Evaluation of Antibiogram, Genotype and Phylogenetic Analysis of Biofield Treated Nocardia otitidis

Text-only Preview

B
i
o
l
o
g
i
c
a
l
S
y
s
t
e
m
s
:
O
p
e
n
A
c
c
e
s
s
ISSN: 2329-6577
Biological Systems: Open Access
Trivedi et al,, Biol Syst Open Access 2015, 4:2
http://dx.doi.org/10.4172/2329-6577.1000143
Open Access
Research Article
Volume 4 • Issue 2 • 1000143
Biol Syst Open Access
ISSN: 2329-6577 BSO, an open access journal
Evaluation of Antibiogram, Genotype and Phylogenetic Analysis of
Biofield Treated
Nocardia otitidis
Mahendra Kumar Trivedi1, Alice Branton1, Dahryn Trivedi1, Gopal Nayak1, Sambhu Charan Mondal2 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, Hoshangabad Rd., Bhopal- 462026, Madhya Pradesh, India
Abstract
Nocardiosis is a soil-borne aerobic infection caused by Nocardia species commonly affects the respiratory tract.
Nocardia otitidis (N. otitidis) is the key organism for non-mycobacterial tuberculosis. The current study was attempted to
investigate the effect of Mr. Trivedi’s bioeld energy treatment on N. otitidis and analyzed for antimicrobial susceptibility
pattern, minimum inhibitory concentration (MIC), DNA polymorphism by Random Amplied Polymorphic DNA (RAPD)
and 16S rDNA sequencing. The strain of N. otitidis (ATCC 14630) was divided into two parts, control and treated.
Antimicrobial susceptibility was studied using the broth microdilution technique. Overall, the MIC values of 16.67%
antimicrobials were changed in the treated group of N. otitidis as compared to the control. Moreover, MIC value of
trimethoprim/sulfamethoxazole was reduced by two-fold (0.5/9.5 to 0.25/4.75 µg/mL) in the bioeld energy treated
sample as compared to the control without alteration in the sensitivity spectrum. The 16S rDNA analysis showed that
the treated sample was detected as Enterobacter aerogenes strain NCTC10006T (GenBank Accession No: AJ251468)
with 98% identity of gene sequencing data. However, the nearest homolog genus-species was found as Kluyvera
cryocrescens (GenBank Accession No: AM184245). Using RAPD biomarkers, the sample showed an average range of
34 to 53% of polymorphism among treated samples as compared to the control. The 16S rDNA sequencing of treated
sample was carried out to correlate the phylogenetic relationship of N. otitidis with other bacterial species. These
results suggested that Mr. Trivedi’s bioeld energy treatment has a signicant impact on N. otitidis.
*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:
Received August 31, 2015; Accepted September 21, 2015; Published September
29, 2014
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015)
Evaluation of Antibiogram, Genotype and Phylogenetic Analysis of Bioeld Treated
Nocardia otitidis. Biol Syst Open Access 4: 143. doi:10.4172/2329-6577.1000143
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.
nocardiosis [9] in immunocompromised patients. Infectious Diseases
Society of America (IDSA) reported that between 500 and 1,000 cases
of Nocardial infections are recognized in the United States each year, of
which 85% are serious pulmonary or systemic infections [10].
Since 1940s, the sulfonamides have been the drugs of choice for
the treatment of nocardiosis [11]. Due to high mortality rate (50%)
in patients with central nervous system (CNS) Nocardia infections
(Nocardia brain abscess) and patients with non-CNS overwhelming or
disseminated disease, the treatment strategy is inadequate. However, the
combination of sulfamethoxazole with trimethoprim is oen used as the
drug of choice for the treatment of nocardiosis [12]. erefore, some
alternative strategies are needed to treat against nocardiosis. Bioeld
energy has been known as an alternative approach which may be useful
as an alternative treatment to Nocardia infected patients. National
Institute of Health/National Center for Complementary and Alternative
Medicine (NIH/NCCAM) have reported that bioeld (putative energy
elds) or electromagnetic based energy therapies were commonly
used to promote the health and healing [13]. Harold Saxton Burr had
performed the detailed studies on the correlation of electric current
with the physiological process and concluded that every single process
in the human body had an electrical signicance [14]. Recently, it was
discovered that all the electrical processes happening in the human
body have strong relationship with the magnetic eld as required by
Keywords: Nocardia otitidis; Nocardiosis; Antimicrobial
susceptibility; Bioeld energy treatment; 16S rDNA sequencing;
Random amplied polymorphic DNA
Abbreviations: NIH/NCCAM: National Institute of Health/
National Center for Complementary and Alternative Medicine;
ATCC: American Type Culture Collection; MIC: Minimum Inhibitory
Concentration; OTUs: Operational Taxonomic Units; NCBI: National
Center for Biotechnology Information; MEGA: Molecular Evolutionary
Genetics Analysis; PCR: Polymerase Chain Reaction; RDP: Ribosomal
Database Project; RAPD: Random Amplied Polymorphic DNA; CNS:
Central Nervous System
Introduction
e genusNocardiais associated with the group of microorganisms
known as the aerobic actinomycetes and belongs to the family of
Mycobacteriaceae. Nocardia contains tuberculostearic acids but that
dier from the mycobacteria by the possession of short-chain (40 to
60 carbons) mycolic acids [1]. Nocardia otitidis (N. otitidis) is a weak
lamentous Gram-positive, catalase-positive, branching rods shaped
bacterium that appears similar to Actinomyces species. However, it
can usually be dierentiated from Actinomyces by acid-fast staining
[2]. e taxonomic history of the genus Nocardia is controversial [3].
Nocardia typically exhibits varying degrees of acid fastness due to the
presence of cell wall mycolic acid. e genus is typically similar to the
genus of Mycobacterium. Mordarska et al. had studied the short-chain
fatty acids content in the cell wall of Nocardia and Mycobacterium
genera based on gas-liquid chromatography analysis did not nd
any dierence between two genera [4]. Based on immunoblot and
enzyme-linked immunosorbent assay (ELISA) techniques that detect
specic antibodies that appear as common in various Nocardia and
Actinomadura species. ese antigens do not react with the antibodies
produced in response to Mycobacterium tuberculosis infections [5].
Most of Nocardial infections occur in the United States due to inhalation
of airborne spores or mycelial fragments from the environmental
sources [6]. e most common manifestation of Nocardial disease is
pulmonary nocardiosis [7], extrapulmonary disease [8], and ocular
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015) Evaluation of Antibiogram, Genotype and Phylogenetic Analysis of
Bioeld Treated Nocardia otitidis. Biol Syst Open Access 4: 143. doi:10.4172/2329-6577.1000143
Page 2 of 6
Volume 4 • Issue 2 • 1000143
Biol Syst Open Access
ISSN: 2329-6577 BSO, an open access journal
Ampere’s law, which states that the moving charge produces magnetic
elds in the surrounding space [15,16]. us, a human body emits the
electromagnetic waves in the form of bio-photons that is also known
as ultra-weak photon emissions (UPE). It surrounds the body and it
is commonly known as bioeld. erefore, the bioeld consists of an
electromagnetic eld, being generated by moving electrically charged
particles (ions, cell, molecule, etc.) inside the human body [17]. e
transfer of information from cell to cell or DNA or storage by biophotons
has been demonstrated in plants, bacteria, animal neutriophil
granulocytes and kidney cells [18]. Prakash et al. in 2015 reported that the
various scientic instruments such as Kirlian photography, polycontrast
interference photography (PIP) and resonance eld imaging (RFI) can
be extensively used to measure the bioeld of human body [19]. us,
human has the ability to harness the energy from the 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
the useful way that is called bioeld energy and the process is known as
bioeld energy treatment. Mr. Mahendra Trivedi’s unique bioeld energy
treatment (e Trivedi Eect®) has been known to improve the overall
productivity of crops [20,21], altered characteristics features of microbes
[22-24], alter the structural, physical and thermal properties of several
metals [25,26], and improved growth and anatomical characteristics of
various medicinal plants [27,28].
Based on clinical signicance of N. otitidis and signicant impact
of Mr. Trivedi’s bioeld energy modality on microbes, the present work
was undertaken to evaluate the impact of Mr. Trivedi’s bioeld energy
on N. otitidis in relation to antimicrobials susceptibility, minimum
inhibitory concentration (MIC), random amplied polymorphic DNA
analysis (RAPD) and 16S rDNA sequencing.
Materials and Methods
N. otitidis, American Type Culture Collection (ATCC 14630) strain
was procured from Bangalore Genei, Bangalore-India in two vials A
and B. Two dierent sealed packs were stored with proper storage
conditions until further use. All the tested antimicrobials were procured
from Sigma-Aldrich, India. e antimicrobial susceptibility and
MIC were estimated with the help of broth micro dilution technique
as per the Clinical Laboratory Standards Institute (CLSI) guidelines
document number M24-A [29]. e 16S rDNA sequencing and DNA
ngerprinting (RAPD) studies were carried out using Ultrapure
Genomic DNA Prep Kit; Cat KT 83 (Bangalore Genei, India).
Experimental design and bioeld treatment strategy
N. otitidis strain was divided into two groups, i.e., control and
treated. e treated group was in sealed pack and handed over to Mr.
Trivedi for bioeld energy treatment under laboratory conditions. Mr.
Trivedi provided the treatment through his energy transmission process
to the treated group without touching the sample. Aer treatment,
control and treated groups were assessed on day 10 for antimicrobial
susceptibility, and MIC. For RAPD analysis three inoculums (one for
control and the other two for treated named as treated A and B) were
prepared from N. otitidis samples. ese two bioeld treated samples
A and B 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. e result of treated sample was compared with respect to
the control. e 16S rDNA analysis was performed on bioeld treated
samples A and its subcultured sample A1.
Antimicrobial susceptibility test
e antimicrobial susceptibility testing is important for clinically
signicant species. e investigation of antimicrobial susceptibility of N.
otitidis was carried out with the help of broth micro dilution technique,
as per CLSI guidelines. Broth micro dilution was recommended
for isolates of rapidly growing mycobacteria (RGM) based on CLSI
published guidelines and recommendations for testing of non-
tuberculous mycobacteria (CLSI, M24-A, 2003) [29]. e detailed
experimental procedure and conditions were followed as per the
manufacturer’s instructions. e antimicrobial susceptibility pattern
(S: Susceptible, R: Resistant) and minimum inhibitory concentration
(MIC) were determined by observing the lowest antimicrobial
concentration showing inhibition of growth.
Random amplied polymorphic DNA (RAPD) analysis
For DNA ngerprinting (RAPD) analysis all the treated samples
(A, A1, B, and B1) were incubated at 37°C with 160 rpm for 18 h.
Subsequently, the cultures were spun down, pelleted at 5000 rpm at
40°C for 10 minutes and the genomic DNA was isolated for control
and treated samples using Genomic DNA Prep Kit (Bangalore Genei,
India). RAPD was performed with all samples of N. otitidis using ve
RAPD primers, which were labelled as RBA8A, RBA13A, RBA20A,
RBA10A and RBA15A were adopted from earlier studies. e PCR
mixture contained 2.5 μL each of buer, 4.0 mM each of dNTP, 2.5 μM
each of primer, 5.0 μL (approximately 20 ng) of each genomic DNA,
2U 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 [30]. e
percentage of polymorphism was calculated using following equation:
Percent polymorphism=A/B × 100
Where, A=Number of polymorphic bands in treated sample; and
B=Number of polymorphic bands in control.
Amplication and gene sequencing of 16S rDNA
Genomic DNA was isolated from N. otitidis cells (set A, sample coded
as 9A) using genomic purication kit, according to the manufacturer
instructions. 16S rDNA gene (~ 1.5 kb) fragment was amplied with the
help of high-delity polymerase chain reaction (PCR) using universal
primers; forward primer (5-AGAGTTTGATCCTGGCTCAG-3) and
reverse primer (3-ACGGTCATACCTTGTTACGACTT-5). Amplied
products were subjected to gel 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. Sequencing of amplied product was done on a commercial basis
from Bangalore Genei, India. e 16S rDNA sequences obtained were
aligned and compared with the sequences stored in GenBank database
available from National Center for Biotechnology Information (NCBI)
using the algorithm BLASTn program. Multiple sequence alignment/
phylogenetic tree were established using MEGA3.1 molecular soware
[31].
Results and Discussion
Antimicrobial susceptibility test
Antimicrobial susceptibility pattern of Nocardia can vary from
Citation: Trivedi MK, Branton A, Trivedi D, Nayak G, Mondal SC, et al. (2015) Evaluation of Antibiogram, Genotype and Phylogenetic Analysis of
Bioeld Treated Nocardia otitidis. Biol Syst Open Access 4: 143. doi:10.4172/2329-6577.1000143
Page 3 of 6
Volume 4 • Issue 2 • 1000143
Biol Syst Open Access
ISSN: 2329-6577 BSO, an open access journal
species to species. e therapeutic eectiveness depends on the proper
identication of species in infected patients and on in vitro sensitivity
studies [32]. e antimicrobial sensitivity assay against Nocardia
species had been especially considered in refractory cases. A standard
test for antimicrobials sensitivity assessed by broth micro dilution and
with cation-supplemented Mueller-Hinton broth has been approved by
the National Committee for Clinical Laboratory Standards (NCCLS)
[29]. e outcome of N. otitidis susceptibility pattern and MIC values of
tested antimicrobials aer bioeld energy treatment are summarized in
Table 1 and 2 respectively. e data were analyzed and compared with
respect to the control. Study was carried out in twelve antimicrobials
for assessment of antimicrobial susceptibility and MIC value. e
treated cells of N. otitidis did not show any alteration with respect to
antimicrobial susceptibility pattern as compared to the control (Table
1). e susceptible nature of N. otitidis to amikacin, sulfamethoxazole,
and ciprooxacin and resistance pattern to ceriaxone, amoxicillin-
clavulanic acid, and imipenem were well corroborated with the
literature data [3]. Beside this, the MIC value of trimethoprim/
sulfamethoxazole was reduced by two-fold (0.5/9.5 to 0.25/4.75 µg/mL)
in bioeld energy treated sample as compared to the control sample.
Moreover, the MIC value was slightly reduced (>32/16 to 32/16 µg/mL)
in amoxicillin/k-clavulanate aer the bioeld therapy. Overall, 16.67%
out of twelve antimicrobials showed an alteration of MIC values. Rest
of the antimicrobials did not show any alteration of MIC values with
respect to the control sample. Based on the literature, a combination
of sulfa and antimalarial drugs, i.e., trimethoprim/sulfamethoxazole
are the drug of choice against Nocardia infections as compared to the
single sulfa drug [11,12]. In this experiment, the susceptibility nature
of trimethoprim/sulfamethoxazole was constant in both controls as
well as in treated sample, while the MIC value was reduced by two-
fold in treated sample aer Mr. Trivedi’s bioeld energy treatment.
Hence, authors assumed that this improvement of MIC value without
alteration of sensitivity pattern may be due to the eect of putative
energy transmit through bioeld healing (Table 2).
Random amplied polymorphic DNA (RAPD) analysis
e treated and control samples were identied on the basis of
their dierent and discriminative RAPD patterns. RAPD is a preferred
tool that is being used now days to correlate the genetic similarity
or mutations between species. e simplicity and wide applicability
of RAPD analysis mainly depend upon the use of short nucleotide
primers, which were unrelated to known DNA sequences of the target
organism [33]. DNA polymorphisms can be eciently detected using
the PCR primers and identify inter-strain variations among species in
treated samples [34]. e degree of relatedness and genetic mapping
can be correlated between similar or dierent treated sample species
[35].
e DNA ngerprinting by RAPD analysis using ve primers was
carried out on the control and treated samples. DNA ngerprinting by
RAPD analysis of the control and treated samples are shown in Figure 1,
and the polymorphic bands are marked by arrows. e RAPD patterns
of treated samples showed some unique and dissimilar patterns. DNA
polymorphism analyzed by the RAPD analysis was presented in Table 3.
e level of polymorphism between control and treated samples (A, A1,
B, and B1) are summarized in Table 4. e level of polymorphism was
found in an average range of 34 to 53% in treated samples as compared
to control in N. otitidis aer the bioeld treatment. e highest change
in DNA sequence was observed in treated groups with RBA 13A primer
as compared to the control; a negligible change was found in treated
group with RBA 8A primer as compared to the control (Table 3 and 4).
16S rDNA genotyping
e bacteria that are poorly dierentiated by conventional methods
needs molecular analysis method like 16S rDNA sequence [36]. is
molecular-based technique is a suitable tool for identication of most
1000
900
800
700
600
500
400
300
200
100
bp
Figure 1: Random amplied polymorphic-DNA fragment patterns of Nocardia
otitidis generated using ve RAPD primers, RBA 8A, RBA 13A, RBA 20A, RBA
10A and RBA 15A. 1: Control; 2: Treated A; 3: Treated A-1; 4: Treated B; 5:
Treated B-1; M: 100 bp DNA Ladder.
S. No. Antimicrobial Control Treatment
1. Linezolid S S
2. Clarithromycin R R
3. Amikacin S S
4. Cefoxitin R R
5. Ceftriaxone R R
6. Imipenem R R
7. Minocycline S S
8. Tobramycin S S
9. Ciprooxacin S S
10. Gatioxacin S S
11. Amoxicillin/k-clavulanate R R
12. Trimethoprim/sulfamethoxazole S S
R: Resistant; S: Susceptible; Control: ATCC strain of N. otitidis (without bioeld
energy treatment); Treatment: ATCC strain of N. otitidis (with Mr. Trivedi’s bioeld
energy treatment)
Table 1: Antimicrobial susceptibility pattern of antimicrobials against ATCC strain of
Nocardia otitidis after bioeld treatment on day 10 as per CLSI guidelines.
S. No. Antimicrobial Control Treatment
1. Linezolid 2.0 2.0
2. Clarithromycin >32.0 >32.0
3. Amikacin <1.0 <1.0
4. Cefoxitin 256.0 256.0
5. Ceftriaxone 64.0 64.0
6. Imipenem 64.0 64.0
7. Minocycline 1.0 1.0
8. Tobramycin 8.0 8.0
9. Ciprooxacin 4.0 4.0
10. Gatioxacin 0.12 0.12
11. Amoxicillin/k-clavulanate >32/16 32/16
12. Trimethoprim/sulfamethoxazole 0.5/9.5 0.25/4.75
CLSI: Clinical and Laboratory Standards Institute; MIC values are presented
as µg/mL; Control: ATCC strain of N. otitidis (without bioeld energy treatment);
Treatment: ATCC strain of N. otitidis (with Mr. Trivedi’s bioeld energy treatment)
Table 2: Minimum inhibitory concentration (MIC) of antimicrobials against ATCC
strain of Nocardia otitidis after bioeld energy treatment on day 10 as per CLSI
guidelines.