Synthesis and structural characterization of silver nanoparticles using bacterial exopolysaccharide and its antimicrobial activity against food and multidrug resistant pathogens

A green, simple, and effective approach was performed to synthesize potent silver nanoparticles (SNPs) using bacterial exopolysaccharide as both a reducing and stabilizing agent. The synthesized SNPs were characterized using UV-vis spectroscopy, transmission electron microscopy, energy dispersive X-ray analysis, X-ray diffraction, and Fourier-transform-infrared spectra analyses. The SNPs varied in shape and were multidispersed with a mean diameter of 10 nm ranging from 2 to 15 nm and were stable up to 2 months at room temperature. The antimicrobial activity of the SNPs was analyzed against bacterial and fungal pathogens using the agar well diffusion method. Dose dependent inhibition was observed for all bacterial pathogens. The multidrug resistant pathogens P. aeruginosa and K. pneumonia were found to be more susceptible to the SNPs than the food borne pathogen L. monocytogenes. The fungi Aspergillus spp. exhibited a maximum zone of inhibition compared to that of Penicillum spp. These results suggest that exopolysaccharide-stabilized SNPs can be used as an antimicrobial agent for various biomedical applications.     

Eradication of methicillin-resistant Staphylococcus aureus with an antiseptic soap and nasal mupirocin among colonized patients – an open uncontrolled clinical trial

Resistance to β-lactam antibiotics in enteric Gram-negative bacilli may be difficult to detect using standard methods of either Kirby-Bauer disc diffusion (KBDD) or broth dilution for minimal inhibitory concentration (MIC). This difficulty is due to genetic differences in resistance determinants, differences in levels of gene expression, and variation in spectra of enzymatic activity against the substrate β-lactams used for susceptibility testing. We have examined 95 clinical isolates reportedly susceptible to ceftazidime and ceftriaxone, as originally determined by either KBDD or MIC methods. The organisms studied here were isolated in 2002 from two pediatric hospital centers (Seattle, USA and Shanghai, China). They belong to the inducible β-lactamase producing Gram-negative bacilli, such as Enterobacter spp., Citrobacter spp., Serratia spp., Morganella spp., Providencia spp., and Proteus vulgaris. A Kirby-Bauer disc approximation (KBDA) method identified inducible phenotypes of third-generation cephalosporin resistance in 76% of isolates, which would otherwise be considered susceptible by standard KBDD methods.     

Effect of toxaphene on carbon dioxide assimilation and translocation in avena secale

In susceptible oat, toxaphene inhibits photosynthetic electron flow and concomitant ATP synthesis. Although the rate of ¹⁴CO₂ assimilation is apparently not affected markedly there is an increase in dry weight of leaves contacting the pesticide. The labelling patterns in leaf sections exposed to ¹⁴CO₂ are similar for both toxaphene-treated and untreated seedlings. However, if given a period in darkness before extraction it is evident that assimilation products in leaf sections from toxaphene-treated leaves remain as small M, materials, including substantial amounts of sugars, whereas in untreated controls these were converted to polymeric materials. In toxaphene-treated seedlings the translocation of assimilation products to the roots is decreased and sucrose accumulates in the leaves.     

Modification of Herbicide Binding to Photosystem II in Two Biotypes of Senecio vulgaris L

The present study compares the binding and inhibitory activity of two photosystem II inhibitors: 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron [DCMU]) and 2-chloro-4-(ethylamine)-6-(isopropyl amine)-S-triazene (atrazine). Chloroplasts isolated from naturally occurring triazine-susceptible and triazine-resistant biotypes of common groundsel (Senecio vulgaris L.) showed the following characteristics. (a) Diuron strongly inhibited photosynthetic electron transport from H₂O to 2,6-dichlorophenolindophenol in both biotypes. Strong inhibition by atrazine was observed only with the susceptible chloroplasts. (b) Hill plots of electron transport inhibition data indicate a noncooperative binding of one inhibitor molecule at the site of action for both diuron and atrazine. (c) Susceptible chloroplasts show a strong diuron and atrazine binding (¹⁴C-radiolabel assays) with binding constants (K) of 1.4 × 10⁻⁸ molar and 4 × 10⁻⁸ molar, respectively. In the resistant chloroplasts the diuron binding was slightly decreased (K = 5 × 10⁻⁸ molar), whereas no specific atrazine binding was detected. (d) In susceptible chloroplasts, competitive binding between radioactively labeled diuron and non-labeled atrazine was observed. This competition was absent in the resistant chloroplasts.     

Determination of MIC Distribution of Arbekacin,Cefminox, Fosfomycin,Biapenem and Other Antibiotics against Gram-Negative Clinical Isolates in South India: A Prospective Study

Objectives

To determine the in vitro activity of antibiotics, including arbekacin, cefminox, fosfomycin and biapenem which are all still unavailable in India, against Gram-negative clinical isolates.

Methods

We prospectively collected and tested all consecutive isolates of Escherichia coli, Klebsiella spp., Pseudomonas aeruginosa and Acinetobacter spp. from blood, urine and sputum samples between March and November 2012. The minimum inhibition concentration (MIC) of 16 antibiotics was determined by the broth micro-dilution method.

Results

Overall 925 isolates were included; 211 E. coli, 207 Klebsiella spp., 153 P. aeruginosa, and 354 Acinetobacter spp. The MIC₅₀ and MIC₉₀ were high for cefminox, biapenem and arbekacin for all pathogens but interpretative criteria were not available. The MIC₅₀ was categorized as susceptible for a couple of antibiotics, including piperacillin/tazobactam, carbapenems and amikacin, for E. coli, Klebsiella spp. and P. aeruginosa. However, for Acinetobacter spp., the MIC₅₀ was categorized as susceptible only for colistin. On the other hand, fosfomycin was the only antibiotic that inhibited 90% of E. coli and Klebsiella spp. isolates, while 90% of P. aeruginosa isolates were inhibited only by colistin. Finally, 90% of Acinetobacter spp. isolates were not inhibited by any antibiotic tested.

Conclusion

Fosfomycin and colistin might be promising antibiotics for the treatment of infections due to E. coli or Klebsiella spp. and P. aeruginosa, respectively, in India; however, clinical trials should first corroborate the in vitro findings. The activity of tigecycline should be evaluated, as this is commonly used as last-resort option for the treatment of multidrug-resistant Acinetobacter infections.     

Genotoxic activity of a technical toxaphene mixture and its photodegradation products in SOS genotoxicity tests

Toxaphene (CAS No. 800-35-2) is a complex mixture of several hundred components that was used worldwide primarily as an agricultural pesticide with insecticide effects in the second half of the 20th century. In vitro investigations of the genotoxicity and mutagenicity of toxaphene were generally described in the literature, but they provided somewhat equivocal results. We re-evaluated the genotoxicity of technical toxaphene in two prokaryotic systems. The SOS Chromotest showed high sensitivity to toxaphene: three concentrations (40, 20 and 10 mg/l) were clearly positive and the dose–response effect was evident. In the umuC assay, a dose-dependent increase in genotoxic activity was observed at toxaphene concentrations from 2.5 to 40.0 mg/l, but these results were found to be not significant. The genotoxicity of toxaphene and its photodegradation products after UV-irradiation (3–6–9 h) at concentrations ranging from 7.5 to 60.0 mg/l was also examined in this study. An irradiated solution of technical toxaphene after 3 h showed no significant evidence of bacterial growth inhibition. However, exposure of Salmonella to 6 h UV-irradiated toxaphene showed a toxic effect compared with the negative control. After 9 h irradiation, a decrease of bacterial growth was observed. Activity of β-galactosidase in the presence of a toxaphene solution was significantly increased after 6 and 9 h irradiation, reaching values that were 2.4- and 3.1-fold higher, respectively, than the control, which exceeded the criteria of significant genotoxicity. These results show that while technical toxaphene is a weak, direct-acting mutagen in some bacterial tests, a dose-dependent toxicity and genotoxicity of its photoproducts could be conclusively demonstrated by the umuC test.     

Response of Resistant and Susceptible Bell Pepper (Capsicum annuum) to a Southern California Meloidogyne incognita Population from a Commercial Bell Pepper Field

To determine the presence and level of root-knot nematode (Meloidogyne spp.) infestation in Southern California bell pepper (Capsicum annuum) fields, soil and root samples were collected in April and May 2012 and analyzed for the presence of root-knot nematodes. The earlier samples were virtually free of root-knot nematodes, but the later samples all contained, sometimes very high numbers, of root-knot nematodes. Nematodes were all identified as M. incognita. A nematode population from one of these fields was multiplied in a greenhouse and used as inoculum for two repeated pot experiments with three susceptible and two resistant bell pepper varieties. Fruit yields of the resistant peppers were not affected by the nematodes, whereas yields of two of the three susceptible pepper cultivars decreased as a result of nematode inoculation. Nematode-induced root galling and nematode multiplication was low but different between the two resistant cultivars. Root galling and nematode reproduction was much higher on the three susceptible cultivars. One of these susceptible cultivars exhibited tolerance, as yields were not affected by the nematodes, but nematode multiplication was high. It is concluded that M. incognita is common in Southern California bell pepper production, and that resistant cultivars may provide a useful tool in a nonchemical management strategy.     

Effects of DDT on photosynthetic electron flow in Secale species

Following a survey of a range of varieties of rye, mainly Secale cereale, for reaction to DDT, the mode of action of the pesticide in a susceptible variety was studied. Two sites of interaction of DDT with the photosynthetic electron transport chain were demonstrated. The first site of inhibition was on the oxidizing side of photosystem 2, between the sites of electron donation from diphenylcarbazide at pH 6.0 and pH 8.0 in Tris-washed chloroplasts. The second site of DDT inhibition was in the intermediate electron transport chain, and was demonstrated by using dichlorophenol-indophenol and phenyldiamines as electron donors in chloroplasts where electron flow from photosystem 2 was inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea. The sites are distinct from those characteristic of herbicides which affect photosynthetic electron flow.     

Antifungal Activity of Geldanamycin Alone or in Combination with Fluconazole Against Candida species

A standardized broth microdilution method was used to test the antifungal activity of geldanamycin (GA), an inhibitor of heat shock protein 90 (Hsp90), alone or in combination with the antifungal agent fluconazole (FLC) against 32 clinical isolates of Candida spp. In addition, a disk diffusion test was also used to evaluate the antifungal effect of these two drugs against Candida spp. by measuring the inhibition zone diameters. We found that the range of minimal inhibitory concentrations (MICs) for GA alone against Candida spp. was 3.2–12.8 mg/L and the geometric mean of MICs was 6.54 mg/L. In addition, the combination of GA with FLC showed synergistic effects in vitro against 2 FLC-susceptible and 6 FLC-resistant isolates of C. albicans. As for the other isolates, indifference but no antagonism was observed. In the disk diffusion assay, the diameter of inhibition zones for FLC combined with GA against FLC-resistant C. albicans isolates was 30 mm, while no inhibition was observed with FLC alone. These results demonstrate that GA possesses antifungal activity against Candida spp., and the combination of GA with FLC shows in vitro synergistic activity against some C. albicans isolates, especially those resistant to FLC.     

Effects of Helminthosporium maydis Race T Toxin on Electron Transport in Susceptible Corn Mitochondria and Prevention of Toxin Actions by Dicyclohexylcarbodiimide

The effect of Helminthosporium maydis race T toxin on electron transport in susceptible cytoplasmic male-sterile Texas corn (Zea mays L.) mitochondria was investigated, using dichlorophenol indophenol and ferricyanide as electron acceptors. Succinate-dependent electron transport was stimulated by the toxin, consistent with the well described increase in membrane permeability induced by the toxin. Malate-dependent electron transport was inhibited. This inhibition of electron transport increased as a function of time of exposure to the toxin. Mitochondria from normal-fertile (N) corn were not affected by the toxin. Both the inhibition of electron transport and the increase in ion permeability, such as dissipation of membrane potential and Ca²⁺ gradients, induced by the toxin in T corn was prevented by N,N′-dicyclohexylcarbodiimide, a hydrophobic carbodiimide. A water-soluble carbodiimide, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide, was ineffective in preventing dissipation of membrane potential by the toxin. These results suggest that the various toxin actions are mediated via interaction of the toxin with one target site, most probably a 13 kilodalton polypeptide unique to T mitochondria. N,N′-dicyclohexylcarbodiimide may confer protection by modifying an amino acid residue in a hydrophobic portion of the target site.     

Biological control of bayoud disease in date palm: Selection of microorganisms inhibiting the causal agent and inducing defense reactions

Twenty-one isolates of microorganisms, including Bacillus spp., Rhizobium spp., Ulocladium atrum, Candida guilliermondii, Pseudomonas sp., Rahnella aquatilis and other bacteria not yet identified, were tested to determine their effects on the mycelial growth and the sporulation of Fusarium oxysporum f.sp. albedinis (Foa), the causal agent of bayoud on date palm. The potential of these antagonists in the induction of defense reactions in date palm seedlings was also studied. Four bacteria, B. pumilus W1, R. aquatilis W2, B. cereus X16 and n.d. S1, have exhibited a high inhibition toward mycelial growth of Foa (70–77%), and its sporulation (80–95% of the control). Moreover, cytological alterations have been detected in the Foa mycelium grown in the inhibition zone. Application of these antagonists into date palm seedlings has led to trigger defense reactions with an accumulation of non-constitutive hydroxycinnamic acid derivatives, such as the sinapic derivative I2, known to play a crucial role in resistance of date palm to Foa. This reaction was more pronounced in resistant cultivar (BSTN) than in susceptible (JHL). The combined effects of direct and indirect actions of Foa antagonists are discussed in the hope of providing a biocontrol strategy against bayoud.     

Microbial Populations Stimulated for Hexavalent Uranium Reduction in Uranium Mine Sediment

Uranium-contaminated sediment and water collected from an inactive uranium mine were incubated anaerobically with organic substrates. Stimulated microbial populations removed U almost entirely from solution within 1 month. X-ray absorption near-edge structure analysis showed that U(VI) was reduced to U(IV) during the incubation. Observations by transmission electron microscopy, selected area diffraction pattern analysis, and energy-dispersive X-ray spectroscopic analysis showed two distinct types of prokaryotic cells that precipitated only a U(IV) mineral uraninite (UO₂) or both uraninite and metal sulfides. Prokaryotic cells associated with uraninite and metal sulfides were inferred to be sulfate-reducing bacteria. Phylogenetic analysis of 16S ribosomal DNA obtained from the original and incubated sediments revealed that microbial populations were changed from microaerophilic Proteobacteria to anaerobic low-G+C gram-positive sporeforming bacteria by the incubation. Forty-two out of 94 clones from the incubated sediment were related to sulfate-reducing Desulfosporosinus spp., and 23 were related to fermentative Clostridium spp. The results suggest that, if in situ bioremediation were attempted in the uranium mine ponds, Desulfosporosinus spp. would be a major contributor to U(VI) and sulfate reduction and Clostridium spp. to U(VI) reduction.     

Studies on the Natural Durability of Tropical Timbers to termite attack

Small samples of 28 tropical timbers were tested with the dry-wood termite Cryptotermes cynocephalus and the subterranean termite Coptotermes curvignathus using several methods of testing. The results revealed that four among 28 species of wood were completely durable to the two species of termite. They are Dalbergia latifolia, Eusideroxylon zwageri, Intsia bijuga and Tectona grandis. Agathis alba, Dipterocarpus spp. and Mangifera spp. were classified as the most susceptible wood species to termites.     

Sensibilidad a fluconazol y voriconazol de especies de Candida aisladas de pacientes provenientes de unidades de cuidados intensivos en Medellín,Colombia (2001–2007)

BackgroundDisseminated candidiasis is caused by different Candida species and mainly affects immunocompromised patients and those hospitalized in intensive care units (ICU).ObjectiveOur aim was to determine the frequency and susceptibility of Candida spp. isolates to fluconazole and voriconazole, obtained from patients hospitalized in ICU in the city of Medellin during the years 2001–2007.MethodsThe agar diffusion technique based on the protocols recommended by the CLSI from the United States (M44A) was used. The Chi² test and the Kruskal Wallis statistical methods were used to compare changes in the frequency of Candida spp. isolates and their susceptibility to azoles by year of isolation.ResultsA total of 337 isolates were analyzed, 147 (43.6%) of which corresponded to Candida albicans, followed by 79 (23.4%) Candida tropicalis, 47 (13.9%) Candida parapsilosis, 32 (9.5%) Candida glabrata, 12 (3.6%) Candida guilliermondii and 11 (3.3%) Candida krusei. The remaining isolates (2.7%) were distributed among other species (Candida famata, Candida lusitaniae, Candida lipolytica, Candida pelliculosa and Candida spp.) Most of these isolates (78.3%) were susceptible; 11.9% were dose-dependent susceptible (DDS) and 9.8% resistant to fluconazole. For voriconazole, we observed that 94.1% of the isolates were susceptible, 2.4% DDS and 3.6% resistant.ConclusionsThese data indicate a notable change in the species frequency, as well as a new susceptibility patterns that requires the precise identification of the causative organism and susceptibility testing in order to determine the characteristics of the isolates circulating in ICUs and then to treat them appropriately.     

Studies on the Sites of Inhibition of Photosynthetic Electron Transport in Barley by DDT (1,1,1-trichloro-2,2-bis(p-chlorophenyl)ethane)

The effect of DDT in resistant and susceptible barley on variousphotosynthetic electron transport activities involving photosystems1 and 2 functioning alone and in series is reported. Whereasnone of the measured activities in resistant barleys were affectedby DDT treatment, in susceptible barley two sites of interactionof DDT with the photosynthetic electron transport chain weredemonstrated. The first site of inhibition was located beforephotosystem 2, between the sites of electron donation from diphenylcarbazideat pH 6·0 and 8·0, and on the oxidizing side ofthe inhibitions resulting from tris washing or heat treatment.Mn²⁺ ions, which can act as donor before photesystem 2, appearedto donate electrons on the H₂O side of the site of inhibitionby DDT. The second site of DDT inhibition was located in thepath of electron flow from photosystem 2 to NADP⁺ or diquat,and was demonstrated by using dichlorophenolindophenol and phenylenediaminesas electron donors.     

A novel GFP-based approach for screening biocontrol microorganisms in vitro against Dothistroma septosporum

Dothistroma septosporum is the causal agent of Dothistroma needle blight of pine trees. A novel green fluorescent protein (GFP)-based screening method was developed to assess the potential of microorganisms for biocontrol of Dothistroma. The screen utilizes GFP expression as an indicator of metabolic activity in the pathogen and hygromycin resistance selection to determine if the interaction is fungistatic or fungicidal. Results suggested that six of eight Trichoderma isolates tested have the potential to control Dothistroma in vitro, via a fungicidal action. Because D. septosporum produces a broad-spectrum toxin, dothistromin, the inhibition of Trichoderma spp. by D. septosporum was determined by growth rate measurements compared to controls. Inhibition of the Trichoderma spp. ranged from no inhibition to 30% inhibition and was influenced by the assay medium used. The GFP screening method was also assessed to determine if it was suitable for screening bacteria as potential biocontrol candidates. Although a method involving indirect-contact had to be used, two of four Bacillus strains showed antagonistic activity against D. septosporum in vitro, via a fungistatic interaction. The four bacterial strains inhibited D. septosporum growth by 14.0 to 39.8%. This GFP-based method represents a novel approach to screening fungi and bacteria for antagonistic activity.     

Susceptibility of Phelipanche and Orobanche species to AAL-toxin

Fusarium and Alternaria spp. are phytopathogenic fungi which are known to be virulent on broomrapes and to produce sphinganine-analog mycotoxins (SAMs). AAL-toxin is a SAM produced by Alternaria alternata which causes the inhibition of sphinganine N-acyltransferase, a key enzyme in sphingolipid biosynthesis, leading to accumulation of sphingoid bases. These long chain bases (LCBs) are determinant in the occurrence of programmed cell death (PCD) in susceptible plants. We showed that broomrapes are sensitive to AAL-toxin, which is not common plant behavior, and that AAL-toxin triggers cell death at the apex of the radicle as well as LCB accumulation and DNA laddering. We also demonstrated that three Lag1 homologs, encoding components of sphinganine N-acyltransferase in yeast, are present in the Orobanche cumana genome and two of them are mutated leading to an enhanced susceptibility to AAL-toxin. We therefore propose a model for the molecular mechanism governing broomrape susceptibility to the fungus Alternaria alternata.     

Comparison of Five Populations of Tylenchulus semipenetrans to Citrus,Poncirus, and their Hybrids

The infectivity of five populations of Tylenchulus semipenetrans were compared and differentiated on 10 hosts (5 Citrus spp., 1 Poncirus trifoliata, and 4 hybrids of Citrus spp. X P. trifoliata). Differences in levels of infection and development (P = 0.01) occurred between Citrus spp. and P. trifoliata cv. ''Pomeroy'' and their three hybrids, C. paradisi X P. trifoliata cv. ''Swingle'' citruntelo and C. sinensis, cv. ''Ruby'' orange X P. trifoliata cv. ''Webber Fawcett 14-7'', and ''15-7''. Poncirus trifoliata cv. Pomeroy was susceptible to a California biotype 3 and highly resistant to the other citrus nematode populations. Low infection levels with California biotype 1, Arizona, and Florida populations on Swingle citrumelo, and the two Ruby orange hybrids indicated inherited resistance. Reproduction of the nematode population from Texas was greatest on the three hybrids, Swingle citrumelo, Ruby orange 14-7, and 15-7, from the California 1, Arizona, and Florida populations, but its comparable densities on P. trifoliata and Citrus spp. were not sufficiently different from these populations to consider it a separate biotype. California biotype 3 was sufficiently different from all other populations to be considered a different biotype, and it was named the "Poncirus biotype."     

Fructophilic Characteristics of Fructobacillus spp. may be due to the Absence of an Alcohol/Acetaldehyde Dehydrogenase Gene (adhE)

Fructophilic strains of Leuconostoc spp. have recently been reclassified to a new genus, i.e., Fructobacillus. Members of the genus are differentiated from Leuconostoc spp. by their preference for fructose on growth, requirement of an electron acceptor for glucose metabolism, and the inability to produce ethanol from the fermentation of glucose. In the present study, enzyme activities and genes involved in ethanol production were studied, since this is the key pathway for NAD⁺/NADH cycling in heterofermentative lactic acid bacteria. Fructobacillus spp. has a weak alcohol dehydrogenase activity and has no acetaldehyde dehydrogenase activity, whereas both enzymes are active in Leuconostoc mesenteroides. The bifunctional alcohol/acetaldehyde dehydrogenase gene, adhE, was described in Leuconostoc spp., but not in Fructobacillus spp. These results suggested that, due to the deficiency of the adhE gene, the normal pathway for ethanol production is absent in Fructobacillus spp. This leads to a shortage of NAD⁺, and the requirement for an electron acceptor in glucose metabolism. Fructophilic characteristics, as observed for Fructobacillus spp., are thus due to the absence of the adhE gene, and a phenotype that most likely evolved as a result of regressive evolution.     

Focus: Zoonotic Disease: Molecular Detection of Zoonotic Pathogens in the Blood and Tissues of Camels (Camelus dromedarius) in Central Desert of Iran

Dromedary camels (Camelus dromedarius) play a major economic role in many countries in Africa and Asia. Although they are resistant to harsh environmental conditions, they are susceptible to a wide range of zoonotic agents. This study aimed to provide an overview on the prevalence of selected zoonotic pathogens in blood and tissues of camels in central Iran. Blood, liver, portal lymph node, and brain were collected from 100 apparently healthy camels at a slaughterhouse in Qom city to assess the presence of DNA of Brucella spp., Trypanosoma spp., Coxiella burnetii, and Bartonella spp. PCR products were sequenced bidirectionally and phylogenetic analyses were performed. Eleven percent of camels tested positive for Brucella abortus (3%) and Trypanosoma evansi (8%). Coxiella burnetii and Bartonella spp. DNA was not detected. Our data demonstrate that camels from Iran contribute to the epidemiology of some zoonotic pathogens. Performing proper control strategies, such as vaccination of camels and humans in contact with them, test-and-slaughter policy, and education of the general population is necessary for minimizing the risk of zoonotic infection.