Characterization of Antimicrobial Susceptibility Profile of Biofield Treated Multidrug-resistant Klebsiella oxytoca

Klebsiella are opportunistic pathogens that cause a wide spectrum of severe diseases. The aim of the present study was to investigate the impact of biofield treatment on multidrug resistant strain of K. oxytoca with respect to antibiogram pattern along with biochemical study and biotype number. Clinical lab isolate of K. oxytoca was divided into two groups i.e. control and treated. Control group remain untreated and treated group was subjected to Mr. Trivedi’s biofield. The analysis was done on day 10 after biofield treatment and compared with control group. Control and treated groups were analyzed for antimicrobial susceptibility pattern, minimum inhibitory concentration (MIC), biochemical reactions and biotype number using MicroScan Walk-Away® automated system. Experimental results showed the impact of biofield treatment on K. oxytoca and found alteration in both antimicrobial sensitivity and MIC values as compared with untreated group. Antimicrobial sensitivity of about 26.67% tested antimicrobials out of thirty was altered with respect to control. MIC results showed about 12.50% alterations in tested antimicrobials as compared to control. Biochemical study showed 24.24% alteration in tested biochemical reactions after biofield treatment. A significant change in biotype number (7713 5272) was identified after biofield treatment as compared to control (7775 4332). In treated group, a new species was identified as Kluyvera ascorbata, as compared to control, K. oxytoca. Study findings suggest that biofield treatment has a significant effect in altering the antimicrobial sensitivity, MIC values, biochemical reactions and biotype number of multidrug resistant strain of K. oxytoca. Biofield treatment could be applied to alter the antibiogram-resistogram pattern of antimicrobials.


Introduction
Klebsiella oxytoca (K. oxytoca) is a Gram-negative pathogen, cylindrical rod shaped, non-motile in nature, and belongs to Enterobacteriaceae family. Klebsiella spp. are ubiquitous in environment [1], but K. oxytoca can be cultured from intestines of healthy humans and animals, oropharynx, mucous membrane and skin. K. oxytoca initially named as Aerobacter aerogens, which was identified as Klebsiella pneumoniae, but recent report classified it as K. oxytoca, on the basis of indole-positive test and ability to grow on melezitose, not in 3-hydroxybutyrate [2]. It is considered as an opportunistic pathogen, as most of the cases K. oxytoca-infected persons remain asymptomatic. However, K. oxytoca is now recognized as important clinical pathogen in hospitalized patients causing major nosocomial infections in children and neonates [3]. It is reported in many etiological human infections such as urinary tract infection, septic arthritis, bacteremia, septicemia, cholecystitis, soft tissue infections, and most recently in colicky neonates [1,[4][5][6][7]. During last few years, incidence of extended spectrum β-lactamase producing multi-drug-resistance (MDR) Klebsiella spp. had increased. Cases of MDR infections had been increased suddenly, which resulted in ineffective antimicrobials treatment. Clinicians prefer multiple combination bactericidal therapy against infection instead of single drug. Recently, an alternate approach called biofield treatment on pathogenic microorganism is reported to alter the antimicrobial susceptibility.
Biofield is a cumulative outcome of electric and magnetic field energy, exerted by the human body. However, the energy can exist in several forms such as kinetic, potential, electrical, magnetic, and nuclear. Similarly, the human nervous system consists of the energy and chemical information in the form of electrical signals. 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 via biofield energy. Mr. Trivedi's unique biofield treatment is also known as The Trivedi Effect ® . In spite of countless study reports on biofield therapies [8,9], there are very few well controlled and peerreviewed experimental studies on pathogenic or MDR microbes. According to law of mass-energy inter-conversion [10], the conversion of mass into energy is well established, but its inversion i.e. energy into mass has not yet proved scientifically. Whenever these electrical signals fluctuate with time, the magnetic field generates as per the Ampere-Maxwell law, and cumulatively known as electromagnetic field. Mr. Trivedi's biofield treatment is well-known to change the various physicochemical characteristics of metals and ceramics [11][12][13][14]. In addition, his unique biofield treatment has considerably altered the antimicrobials susceptibility and biochemical reactions of pathogenic microbes [15][16][17]. In agricultural science, biofield treatment altered the growth, characteristics and yield of important medicinal plants [18][19][20][21]. On the basis of several reports on biofield treatment, present study was designed to study the impact of biofield on MDR isolate of K. oxytoca, for its antimicrobials susceptibility pattern, minimum inhibitory concentration (MIC), along with biotyping based on variation in biochemical reactions.

Materials and Methods
Experimental design and biofield treatment MDR clinical strain of K. oxytoca was collected from stored stock cultures of clinical sample in Microbiology Lab, Hinduja Hospital, Mumbai. MDR strain was divided in two groups i.e. control and treatment. Treatment group, in sealed pack was handed over to Mr. Trivedi for biofield treatment under laboratory conditions. Mr. Trivedi provided the treatment through his energy transmission process to the treated groups without touching the samples. The biofield treated sample was returned in the similar sealed condition for further analysis on day 10 with respect to control using the standard protocols. After biofield treatment, treated sample was analyzed for antimicrobial susceptibility, biochemical reactions and biotype number using MicroScan Walk-Away ® (Dade Behring Inc., USA) and Negative Break Point Combo (NBPC 30) panel with respect to control groups. The antimicrobials and biochemicals were procured from Sigma Aldrich, MA, USA.

Evaluation of antimicrobial susceptibility assay
Antimicrobial susceptibility pattern of K. oxytoca was studied using MicroScan Walk-Away ® NBPC 30 as per manufacturer's instructions. The antimicrobial susceptibility pattern (S: Susceptible, I: Intermediate, and R: Resistant) and MIC were determined by observing the lowest antimicrobial concentration showing growth inhibition [22].

Biochemical reaction study
Biochemical study of K. oxytoca was determined by MicroScan Walk-Away ® system in both control and treated groups [22].

Identification by biotype number
The biotype number of K. oxytoca control and treated samples were determined by MicroScan Walk-Away ® processed panel data report with the help of biochemical reactions data [22].

Antimicrobial susceptibility test
Results of antimicrobial sensitivity pattern and MIC of K. oxytoca isolate are summarized in Tables 1 and 2 (Table 3).
This study investigated the influence of biofield treatment on MDR strain of K. oxytoca with respect to antimicrobial sensitivity assay, and results found that biofield treatment has the potential to alter the sensitivity and MIC values of antimicrobials against biofield treated pathogen. The increased emergence of MDR strains of Klebsiella spp., in immunocompromised patients and increased infections lead to serious matter of concern worldwide. Extended spectrum β-lactamase (ESBL) producing species have still the serious problem worldwide, which may be due to continuous new drug discovery [2]. Results suggest the natural resistant pattern of MDR strain of K. oxytoca against most of the tested antimicrobials. Antimicrobial sensitivity of K. oxytoca is well supported with literature data [23]. Biofield treatment group showed significant effect on ESBL producing antimicrobials, as sensitivity after biofield treatment changed to resistant in case of aztreonam, cefotaxime, ceftazidime, and ceftriaxone. Most of the clinical strains of K. oxytoca produced chromosomal and plasmid mediated β-lactamase. Chromosomal mediated β-lactamases had the capacity to hydrolyze extended spectrum antimicrobials such as cephalosporin, and aztreonam. Mutational hyper production of β-lactamase results in a characteristic antibiogram with resistant pattern against piperacillin, cefuroxime, and aztreonam [24]. However, most of the clinical isolates of K. oxytoca have been associated with low production of β-lactamase. Biofield treatment might induce some enzymatic changes which result in significant alteration in antimicrobial sensitivity and MIC values. Resistant pattern in MDR is also associated with alteration in cell membrane, which may causes decrease uptake of antimicrobial, drug target enzyme overexpression, or alteration in drug efflux pump [25][26][27]. Biofield treatment in MDR K. oxytoca might alter the cell membrane permeability which results in alteration in sensitivity of tested antimicrobials.

S. No.
Antimicrobial Control Treated

Identification of organism by biochemical reactions
Several phenotypic identification tests are available to differentiate the Klebsiella species. Experimental identification of K. oxytoca was performed using different standard biochemical reaction analysis. Adonitol, inositol, urea, and Voges-Proskauer showed negative reaction i.e. positive to negative, while cetrimide, citrate, hydrogen sulfide, and ornithine showed positive reaction i.e. negative to positive after biofield treatment as compared to control. Rest of the biochemical reactions were not altered after biofield treatment with respect to control. Overall, biochemical reactions showed the alteration of 24.24% after biofield treatment. Experimental control biochemical reaction data of K. oxytoca are well supported with literature data [2]. Biofield treatment showed a significant alteration in positive as well as negative reactions in tested biochemical, which are the basic characteristics of K. oxytoca. The standard positive biochemical reactions of K. oxytoca were reported in case of indole, lysine decarboxylase, L-sorbose, malonate, urea and Voges-Proskauer while negative reactions in ornithine decarboxylase, gas production, and citrate.

Identification of organism by biotype number
On the basis of above biochemical changes, biotyping was performed to check the identity of microorganism after biofield treatment using an automated system. Results of biotyping found a significant changed in biotype number (7713 5272) in treated group on day 10, with respect to control (7775 4332). The organism was identified as Kluyvera ascorbata in treated group after biofield treatment as compared to control organism, K. oxytoca (Table 4).
Biofield treatment on pathogenic microorganism showed significant alteration in biochemical reactions followed by altered biotype number, which are well supported with literature reports [15][16][17].
Biofield therapies in biomedical health care system are very popular and reported to improve human well-being with respect to several diseased conditions [28]. Increased emergence of resistant microorganisms due to widespread uses of antibiotics contributed to the spread of multidrug resistant organisms [29]. Biofield treatment is practiced by many heath care professionals as it was accepted by National Center for Complementary and Alternative Medicine (NCCAM), in complementary and alternate medicine [30]. Biofield treatment in pathogenic microorganisms had been reported to alter the antimicrobial sensitivity, phenotypic characteristics, and growth of microorganism [16,17]. It results in altered sensitivity of antimicrobials which may involve cellular changes in biofield treated K. oxytoca at molecular and/or genetic level [31]. Results showed that, biofield treatment induced changes in susceptibility pattern of antimicrobials, MIC values biochemical reactions, and biotype number of MDR strain of K. oxytoca.

Feature Control Treated
Biotype 7775 4332 7713 5272 Organism Identification Klebsiella oxytoca Kluyvera ascorbata Table 4: Effect of biofield treatment on multidrug resistant strain of Klebsiella oxytoca to its biotype number.

Conclusion
Altogether, the biofield treatment on MDR strain of K. oxytoca showed alteration of antimicrobial sensitivity pattern, MIC, biochemical reactions followed by biotype number. Altered biochemical reactions may be responsible for changed biotype number, and a new species was identified as Kluyvera ascorbata, as compared to control. Alteration in above standard microbiological techniques after biofield treatment might involve the changes at enzymatic or genetic level of K. oxytoca, which can be further studied at molecular level with respect to altered antimicrobial sensitivity and biotype number. Based on the study outcomes, biofield treatment could be applied to alter the sensitivity pattern of antimicrobials, against multidrug resistance strain of K. oxytoca.