Molecular typing, serotyping and cytotoxicity testing of Campylobacter jejuni strains isolated from commercial broilers in Puerto Rico

Aims: Thirty Campylobacter jejuni strains isolated from fecal samples (n = 94; 32%) from 13 positive farms (n = 17; 76%) from commercial broiler chickens in Puerto Rico were analysed by molecular methods. Methods and Results: Isolates were identified with multiplex polymerase chain reaction assays, tested for their antimicrobial susceptibility and characterized with pulsed‐field gel electrophoresis (PFGE), multilocus sequence typing (MLST), serotyping and bacterial cytotoxicity in mammalian cells. Isolates exhibited high resistance to vancomycin (minimum inhibitory concentration, MIC of >256 μg ml^?1) and trimethoprim (MIC of >32 μg ml^?1); few were resistant to clindamycin (MIC₉₀ 4 μg ml^?1), erythromycin (MIC₉₀ 8 μg ml^?1) and tetracycline (MIC₉₀ 8 μg ml^?1); but none was resistant to azithromycin (MIC₉₀ 4 μg ml^?1), ciprofloxacin (MIC₉₀ 1 μg ml^?1) or gentamycin (MIC₉₀ 4 μg ml^?1). Most strains restricted with SmaI, but a combination of SmaI–KpnI digestion was more discriminatory. MLST analysis yielded four sequence types (ST), and ST‐2624 was the predominant one. Phylogenetic analysis revealed a high degree of recombination for glnA and pgm genes. The predominant serotypes were O:3 and O:5. Most strains had lowest cytotoxicity potential with Caco‐2 cells, medium cytotoxicity with INT‐407 and Hep‐2 cells and high cytotoxicity with CHO cells. Conclusion: A low degree of antimicrobial resistance, 13 PFGE profiles, 4 ST and a large variability in cytotoxicity assays were found for these strains. Significance and Impact of the Study: This is the first characterization of C. jejuni strains isolated from broilers in Puerto Rico. The genetic diversity of these strains suggests that several techniques are needed for strain characterization.     

The effect of simulated gastric environments on the anti-Helicobacter activity of garlic oil

Aims: To investigate the effects of simulated gastric conditions upon the anti-Helicobacter pylori effects of garlic oil (GO). Methods and Results: Time course viability experiments assessed the anti-H. pylori activity of GO (16 and 32 μg ml⁻¹) in simulated gastric environments. Rapid anti-H. pylori action of GO was observed in artificial gastric juice. Mucus (1–5%) was strongly protective of H. pylori both alone and in the presence of GO, but its protective effect was antagonized by GO. Peptone (5–15 g l⁻¹) caused a dose-dependent reduction in the anti-H. pylori activity of GO. Rapeseed oil (5·7–17 g l⁻¹) greatly diminished the anti-H. pylori activity of GO. Dextrin (44 and 133 g l⁻¹) exhibited direct anti-H. pylori effects and added to those of GO. Simulated meal mixtures decreased but did not eliminate the anti-H. pylori activity of 32 μg ml⁻¹ GO. Conclusions: The anti-H. pylori activity of GO was noticeably affected by food materials and mucin. However, substantial activity remained under simulated gastric conditions. Further investigation of the therapeutic potential of GO against H. pylori is therefore warranted. Significance and Impact of the Study: Garlic oil may be useful as an alternative treatment against H. pylori, a major cause of gastrointestinal infections in humans.     

In vitro assays on the susceptibility of four species of nematophagous fungi to anthelmintics and chemical fungicides/antifungal drug

Using nematophagous fungi for the biological control of animal parasitic nematodes will become one of the most promising strategies in the search for alternative chemical drugs. The purpose of this study was to check the in vitro activity of four anthelmintics, four chemical fungicides and two antifungal drugs on the spore germination of nematophagous fungi: Duddingtonia flagrans (SF170), Arthrobotrys oligospora (447), Arthrobotrys superba (435) and Arthrobotrys sp. (PS011). A modified 24-well cell culture plate assay was conducted to evaluate the susceptibility of nematophagous fungi against drugs tested by calculating the effective middle concentrations (EC₅₀) of each tested drug to inhibit the germination of fungal spores. EC₅₀ ranged between 0·7 and 47·2 μg ml⁻¹ for fenbendazole, thiabendazole and ivermectin, except levamisole (546·5–4057·8 μg ml⁻¹). EC₅₀ of tested fungicides was 0·6–2·3 μg ml⁻¹ for carbendazim, 55·9–247·4 μg ml⁻¹ for metalaxyl, 24·4–45·2 μg ml⁻¹ for difenoconazole, and 555·9–1438·3 μg ml⁻¹ for pentachloronitrobenzene (PCNB). EC₅₀ of two antifungal drugs was 0·03–3·4 μg ml⁻¹ for amphotericin B and 0·3–10·9 μg ml⁻¹ for ketoconazole. The results showed that 10 tested drugs, except for levamisole and PCNB, had in vitro inhibitory effects on nematophagous fungi. The chlamydospores of D. flagrans had the highest sensitivity to nine tested drugs, except for ketoconazole.     

Prevalence of Prototheca spp. on dairy farms in Poland – a cross-country study

The Prototheca algae have recently emerged as an important cause of bovine mastitis globally. Here, we present results of a first large-scale, cross-country survey on the prevalence of Prototheca spp. in dairy cows, and their environment in Poland. A total of 1211 samples were collected and microbiologically analysed. Included within this number were milk (n = 638), body swabs (n = 374) and environmental samples (n = 199), originating from 400 dairy cows and their surroundings, on 16 dairy farms, based in all major provinces of the country. Prototheca spp. were the third, after Streptococcus and Staphylococcus spp., most common mastitis pathogens. The overall prevalence of protothecal mastitis was 8.3% (33/400), with the majority (75.8%) of cases having a subclinical course, and all but one attributable to P. zopfii genotype 2. Prototheca spp. were cultured from body swabs of both healthy and mastitic cows, yet the isolation rate among the latter was conspicuously lower (12.3% vs. 17.8%). Forty-two (21.2%) environmental samples yielded growth of Prototheca spp. However, no clear association between Prototheca mastitis in dairy cows and the algal isolation from the herd environment was found. Nor was there any association between the environmental recovery of the algae and farm management practices.     

In vitro and ex vivo antimycobacterial potential of azole drugs against Mycobacterium tuberculosis H37Rv

The antimycobacterial activity of two clinically approved antifungal azole drugs, clotrimazole and econazole, was evaluated against Mycobacterium tuberculosis H₃₇Rv under in vitro and ex vivo conditions. The minimum inhibitory concentration (MIC₉₀) was 0.120 μg ml⁻¹, whereas the minimum bactericidal concentration and effective concentration was 0.125 μg ml⁻¹ for both the drugs demonstrating their potent antimycobacterial activity. Further, the azole drugs exhibited a synergistic activity with either rifampicin or isoniazid as evaluated on the basis of reduction of colony forming units. The results suggest that azole compounds bear the potential to enhance the efficacy of currently prescribed antitubercular drugs.     

Inhibition of methicillin-resistant Staphylococcus aureus (MRSA) biofilm by cationic poly (D,L-lactide-co-glycolide) nanoparticles

Abstract

The emergent need for new treatment methods for multi-drug resistant pathogens such as methicillin-resistant Staphylococcus aureus (MRSA) has focused attention on novel potential tools like nanoparticles (NPs). In the present study, a drug-free cationic nanoparticles (CNPs) system was developed and its anti-MRSA effects were firstly investigated. The results showed that CNPs (261.7?nm, 26.1?mv) showed time- and concentration-dependent activity against MRSA growth, killing ～ 90% of planktonic bacterial cells in 3?h at 400?μg ml^?1, and completely inhibiting biofilm formation at 1000?μg ml^?1. Moreover, CNPs at 400?μg ml^?1 reduced the minimum inhibitory concentration (MIC) of vancomycin on inhibition of planktonic MRSA growth (～ 25%) and biofilm formation (～ 50%). The CNPs–bacteria interaction force was up to 22 nN. Overall, these data suggest that CNPs have a good potential in clinical applications for the prevention and treatment of MRSA infection.     

Spatiotemporal dynamics of the total and active Vibrio spp. populations throughout the Changjiang estuary in China

Vibrio is ubiquitously distributed in marine environments and is the most extensively characterized group within Gammaproteobacteria. Studies have investigated Vibrio spp. worldwide, but mostly focused on pathogenic vibrios and based on cultivation methods. Here, using a combination of molecular and culturing methods, we investigated the dynamics of the total and active Vibrio spp. throughout the Changjiang estuary in China. The total Vibrio abundance was higher in summer (~6.59 × 10³ copies ml⁻¹) than in winter (~1.85 × 10³ copies ml⁻¹) and increased from freshwater to saltwater (e.g. 8.04 × 10¹ to 9.39 × 10³ copies ml⁻¹ in summer). The ratio of active to total Vibrio (Va/Vt) revealed a high activity of vibrios, with remarkable differences between freshwater and saltwater (p < 0.05). Based on the community compositions of the culturable, total and active Vibrio, Vibrio atlanticus and Vibrio owensii were the dominant and active species in winter and summer, respectively. The distribution of Vibrio was governed by the effects of diverse environmental factors, such as temperature, salinity, pH, dissolved oxygen and SiO₃²⁻. Our study clearly demonstrates the spatiotemporal dynamics of total and active Vibrio spp. and lays a foundation for fully understanding the ecological roles of marine Vibrio.     

Surfactin inhibits the growth of Propionibacterium acnes by destroying the cell wall and membrane

Propionibacterium acnes plays a major role in acne vulgaris. In the pre-experiment, the growth of P. acnes was inhibited effectively using surfactin; however, the antibacterial mechanism has not been described. Therefore, the aim of this study was to evaluate antibacterial activity and analyse the mechanism of surfactin against P. acnes. Minimum inhibitory concentration, time-killing kinetics and scanning electron microscopy were used to evaluate the activity of surfactin against P. acnes, which showed that 128 μg ml⁻¹ effectively inhibited growth. Cell wall permeability was evaluated by detecting the extracellular alkaline phosphatase activity, which increased to 1·83- and 2·32-fold after incubating with 128 and 256 μg ml⁻¹ of surfactin for 10 h, respectively. Propidium iodide fluorescence, leakage of nucleic acid, protein, K⁺, and Ca²⁺, membrane potential and the leakage of calcein from small unilamellar vesicles all increased after incubation with surfactin, indicating that its strong biological activities act mainly by altering membrane integrity. In a mouse model of acne, surfactin significantly reduced P. acnes–induced epidermal swelling and erythema. These results indicate that surfactin effectively inhibited the growth of P. acnes by destroying the cell wall and membrane, and is a potential candidate for acne treatment.     

Fluorescence detection of carbofuran in aqueous extracts based on dual-emission SiO2@Y2O3:(Eu3+,Tb3+)@MIP core-shell structural nanoparticles

A novel double-windows fluorescence sensor for carbofuran (CF) detection was successfully developed based on rare-earth Eu,Tb-doped Y₂O₃@SiO₂-based molecularly imprinted nanoparticles (MINs) with a multilayer core-shell structure. The recognition process of the MINs for CF was fairly fast and needed only ~8 min to reach a dynamic equilibrium. Interestingly, one fluorescence attenuation window was found with an increase in CF concentration (Q) from 0.1 to 10 μg ml⁻¹ and with a limit of detection (LOD) of 0.04 μg ml⁻¹ at 544 nm belonging to the Tb³⁺ emission, as well as another fluorescence enhanced window within the CF concentration range 10–100 μg ml⁻¹ (LOD = 4 μg ml⁻¹) at 617 nm of Eu³⁺ emission in the dispersed rare-earth-doped MIN colloidal aqueous solution. Luminescence resonance energy transfer from CF to Eu³⁺ and an inner filter effect of CF towards Tb³⁺, as well from the two independent detection windows were clearly observed simultaneously. The competition experiment displayed hardly any marked interference during detection of CF following addition of its analogues (carbaryl, isoprocarb, aldicarb, methomyl, and etofenprox). Moreover, the MINs could also be applied to accurately detect CF in rhubarb and wolfberry samples with recoveries of 85.7–92.2%. This sensing system has high specific recognition and a wide detection range for CF and provides new opportunities for pesticide detection.     

Enhanced systemic resistance to bacterial speck disease caused by Pseudomonas syringae pv. tomato by dl-β-aminobutyric acid under salt stress

Plants have evolved different but interconnected strategies to defend themselves against microbial pathogens and stress conditions. The defense responses of tomato (Lycopersicon esculentum Mill) seedlings treated with dl ‐β‐aminobutyric acid (BABA) were investigated with and without abiotic stress (100 mM NaCl) against bacterial speck disease caused by Pseudomonas syringae pv. tomato. The plants were sprayed with 50, 125, 250 or 500 μg ml⁻¹ BABA and were inoculated with 10⁸ colony‐forming units ml⁻¹ bacterial suspension 1 day after treatment. Abiotic stress led to an increase in plant resistance. When BABA was additionally applied as a foliar spray at 125 μg ml⁻¹, the effect on plants was almost identical to that on plants that were sprayed with BABA at 500 μg ml⁻¹ alone. The bacterial multiplication in the plants was 250‐fold lower than in the water‐treated (control) plants and in plants that were sprayed with 500 μg ml⁻¹ BABA alone within 48 h postinoculation (hpi). Physiological studies were carried out in the plants treated with BABA in order to investigate the reason for this synergistic effect. Abiotic stress with BABA spray resulted in high H₂O₂ generation and guaiacol peroxidase activity in the plants. The activity of the enzymatic antioxidative protective system of the plants, superoxide dismutase, ascorbate peroxidase and catalase (CAT), also showed a significant delayed increase in BABA‐treated plants under abiotic stress conditions. These increases in enzyme activity coincided with the initiation of the most suppressive effect of BABA on bacterial growth by 24 hpi, which were significantly higher than the control. Salt stress alone did not lead to any significant increase in CAT activity, but salt stress with BABA did. These findings indicate a synergistic effect between salt stress and BABA at low concentrations, resulting in induced plant resistance. Furthermore, a stress regulation effect of BABA under abiotic stress can be associated with plant resistance.     

Activity and mechanism of the action of zeylasterone against Bacillus subtilis

Aims:  To investigate the antimicrobial properties of 6-oxophenolic triterpenoids isolated from Maytenus blepharodes against different micro-organisms and the mode of action on Bacillus subtilis. Methods and Results:  The activity of zeylasterone and demethylzeylasterone was evaluated by microdilution method. Zeylasterone showed a higher activity, being active against Gram-positive bacteria (minimum inhibitory concentration 3–20 μg ml⁻¹) and Candida albicans (10 μg ml⁻¹). Killing curves revealed a bacteriostatic effect on B. subtilis that was dependent on the growth phase and inoculum size. Zeylasterone caused cell membrane alterations in B. subtilis, as shown by potassium leakage and formation of mesosome-like structures. However, membrane disruption was not revealed by either LIVE/DEAD Baclight assay or measurement of intracellular constituent efflux. Zeylasterone showed an early effect on N-acetyl-glucosamine and uridine incorporation and later on that of thymidine and leucine. Conclusions:  Diverse micro-organisms exhibit sensitivities towards compounds studied. The permeability changes in the cytoplasmic membrane and nonsimultaneous ceasing of macromolecular synthesis suggest that zeylasterone could act on multiple targets on B. subtilis. Significance and Impact of the Study:  The activity showed against B. subtilis as a model of spore-forming bacteria would provide valuable information for further studies in the development of 6-oxophenolic triterpenoids as antiseptic and disinfectant properties.     

ALKALINE PHOSPHATASE ACTIVITIES AMONG POPULATIONS OF THE COLONY-FORMING DIAZOTROPHIC CYANOBACTERIUM TRICHODESMIUM SPP. (CYANOBACTERIA) IN THE RED SEA

Alkaline phosphatase activities of the diazotrophic marine cyanobacterium Trichodesmium were studied among natural populations in the northern Red Sea and in laboratory cultures of Trichodesmium sp. strain WH9601. Open-water tuft-shaped colonies of Trichodesmium showed high alkaline phosphatase activities with 2.4–11.7 μmol p-nitrophenylphosphate (PNPP) hydrolyzed·μg chl a ^{− 1}·h ^{− 1}, irrespective of date or origin of the sample. Coastal populations of the Trichodesmium tuft colonies had low alkaline phosphatase activities with 0.2–0.5 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. An exception was the Trichodesmium fall maximum, when both tuft colonies and the plankton community (<100 μm) had alkaline phosphatase activities of 0.6–7.4 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. Likewise, the more rare puff and bow-tie colonies of Trichodesmium spp. in coastal waters had elevated alkaline phosphatase activities (0.8–1.6 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}) as compared with tuft colonies coinhabiting the same waters. Intact filaments of tuft-forming Trichodesmium sp. strain WH9601 from phosphate-replete cultures had a base alkaline phosphatase activity of 0.5 μmol PNPP·μg chl a ^{− 1}·h ^{− 1}. This activity underwent a 10-fold increase in phosphate-deplete cultures and in cultures supplied with glycerophosphate as the sole P source. The elevated level of alkaline phosphatase activity was sustained in P-deplete cultures, but it declined in cultures with glycerophosphate. The decline is suggested to result from feedback repression of alkaline phosphatase synthesis by the phosphate generated in the glycerophosphate hydrolysis. The enhanced alkaline phosphatase activities of Trichodesmium spp. populations provide evidence that P stress is an important factor in the ecology of Trichodesmium in the northern Red Sea.     

Fouling deterrent chemical defence in three muricid gastropod egg masses from the Southeast coast of India

Abstract

The egg masses of the marine muricid gastropod molluscs Chicoreus virgineus, Chicoreus ramosus and Rapana rapiformis were studied for antifouling activities. The minimum inhibitory concentrations of crude extracts for the inhibition of byssal production and attachment of the brown mussel Perna indica were 650 μg ml^?1, 1150 μg ml^?1 and 925 μg ml^?1 from the three muricid gastropods, respectively. Higher LC₅₀ values than EC₅₀ values and 100% recovery of the mussels in the toxicity assay indicated the non-toxic nature of the extracts. The gradient partitioning of the egg mass extracts and subsequent antimicrofouling screening against 40 biofilm bacteria showed wide-spectrum antibacterial activity of the medium polar fraction from C. virgineus; the non-polar fraction from R. rapiformis and both non-polar and medium polar fractions from C. ramosus. The antimicrofouling activity from extracts of the three egg masses was found to be more prominent than antimacrofouling activity. This may be attributed to the targeting of a defence strategy against microbes in order to protect the developing mollusc embryos.     

Evaluation of the antifouling properties of 3-alyklpyridine compounds

One of the most promising alternative technologies to antifouling (AF) biocides based on toxic heavy metals lies in the development of natural eco-friendly biocides. The present study evaluates the AF potential of structurally different compounds containing a 3-alkylpyridine moiety. The products, namely poly 3-alkylpyridinium salts, saraine, and haminols, were either extracted or derived from natural sources (the sponges Haliclona sp. and Reniera sarai and the mollusc Haminoea fusari), or obtained by chemical synthesis. All the molecules tested showed generally good anti-settlement activity against larvae of the barnacle Amphibalanus (=Balanus) amphitrite (EC₅₀ values between 0.19 and 3.61?μg?ml^?1) and low toxicity (LC₅₀ values ranging from 2.04 to over 100?μg?ml^?1) with non-target organisms. For the first time, the AF potential of a synthetic monomeric 3-alkylpyridine was demonstrated, suggesting that chemical synthesis is as a realistic way to produce large amounts of these compounds for future research and development of environmentally-friendly AF biocides.     

Enantioselective analysis of ibuprofen enantiomers in mice plasma and tissues by high‐performance liquid chromatography with fluorescence detection: Application to a pharmacokinetic study

A direct fluorometric high‐performance liquid chromatography (HPLC) method was developed and validated for the analysis of ibuprofen enantiomers in mouse plasma (100 μl) and tissues (brain, liver, kidneys) using liquid–liquid extraction and 4‐tertbutylphenoxyacetic acid as an internal standard. Separation of enantiomers was accomplished in a Chiracel OJ‐H chiral column based on cellulose tris(4‐methylbenzoate) coated on 5 μm silica‐gel, 250 x 4.6 mm at 22 °C with a mobile phase composed of n‐hexane, 2‐propanol, and trifluoroacetic acid that were delivered in gradient elution at a flow rate of 1 ml min⁻¹. A fluorometric detector was set at: λ_excit. = 220 nm and λ_emis. = 290 nm. Method validation included the evaluation of the selectivity, linearity, lower limit of quantification (LLOQ), within‐run and between‐run precision and accuracy. The LLOQ for the two enantiomers was 0.125 μg ml⁻¹ in plasma, 0.09 μg g⁻¹ in brain, and 0.25 μg g⁻¹ in for liver and kidney homogenates. The calibration curves showed good linearity in the ranges of each enantiomers: from 0.125 to 35 μg ml⁻¹ for plasma, 0.09–1.44 μg g⁻¹ for brain, and 0.25–20 μg g⁻¹ for liver and kidney homogenates. The method was successfully applied to a pharmacokinetic study of ibuprofen enantiomers in mice treated i.v. with 10 mg kg⁻¹ of racemate.     

Anaerobic Bacterial Flora of Intra-abdominal Infections and their Antimicrobial Susceptibility Pattern in Kuwait

Clinical samples obtained from 200 patients with intra-abdominal infections were investigated for the presence of anaerobic bacteria. The majority of samples were from patients with appendicitis (108, 54%) followed by peritoneal abscess/peritonitis (37, 18.5%). A total of 153 anaerobes were isolated from 83 culture positive specimens with an isolation rate of 1.8 per sample. Ninety (59%) yielded Bacteroides fragilis group and B. fragilis stricto sensu accounted for half of them. Other isolates were 36 (23.5%) Prevotella species and 15 (9.8%)Peptostreptococcus micros . The susceptibility of the 153 isolates against eight antibiotics was determined by the E-test. All the isolates were susceptible to metronidazole, MIC₉₀s varying between 1–2 μg/mL. ThePrevotella spp., Peptostreptococcus spp., Fusobacterium spp. and Porphyromonas spp. were all susceptible to clindamycin (MIC₉₀s=0.25–2 μg/mL respectively), imipenem (MIC₉₀s=0.12–0.5μg/mL respectively) and meropenem (MIC₉₀=0.25 μg/mL each). About 25% of the B. fragilis group were resistant to clindamycin with MIC more than 256 μg/mL. Piperacillin-tazobactam also exhibited excellent in vitro activity against all the isolates (MIC₉₀=0.25 μg/mL).     

Bioprocess strategies for enhancing biomolecules productivity in Chlorella fusca LEB 111 using CO2 a carbon source

The aim of this study was to evaluate the influence of different carbon dioxide (CO₂) concentrations on the distribution of carbon forms in the culture medium and the biomass production and biomolecules productivity of the strain Chlorella fusca LEB 111. In this study, experiments were carried out in which C. fusca cultures were exposed to different CO₂ concentrations, 0.03% (0.08 ml_CO2 ml_medium⁻¹ days⁻¹), 5% (0.18 ml_CO2 ml_medium⁻¹ days⁻¹), and 15% vol/vol CO₂ (0.54 ml_CO2 ml_medium⁻¹ days⁻¹). Among the carbon chemical species distributions in the culture medium, bicarbonate was predominant (94.2–98.9%), with the highest quantitative percentage in the experiment receiving a 15% CO₂ injection. C. fusca LEB 111 cultivated with 15% CO₂ showed the highest biomass productivity (194.3 mg L⁻¹ days⁻¹) and CO₂ fixation rate (390.9 mg L⁻¹ days⁻¹). The carbohydrate productivity in the culture that received 15% CO₂ was 46.2% higher than the value verified for the culture with the addition of CO₂ from the air (0.03% CO₂). In addition, CO₂ concentration providing increases of 0.03–15% to C. fusca cultures resulted in a 31.6% increase in the lipid productivity. These results showed that C. fusca can be used for CO₂ bioconversion and for producing biomass with potential applications for biofuels and bioproducts.     

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro anti‐enterovirus 71 activity of gallic acid from Woodfordia fruticosa flowers

Aims: The anti‐enterovirus 71 (EV71) activity of six Nepalese plants’ extracts and gallic acid (GA) isolated from Woodfordia fruticosa Kurz (family; Lythaceae) flowers were evaluated in Vero cells. Methods and Results: The anti‐EV71 activity of tested compounds was evaluated by a cytopathic effect reduction method. Our results demonstrated that flowers’ extracts of W. fruticosa exerted strong anti‐EV71 activity, with a 50% inhibitory concentration (IC₅₀) of 1·2 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived therapeutic index (TI) was more than 83·33. Rivabirin showed no antiviral activity against EV71. Furthermore, GA isolated from W. fruticosa flowers exhibited a higher anti‐EV71 activity than the extract of W. fruticosa flowers, with an IC₅₀ of 0·76 μg ml^?1 and no cytotoxicity at a concentration of 100 μg ml^?1, and the derived TI was 99·57. Conclusions: This study demonstrated that flower extracts of W. fruticosa possessed anti‐EV71 activity and GA isolated from these flowers showed stronger anti‐EV71 activity than that the extracts. Significance and Impact of the Study: Our results suggest that the GA from W. fruticosa flowers may be used as a potential antiviral agent.