Acinetobacter sp. mediated synthesis of AgNPs, its optimization, characterization and synergistic antifungal activity against C. albicans.

AIMS: To synthesize silver nanoparticles (AgNPs) with cell free extract of Acinetobacter sp. and evaluate antifungal activity against planktonic and biofilm of Candida. Also, to study mechanism of antifungal action of AgNPs. METHODS AND RESULT: Acinetobacter spp were screened for synthesis of AgNPs. Physio-chemical parameters were optimized to obtained monodispersed nanoparticles. Optimized nanoparticles were characterized using spectroscopic, microscopic and diffraction techniques. Antifungal and biofilm disruption activity of AgNPs (10 ± 5 nm) were investigated against C. albicans. Mechanism of antifungal activity of nanosilver was deduced by growth curve, reactive oxygen species generation, thiol interaction and microscopic analysis. Acinetobacter sp. GWRFH 45 gave maximum synthesis of AgNPs. At optimized condition monodispersed, spherical nanoparticles were obtained which were crystalline with negative surface charge. AgNPs exhibited antifungal activity against planktonic cells and biofilm of Candida. AgNPs showed synergistic effect with amphotericin B as well as fluconazole against biofilm disruption. AgNPs were found to affect growth of Candida, generate reactive oxygen species and disrupt cellular morphology. CONCLUSIONS: Cell free extract of A. calcoaceticus GWRFH 45 has ability to synthesize AgNPs. AgNPs alone and in combination with drugs have potential to inhibit C. albicans. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report of bacteriogenic AgNPs used in combination with antifungal drugs against Candida.

Characterization and Fungal Inhibition Activity of Siderophore from Wheat Rhizosphere Associated Acinetobacter calcoaceticus Strain HIRFA32.

Acinetobacter calcoaceticus HIRFA32 from wheat rhizosphere produced catecholate type of siderophore with optimum siderophore (ca. 92 % siderophore units) in succinic acid medium without FeSO4 at 28 °C and 24 h of incubation. HPLC purified siderophore appeared as pale yellow crystals with molecular weight [M(+1)] m/z 347.18 estimated by LCMS. The structure elucidated by (1)H NMR, (13)C NMR, HMQC, HMBC, NOESY and decoupling studies, revealed that siderophore composed of 2,3-dihydroxybenzoic acid with hydroxyhistamine and threonine as amino acid subunits. In vitro study demonstrated siderophore mediated mycelium growth inhibition (ca. 46.87 ± 0.5 %) of Fusarium oxysporum. This study accounts to first report on biosynthesis of acinetobactin-like siderophore by the rhizospheric strain of A. calcoaceticus and its significance in inhibition of F. oxysporum.

Microbial synthesis of gold nanoparticles: current status and future prospects.

Gold nanoparticles have been employed in biomedicine since the last decade because of their unique optical, electrical and photothermal properties. Present review discusses the microbial synthesis, properties and biomedical applications of gold nanoparticles. Different microbial synthesis strategies used so far for obtaining better yield and stability have been described. It also includes different methods used for the characterization and analysis of gold nanoparticles, viz. UV-visible spectroscopy, Fourier transform infrared spectroscopy, X ray diffraction spectroscopy, scanning electron microscopy, ransmission electron microscopy, atomic force microscopy, electron dispersive X ray, X ray photoelectron spectroscopy and cyclic voltametry. The different mechanisms involved in microbial synthesis of gold nanoparticles have been discussed. The information related to applications of microbially synthesized gold nanoparticles and patents on microbial synthesis of gold nanoparticles has been summarized.

The nucleotide sequence of Shigella flexneri 1A: A common Indian isolate.

We report complete sequences of 2 genes of S.flexneri 1a an Indian isolate for the first time. Shigella is causative agent of shigellosis or bacillary dysentery. Genomic library was constructed by shotgun approach. Sequencing was carried out using Big Dye terminator chemistry using ABI 3730 48 capillary DNA analyzer. 170 recombinants were subjected to nucleotide sequencing. Sequence data was analyzed, of these 2 clones showed presence of complete genes out of the total clones sequenced. Annotations were done using various bioinformatics tools. Gene Sfo676 on contig SF21B11, 513 bp long codes for a protein 170 aa long with molecular weight of 18836.5 daltons. The protein is 99 % identical to S. flexneri 2a 301 and not with any other strain of Shigella. It has 7 different sites for phosphorylation, myristoylation and glycosylation. Predicted cellular localization is cytoplasmic membrane. SF0368 is another full-length gene SF0368 on contig SF69C1 is a 312 nucleotide long. It is 103 aa long with molecular weight 11394.0 daltons. Protein is 100% identical to S. flexneri 2a 301 and 99% with S. sonnei strain 046. The gene shows difference when compared with S.sonnei in mono and dinucleotide frequency as well as amino acid composition.

A study on nosocomial pathogens in ICU with special reference to multiresistant Acinetobacter baumannii harbouring multiple plasmids.

BACKGROUND & OBJECTIVES: Antibiotic resistant bacterial nosocomial infections are a leading problem in intensive care units (ICU). Present investigation was undertaken to know antibiotic resistance in Acinetobacter baumannii and some other pathogens obtained from clinical samples from ICU causing nosocomial infections. Special emphasis was given on plasmid mediated transferable antibiotic resistance in Acinetobacter. METHODS: The clinical specimens obtained from ICU, were investigated to study distribution of nosocomial pathogens (272) and their antibiotic resistance profile. Acinetobacter isolates were identified by API2ONE system. Antimicrobial resistance was studied with minimum inhibitory concentration (MIC) by double dilution agar plate method. The plasmid profile of 26 antibiotic resistant isolates of Acinetobacter was studied. Curing of R-plasmids was determined in three antibiotic resistant plasmid containing A. baumannii isolates. Plasmid transfer was studied by transformation. RESULTS: Major infections found in ICU were due to Acinetobacter baumannii, Escherichia coli, Klebsiella pneumoniae, Pseudomonas aeruginosa, Staphylococcus aureus and Streptococcus pyogenes. The infection rate was maximum in urinary tract (44.4%) followed by wound infections (29.4%), pneumonia (10.7%) and bronchitis (7.4%). Acinetobacter isolates displayed high level of antibiotic resistance (up to 1024microg/ml) to most of antibiotics. More than 90 per cent isolates of Acinetobacter were resistant to a minimum of 23 antibiotics. Plasmid profile of Acinetobacter isolates showed presence of 1-4 plasmids. Ethidium bromide cured plasmids pUPI280, pUPI281, pUPI282 with curing efficiencies 20, 16 and 11 per cent respectively while acridine orange cured plasmids pUPI280, pUPI281 with curing efficiencies 7 and 18 per cent retrospectively. Transformation frequency of E. coli HB101 with pUPI281 was 4.3 x 10(4) transformants/microg plasmid DNA. INTERPRETATION & CONCLUSIONS: A. baumannii was found to be associated with urinary tract infections, respiratory tract infections, septicaemia, bacteraemia, meningitis and wound infections. A. baumannii displayed higher resistance to more number of antibiotics than other nosocomial pathogens from ICU. Antibiotic sensitivity of A. baumannii cured isolates confirmed plasmid borne nature of antibiotic resistance markers. Transfer of antibiotic resistant plasmids from Acinetobacter to other nosocomial pathogens can create complications in the treatment of the patient. Therefore, it is very important to target Acinetobacter which is associated with nosocomial infections.

Species distribution and physiological characterization of Acinetobacter genospecies from healthy human skin of tribal population in India.

BACKGROUND: Various reports on distribution of Acinetobacter spp. from healthy human skin restricted to urban population. However, no such data is available from healthy human skin of tribal population not exposed to modern antibiotics during their life time. PURPOSE: Isolation, biotyping, distribution and physiological characterisation of Acinetobacter spp. from healthy human skin of tribal population. METHODS: Tribal population of Toranmal area of Satpuda Ranges, Maharashtra, India were sampled for ten body sites. Tentative Acinetobacter isolates were confirmed to the genus level by chromosomal DNA transformation assay and to species level using Bouvet and Grimont system. Novel physiological characteristics like pH, temperature and salt tolerance were studied. All strains were screened for production of various enzymes. RESULTS: One hundred and eighteen strains were isolated, which belonged to nine Acinetobacter genospecies. A. haemolyticus was most abundant followed by A. calcoaceticus and A. genospecies 1-3. Higher percentage of Acinetobacter was recovered from skin of nose, Pawara tribe and female volunteers. They showed wide variation in temperature, salt and pH tolerance. Most of the strains could produce enzymes viz, lipase, esterase, urease and amylase. CONCLUSIONS: Acinetobacter spp. belonging to nine genospecies were obtained in the present study. Physiological characteristics including high salt, temperature and acidic pH tolerance were helpful to differentiate between the commensal and pathogenic species of Acinetobacter genus.

Isolation, characterization, and plasmid pUPI126-mediated indole-3-acetic acid production in acinetobacter strains from rhizosphere of wheat.

Thirty-seven strains of Acinetobacter isolated and characterized from rhizosphere of wheat were screened for indole-3-acetic acid (IAA) production. Only eight Acinetobacter strains showed IAA production. The genus Acinetobacter was confirmed by chromosomal DNA transformation assay. Biotyping of eight strains was carried out and they were found to be genospecies of A. junii, A. baumannii, A. genospecies 3, and A. haemolyticus. Five of eight strains produced IAA at the early stationary phase: A. haemolyticus (A19), A. baumannii (A18, A16, A13), and Acinetobactergenospecies 3 (A15). A. junii A6 showed maximum IAA production at log phase and A. genospecies 3 and A. baumannii (A28, A30) at late stationary phase. IAA was extracted by ethyl acetate and purified by preparative thin-layer chromatography. Purified IAA was confirmed by 1H-nuclear magnetic resonance and infrared spectrum analysis. Pot experiments showed a significant increase in plant growth inoculated with eight Acinetobacter genospecies as compared to control plants. IAA production was found to be encoded by plasmid pUPI126. All eight strains of Acinetobacter contain a plasmid pUPI126 with a molecular weight of 40 kb. Plasmid pUPI126 was transformed into Escherichia coli HB101 at a frequency of 5 x 10(-5), and E. coli HB101 (pUPI126) transformants also showed IAA activity. PUPI126 also encoded resistance to selenium, tellurium, and lead. This is the first report of plasmid-encoded IAA production in the genus Acinetobacter.

Distribution and in vitro antimicrobial susceptibility of Acinetobacter species on the skin of healthy humans.

BACKGROUND: Acinetobacter spp. are ubiquitous in the environment and have emerged as important nosocomial pathogens. The distribution of Acinetobacter spp. In some temperate European countries has been reported. However, similar data from a tropical country such as India are not available. METHODS: Six body sites (antecubital fossa, axilla, forehead with hairline, neck, outer surface of nose and toe webs) from men and women volunteers were sampled with saline-soaked cotton swabs enriched in Baumann's enrichment medium. The isolates were identified to the genus level by chromosomal DNA transformation assay and to the species level by a 16-test biochemical system. The minimum inhibitory concentration for 39 antibiotics was determined by the two-fold agar dilution method. RESULTS: Seven genospecies of Acinetobacter were found at 6 body sites on healthy human skin. Acinetobacter lwoffii was the most dominant comprising 40% of the total number of isolates, followed by A. junii (35%) and A. haemolyticus (16.5%). The antecubital fossa had the highest colonization frequency (48.5%). The overall positivity rate of samples was higher from women (26.3%) compared to men volunteers (25%). Only two Acinetobacter genospecies 1-3 isolates were isolated while no A. radioresistens were isolated. Susceptibility testing revealed no major differences among the 7 Acinetobacter spp. tested. Fluoroquinolones were the most active, while low-to-intermediate resistance was exhibited towards beta-lactams and aminoglycosides. Acinetobacter spp. isolated from the skin showed susceptibility to commonly used antibiotics. CONCLUSION: Seven Acinetobacter genospecies were isolated from 6 different body sites from the skin of healthy human volunteers. Acinetobacter lwoffii was the dominant isolate. The rate of skin carriage was higher in men than in women and the maximally colonized site was the antecubital fossa. All the genospecies displayed susceptibility to most of the commonly used antimicrobials.

Studies on bioemulsifier production by Acinetobacter strains isolated from healthy human skin.

AIMS: In recent years, interest has been growing in the search for novel bioemulsifiers. Many bacterial genera including Acinetobacter have been reported to produce bioemulsifiers. The present study aims to screen Acinetobacter isolates from healthy human skin for bioemulsifier production. METHODS AND RESULTS: Acinetobacter junii SC14 produced maximum bioemulsifier in the presence of almond oil during stationary growth phase at 37 degrees C and pH 7.2. Partially purified, nondialysable bioemulsifier from SC14 was a proteoglycan. The protein and polysaccharide fractions resulted in 95.2% reconstitution of the emulsification activity. The role of esterase in the release of cell-bound emulsifier and the contribution of capsular polysaccharide to the emulsification activity were observed. CONCLUSION: Acinetobacter strains from human skin exhibited better emulsification activity than that by burn wound or soil isolates, owing to the inherent differences in chemical microenvironment of their habitats. SIGNIFICANCE AND IMPACT OF THE STUDY: Investigation of skin commensals, especially acinetobacters, would lead to the discovery of novel bioemulsifiers with interesting properties. Attempts of screening and strain improvement directed towards skin commensals will open up new avenues for strains producing bioemulsifier on a commercial scale.

Removal of silver from photographic wastewater effluent using Acinetobacter baumannii BL54.

Acinetobacter baumannii BL54, a silver (Ag) resistant micro-organism was isolated from clinical samples collected at the Armed Forces Medical College hospital in Pune, India. The strain BL54 removed a high quantity of silver (2.85 mg/g biomass) from photographic wastewater effluent. Treatment of the cells with 10 mM EDTA or agitating the culture did not affect the removal process, while altering pH of the wastewater or pre-treating the cells with 0.5 mM 2,4-dinitrophenol (DNP), 20 microM N,N'-dicyclohexylcarbodiimide (DCC), 25 micrograms/mL cefotaxime, and polymyxin-B resulted in considerable decrease in removal of silver by the organism. Dead cells, or a Ags plasmid-cured derivative (BL54.1) removed little silver, which was mainly surface bound. The results, compared with accumulation of Ag by a sensitive culture of Escherichia coli K12 J53.2, suggest that A. baumannii BL54 has good potential for bioremediation of silver from photographic wastewater effluents.

Characterization of membrane-associated Pseudomonas aeruginosa Ras-like protein Pra, a GTP-binding protein that forms complexes with truncated nucleoside diphosphate kinase and pyruvate kinase to modulate GTP synthesis.

We report the purification and characterization of a protein from the membrane fraction of Pseudomonas aeruginosa showing intrinsic guanosine triphosphatase (GTPase) activity. The protein was purified as a 48-kDa polypeptide capable of binding and hydrolyzing GTP. The N-terminal sequence of the purified protein revealed its similarity to the Escherichia coli Ras-like protein (Era), and the protein cross-reacted with anti-Era antibodies. This protein was named Pseudomonas Ras-like protein (Pra). Anti-Pra antibodies also cross-reacted with E. coli Era protein. Pra is autophosphorylated in vitro, with phosphotransfer of the terminal phosphate from [gamma-32P]GTP but not [gamma-32P]ATP. Pra is capable of complex formation with the truncated 12-kDa form of nucleoside diphosphate kinase (Ndk) but not with the 16-kDa form. Purified Pra was also shown to physically interact with pyruvate kinase (Pk); Pk and Pra can form a complex, but when the 12-kDa Ndk, Pk, and Pra are all present, Pk has a higher affinity than Pra for forming a complex with the 12-kDa Ndk. The 12-kDa Ndk-Pra complex catalyzed increased synthesis of GTP and dGTP and diminished synthesis of CTP and UTP or dCTP and dTTP relative to their synthesis by uncomplexed Ndk. Moreover, the complex of Pra with Pk resulted in the specific synthesis of GTP as well when Pra was present in concentrations in excess of that of Pk. Membrane fractions from cells harvested in the mid-log phase demonstrated very little nucleoside triphosphate (NTP)-synthesizing activity and no detectable Ndk. Membranes from cells harvested at late exponential phase showed NTP-synthesizing activity and the physical presence of Ndk but not of Pk or Pra. In contrast, membrane fractions of cells harvested at early to late stationary phase showed predominant GTP synthesis and the presence of increasing amounts of Pk and Pra. It is likely that the association of Pra with Ndk and/or Pk restricts its intrinsic GTPase activity, which may modulate stationary-phase gene expression and the survival of P. aeruginosa by modulating the level of GTP.

Nucleoside diphosphate kinase from Pseudomonas aeruginosa: characterization of the gene and its role in cellular growth and exopolysaccharide alginate synthesis.

We report the cloning and determination of the nucleotide sequence of the gene encoding nucleoside diphosphate kinase (Ndk) from Pseudomonas aeruginosa. The amino acid sequence of Ndk was highly homologous with other known bacterial and eukaryotic Ndks (39.9 to 58.3% amino acid identity). We have previously reported that P. aeruginosa strains with mutations in the genes algR2 and algR2 algH produce extremely low levels of Ndk and, as a consequence, are defective in their ability to grow in the presence of Tween 20, a detergent that inhibits a kinase which can substitute for Ndk. Hyperexpression of ndk from the clone pGWS95 in trans in the P. aeruginosa algR2 and algR2 algH double mutant restored Ndk production to levels which equalled or exceeded wild-type levels and enabled these strains to grow in the presence of Tween 20. Hyperexpression of ndk from pGWS95 in the P. aeruginosa algR2 mutant also restored alginate production to levels that were approximately 60% of wild type. Nucleoside diphosphate kinase activity was present in both the cytosolic and membrane-associated fractions of P. aeruginosa. The cytosolic Ndk was non-specific in its transfer activity of the terminal phosphate from ATP to other nucleoside diphosphates. However, the membrane form of Ndk was more active in the transfer of the terminal phosphate from ATP to GDP resulting in the predominant formation of GTP. We report in this work that pyruvate kinase and Ndk form a complex which alters the specificity of Ndk substantially to GTP. The significance of GTP in signal transduction events within the cell and in the production of GDP-mannose, an essential alginate precursor, clearly indicates the importance of Ndk in cellular processes as well as in alginate synthesis.

Plasmid mediated silver resistance in Acinetobacter baumannii.

Acinetobacter baumannii BL88, an environmental isolate, was resistant to 13 metals and 10 antibiotics. Plumbagin cured resistance to silver, cadmium, antimony, streptomycin and ampicillin at varying frequencies. However, only silver resistance transferred (1 x 10(-6) recepient-1) to Escherichia coli K12 during conjugation. Correspondingly there was transfer of a 54 kb plasmid (pUPI199) from A. baumannii BL88. The plasmid transformed E. coli DH5 alpha cells at a frequency of 1 x 10(-8) recepient-1. The growth rate of E. coli DH5 alpha (pUPI199) was slower as compared with E. coli DH5 alpha. Plasmid pUPI199 was 76 and 9.6% stable in the host A. baumannii BL88 in the presence and absence of selection pressure, respectively. A. baumannii BL88 was found to accumulate and retain silver whereas E. coli DH5 alpha (pUPI199) effluxed 63% of the accumulated silver ions.

High levels of multiple metal resistance and its correlation to antibiotic resistance in environmental isolates of Acinetobacter.

Forty strains of Acinetobacter were isolated from different environmental sources. All the strains were classified into four genospecies, i.e., A. baumannii (33 isolates), A. calcoaceticus (three isolates), A. junii (three isolates) and A. genospecies3 (one isolate). Susceptibility of these 40 strains to salts of 20 heavy metals and 18 antibiotics was tested by the agar dilution method. All environmental isolates of Acinetobacter were resistant to multiple metal ions (minimum 13 metal ions) while all but one of the strains were resistant to multiple antibiotics (minimum four antibiotics). The maximum number of strains were found to be sensitive to mercury (60% strains) while all strains were resistant to copper, lead, boron and tungsten even at 10 mM concentration. Salts of these four metal ions may be added to the growth medium to facilitate selective isolation of Acinetobacter. Rifampicin and nalidixic acid were the most toxic antibiotics, inhibiting 94.5 and 89.5% of the acinetobacters, respectively. A. genospecies3 was found to be the most resistant species, tolerating high concentrations of all the 20 metal ions and also to a greater number of antibiotics than any other species of Acinetobacter tested. An inhibitory concentration (10 mM) of Ni(2+) and Zn(2+) was observed to inhibit the growth of all of the clinical isolates but allowed the growth of the environmental isolates, facilitating the differentiation between pathogenic and non-pathogenic acinetobacters.

Metal resistance in Acinetobacter and its relation to beta-lactamase production.

Thirty nine clinical isolates of Acinetobacter belonging to six species were tested for resistance to 20 metal ions and their ability to produce beta-lactamase. Fifty two percent of the strains produced beta-lactamase. beta-Lactamase producers and non-producers were almost equally distributed in the different species. A. baumannii was the predominant biotype and was found to be most resistant to metals. Resistance to mercury was prevalent in beta-lactamase-producing A. baumannii only. Silver resistant strains of A. baumannii produced beta-lactamase. Sensitivity and resistance to copper and cadium was equally distributed between beta-lactamase producers and non-producers. beta-Lactamase-producer and -non-producer strains were uniformly sensitive to cadmium except Acinetobacter genospecies 1.

Novel broad-spectrum metal-based antifungal agents. Correlations amongst the structural and biological properties of copper (II) 2-acetylpyridine N4-dialkylthiosemicarbazones.

Copper(II) complexes of the type [Cu(L)X], where L = tridentate anion of 2-acetylpyridine N4-diethyl thiosemicarbazone and X = C1 or Br, were screened against seven fungal strains pathogenic to man viz. Aspergillus niger, Aspergillus fumigatus, Candida albicans, Cryptococcus neoformans, Tricophyton rubrum, Epidermophyton floccosum and Microsporum canis. The greater growth inhibition exhibited by the bromo complex can be explained on the basis of its lower Cu(II)/Cu(I) redox couple and greater covalent bonding. These compounds represent a novel class of metal-based antifungal agents which provide opportunities for a large number of synthetic variations for modulation of the activities.

Biotyping of Acinetobacter calcoaceticus using the API 2ONE system.

The API 2ONE system for the identification of non-fermentative Gram-negative bacilli enables the discrimination of a possible 209 different biotypes of Acinetobacter spp. and consequently has potential for use as an Acinetobacter spp. typing system. A total of 122 separate strains of Acinetobacter spp. isolated in Nottingham hospitals over a 4 year period from a wide variety of clinical specimens, divided into 31 different biotypes which were stable over a 1 year storage period. Two biotypes predominated, one of which was a multiply resistant strain of A. calcoaceticus variant anitratus. The API 2ONE system was found to be a rapid method for biotyping strains of Acinetobacter spp. and was helpful in monitoring cross-infection and spread of particular strains of Acinetobacter spp. in the hospital environment.

Plasmid transfer and behaviour in Acinetobacter calcoaceticus EBF65/65.

At least one plasmid from each of the incompatibility groups B, C, FIV, H2/S, I alpha, I delta, P, W and X was shown to be capable of transfer from Escherichia coli K12 to Acinetobacter calcoaceticus EBF65/65. Transfer was influenced by the presence of pAV2 (thought to encode a restriction-modification system) in the recipient strain; however, not all plasmids belonging to a particular incompatibility group behaved identically. All plasmids were unstable to varying degrees in A. calcoaceticus EBF65/65, but under suitable conditions were capable of transfer to further strains of EBF65/65 and re-transfer to E. coli K12. Of 40 recently isolated trimethoprim R plasmids 31 transferred successfully from E. coli K12 to A. calcoaceticus EBF65/65, but 17 of these 31 required the introduction of a second mobilizing plasmid for re-transfer to occur.