Resistant Enterococcus Faecium Research Articles

Sequential steps of daptomycin resistance in Enterococcus faecium and reversion to hypersusceptibility through IS-mediated inactivation of the liaFSR operon.

To improve understanding of mechanisms of daptomycin resistance and to dissect the genetic basis of reversion to daptomycin hypersusceptibility in Enterococcus faecium. Daptomycin-resistant mutants (Mut4, Mut8, Mut16, Mut32, Mut64 and Mut128 with MICs from 4 to 128 mg/L) were obtained in vitro from E. faecium strain Aus0004 (MIC at 2 mg/L). The entire genome sequences of Mut64 and Mut128 were determined as well as those of liaFSR and cls genes for other mutants and corresponding revertants (named Rev4 to Rev128). The study of daptomycin resistance stability was performed without any selective pressure. The expression of liaF, liaS and liaR genes was quantified by quantitative RT-PCR. By comparative genomic analysis, substitutions Asn13Ser in cls and Gly92Asp in liaS were identified in Mut64 and Mut128. Only the liaS mutation was found in Mut16 and Mut32 while Mut4 and Mut8 were devoid of any mutation. After 15 days, all mutants except Mut4 reverted to daptomycin hypersusceptibility (MICs from 0.12 to 0.25 mg/L). In all revertants (except Rev4 and Rev8), an IS was found in the liaFSR operon with a dramatic decrease of its expression: IS66 in the promoter region of liaF (Rev16 and Rev64), IS30 in liaR (Rev32) and IS982 in liaF (Rev128). We demonstrated the stepwise and sequential acquisition of mutations in liaS and in cls leading to daptomycin resistance in E. faecium, and the instability of daptomycin resistance as well as the role of liaFSR inactivation in reversion to daptomycin hypersusceptibility.

Real time application of whole genome sequencing for outbreak investigation – What is an achievable turnaround time?

Whole genome sequencing (WGS) is increasingly employed in clinical settings, though few assessments of turnaround times (TAT) have been performed in real-time. In this study, WGS was used to investigate an unfolding outbreak of vancomycin resistant Enterococcus faecium (VRE) among 3 patients in the ICU of a tertiary care hospital. Including overnight culturing, a TAT of just 48.5 h for a comprehensive report was achievable using an Illumina Miseq benchtop sequencer. WGS revealed that isolates from patient 2 and 3 differed from that of patient 1 by a single nucleotide polymorphism (SNP), indicating nosocomial transmission. However, the unparalleled resolution provided by WGS suggested that nosocomial transmission involved two separate events from patient 1 to patient 2 and 3, and not a linear transmission suspected by the time line. Rapid TAT’s are achievable using WGS in the clinical setting and can provide an unprecedented level of resolution for outbreak investigations.

Screening municipal wastewater effluent and surface water used for drinking water production for the presence of ampicillin and vancomycin resistant enterococci.

The emergence of clinical enterococcal isolates that are resistant to both ampicillin and vancomycin is a cause of great concern, as therapeutic alternatives for the treatment of infections caused by such organisms are becoming limited. Aquatic environments could play a role in the dissemination of antibiotic resistant enterococci. This study investigated the presence of ampicillin and vancomycin resistant enterococci in the treated effluent of six wastewater treatment plants (WWTPs) and in surface water used as a source for drinking water production in the Netherlands. Membrane filtration in combination with selective media with ampicillin or vancomycin was applied to determine the presence of ampicillin resistant Enterococcus (ARE) and vancomycin resistant Enterococcus (VRE) species. Ampicillin resistant Enterococcus faecium (minimal inhibitory concentration (MIC) >16μg/mL; n=1033) was observed in all studied WWTP effluents. In surface water used for drinking water production (intake locations), no ARE or VRE were observed. At both types of location, intrinsic vancomycin resistant Pediococcus spp., Leuconostoc spp. and Lactobacillus spp. were isolated with the vancomycin medium. The ampicillin resistant E. faecium (AREfm) isolates (n=113) did not contain the vanA or vanB gene, but MIC testing for vancomycin showed intermediate vancomycin resistance (2-8μgmL(-1)) to occur in these AREfm strains. This study documents the discharge of ampicillin resistant E. faecium strains with intermediate vancomycin resistance by the WWTPs into the surface water, but no presence of these strains downstream at intake locations for drinking water production.

Antibacterial Activity of Plants Extracts against Methicillin-Resistant Staphylococcus aureus and Vancomycin-Resistant Enterococcus faecalis

The antibiotics resistant micro-organism has increased in past three decades. The Methicillin Resistant Staphylococcus aureus (M.R.S.A) and Vancomycin resistant Enterococcus faecalis (VRE) is a problematic in the field of medical science. This organism tends to cause serious infection in the burnt patients and mortality. MRSA is a major nosocomial pathogen. Thereby our study aims to control these micro-organisms using various plants extracts. Four plant species Ageratum conyzoides, Phyllanthus emblica, Camellia sinensis and Mentha longifolia were collected and subjected to ethanolic extraction. The ethanolic extracts were tested against clinically isolated multidrug resistance Enterococcus faecalis and Staphylococcus aureus. The ethanolic plants extracts were prepared at variable concentrations for Staphylococcus aureus and Enterococcus faecalis. In view of MRSA against clinically isolated Staphylococcus aureus and Enterococcus faecalis the concentration based analysis showed the zone of inhibition against the selected micro-organism in which Camellia sinensis was found potent against Staphylococcus aureus and Mentha longifolia against Enterococcus faecalis. In future after the detailed study it can be used as a potent biochemical drugs to overcome the infections caused by these organisms.

Community and hospital acquired methicillin resistant Staphylococcus aureus efficiently retain the Van A determinant

Dissemination of vancomycin resistance from hospital to community strains is a serious threat to public health. Our study aimed to provide evidence for transmission of Van A type resistance from the hospital to the community. Wild-type community and hospital associated methicillin resistant Staphylococcus aureus strains were studied in vitro and in a model that mimicked a natural environment to ascertain their ability to acquire and maintain the vancomycin resistance determinant (Van A gene) from vancomycin resistant Enterococcus faecalis. Fitness was assessed and the cost of Van A acquisition and retention was estimated. In vitro mating experiments were carried out using a filter mating technique and a model of a natural water body environment. Transfer of resistance was carried out in two different conditions: restricted and favorable. Transconjugants were confirmed by E test and PCR using specific primer sets. Growth kinetics and fitness measurements were done by spectrometric analysis. Using the in vitro filter mating technique, high transfer frequencies that ranged from 0.7 × 10–3(0.0006) to 3.1 × 10–4(0.00011) were recorded, with the highest transfer frequencies for CA MRSA (thermosensitively homogenous) (0.7 × 10–3), and 1.2 × 10–4 to 2.4 × 10–6 in the model. HA MRSA (homogenous) showed a greater capacity (3.6 × 10–4) to receive the Van A gene, while CA MRSA showed a reduced ability to maintain the gene after serial subcultures. CA and HA thermosensitively heterogeneous MRSA transconjugants exhibited higher growth rates. The present study provides evidence for the enhanced ability of CA and HA MRSA clones to acquire and maintain Van A type resistance.

High-level aminoglycoside resistance in Enterococcus faecalis and Enterococcus faecium causing invasive infection: Twelve-year surveillance in the Minami Ibaraki Area.

We examined prevalence of high-level aminoglycoside resistance (HLAR) in Enterococcus faecalis and Enterococcus faecium causing invasive infection in the Minami Ibaraki Area. Ten strains of both species each, recovered from the blood or the cerebrospinal fluid between 2003 and 2014, were randomly selected every year. High-level resistance to gentamicin (HLR-GM) and streptomycin (HLR-SM) was detected in 34% (41 of 120 strains) and 18% (21) of E.faecalis and 9% (11) and 39% (48) of E.faecium, respectively. In comparisons of the proportions among three four-year periods, HLR-SM among E.faecium was significantly lower in the 2011-2014 period. All strains with HLR-GM were positive for the aac(6')-Ie-aph(2″)-Ia gene. The ant(6')-Ia gene was detected in all with HLR-SM except for one E.faecalis strain. The present study showed that prevalence of HLR-GM among E.faecalis and E.faecium causing invasive infection in this area was nearly equivalent to that described in previous studies in Japan and that proportions of strains with HLAR did not vary during the study period except for that of HLR-SM among E.faecium.

Antibiotic resistance and virulence of enterococci isolates from healthy humans in Tunisia

The aim of this work was to determine the occurrence of different enterococcal species and the prevalence of antimicrobial resistance and virulence genes in enterococci isolates recovered from faecal samples of 98 healthy human volunteers in Tunisia. Isolates were tested for antibiotic resistance phenotypes, genotypes and virulence genes. In addition, high-level aminoglycoside resistant (HLAR) Enterococcus faecalis and Enterococcus faecium isolates were tested for clonal diversity by pulsed field gel electrophoresis (PFGE). The following species were detected among the 98 enterococci obtained: 51 Enterococcus faecalis, 40 Enterococcus faecium, 4 Enterococcus mundtii and 3 Enterococcus gallinarum. Antibiotic resistance was detected as follows (% in E. faecalis/E. faecium): erythromycin (51 %/55 %), tetracycline (51 %/15 %), pristinamycin (51 %/27.5 %), gentamicin (19.6 %/10 %), kanamycin (25.5 %/12.5 %), streptomycin (29.4 %/7.5 %), chloramphenicol (7.8 %/2.5 %). The tet(M)+/−tet(L) genes were found in 92 % and 83 % of tetracycline-resistant E. faecalis and E. faecium isolates, respectively. High-level resistance for kanamycin and gentamicin was mediated by aph(3′)-IIIa and aac(6′)-aph(2″) genes. Of erythromycin-resistant enterococci, 85 % harboured the erm(B) gene. The erm(C) gene was found in one erythromycin-resistant E. mundtii isolate. Most high-level gentamicin and/or streptomycin E. faecalis and E. faecium isolates showed unrelated patterns by PFGE. Fifty-two percent of enterococci showed a multidrug-resistant phenotype. The following virulence genes were detected among the 98 enterococci: gelE (13.2 %), esp (20.4 %), cyl (9 %, showing haemolytic activity), ace (48.9 %), and hyl (0 %). In conclusion, E. faecalis and E. faecium are the predominant enterococcal species in the faecal environment of healthy humans, and they present high rates of resistance for antibiotics of clinical relevance, such as aminoglycosides.

Studies on the drug resistance profile of Enterococcus faecium distributed from poultry retailers to hospitals

The multidrug resistant Enterococcus faecium (MEF) strains originating from farm animals are proliferating at a substantial pace to impact downstream food chains and could reach hospitals. This study was conducted to elucidate the drug susceptibility profile of MEF strains collected from poultry products in Ann Arbor, MI area and clinical settings from Michigan State Lab and Moffitt Cancer Center (MCC) in Florida. Presumptive positive Enterococcus isolates at species level were identified by Matrix Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) analysis. The antibiotic susceptibility profile for both poultry and clinical strains was determined by the Thermo Scientific's Sensititre conform to the National Committee for Clinical Laboratory Standards (NCCLS) and validated via quantitative real-time PCR (qPCR) methods. Out of 50 poultry samples (Turkey: n = 30; Chicken: n = 20), 36 samples were positive for Enterococcus species from which 20.83% were identified as E. faecium. All the E. faecium isolates were multidrug resistant and displayed resistance to the last alternative drug, quinupristin/dalfopristin (QD) used to treat vancomycin resistant E. faecium (VRE) in hospitals. Results indicate the presence of MEF strains in food animals and clinical settings that are also resistant to QD.

Healthcare-associated vancomycin resistant Enterococcus faecium infections in the Mansoura University Hospitals intensive care units, Egypt.

Vancomycin resistant Enterococcus faecium (VREF) ia an emerging and challenging nosocomial pathogen. This study aimed to determine the prevalence, risk factors and clonal relationships between different VREF isolates in the intensive care units (ICUs) of the university hospitals in our geographic location. This prospective study was conducted from July, 2012 until September, 2013 on 781 patients who were admitted to the ICUs of the Mansoura University Hospitals (MUHs), and fulfilled the healthcare-associated infection (HAI) criteria. Susceptibility testing was determined using the disk diffusion method. The clonal relationships were evaluated with pulsed field gel electrophoresis (PFGE). Out of 52 E. faecium isolates, 12 (23.1%) were vancomycin resistant. The significant risk factors for the VREF infections were: transfer to the ICU from a ward, renal failure, an extended ICU stay and use of third-generation cephalosporins, gentamicin, or ciprofloxacin. PFGE with the 12 isolates showed 9 different patterns; 3 belonged to the same pulsotype and another 2 carried a second pulsotypes. The similar pulsotypes isolates were isolated from ICUs of one hospital (EICUs); however, all of the isolates from the other ICUs had different patterns. Infection control policy, in conjunction with antibiotic stewardship, is important to combat VREF transmission in these high-risk patients.

Deletions in a ribosomal protein-coding gene are associated with tigecycline resistance in Enterococcus faecium

Enterococcus faecium is an emerging nosocomial pathogen associated with antibiotic therapy in the hospital environment. Whole-genome sequences were determined for three pairs of related, consecutively collected E. faecium clinical isolates to determine putative mechanisms of resistance to tigecycline. The first isolates (1S, 2S and 3S) in each of the three pairs were sensitive to tigecycline [minimum inhibitory concentration (MIC) of 0.125mg/L]. Following tigecycline therapy, the second isolate in each pair demonstrated increased resistance to tigecycline. Two isolates (1R and 2R) were resistant (MIC of 8mg/L) and one isolate (3I) demonstrated reduced susceptibility (MIC of 0.5mg/L). Mutations distinguishing each pair of sensitive and resistant isolates were determined through alignment to a reference genome and variant detection. In addition, a de novo assembly of each isolate genome was constructed to confirm mutations. A total of 16 mutations in eleven coding sequences were determined. Mutations in the rpsJ gene, which encodes a structural protein forming part of the 30S ribosomal subunit, were detected in each of the pairs. Mutations were in regions proximal to the predicted tigecycline-binding site. Predicted amino acid substitutions were detected in 1R and 3I. The resistant strains were additionally associated with deletions of 15 nucleotides (2R) and 3 nucleotides (1R). This study confirms that amino acid substitutions in rpsJ contribute towards reduced susceptibility to tigecycline and suggests that deletions may be required for tigecycline resistance in E. faecium.

Microbiological toxicity of tilmicosin on human colonic microflora in chemostats

To evaluate the microbiological safety of tilmicosin on human intestinal microflora, four chemostat models of healthy human colonic ecosystems were exposed to tilmicosin (0, 0.436, 4.36, and 43.6 μg/mL) for 7 days. Prior to and during drug exposure, three microbiological endpoints were monitored daily including short-chain fatty acids, bacterial counts and macrolide susceptibility. Colonization resistance of each community was determined by 3 successive daily challenges of Salmonella typhimurium. Genes associated with virulence and macrolide resistance in Enterococcus faecalis were determined by PCR. Transcriptional expression of the virulence gene (gelE) in E. faecalis was determined by real-time RT-PCR. Our results showed that different concentrations of tilmicosin did not disrupt the colonization resistance in each chemostat. During exposure to 4.36 and 43.6 μg/mL tilmicosin, the Bacteroides fragilis population was significantly decreased while the proportion of resistant Enterococci increased. After long-term exposure to the highest concentration (43.6 μg/mL) of tilmicosin, the gelE gene was significantly up-regulated in the high-level macrolide resistant strains that also contained the ermB resistance gene. This study was the first of its kind to evaluate the microbiological toxicity of tilmicosin using a chemostat model. These findings also provide new insight into the co-occurrence of macrolide resistance and virulence in E. faecalis under tilmicosin selective pressure.

Genome sequences of copper resistant and sensitive Enterococcus faecalis strains isolated from copper-fed pigs in Denmark.

Six strains of Enterococcus faecalis (S1, S12, S17, S18, S19 and S32) were isolated from copper fed pigs in Denmark. These Gram-positive bacteria within the genus Enterococcus are able to survive a variety of physical and chemical challenges by the acquisition of diverse genetic elements. The genome of strains S1, S12, S17, S18, S19 and S32 contained 2,615, 2,769, 2,625, 2,804, 2,853 and 2,935 protein-coding genes, with 41, 42, 27, 42, 32 and 44 genes encoding antibiotic and metal resistance, respectively. Differences between Cu resistant and sensitive E. faecalis strains, and possible co-transfer of Cu and antibiotic resistance determinants were detected through comparative genome analysis.

Anti-Microbial Activity of Chloramphenicol from Streptomyces sp.10CM9

An antimicrobial agent, chloramphenicol, was isolated from Actinobacteria 10CM9. The strain was isolated from lake sediment collected from Erçek Lake, Van (38°29’57.76“N and 43°33’10.98”E) and AIA medium was used as a isolation medium. 16S rRNA gene sequence analysis that showed the strain belongs to the genus Streptomycetes. A fermentation study in AIA broth medium (3L) followed by extraction with EtOAc afforded 105 mg of crude extract. A bioactivity-guided fractionation by Open Column chromatography on silica gel led to the isolation of one secondary metabolite (10CM19-01, 31 mg). Structures of the metabolites were established as chloramphenicol, by means of NMR (1H, 13C and COSY) data. Antimicrobial activity of 10CM9 was tested by microdilution method against enteropathogenic Escherichia coli 0157:H7 (RSKK 234), methicillin resistant Staphylococcus aureus ATCC 43300, vancomycin resistant Enterococcus faecium DSMZ 13590, Candida albicansDSMZ 5817 and Pseudomonas aeruginosa ATCC 27853. As a result, 0.5 μg/ml, 0.5 μg/ml and 1 μg/ml MIC values were observed against Escherichia coli 0157:H7, Enterococcus faecium and Staphylococcus aureus, respectively.

Spread of Enterococcal Surface Protein in Antibiotic Resistant Entero-coccus faecium and Enterococcus faecalis isolates from Urinary Tract Infections.

Enterococci rank among leading cause of nosocomial bacteremia and urinary tract infection in hospital and community acquired infections. Several traits that may contribute to enhanced virulence have been identified in Enterococci. Extracellular surface protein (Esp) is a virulence factor that contributes in biofilm formation and resistance to environmental stresses. In this study we aimed to determine occurrence of esp in E. faecium and E. faecalis isolates isolated from urinary tract infections and to investigate whether there is any correlation between presence of esp and antibiotic resistance. One hundred and sixty six isolates were collected from patients with UTI and after identification by biochemical and PCR, antibiotic resistances were examined. The presence of esp was investigated by primer-specific PCR. 43.3% of isolates identified as E. faecium and 56.7% as E. faecalis. The esp gene was found in 76.1% of E. faecium isolates and 77.9% of E. faecalis isolate. There were significant correlation between esp positive E. faecium and resistance to Vancomycin (p<0.01), also in E.faecalis we found correlation between esp positive and resistance to Ampicillin, Chloramphenicol and Tetracycline (p<0.01, p<0.01, p<0.01 respectively). Occurrence of esp in our isolates from urinary tract infection was high that indicates importance of this gene in urinary tract infections and shows importance of ability to forming biofilm and hydrophobicity of surface of Enterococci for causing urinary infection by Enterococci. Also, our finding showed significant correlation between resistance to antibiotics and presence of esp in Enterococci.

MiBalert, a realtime alert of multidrug-resistant bacteria in the electronic medical record

When patients are readmitted or transferred between departments or hospitals, information on multiresistant microorganisms is often recognized late or lost, resulting in infection or colonization of fellow patients or even hospital outbreaks. Currently, the hospitals of the Capital region, covering 1.8 million inhabitants, face an outbreak of vancomycin resistant Enterococcus faecium (VRE). All hospitals share the same EHR system.

Successful control of vancomycin resistant Enterococcus faecium in eastern Taiwan

Successful control of vancomycin resistant Enterococcus faecium in eastern Taiwan

Clonal diversity and antimicrobial resistance of Enterococcus faecalis isolated from endodontic infections

article i nfo Background: Enterococcus faecalis is considered to be one of most prevalent species in the oral cavity, particularly in endodontic infections. The aim of the present study was to investigate the prevalence of E. faecalis in dental root canals, clonal diversity by restriction fragment length polymorphism (RFLP) and randomly amplified polymorphic DNA (RAPD-PCR) analysis, and the antibiotic susceptibility of E. faecalis isolates. Results: Among the bacterial strains isolated from dental root canal specimens (n = 82), E. faecalis was determined to have the highest prevalence followed by Streptococcus viridians, Leuconostoc mesenteroides, Staphylococcus aureus, Streptococcus mitis ,a ndPediococcus pentosaceus. Cluster analysis of RAPD-PCR and RFLP patterns of the E. faecalis isolates discriminated five and six different genotypes, respectively. Among the tested strains, 43%, 52% and 5% were susceptible, intermediate resistant, and resistant to erythromycin, respectively. In addition, one strain (E-12) was intermediate resistant to linezolid, and one isolate (E-16) was resistant to tetracycline. Interestingly, many of the intermediate resistant/resistant strains were grouped in clusters 5 and 6, according RAPD and to RFLP, respectively. Conclusions: E. faecalis demonstrated the highest prevalence in the tested dental root canal specimens collected from Saudi patients and were grouped into five to six different genotypes. Different levels of antimicrobial susceptibility were observed in the tested E. faecalis strains, which clearly indicated that although bacterial strains may be similar, point mutations can result in extreme susceptibility or resistance to various antibiotics. This phenomenon is a cause for concern for clinicians in the treatment of dental infections caused by E. faecalis.

Increased membrane surface positive charge and altered membrane fluidity leads to cationic antimicrobial peptide resistance in Enterococcus faecalis

To understand the role of cell membrane phospholipids during resistance development to cationic antimicrobial peptides (CAMPs) in Enterococcus faecalis, gradual dose-dependent single exposure pediocin-resistant (Pedr) mutants of E. faecalis (Efv2.1, Efv3.1, Efv3.2, Efv4.1, Efv4.2, Efv5.1, Efv5.2 and Efv5.3), conferring simultaneous resistance to other CAMPs, selected in previous study were characterized for cell membrane phospholipid head-groups and fatty acid composition. The involvement of phospholipids in resistance acquisition was confirmed by in vitro colorimetric assay using PDA (polydiacetylene)-biomimetic membranes. Estimation of ratio of amino-containing phospholipids to amino-lacking phospholipids suggests that phospholipids in cell membrane of Pedr mutants loose anionic character. At moderate level of resistance, the cell-membrane becomes neutralized while at further higher level of resistance, the cell-surface acquired positive charge. Increased expression of mprF gene (responsible for lysinylation of phospholipids) was also observed on acquiring resistance to pediocin in PedrE. faecalis. Decreased level of branched chain fatty acids in Pedr mutants might have contributed in enhancing rigidification of cell membrane and contributing towards resistance. The interaction of pediocin with PDA-biomimetic membranes prepared from wild-type and Pedr mutants was monitored by measuring percent colorimetric response (%CR). Increased %CR of pediocin against PDA-biomimetic membranes prepared from Pedr mutants confirmed that cell membrane phospholipids are involved in the interactions of pore formation by CAMPs. There was a direct linear relationship between percent colorimetric response and IC50 of CAMPs for wild-type and Pedr mutants. This relationship further reveals that in vitro colorimetric assay can be used effectively for quantification of resistance to CAMPs.

Detection of vancomycin resistances in enterococci within 3 ½ hours.

Vancomycin resistant enterococci (VRE) constitute a challenging problem in health care institutions worldwide. Novel methods to rapidly identify resistances are highly required to ensure an early start of tailored therapy and to prevent further spread of the bacteria. Here, a spectroscopy-based rapid test is presented that reveals resistances of enterococci towards vancomycin within 3.5 hours. Without any specific knowledge on the strain, VRE can be recognized with high accuracy in two different enterococci species. By means of dielectrophoresis, bacteria are directly captured from dilute suspensions, making sample preparation very easy. Raman spectroscopic analysis of the trapped bacteria over a time span of two hours in absence and presence of antibiotics reveals characteristic differences in the molecular response of sensitive as well as resistant Enterococcus faecalis and Enterococcus faecium. Furthermore, the spectroscopic fingerprints provide an indication on the mechanisms of induced resistance in VRE.

Endophytic fungi: a reservoir of antibacterials.

Multidrug drug resistant bacteria are becoming increasingly problematic particularly in the under developed countries of the world. The most important microorganisms that have seen a geometric rise in numbers are Methicillin resistant Staphylococcus aureus, Vancomycin resistant Enterococcus faecium, Penicillin resistant Streptococcus pneumonia and multiple drug resistant tubercule bacteria to name a just few. New drug scaffolds are essential to tackle this every increasing problem. These scaffolds can be sourced from nature itself. Endophytic fungi are an important reservoir of therapeutically active compounds. This review attempts to present some data relevant to the problem. New, very specific and effective antibiotics are needed but also at an affordable price! A Herculean task for researchers all over the world! In the Asian subcontinent indigenous therapeutics that has been practiced over the centuries such as Ayurveda have been effective as “handed down data” in family generations. May need a second, third and more “in-depth investigations?”