAPD analysis (using Ultrapure Genomic DNA Prep Kit; Cat KT 83) and the 16S rDNA sequencing studies were carried out using Ultrapure Genomic DNA Prep Kit; Cat KT 83 (Bangalore Genei, India). All the tested antimicrobials, biochemicals and other reagents were procured from Sigma-Aldrich.

Study design
The microorganisms were grouped as per study design like bacterial cell were divided in to three groups G1 (control), G2 (treatment, revived), and G3 (treatment, lyophilized). The treatment groups (G1 and G2) were in sealed pack and handed over to Mr. Trivedi for biofield treatment under laboratory condition. Mr. Trivedi provided the treatment through his energy transmission process to the treated groups without touching the samples. After that, G2 group was assessed for antimicrobial susceptibility and biochemical reactions on 5 th and 10 th day of incubation; and G3 group was assessed on 10 th day of treatment. The treated groups were compared with respect to control.

Biotype number
The biotype number of S. marcescens was determined by MicroScan Walk-Away® processed panel data utilizing biochemical reactions data [25].

Random Amplified Polymorphic DNA (RAPD) analysis
Three inoculums (one for control and other two for treatment named as treatment A and B) were prepared of S. marcescens samples.
Two inoculums (treatment samples A and B) were subjected to Mr. Trivedi's biofield treatment. After that, the treated samples were subcultured by taking 1% inoculum and inoculated to fresh 5 mL medium and labeled as treatment A-1 and treatment B-1, respectively. All samples were incubated at 37°C with 160 rpm for 18 h. Subsequently, the cultures were spun down, and genomic DNA was isolated for control and treated samples using Genomic DNA Prep Kit (Bangalore Genei, India). RAPD was performed with all samples of S. marcescens using five RAPD primers, which were labelled as RBA8A, RBA13A, RBA20A, RBA10A and RBA15A. The PCR mixture contained 2.5 µL each of buffer, 4.0 mM each of dNTP, 2.5 μM each of primer, 5.0 μL each of genomic DNA, 2 U each of Taq polymerase, 1.5 μL of MgCl 2 and 9.5 μL of water in a total of 25 μL with the following PCR amplification protocol; initial denaturation at 94°C for 7 min, followed by 8 cycles of denaturation at 94°C for 1 min, annealing at 35°C for 1 min, and extension at 72°C for 2 min; and 35 cycle of denaturation at 94°C for 1 min, annealing at 38°C for 1 min, and extension at 72°C for 1.5 min; and the final extension at 72°C for 7 min. Amplified PCR products from all samples (control and treated) were separated on 1.5 % agarose gels at 75 volts, stained with ethidium bromide and visualized under UV illumination.

Amplification and gene sequencing of 16S rDNA
Genomic DNA was isolated from S. marcescens cells by using genomic purification Kit, according to the instructions of manufacturer. 16S rDNA gene (~1.5 kb) was amplified by universal primers; forward primer (5ˊ-AGAGTTTGATCCTGGCTCAG-3ˊ) and reverse primer (3ˊ-ACGGTCATACCTTGTTACGACTT-5ˊ). Amplified products were subjected to electrophoresis in 1.0% agarose gel, stained with ethidium bromide and visualized under UV light in a gel documentation unit (BioRad Laboratories, USA). The PCR amplified fragment was purified from the agarose gel using a DNA Gel Extraction Kit. Sequencing of amplified product was done on commercial basis from Bangalore Genei, India. The 16S rDNA sequences obtained were aligned and compared with the sequences stored in Gene Bank data base available from National Center for Biotechnology Information (NCBI) using the algorithm BLASTn program. Multiple sequence alignment/phylogenetic tree were established using MEGA3.1 molecular software [29]. and 2, respectively. The data were analyzed and compared with respect to control. The treated cells of S. marcescens showed an alteration of 10.34% and 34.48% in G2 and G3 group on 10 th day, respectively of antimicrobials susceptibility among all tested antimicrobials as compared to control. Studying the effect of biofield treatment in the antibiogram of S. marcescens, revealed that the amikacin and tobramycin were converted from resistance to susceptible on 10 th day of G3 group as compared to control. Aztreonam, cefotetan, ceftazidime, cefuroxime and chloramphenicol were converted from resistance to intermediate on 10 th day of biofield treatment of G3 group as compared to control. The cefepime and cefotaxime were converted from resistance to intermediate on 10 th day of G2 treated cells and complete susceptibility was observed for gentamycin and cefepime on 10 th day of G3 treated cells as compared to control (Table  1). It was also observed that there was reduced activity of inducible βlactamase of aztreonam, cefotaxime, cefotetan, ceftazidime, and ceftriaxone antimicrobials. The MIC values of amikacin, aztreonam, cefepime, cefotetan, ceftazidime, gentamicin and tobramycin were decreased about two-folds; whereas about four-folds decrease in MIC values of cefotaxime and ceftriaxone on 10 th day of G2 treated cells as compared to control (

Organism identification by biochemical reactions
The biochemical reactions of S. marcescens are presented in Table 3.
In the present study, acetamide, cetrimide, indole, inositol, and oxidase biochemical reactions of control and treated cells of S. marcescens showed negative biochemical reactions.  Twenty-four of thirty-one biochemical reactions were showed positive reaction for control and two treatment groups. Arginine reaction of treated G2 cells on 10 th day was negative and tartrate reaction was positive for the treatment G2 cells on both 5 th and 10 th day as compared to control. Ten out of thirty one biochemical reactions (32.25 %) of treated cells in G3 were converted from positive to negative reaction, and tartrate biochemical reaction was remain unchanged as negative as compared to control (Table 3).

Organism identification by biotype number
The biotype number of S. marcescens was determined by MicroScan Walk-Away® processed panel, using biochemical reactions data. There was no change in biotype number observed in treated G2 cells on 5 th day of incubation. However, the significant changes in the biotype number of S. marcescens were observed in G2 and G3 on 10 th day of incubation as compared to control (

Random Amplified Polymorphic DNA (RAPD) analysis
The DNA polymorphic photograph is shown in Figure 1, and the polymorphic bands are marked by arrows.

16S rDNA genotyping
The 16S rDNA sequence was determined in S. marcescens. The alignment and comparison of the gene sequences were performed with the sequences stored in Gene Bank database available from NCBI using the algorithm BLASTn program. The nearest homolog genusspecies of S. marcescens was found to be P. fluorescens (Accession No. DQ439976). Some other close homologs of S. marcescens were can be found from the alignment as shown in Table 7.  The distance matrix based on nucleotide sequence homology data are presented in Figure 2. Based on nucleotides homology and phylogenetic analysis the microbe (Sample 8A) was detected to be S. marcescens (GenBank Accession Number: EU233275). Phylogenetic tree was established using BLAST-Webpage (NCBI). According to Figure 2, ten different related bacterial species and S. marcescens were selected as Operational Taxonomic Units (OTUs) in order to investigate the phylogenetic relationship of S. marcescens among other ten other bacterial species. There were 1506 base nucleotides of 16S rDNA gene sequences were analyzed and multiple alignment were constructed using ClustalW in MEGA3.1. The numbers of base substitutions per site from pairwise distance analysis between sequences are shown in Table 7. All results are based on the pairwise analysis of 11 sequences. According to the data in Figure 2, the lowest value of genetic distance from S. marcescens was 0.000 base substitutions per site. All pairwise distance analysis was carried out using the p-distance method in MEGA3.1. The proportion of remarked distance, sometimes also called p-distance and showed as the number of nucleotide distances site. Values in Table 7 were programmed into Figure 2 with optimal bootstrap consensus tree. In the phylogram, there were eleven OTUs. Based on the phylogenetic tree and 16S rDNA sequencing, the nearest homolog genus-species of S. marcescens was found to be P. fluorescens (Figure 3).

Discussion
The increasing incidence of antimicrobial resistance is getting more global attention. Antibiotic multi-resistant Gram-negative bacteria pose a risk to public health [30]. Extended spectrum β-lactam antibiotics have been extensively used for treatment of severe Gram-negative infections since four decades. Although, bacterial resistance has emerged rapidly due to the production of ESBLs [31]. Enterobacteriaceae producing ESBL are emerging as a threatening cause of both hospital and community acquired infection, as they are often resistant to standard antimicrobial choices [32,33]. It is generally thought that patients infected by an ESBL-producing organism are at an increased risk of treatment failure [34]. Recently, an increasing percentage of ESBL-producing S. marcescens has been detected worldwide with a significant impact on the clinical course of disease. ESBL are enzymes produced by some S. marcescens species that inactivate many antimicrobials such as penicillins, expanded spectrum cephalosporins, monobactams including older β-lactam antimicrobial agents and are inhibited by clavulanic acids, imipenem, sulbactam or monobactam [35,36].
In the present work, we investigated the impact of biofield treatment on S. marcescens and evaluated the antimicrobials susceptibility pattern, biochemical reactions, biotype number, and DNA polymorphism of this microbe. The treated cells of S. marcescens showed an alteration in susceptibility of 10.34% and 34.48% antimicrobials of G2 and G3 group on 10 th day, respectively, as compared to control (Table 1). A significant change was found for a few antimicrobials to their antimicrobial susceptibility from resistant to intermediate and resistant to susceptible at 10 th day of treated G2 and G3 group, respectively (Table 1). MIC values of about 10.34% and 34.48% antimicrobials were decreased in G2 and G3 group, respectively on 10 th day ( Table 2). Studying the effect of biofield treatment in the antibiogram of S. marcescens revealed that the amikacin converted from resistance to susceptible on 10 th day of G2 cells as compared to control. The MIC values of some antimicrobials such as cefotetan, ceftazidime, gentamicin, and tobramycin were decreased by about two-folds in treated G2 on 10 th day and four-folds decrease were observed for cefotaxime and ceftriaxone at 10 th day of treated G3 ( Table 2). The S. marcescens also showed the substantial changes in biochemical reactions pattern towards a few biochemicals on 10 th day of treated G3 group as mentioned in Table 3. The alterations in biochemical reactions pattern were further supported by the determination of biotype number of S. marcescens, which was changed from 7736 7376 (control) to 7736 5376 and 7020 5356 for treated G2 and G3 on 10 th day, respectively (Table 4). DNA fingerprinting by RAPD analysis using five primers was carried out on control and treated samples. DNA profiles were compared within and across control and treated groups. The RAPD data showed an average range of 16-49.2% of polymorphism in treated samples as compared to control, indicated polymorphism occurred in treated groups. The highest change in DNA sequence was observed in treated groups with RBA 8A primer as compared to control; a negligible change was found in treated group with RBA 20A primer as compared to control. BLASTn analysis revealed studied sample (8A) gene sequence shared 99% identity to the sequence of S. marcescens. The phylogenetic tree diagram predicted the closest species of S. marcescens was found to be as P. fluorescens (Figure 3). Based on these results, it is expected that biofield treatment has the scope to be a cost effective and alternative approach than the existing antimicrobial therapy in near future.

Conclusion
The results suggest that there has an impact of biofield treatment on antimicrobial susceptibility, biochemical reactions, and DNA polymorphism of S. marcescens. These changes were found in the enzymatic level after biofield treatment. Therefore, biofield treatment could be applied to improve the sensitivity of antimicrobials against microbial resistance.