Fighting antimicrobial resistance (AMR): Chinese herbal medicine as a source of novel antimicrobials - an update.

Antimicrobial resistance (AMR) has now emerged as a global public health crisis, requiring the discovery of new and novel antimicrobial compounds, that may be precursors of future therapeutic antibiotics. Chinese Herbal Medicine (CHM) comes with a rich pedigree of holistic and empirical usage in Asia for the last 5000 years. Extracts of Anemarrhena asphodeloides Bunge, Angelica sinensis (Oliv.) Diels, Dianthus superbus L. Forsythiae fructus (Lian Qiao), Lonicerae flos (Jin Yin Hua), Naemorhedi cornu, Platycladus orientalis Franco, Polygonum aviculare, Polygonum cuspidatum, Poria cocos (Schw.), Rehmannia glutinosa (Gaertn.) DC, Rheum palmatum, Salvia miltiorrhiza Bunge, Scutellaria barbata, Scutellariae radix (Huang Qin) and Ursi fel (Xiong Dan) have shown to have antimicrobial properties against clinically significant Gram-negative and Gram-positive bacterial pathogens, as well as the mycobacteria (TB and non-tuberculous mycobacteria). Evidence is now beginning to emerge through systematic reviews of the outcomes of clinical studies employing CHM to treat infections. Of the 106 Cochrane systematic reviews on CHM, 16 (ca 15%) reviews examine CHM in the context of treating a specific infection disease or state. This update examines direct antimicrobial effect of CHM on bacterial pathogens, as well as synergistic effects of combining CHM with conventional antibiotics.

A microscopic evaluation of collagen-bilirubin interactions: in vitro surface phenomenon.

This study is carried out to understand the morphology variations of collagen I matrices influenced by bilirubin. The characteristics of bilirubin interaction with collagen ascertained using various techniques like XRD, CLSM, fluorescence, SEM and AFM. These techniques are used to understand the distribution, expression and colocalization patterns of collagen-bilirubin complexes. The present investigation mimic the in vivo mechanisms created during the disorder condition like jaundice. Fluorescence technique elucidates the crucial role played by bilirubin deposition and interaction during collagen organization. Influence of bilirubin during collagen fibrillogenesis and banding patterns are clearly visualize using SEM. As a result, collagen-bilirubin complex provides different reconstructed patterns because of the influence of bilirubin concentration. Selectivity, specificity and spatial organization of collagen-bilirubin are determined through AFM imaging. Consequently, it is observed that the morphology and quantity of the bilirubin binding to collagen varied by the concentrations and the adsorption rate in protein solutions. Microscopic studies of collagen-bilirubin interaction confirms that bilirubin influence the fibrillogenesis and alter the rate of collagen organization depending on the bilirubin concentration. This knowledge helps to develop a novel drug to inhibit the interface point of interaction between collagen and bilirubin.

Molecular cloning, sequencing, and biological characterization of GRA4 gene of Toxoplasma gondii.

In the present study, GRA4 (dense granule antigen) gene of Toxoplasma gondii was cloned, sequenced, and biologically characterized. The nucleotide sequence data obtained were analyzed and submitted in GenBank database (accession no. EU660037). Analysis of nucleotide sequence of GRA4 gene revealed 99.2 % homology with the published sequence (accession no. M76432). The gene segment (open reading frame) of 1,054 bp was further amplified and re-cloned in expression vector pET-32a. The recombinant protein obtained following the expression in prokaryotic system had a molecular mass of approx. 50 kDa and showed good immunoreactivity with T. gondii sera collected from infected goats. The immunization study of the recombinant protein performed in laboratory mice and live challenge with T. gondii revealed a high level of IgG response against the tachyzoite lysate antigen (TLA) by an indirect ELISA. Protection against T. gondii challenge infection was not evident in immunized mice except for the prolongation of survival period by 2 days. Humoral immune response profile revealed initially a high level of IgG antibody, but at 1 week post-challenge, a sudden drop in the level of the antibody was appreciable. Cytokine profiling by enzyme-linked immunosorbent spot method revealed relatively high level of IFN-γ production by the rodent spleen cells followed by IL-10 and IL-4. Increase in IFN-γ production by spleen cells of immunized mice following TLA stimulation suggested direct correlation to the up-regulated Th1 cells. However, the present immunization trial failed to show any positive relationship with the protection of mice following T. gondii challenge infection.

Adiponectin increases insulin content and cell proliferation in MIN6 cells via PPARγ-dependent and PPARγ-independent mechanisms.

AIMS: Adiponectin is an important adipokine whose levels are decreased in obesity despite increases in adipocyte mass. Studies in animal models implicate adiponectin as an insulin sensitizer in skeletal muscle and liver. Thiazolidinediones (TZDs) are insulin sensitizers and ligands for peroxisome proliferator-activated γ receptors (PPARγ) and these receptors are expressed in ß cells where their activation promotes cell survival. We hypothesize that adiponectin promotes ß cell survival by activating PPARγ. METHODS: We used MIN6 cells to investigate the effect of adiponectin on PPARγ expression, ß-cell proliferation, insulin synthesis and insulin secretion. RESULTS: We demonstrate that MIN6 cells contain adiponectin receptors and that adiponectin activates PPARγ mRNA and protein expression. This increase in PPARγ expression is blocked by the PPARγ antagonist, GW9662, indicating a transcriptional feedback loop involving PPARγ activation of itself. Adiponectin causes a significant increase in insulin content and secretion and this occurs also via PPARγ activation due to the inhibitory effect of GW9662. Adiponectin also promotes MIN6 cell proliferation, however, this effect is independent of PPARγ activation. CONCLUSIONS: Our results identify novel roles for the adipokine, adiponectin, in ß-cells function. Adiponectin upregulates PPARγ expression, insulin content and insulin secretion through PPARγ-dependent mechanisms. Reductions in circulating adiponectin levels in obese individuals could therefore result in negative effects on ß-cell function and this may have direct relevance to ß-cell dysfunction in type 2 diabetes.

Haemoprotozoa of cattle in Northern Kerala, India.

A cross-sectional study was conducted using 150 blood samples collected from apparently normal / healthy crossbred cattle of Northern Kerala, South India, for detection of haemoprotozoan infections using staining techniques (Giemsa and Acridine Orange) and specific PCR. Theileria like piroplasms and Babesia bigemina were the only protozoan organisms detected in blood smears. Polymerase chain reaction using specific primers revealed amplification of products specific for Trypanosoma evansi (34.6%), Theileria sp. other than T. annulata (16%) and B. bigemina (0.6%). The higher prevalence rate of Trypanosoma evansi indicated that the subclinical parasitism can be due to higher prevalence of tabanid flies. The study also revealed the presence of a theilerial piroplasm other than T. annulata in North Kerala, which needs further investigation.

Non-coding small (micro) RNAs of Pseudomonas aeruginosa isolated from clinical isolates from adult patients with cystic fibrosis.

MicroRNAs are a class of small non-coding RNAs widely reported in eukaryotic multicellular organisms. In this study, a number of small non-coding micro (mi)RNA species in clinical isolates of prokaryote Pseudomonas aeruginosa were obtained from the sputum of adult patients with cystic fibrosis (CF) utilising a DynaExpress miRNA cloning kit, and five miRNAs of 16-47 nucleotides that were smaller than those encountered or described (80-100 nucleotides) previously in bacterial systems were described. This report presents data on these unknown cellular miRNAs cloned from P. aeruginosa isolates from CF patients. Adapting a computational miRNA prediction model that takes advantage of the highly conserved known miRNA hair pin stems regions, the results revealed that the fold structure of the microRNAs had a high homology to the recently reported human bacterial infection response (BiR)-related microRNA, mi-146, associated with the Toll-like receptor (TLR) family, which is the primary evolutionarily conserved sensors of pathogen-associated molecular patterns (PAMPs), and known to trigger host inflammatory and immune responses.

Comparative evaluation and economic assessment of coprological diagnostic methods and PCR for detection of Cryptosporidium spp. in bovines.

The role of Cryptosporidium spp. as a major cause of diarrhoea and gastrointestinal illness of protozoan origin in neonatal calves has been established. Many coprological and serological techniques have been described for detection of the parasites with the limitations of sensitivity and specificity. Polymerase chain reaction (PCR) technique offers a useful alternative to conventional diagnosis of Cryptosporidium spp. in bovines from both clinical and environmental samples. We compared four conventional coprological techniques, viz., direct faecal smear staining (DFSS), normal saline sedimentation staining (NSSS), Sheather's flotation (SF) and Sheather's flotation sedimentation staining (SFSS) with PCR directed against the 18S SSU rRNA gene as standard reference test for the diagnosis of cryptosporidiosis in bovines. Out of 457 faecal samples collected from neonatal bovine calves, specific PCR amplification was achieved in 138 samples, whereas, 65 samples turned positive by DFSS. Normal saline sedimentation staining, SF and SFSS could detect 92, 82 and 109 samples as positive, respectively. Sheather's flotation sedimentation staining was found to be the most sensitive (82.6%) and specific (98.76%) among the coprological techniques. On per sample processing based cost analysis, DFSS was found to be the most economical method (15 cents) followed by NSSS (19.6 cents), SF (23.6 cents) and SFSS (33.9 cents). The time taken for complete processing and diagnosis varied between 70 and 100 min. PCR based diagnosis of a sample took about 7.5-8h for completion and cost of diagnosis was estimated as approximately 7.604 US$ per sample. Among the conventional coprological methods, SFSS provided the required sensitivity and specificity along with nominal cost for diagnosis on per sample basis, and may be considered as a viable diagnostic alternative when PCR is not an option for a particular laboratory setting, especially in developing countries. This is the first comparative study describing the sensitivity and specificities of four conventional coprological techniques altogether with respect to PCR along with the economic assessment and per sample diagnosis time of all the techniques for the diagnosis of cryptosporidiosis in bovines.

Adiponectin regulate growth hormone secretion via adiponectin receptor mediated Ca(2+) signalling in rat somatotrophs in vitro.

Obesity is associated with reduced levels of growth hormone (GH) and the disruption of pulsatile GH secretion. This results in relative GH deficiency. It is likely that a regulatory relationship between GH secretion and adipose tissue exists as the secretion of GH recovers to normal levels after a reduction in body weight. This report characterise the expression and interaction of adiponectin receptors 1 and 2 (AdipoR1 and AdipoR2) and adiponectin, respectively, in regulating the activity of GH secreting cells. Polymerase chain reaction analysis of the GH3 cell line, rat anterior pituitary gland and isolated somatotroph cells from transgenic GFP expressing mice confirmed the expression of both AdipoR1 and AdipoR2 in GH secretory cells. Because GH cells expressed both receptors, it is likely that the measured increase in GH secretion, observed in primary cultured rat pituitary cells after 30 min of incubation with full-length murine adiponectin, was mediated by a direct receptor regulated process. Adiponectin induced an increase in intracellular Ca(2+) through both the influx of extracellular Ca(2+) and the release of intracellular Ca(2+) stores resulting in the secretion of GH. Furthermore, results confirm that this increase in GH secretion depended mainly on an increase in Ca(2+) influx through L-type Ca(2+) channels. It is concluded that adiponectin directly regulates GH secretion from somatotrophs by binding to either adiponectin receptor, and that this is mediated via a similar process observed after the stimulation of GH secretion by GH-releasing hormone.

Prevalence of Cryptosporidium andersoni: a molecular epidemiological survey among cattle in India.

Cryptosporidiosis is an important and established cause of calfhood morbidity in bovines. The present communication reports the prevalence of Cryptosporidium infection among juvenile and adult cattle (6-24 months old) in India based on examination of faecal samples collected from 350 animals across three different agro-climatic regions of the country and further confirmation by a two-step nested PCR assay targeting 18S ssu rRNA gene. A total of 45 samples were positive for Cryptosoridium species by nested PCR assay. The PCR products were subjected to restriction fragment length polymorphism (RFLP) analysis using SspI and VspI restriction enzymes for species differentiation. The results showed that the species involved in all the samples found positive was Cryptosporidium andersoni. The overall prevalence rate was 12.85%, with highest occurrence in the northern states (14.37%) of the country. The animals between age group of 6-12 months were mostly affected (21.67%) and the season wise prevalence of infection was more during the hot and humid monsoon season (20.16%). The results clearly demonstrated that C. andersoni is the major Cryptosporidium species affecting juvenile and adult cattle in three agro-climatically different geographical regions of India. This is the first report on prevalence of C. andersoni in bovines from India the confirmation of which is based on application of nested PCR and PCR-RFLP based molecular tools.

An examination of antibacterial and antifungal properties of constituents of Shiitake (Lentinula edodes) and oyster (Pleurotus ostreatus) mushrooms.

BACKGROUND: Antibiotic agents have been in widespread and largely effective therapeutic use since their discovery in the 20th century. However, the emergence of multi-drug resistant pathogens now presents an increasing global challenge to both human and veterinary medicine. It is now widely acknowledged that there is a need to develop novel antimicrobial agents to minimize the threat of further antimicrobial resistance. With this in mind, a study was undertaken to examine the antimicrobial properties of aqueous extracts of 'exotic' Shiitake and Oyster mushrooms on a range of environmental and clinically important microorganisms. METHOD: Several batches of Shiitake and oyster mushrooms were purchased fresh from a local supermarket and underwent aqueous extraction of potential antimicrobial components. After reconstitution, aqueous extracts were tested qualitatively against a panel of 29 bacterial and 10 fungal pathogens, for the demonstration of microbial inhibition. RESULTS: Our data quantitatively showed that Shiitake mushroom extract had extensive antimicrobial activity against 85% of the organisms it was tested on, including 50% of the yeast and mould species in the trial. This compared favourably with the results from both the Positive control (Ciprofloxacin) and Oyster mushroom, in terms of the number of species inhibited by the activity of the metabolite(s) inherent to the Shiitake mushroom. CONCLUSIONS: This small scale study shows the potential antimicrobial effects of Shitake extracts, however further work to isolate and identify the active compound(s) now requires to be undertaken. Once these have been identified, suitable pharmaceutical delivery systems should be explored to allow concentrated extracts to be prepared and delivered optimally, rather than crude ingestion of raw material, which could promote further bacterial resistance.

Comparison of indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) for diagnosis of Babesia bigemina infection in bovines.

An indirect fluorescent antibody test (IFAT) and slide enzyme linked immunosorbent assay (SELISA) were standardized for the detection of antibodies specific to Babesia bigemina in experimentally infected bovine calves and subsequently used for the screening of naturally infected bovine and bubaline sera. In experimentally infected calves positive reactivity was detected in sera at the earliest on day 7 by both the tests. Serological studies for detection of B. bigemina specific antibodies in 180 cow and 120 buffalo serum samples procured from endemic zones of Uttar Pradesh and Punjab revealed 56.11% and 23.33% seropositivity, respectively, both by SELISA and IFAT. Variation in the reactivity pattern between these tests was found to be non significant. The sensitivity of SELISA was determined to be 94.85% whereas the specificity was 90.85% in comparison to IFAT. The agreement between the two tests by kappa statistics at 95% confidence interval revealed kappa- value of 0.853 that depicts almost a perfect degree of agreement. The findings employing experimental as well as test sera from cattle and buffalo from some of the tick infested zones of India suggested that SELISA could be a useful tool for seroprevalence studies on babesiosis, as the test is less cost intensive with high levels of sensitivity and specificity.

Molecular characterization of Echinococcus granulosus of Indian animal isolates on the basis of nuclear and mitochondrial genotype.

Sixty-six isolates of larval stage of Echinococcus granulosus, a known pathogenic parasite of man and animals were collected from cattle, buffalo, sheep, and goats. Single-stranded conformation polymorphism (SSCP) for analysis of variation after denaturation of amplicon of intron of actin II (ACTII) revealed six SSCP phenotypes. Intron portion was analyzed considering introns-early and introns-late theories. Isolates belonging to different conformers were further screened for mitochondrial ATPase subunit 6 (ATP6) and NADH dehydrogenase subunit II (nadII) genotypes. Assignment of each isolate to its specific strain was achieved after comparing with standard genotypes of E. granulosus. Variants deduced by nuclear targets did not match with mitochondrial haplotypes. A possible explanation for this observation can be attributed toward interspecific hybridization since cross-fertilization occurs less frequently in hermaphrodite organisms. A phylogenetic tree drawn on the basis of predicted aminoacid sequence of ATP6 and nadII revealed two distinct clusters i.e. E. granulosus sensu stricto and E. ortleppi/cattle strain (EG5). To the best of our knowledge, this is the first report of genetic characterization of two distinct ATP6 and nadII genotypes of zoonotic importance living in sympatry.

Elucidation of genetic variability among different isolates of Fasciola gigantica (giant liver fluke) using random-amplified polymorphic DNA polymerase chain reaction.

The present communication focuses on molecular characterization of Fasciola gigantica isolates derived from cattle, buffalo, and goat using random-amplified polymorphic DNA (RAPD)-polymerase chain reaction (PCR) analysis to elucidate genetic variability between the three isolates. Seventeen random oligonucleotide primers of 10-11 bases with GC content varying from 50-81.8% were used in the study. Depending upon the F. gigantica isolate-primer combination, one to five fragments in the range of 327-1,973 bp were amplified. It was significant to observe that, out of the 17 primers directing amplification of DNA fingerprints, only two designated as AP9 and AP14 were found to be of potential interest in the generation of polymorphic DNA. On the basis of similarity coefficient data, it may be suggested that cattle and buffalo isolates of F. gigantica show 100% homogeneity against 92.68% similarity coefficient observed between goat and cattle/buffalo isolates. In other words, 7.32% divergence was observed between goat and cattle/buffalo isolates while the primers AP1, AP4, AP10, AP13, and AP17 were able to generate monomorphic DNA fingerprints. Primers AP9 and AP14 are potentially informative in terms of the polymorphic nature of the fingerprints generated in RAPD assays. The finding of the absence of 626-bp DNA fragment in the goat and the uniqueness of the AP14 in generating a single RAPD-PCR product of 1,211 bp as against the product size of 1,162 bp in cattle/buffalo seem to be significant. This is the first report of elucidation of RAPD-PCR based molecular variability in the DNA fingerprinting pattern of F. gigantica isolated from cattle, buffalo, and goat.