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

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Evaluation of Phenotyping and Genotyping Characterization of Serratia
marcescens after Biofield Treatment
Mahendra Kumar Trivedi1, Shrikant Patil1, Harish Shettigar1, Khemraj Bairwa2 and Snehasis Jana2*
1Trivedi Global Inc., 10624 S Eastern Avenue Suite A-969, Henderson, NV 89052, USA
2Trivedi Science Research Laboratory Pvt. Ltd., Hall-A, Chinar Mega Mall, Chinar Fortune City, Bhopal- 462026, Madhya Pradesh, India
*Corresponding author: Snehasis Jana, Trivedi Science Research Laboratory Pvt. Ltd., Hall-A, Chinar Mega Mall, Chinar Fortune City, Hoshangabad Rd., Bhopal-
462026, Madhya Pradesh, India, Tel: +91-755-6660006; E-mail: [email protected]
Received date: July 01, 2015, Accepted date: August 24, 2015, Published date: August 31, 2015
Copyright: © 2015 Trivedi MK, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted
use, distribution, and reproduction in any medium, provided the original author and source are credited.
Abstract
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.
Keywords: Antimicrobials; Bioeld treatment; Polymorphism;
Microbial resistance; RAPD;
S. marcescens
Introduction
Currently, many microorganisms have been acquired the resistance
to number of antibiotics and other antimicrobial agents, which were
eectively used earlier to cure a microbial infections. e antimicrobial
resistant microbes (including bacteria, viruses, fungi, and parasites)
can survive in antimicrobial dr ugs therapy. erefore, regular
treatments are ineective. e 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; uoroquinolones (nalidixic acid,
ciprooxacin, ooxacin, and noroxacin), by proton dependent
multidrug resistance (MDR) eux pumps [2,3]. erefore,
development of eective antimicrobial therapy against
S. marcescens
is
very needful for human health. Recently, bioeld treatment came in
focus that can c ure the microbial infect ion by changing the microbial
susceptibility against the antimicrobial drugs.
e relation between mass-energy was described by Friedrich, then
aer Einstein gave the well-known equation E=mc2 for light and mass
[4,5]. e 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 als o 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 eld, which cumulatively form
electromagnetic eld [7,8]. us, 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. e objects always receive the
energy and responding into useful way that is cal led bioeld energy
and the process is known as bioeld treatment. Mr. Mahendra Trivedi’s
bioeld treatment (e Trivedi Eect®) has been applied to transform
the structural, physical, and chemical properties of materials in several
elds like material science [9-16], agriculture [17-19], and
biotechnology [20,21]. e bioeld treatment has considerably altered
the genotype of the microbes and thereby changed in susceptibility to
antimicrobials [22-24].
Aer consideration of clinical signicance of
S. marcescens
and
signicant impact of bioeld treatment on microbes, we felt a detailed
investigation was required to evaluate the eect of bioeld treatment
on
S. marcescens
. Aer that, the organism was assesse d in relation to
antimicrobials susceptibility and biotyping based on various
Molecular and Genetic Medicine Trivedi et al., J Mol Genet Med 2015, 9:3
http://dx.doi.org/10.4172/1747-0862.1000179
Research Article Open Access
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 3 • 1000179
biochemical reactions. We also explored the genotyping of this
organism using polymerase chain reaction (PCR) based methodologies
of randomly amplied polymorphic DNA (RAPD) and 16S rDNA
sequencing techniques. To the best of our knowledge, this is the rst
report that explores the impact of bioeld 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. e 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
e 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). e treatment groups (G1
and G2) were in sealed pack and handed over to Mr. Trivedi for
bioeld treatment under laboratory condition. Mr. Trivedi provided
the treatment through his energy transmission process to the treated
groups without touching the samples. Aer that, G2 group was
assessed for antimicrobial susceptibility and biochemical reactions on
5th and 10th day of incubation; and G3 group was assessed on 10th day
of treatment. e treated groups were compared with respect to
control.
Investigation of antimicrobial susceptibility of
S. marcescens
Antimicrobial susceptibility of
S. marcescens
was investigated with
the help of automated instrument, MicroScan Walk-Away® using
Negative Breakpoint Combo 30 (NBPC30) panel as per the
manufacturer’s instructions [25]. Briey, aer inoculation and
rehydration with a standardized suspension of
S. marcescens
, were
incubated at 35°C for 16 h. e minimum inhibitory concentration
(MIC) and a qualitative susceptibility like susceptible (S), intermediate
(I), inducible β-lactamases (IB), and resistant (R) were determined by
observing the lowest antimicrobial concentration showing growth
inhibition [26]. In the present study, the following 29 antimicrobials
were used like amikacin, amoxicillin/k-clavulanate, ampicillin/
sulbactam, ampicillin, aztreonam, cefazolin, cefepime, cefotaxime,
cefotetan, cefoxitin, ceazidime, cefuroxime, ceriaxone, cephalothin,
chloramphenicol, ciprooxacin, gatioxacin, gentamicin, imipenem,
levooxacin, meropenem, moxioxacin, nitrofurantoin, noroxacin,
piperacillin, tazobactam, ticarcillin, tobramycin, and vancomycin.
Biochemical studies
e biochemical studies of
S. marcescens
were determined by
MicroScan Walk-Away® where, interpretation of biochemical reactions
for microbial identication of Gram-negative organisms resulted in
high accuracy [27,28]. In this study, the following 31 biochemicals
were used like acetamide, adonitol, arabinose, arginine, cetrimide,
cephalothin, citrate, colistin, esculin hydrolysis, nitrofurantoin,
glucose, hydrogen sulde, indole, inositol, kanamycin, lysine,
malonate, melibiose, nitrate, oxidation, galactosidase, ornithine,
oxidase, ranose, rhamnose, sorbitol, sucrose, tartrate, tobramycin,
urea, and Voges-Proskauer.
Biotype number
e biotype number of
S. marcescens
was determined by MicroScan
Walk-Away® processed panel data utilizing biochemical reactions data
[25].
Random Amplied Polymorphic DNA (RAPD) analysis
ree 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 bioeld treatment. Aer that, the treated samples were sub-
cultured 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 al l samples of
S. marcescens
using ve RAPD primers, which were labelled as RBA8A, RBA13A,
RBA20A, RBA10A and RBA15A. e PCR mixture contained 2.5 µL
each of buer, 4.0 mM each of dNTP, 2.5 μM e ach of primer, 5.0 μL
each of genomic DNA, 2 U each of Taq polymerase, 1.5 μL of MgCl2
and 9.5 μL of water in a total of 25 μL with the following PCR
amplication 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 nal extension at 72°C for 7 min. Amplied 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.
Amplication and gene sequencing of 16S rDNA
Genomic DNA was isolated from
S. marcescens
cells by using
genomic purication Kit, according to the instructions of
manufacturer. 16S rDNA gene (~1.5 kb) was amplied by universal
primers; forward primer (5ˊ-AGAGTTTGATCCTGGCTCAG-3ˊ) and
reverse primer (3ˊ-ACGGTCATACCTTGTTACGACTT-5ˊ).
Amplied 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). e PCR
amplied fragment was puried from the agarose gel using a DNA Gel
Extraction Kit. S equencing of amplied product was done on
commercial basis from Bangalore Genei, India. e 16S rDNA
sequences obtained were aligned and compared with the sequences
stored in Gene Bank data bas e available from National C enter for
Biotechnology Information (NCBI) using the algorithm BLASTn
program. Multiple sequence alignment/phylogenetic tree were
established using MEGA3.1 molecular soware [29].
Results
Assessment of antimicrobial susceptibility
e eect of bioeld treatment on
S. marcescens
to susceptibility
pattern and MIC of selected antimicrobials are summarized in Tables 1
Citation: Trivedi MK, Patil S, Shettigar H, Bairwa K, Jana S (2015) Evaluation of Phenotyping and Genotyping Characterization of Serratia
marcescens after Biofield Treatment. J Mol Genet Med 9: 179. doi:10.4172/1747-0862.1000179
Page 2 of 7
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 3 • 1000179
and 2, respectively. e data were analyzed and compared with respect
to control. e treated cells of
S. marcescens
showed an alteration of
10.34% and 34.48% in G2 and G3 group on 10th day, respectively of
antimicrobials susceptibility among all tested antimicrobials as
compared to control. Studying the eect of bioeld treatment in the
antibiogram of
S. marcescens
, revealed that the amikacin and
tobramycin were converted from resistance to susceptible on 10th day
of G3 group as compared to control. Aztreonam, cefotetan,
ceazidime, cef uroxime and chloramphenicol were converted from
resistance to intermediate on 10th day of bioeld treatment of G3
group as compared to control. e cefepime and cefotaxime were
converted from resistance to intermediate on 10th day of G2 treated
cells and complete susceptibility was obser ved for gentamycin and
cefepime on 10th day of G3 treated cells as compared to control (Table
1). It was also observed that there was reduced activity of inducible β-
lactamase of aztreonam, cefotaxime, cefotetan, ceazidime, and
ceriaxone antimicrobials. e MIC values of amikacin, aztreonam,
cefepime, cefotetan, ceazidime, gentamicin and tobramycin were
decreased about two-folds; whereas about four-folds decrease in MIC
values of cefotaxime and ceriaxone on 10th day of G2 treated cells as
compared to control (Table 2).
S.
No. Antimicrobial
Control G2 G3
10th
day
G1 5th
day
10th
day
1 Amikacin R R R S
2 Amoxicillin/K-clavulanate R R R R
3 Ampicillin/Sulbactam R R R R
4 Ampicillin R R R R
5 Aztreonam R R R IB
6 Cefazolin R R R R
7 Cefepime R R I S
8 Cefotaxime R R I IB
9 Cefotetan R R R IB
10 Cefoxitin R R R R
11 Ceftazidime R R R IB
12 Cefuroxime R R R R
13 Ceftriaxone I I IB IB
14 Cephalothin R R R R
15 Chloramphenicol R R R I
16 Ciprofloxacin S S S S
17 Gatifloxacin S S S S
18 Gentamicin R R R S
19 Imipenem S S S S
20 Levofloxacin S S S S
21 Meropenem S S S S
22 Moxifloxacin S S S S
23 Nitrofurantoin R R R R
24 Norfloxacin S S S S
25 Piperacillin IB IB IB IB
26 Tazobactam IB IB IB IB
27 Ticarcillin IB IB IB IB
28 Tobramycin R R R S
29 Vancomycin S S S S
G stands for group; I: intermediate; S: susceptible; R: resistant; IB: inducible β-
lactamase.
Table 1: Eect of bioeld treatment on
S. marcescens
to susceptibility
pattern of selected antimicrobials.
S. No. Antimicrobial
Control G2 G3
G1 5th day 10th day 10th day
1 Amikacin >32 >32 >32 ≤16
2Amoxicillin/K-
clavulanate ≥16/8 ≥16/8 ≥16/8 ≥16/8
3Ampicillin/
Sulbactam ≥16/8 ≥16/8 ≥16/8 ≥16/8
4 Ampicillin ≥16 ≥16 ≥16 ≥16
5 Aztreonam >16 >16 >16 ≤8
6 Cefazolin ≥16 ≥16 ≥16 ≥16
7 Cefepime >16 >16 16 ≤8
8 Cefotaxime >32 >32 32 ≤8
9 Cefotetan >32 >32 >32 ≤16
10 Cefoxitin ≥16 ≥16 ≥16 ≥16
11 Ceftazidime >16 >16 >16 ≤8
12 Cefuroxime >16 >16 >16 >16
13 Ceftriaxone 32 32 ≤8 ≤8
14 Cephalothin ≥16 ≥16 ≥16 ≥16
15 Chloramphenicol >16 >16 >16 16
16 Ciprofloxacin ≤1 ≤1 ≤1 ≤1
17 Gatifloxacin ≤2 ≤2 ≤2 ≤2
18 Gentamicin >8 >8 >8 ≤4
19 Imipenem ≤4 ≤4 ≤4 ≤4
20 Levofloxacin ≤2 ≤2 ≤2 ≤2
21 Meropenem ≤4 ≤4 ≤4 ≤4
22 Moxifloxacin ≤2 ≤2 ≤2 ≤2
23 Nitrofurantoin ≥64 ≥64 ≥64 ≥64
Citation: Trivedi MK, Patil S, Shettigar H, Bairwa K, Jana S (2015) Evaluation of Phenotyping and Genotyping Characterization of Serratia
marcescens after Biofield Treatment. J Mol Genet Med 9: 179. doi:10.4172/1747-0862.1000179
Page 3 of 7
J Mol Genet Med
ISSN:1747-0862 JMGM, an open access journal Volume 9 • Issue 3 • 1000179