Ranadhir Chakraborty

Antibacterial Activity of Some 3-(Arylideneamino)-2-phenylquinazoline-4(3H)-ones: Synthesis and Preliminary QSAR Studies

Synthesis of ten 3-(arylideneamino)-2-phenylquinazoline-4(3H)-ones is reported. All the compounds contained a common phenyl group at the 2-position, while the substituents on the arylideneamino group were varied. The compounds were investigated for their antimicrobial activity against both Gram-positive (Staphylococcus aureus 6571 and Bacillus subtilis) and Gram-negative bacteria (Escherichia coli K12 and Shigella dysenteriae 6) using a turbidometric assay method. It was found that the incorporation of the 3-arylideneamino substituent enhanced the anti-bacterial activity of the quinazolone system. The preliminary QSAR studies were done using some computer derived property descriptors, calculated values of partition coefficients as well as usual Hammetts sigma constants and the substituents molar refractivity.

Antibiotic Resistance of Bacteria

Antibiotic resistance of bacteria and other microorganisms is one of the most serious and grievous challenges of the twenty-first century. The life-saving drugs, which held a great deal of promises during the 1940s to eradicate all the infectious life-threatening diseases in the world, have ceased to work, because of the increasing emergence of microbial strains invulnerable to them. Many of the previously efficacious antibiotics are no longer usable because of widespread occurrence of multiresistant microbial strains. Lately, discovery of new antibiotics is failing to keep pace with the emergence of (multi)resistance of pathogenic and also environmental bacterial strains. Consequently, the prospect of chemotherapy looks bleak. The trepidation that we might be pushed back to a situation analogous to the preantibiotic era, when no chemotherapeutic agent was available to contain and combat deadly bacterial infections, does not appear to be an overblown imagination. Based on this backdrop, this special issue appears to be an aptly undertaken and well-timed endeavour to address this global problem. The articles contributed by investigators from various research laboratories with different scientific backgrounds have not only portrayed the width of the problem but also displayed some silver lining in the management of the looming crisis. Rapid detection of the profile of resistance is essential for timely application of the right antibiotic to a patient. H. Frickmann et al. summarize the efficacy and limitations of various molecular and mass spectrometric methods for the detection of resistance. The omnipresent nature of the resistant organisms is revealed in a number of articles. F. B. Atique and Md. M. R. Khalil report on the occurrence of antibiotic resistance among bacteria (predominantly skin commensal coagulase-negative staphylococci) isolated from allogenic bone samples for grafting, collected from different hospitals of Bangladesh. Food materials are believed to serve as a vehicle for transmission of resistance. This issue is addressed by F. S. Dehkordi et al. who report on the genotype and resistance-profile of Helicobacter pylori isolated from vegetables and salad samples, picked up from groceries and supermarket in a province of Iran. The high similarity in the genotype pattern of the isolates obtained from vegetables and humans indicates transmission. A. B. Florez et al. reveal tetracycline and erythromycin-resistant bacteria and genes conferring resistance to these antibiotics in 10 Spanish and 10 Italian samples of commercial cheese. P. Krupa et al. report on the population structure (based on spa typing) of oxacillin-resistant Staphylococcus aureus isolated from nasal swabs of pigs, collected from two slaughter houses of Poland. Some meat samples bought from the shops were also included into their studies. D. De Vito et al. characterize multidrug-resistant clinical isolates of Salmonella typhimurium for resistance genes in an area of southern Italy by pulsotyping and phage typing. C. Zhang et al. report on the resistant phenotype and genotype of Streptococcus suis serotype 2, isolated from 62 clinically healthy sows and 34 diseased pigs reared in different farms of China. Antibiotic resistance in the nosocomial isolates is a matter of serious concern. F. Lombardi et al. look into the molecular epidemiology of carbapenemase-producing strains of Klebsiella pneumoniae isolated from the surgery unit at a cardiovascular centre of Italy. D. Ojdana et al. demonstrate the ability of an E. coli strain obtained from a hospital of Poland to produce carbapenemase enzymes and also the presence of genes responsible for the production of carbapenemases and other β-lactamases. Extended-spectrum-β-lactamase (ESBL) is a bacterial enzyme having the ability to hydrolyse even the third-generation cephalosporins and aztreonam. Besides Klebsiella pneumoniae some strains of Escherichia coli are also known to produce this enzyme. This is indicated by M. S. Rezai et al. who performed genotyping of ESBL-producing strains of E. coli, obtained from a paediatric hospital of north Iran. The authors also show the association of ESBL-positive E. coli strains with resistance to various other antimicrobials. Occurrence of ESBL-producing Enterobacteriaceae in iceberg lettuce obtained from the retail market of Rochester (US) is described by N. Bhutani et al. A wide spectrum of diseases is caused by the virulent strains of ESBL-positive isolates of E. coli. Regional difference in the prevalence of virulence genes in 432 phenotypically ESBL-positive patient-isolates of E. coli (obtained from the Baltic Sea region) is shown by J. Lillo et al. Keeping in mind the tremendous challenge posed by drug-resistant tuberculosis, a number of relevant articles are included in this collection. The susceptibility profile of M. tuberculosis isolates to various antitubercular antibiotics varies significantly depending on the test system as revealed by Z. Mei et al. They have also shown that changes in bacterial susceptibility are further caused by mixed infection with particular genotypes of M. tuberculosis strains. Resistance-profile of 100 strains of M. tuberculosis, isolated from patients in northeast Iran, is reported by A. T. Sani et al. Occurrence of nontuberculosis Mycobacterium, in 25 out of 125 patients (20%) surveyed, underscores the need of proper diagnosis before the onset of chemotherapy. Discovery of new drugs and strategies to circumvent antibiotic resistance is the need of the hour to contain the problem. N. Jafari et al. report on the isolation of an antibiotic-producing strain of a soil Actinomycetes belonging to the genus Pseudonocardia. The antibacterial compound produced by it is effective against Staphylococcus aureus. They have also purified and partially characterized this compound. R. D. Wojtyczka et al. demonstrate high antibacterial activity of two new quinoline derivatives of a structure of 3-thioacyl 1-methyl 4-arylaminoquinolinium salts against some nosocomial strains of staphylococci in both planktonic and biofilm form. In view of the widespread nature of the problem caused by inefficacy of the antibiotics produced by fermentation and chemical synthesis, it is necessary to tap alternative sources (e.g., plant kingdom) for novel antibiotics. P. Del Serrone et al. demonstrate antibacterial activity of Neem seed oil (Azadirachta indica A. Juss.) against enteropathogenic strains of E. coli and indicate that some of the ciprofloxacin-resistant isolates lost their virulence following treatment with Neem seed oil. Antimicrobial peptides are considered potential candidates for the management of multidrug-resistant infections. M. Singh and K. Mukhopadhyay evaluate the antimicrobial potential of an anti-inflammatory neuropeptide whereas C. Chen et al. report on the efficacy of recombinant lysostaphin against methicillin-resistant S. aureus (MRSA) in a mouse model. Widespread use of carbapenems is associated with emergence of resistance. The polymyxin antibiotic colistin is not used at present because of its nephrotoxicity. H.-J. Tang et al., however, demonstrate the efficacy of a combination of colistin and imipenem against carbapenem-resistant Klebsiella pneumoniae. Bacteriophages could be suitable alternatives for antibiotics, which currently have lost efficacy because of the emergence of resistant strains. N. Shivshetty et al. demonstrate the potential of a bacteriophage isolated from sewage to protect diabetic mice against Pseudomonas aeruginosa-induced bacteremia. Reversal of bacterial resistance to antibiotics is essential to restore the efficacy of the existing antimicrobials. C. Santiago et al. claim to achieve an increase in susceptibility of a MRSA strain to ampicillin when it was combined with a plant extract. A number of computerized models have been developed during the recent past to assist the physicians with the necessary information to enable prescription of the right antibiotic in the right moment. M. Rodriguez-Maresca et al. report on the efficacy of a new electronic device based on laboratory data on the most probable susceptibility profile of pathogens responsible for infections and also on local epidemiology.

Green synthesis of silver nanoparticles using glucan from mushroom and study of antibacterial activity

This work demonstrates synthesis of silver nanoparticles (AgNPs) using glucan isolated from a mushroom Pleurotus florida blue variant. UV-vis spectroscopy showed maximum absorbance at 425 nm due to surface plasmon resonance of AgNPs. Average diameter of the synthesized AgNPs was 2.445 ± 1.08 nm as revealed from TEM analysis. XRD analysis confirmed the face-centered cubic (fcc) crystalline structure of metallic silver. The synthesized AgNPs-glucan conjugates exhibited antibacterial activity against multiple antibiotic resistant (MAR) bacterium Klebsiella pneumoniae YSI6A and the activity was possibly due to damage of cellular macromolecules by the generation of reactive oxygen species (ROS) which was supported by observed degradation of bacterial DNA. Decrease of bactericidal effect of AgNPs-glucan conjugates in dose-dependent manner in presence of a ROS scavenger histidine further ascertained the involvement of ROS in antibacterial activity. AgNPs-glucan conjugates at LD50 dose caused least damage (0.68% hemolysis) to human RBCs. This particular dose of AgNPs-glucan conjugates in combination with each of the four antibiotics (ampicillin, azithromycin, cefepime and tetracycline) to which K. pneumoniae YSI6A was resistant, showed synergistic effect to inhibit almost 100% bacterial growth. It thus opens an avenue to use antibiotics in combination with minimum dosages of AgNPs-glucan conjugates to control MAR bacteria.

Quorum sensing in metal tolerance of Acinetobacter junii BB1A is associated with biofilm production

Acinetobacter junii strain BB1A, a novel metal-tolerant bacterium, produced biofilm in the presence of added ions such as Ni(2+), AsO(2)(-), Cd(2+) and Hg(2+) on surfaces such as glass and polystyrene. Generation of a metal-sensitive and adhesion-deficient mutant by transposition of Tn5-mob in the A. junii genome has putatively confirmed the association of metal tolerance with the production of biofilm. The requirement of a critical cell density for biofilm formation and presence of acyl-homoserine lactone-like autoinducer molecules in the cell-free supernatant indicated the phenomenon of quorum sensing. Addition of a natural quorum-sensing inhibitor (garlic extract) or synthetic quorum-sensing inhibitor (4-nitro-pyridine oxide) significantly inhibited cell growth and biofilm formation in the presence of metal/metalloid ions.

Bactericidal effect of polyethyleneimine capped ZnO nanoparticles on multiple antibiotic resistant bacteria harboring genes of high-pathogenicity island

Zinc oxide nanoparticles (ZnO-NP) were synthesized by alcoholic route using zinc acetate as the precursor material and lithium hydroxide as hydrolyzing agent. Further ZnO-PEI NP (derivative of ZnO-NP) was made in aqueous medium using the capping agent polyethyleneimine (PEI). The nanoparticles were characterized by XRD measurements, TEM and other techniques; the weight % of coating shell in the polymer-capped particles was determined by TGA. ZnO-PEI NP is more soluble in water than the uncapped ZnO-NP, and forms a colloidal suspension in water. PEI-capped ZnO-NP exhibited better antibacterial activity when compared with that of uncapped ZnO-NP against a range of multiple-antibiotic-resistant (MAR) Gram-negative bacterial strains harboring genes of high-pathogenicity island. ZnO-NP effectively killed these microorganisms by generating reactive oxygen species (ROS) and damaging bacterial membrane. ZnO-PEI NP at LD50 dose in combination with tetracycline showed synergistic effect to inhibit tetracycline-resistant Escherichia coli MREC33 growth by 80%. These results open up a new vista in therapeutics to use antibiotics (which have otherwise been rendered useless against MAR bacteria) in combination with minimized dosage of nanoparticles for the more effective control of MAR pathogenic bacteria.

Guava Leaf Extract Inhibits Quorum-Sensing and Chromobacterium violaceum Induced Lysis of Human Hepatoma Cells: Whole Transcriptome Analysis Reveals Differential Gene Expression

Quorum sensing (QS) is a process mediated via small molecules termed autoinducers (AI) that allow bacteria to respond and adjust according to the cell population density by altering the expression of multitudinous genes. Since QS governs numerous bioprocesses in bacteria, including virulence, its inhibition promises to be an ideal target for the development of novel therapeutics. We found that the aqueous leaf extract of Psidium guajava (GLE) exhibited anti-QS properties as evidenced by inhibition of violacein production in Chromobacterium violaceum and swarming motility of Pseudomonas aeruginosa. The gram-negative bacterium, C. violaceum is a rare pathogen with high mortality rate. In this study, perhaps for the first time, we identified the target genes of GLE in C. violaceum MTCC 2656 by whole transcriptome analysis on Ion Torrent. Our data revealed that GLE significantly down-regulated 816 genes at least three fold, with p value≤0.01, which comprises 19% of the C. violaceum MTCC 2656 genome. These genes were distributed throughout the genome and were associated with virulence, motility and other cellular processes, many of which have been described as quorum regulated in C. violaceum and other gram negative bacteria. Interestingly, GLE did not affect the growth of the bacteria. However, consistent with the gene expression pattern, GLE treated C. violaceum cells were restrained from causing lysis of human hepatoma cell line, HepG2, indicating a positive relationship between the QS-regulated genes and pathogenicity. Overall, our study proposes GLE as a QS inhibitor (QSI) with the ability to attenuate virulence without affecting growth. To the best of our knowledge, this is the first report which provides with a plausible set of candidate genes regulated by the QS system in the neglected pathogen C. violaceum.

Partial characterization and flocculating behavior of an exopolysaccharide produced in nutrient-poor medium by a facultative oligotroph Klebsiella sp. PB12.

A facultative oligotrophic strain from the water sample of River Mahananda, Siliguri India was selected for its property to produce exopolysaccharide (EPS) in nutrient-poor (oligotrophic) medium. Viability assay of the strain was performed in sterile liquid LB, R2A, river water and diluted (10(-3)) LB at 30°C and pH 7 to understand oligotrophy. The selected strain was identified by 16S rRNA gene sequencing and designated as Klebsiella sp. PB12. Phylogenetic analysis showed its closest relationship with Klebsiella variicola ATCC BAA-830(T). Purification of EPS was performed by ethanol precipitation, dialysis and freeze-drying. Chemical analysis revealed that purified EPS was mainly composed of 72.32% (w/w) neutral sugar and 14.12% (w/w) uronic acids. Fourier transform infrared (FT-IR) spectroscopy indicated the presence of hydroxyl, carboxylic and methoxyl functional groups. The optimal dosages for flocculation of activated carbon suspension were 17 mg/l EPS and 4 mM CaCl(2). EPS showed flocculating rate of above 80% over a wide range of pH (pH 3-10) whereas, more than 90% rate was noted in the temperature range (10-50°C) tested in presence of CaCl(2). Moreover, EPS showed characteristic emulsifying activity with toluene (66.6%), n-hexadecane (65%), olive oil (63.3%) and kerosene (50%). The apparent molecular weight of the EPS was ~2 × 10(5) Da.

Characterization of a Novel Trimethoprim Resistance Gene, dfrA28, in Class 1 Integron of an Oligotrophic Acinetobacter johnsonii Strain, MB52, Isolated from River Mahananda, India

The present work has validated the capability of using diluted Luria broth (10(-3)) and its amendment with antibiotics to quantify proportions of antibiotic-resistant oligotrophic bacteria contained in river water. The assay conditions for assessing fractions of antibiotic-resistant oligotrophic bacteria, especially the type and concentration of antibiotic exposure by determining LD(50) concentration for each antibiotic using sensitive oligotrophic strain, have been standardized. The presence of an array of two gene cassettes including a novel dihydrofolate reductase gene, dfrA28, within the variable region of the class 1 integron in an oligotrophic test strain, MB52, in a population of oligotrophic bacteria isolated from Mahananda River water, has been described. The predicted protein consisting of 172 amino acids exhibited 76.4% amino acid identity with DfrA1. A gene for streptomycin resistance, aminoglycoside adenyl transferase aadA1, was located downstream of dfrA28. Phylogenetic analysis of MB52 identified it as a strain of Acinetobacter johnsonii.

Structural and immunological studies of an exopolysaccharide from Acinetobacter junii BB1A.

A water-soluble heteropolysaccharide (PS) was isolated from extracellular polymeric substances (EPS) produced by a novel metal tolerant bacterium, Acinetobacter junii BB1A. Sugar analysis showed that the PS was composed of mannose, galactose and arabinose in a molar ratio of nearly 3:1:1. Structural characterization of the PS was carried out using methylation analysis, periodate oxidation, Smith degradation and 1D/2D NMR experiments. Methylation analysis revealed that the PS was consisted of 2,4-linked-mannopyranosyl, 3,4-linked-mannopyranosyl, 2-linked-galactopyranosyl, terminal mannopyranosyl and arabinopyranosyl residues in a relative proportion of nearly 1:1:1:1:1. Smith degradation of the PS showed the presence of hydrated glyceraldehyde containing disaccharide unit consisting of α-d-Manp-(1→and→4)-α-d-Manp-(1→residues where the later was directly attached to a hydrated glyceraldehyde moiety. This polysaccharide showed significant in vitro splenocyte, thymocyte, and macrophage activations with optimum dose of 100 μg/mL for macrophage and 25 μg/mL both for the splenocyte and thymocyte.

Antibacterial and DNA degradation potential of silver nanoparticles synthesized via green route.

Silver nanoparticles (AgNPs) were synthesized using a hetero polysaccharide (PS) isolated from Lentinus squarrosulus (Mont.) Singer. The polysaccharide fraction (consisting of glucose, fucose and galactose) serves the role of both reducing as well as stabilizing agent. UV-vis spectroscopy showed maximum absorbance at 407 nm due to surface plasmon resonance. High resolution transmission electron microscopy (HRTEM) exhibited that the average diameter of the nanoparticles was 2.78±1.47 nm. The XRD analysis revealed face-centered cubic (fcc) geometry of silver nanoparticles. Antibacterial activity of the AgNPs-PS conjugate was tested against multiple antibiotics resistant (MAR) Escherichia coli strain MREC33 and found that the killing was due to generation of reactive oxygen species (ROS). Internalization of AgNPs-PS conjugate along with its DNA degradation capability was demonstrated using flow cytometry. AgNPs-PS conjugates showed negligible toxicity to human RBCs. This LD50 dosage of AgNPs-PS conjugates in combination with each of the four antibiotics (ampicillin, azithromycin, kanamycin and netilmicin) to which E. coli MREC33 was resistant, showed synergistic effect to inhibit complete bacterial growth.