Antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae isolated from the rearing water of shrimp (Penaeus vannamei) postlarvae

Shrimp farming has experienced rising costs as a result of disease outbreaks associated with Vibrio spp. Suitable strategies for disease prevention and control are therefore urgently needed. This study aimed to evaluate the antimicrobial effect of Moringa oleifera seed powder against Vibrio cholerae in the rearing water of Pacific white shrimp (Penaeus vannamei) postlarvae. In vitro assays included the determination of minimum inhibitory concentration (MIC) of M. oleifera seed powder against V. cholerae, whereas in vivo assays included the effect of M. oleifera seed powder on bacterial load and water quality parameters in the rearing tanks, as well as its effect on shrimp postlarvae survival. M. oleifera seed powder inhibited the growth of V. cholerae with MIC values of 62·5 µg ml⁻¹. Moreover, seawater pH of treated tanks (8·66) was significantly lower (P < 0·01) than pH of the control tanks (9·02), whereas the visibility of treated tanks (37·08 cm) was significantly higher (P < 0·01) as compared to control tanks (35·37 cm). Likewise, V. cholerae load was significantly reduced (P < 0·01) from 4·7 × 10⁴ to 3·1 × 10³ CFU per ml in tanks treated with M. oleifera seed powder. Altogether, this study demonstrates the antimicrobial activity of M. oleifera against V. cholerae in shrimp culture.     

Functional group characterization of lactic bacterial biosurfactants and evaluation of antagonistic actions against clinical isolates of methicillin-resistant Staphylococcus aureus

The present study investigated the antimicrobial and antibiofilm potential of biosurfactants derived from Lactobacillus fermentum Lf1, L. fermentum LbS4 and Lactobacillus plantarum A5 against clinical isolates of methicillin-resistant Staphylococcus aureus (MRSA). The cell wall-bound and intracellular biosurfactants were extracted by solvent extraction method. Fourier-transform infrared spectroscopy-based characterization of biosurfactants revealed the heterogeneous chemical composition involving proteins, fatty acids and carbohydrate moieties in LbS4 and A5, while only the sugar and lipid fractions in Lf1. Fatty acid profiling using Gas chromatography-mass spectrometry indicated hexadecanoic acid and stearic acid as the predominant fatty acids in the biosurfactants of all these strains. Biosurfactants demonstrated dose-dependent antibacterial action against MRSA isolates with the highest inhibition zone diameter (30·0 ± 0·0 to 35·0 ± 0·0 mm) recorded at 400 mg ml⁻¹. Biosurfactants showed an excellent staphylococcal antibiofilm activity by preventing the biofilm formation and disrupting the preformed biofilms. Visual inspection through scanning electron microscopy witnessed the biosurfactants-induced alteration in the cell membrane integrity and subsequent membrane pore formation on staphylococcal cells. Taken together, our findings emphasize the prospects of biomedical applications of biosurfactants as bactericidal and biofilm controlling agents to confront staphylococcal nosocomial infections.     

Effect of plant secondary compounds on in vitro methane,ammonia production and ruminal protozoa population

Aims

The objective of this study was to evaluate the potential of secondary plant metabolites from 38 sources to serve as antimethanogenic additives in ruminant diets. The effect of leaf tannins from these different plant sources on rumen fermentation, protozoal populations and methanogenesis was also studied.

Methods and Results

Samples (200 mg dry matter, DM) were incubated without and with polyethylene glycol (PEG)‐6000 (400 mg DM) as a tannin binder during 24‐h incubation in the in vitro Hohenheim gas system. In the leaf samples, total phenol (g kg^?1 DM) was maximum in Pimenta officinalis (312) followed by Oenothera lamarckiana (185) and Lawsonia inermis (105). Of the 38 samples, condensed tannins exceeded 4·0 g kg^?1 in only Alpinia galanga (7·50), Cinnamomum verum (4·58), Pelargonium graveolens (18·7) and Pimenta officinalis (23·2) and were not detected in seven samples. When the bioactivity of the leaf samples was assessed using the tannin bioassay, the percentage increase in the amount of gas produced during incubation of samples with the tannin‐binding agent PEG‐6000 over the amount produced during incubation without the tannin binder ranged from nil (zero) to 367%, with the highest being recorded with A. galanga leaves. The ratio of methane reduction per ml of total gas reduction was maximum with Rauvolfia serpentina (131·8) leaves, followed by Indigofera tinctoria (16·8) and Withania somnifera (10·2) leaves. Total and differential protozoal counts increased with added PEG in twenty‐two samples, maximum being in Pimenta officinalis. Increased accumulation of total volatile fatty acids during incubation with added PEG‐6000 was recorded, and the values ranged from zero to 61%. However, the increase was significant in only 11 of the 38 tannin sources tested indicating noninterference of tannin on in vitro fermentation of carbohydrates by the majority of samples tested. Conversely, in 26 of 38 plant sources, the leaf tannins reduced N‐digestibility as evidenced by increased accumulation of NH₃‐N with added PEG.

Conclusions

Our study unequivocally demonstrated that plants containing secondary metabolites such as Rauvolfia serpentine, Indigofera tinctoria and Withania somnifera have great potential to suppress methanogenesis with minimal adverse effect of feedstuff fermentation.

Significance and Impact of the Study

It was established that methanogenesis was not essentially related to the density of protozoa population in vitro. The tannins contained in these plants could be of interest in the development of new additives in ruminant nutrition.     

Cytotoxicity in Vero cells and cytokines analyses in Balb/c mice as safety assessments of the probiotic mixture Saccharomyces cerevisiae RC016 and Lactobacillus rhamnosus RC007 for use as a feed additive

The objective was to carry out cytotoxicity assays in Vero cells and cytokines analyses in Balb/c mice as safety assessments to evaluate the probiotic mixture (M) Saccharomyces cerevisiae RC016 (Sc) and Lactobacillus rhamnosus RC007 (Lr) for use as feed additive. Vero cells (10⁴ cells per well) were exposed to Sc (2·08 × 10⁷, 2·08 × 10⁶; 2·08 × 10⁵ cells per ml), Lr (8·33 × 10⁷; 8·33 × 10⁶; 8·33 × 10⁵ cells per ml) and their M (1 : 1). Sc concentrations did not affect the Vero cells viability; in contrast, they were lower when exposed to Lr (P ˂ 0·0001). Vero cells showed increasing viability with M decreasing concentrations (91% viability with M2). Control BALB/c mice received only phosphate buffer saline and the others received the M. The IL-10, IL-6 and TNFα concentrations from intestinal fluid were analysed and no significant differences were observed among treatments. The same occurred with the ratio between IL-10/TNF-α. Beneficial effects of probiotics are associated with the regulation of the excessive inflammatory response; it is desirable they can modulate the cytokines production only under pathological conditions. Here, M administration to healthy mice did not induce negative side effects and expands the knowledge about beneficial effects of using probiotic microorganisms in mixture for feed additives development.     

Fungal pretreatment to enhance the yield of phytochemicals and evaluation of α-amylase and α-glucosidase inhibition using Cinnamomum zeylanicum (L.) quills pressurized water extracts

Bioactive compounds entrapped in plant materials can be effectively recovered using fungal enzymes. Cinnamomum zeylanicum Sri Wijaya (SW) and Sri Gemunu (SG) accessions and commercially available C. zeylanicum (CC) were subjected to fungal pretreatment and extracted with pressured water (PWE, 0·098 MPa). Thirteen fungal species were isolated and the substrate utilization ability of the species was tested using cellulose, pectin and lignin (indirectly). Total phenolic content (TPC, Folin–Ciocalteu method), proanthocyanidin content (PC, vanillin method) and α-amylase and α-glucosidase inhibitory potential of the extracts were evaluated. The anti-diabetic drug, Acarbose was used as the positive control. Trichoderma harzianum (MH298760) showed the highest cell lysis ability and hence was used for the microbial pretreatment process. Extracts of SW treated with T. harzianum species (Pre-SW) gave the highest percentage yield (4·08% ± 0·15%), significantly potent inhibition (P < 0·05) of α-amylase and α-glucosidase activities (IC₅₀ 57 ± 8 and 36 ± 8 μg ml⁻¹ respectively), TPC (2·24 ± 0·02 mg gallic acid equivalent g⁻¹), and PC (48·2 ± 0·4 mg of catechin equivalent g⁻¹) compared to Pre-SG, Pre-CC and nontreated samples. Trichoderma harzianum treatment can enhance the hypoglycaemic properties, PC and TPC of Cinnamon extracts and provide new insights into the recovery of phytochemicals.     

Incidence of Vibrio vulnificus in estuarine waters of the south Texas Coastal Bend region

Aims: To determine the occurrence of the human pathogen, Vibrio vulnificus, in south Texas coastal waters. Methods and Results: Coastal waters were sampled monthly between August 2006 and July 2007. Water temperature, dissolved oxygen, pH, salinity, conductivity and turbidity were measured during each sampling event. Culture‐based techniques utilizing Vibrio vulnificus agar (VVA) and membrane‐Enterococcus indoxyl‐β‐d ‐glucoside agar (mEI) were used to assess the occurrence and levels of V. vulnificus and the faecal contamination indicator group, enterococci, respectively. Vibrio vulnificus isolates were confirmed using colony‐blot hybridization with the species‐specific VVAP probe. Vibrio vulnificus was isolated at all sites throughout the year even when the water temperature dropped to 9·71°C. Significant correlations were found between concentrations of V. vulnificus and the abiotic factors, water temperature (P = 0·002) and dissolved oxygen (P = 0·028), as well as between concentrations of V. vulnificus and enterococci (P < 0·001). Conclusions: This study demonstrated the year‐round presence of V. vulnificus in coastal waters of south Texas. Significance and Impact of the Study: These findings indicate that the potential for human exposure to the pathogen, V. vulnificus, exists throughout the year. It also suggests that routinely monitored data might be used to predict the occurrence of the pathogen.     

Optimization of a solid culture medium based on Monochamus alternatus for Cordyceps militaris fruiting body formation

Monochamus alternatus (Coleoptera: Cerambycidae; M. alternatus), popularly known as the Japanese pine sawyer, is a vector of pinewood nematode (Bursaphelenchus xylophilus) that causes pine wilt disease. A solid medium culture with M. alternatus produced Cordyceps militaris fruiting bodies with the longest strips and the highest biological efficiency. Supplementing the original form of M. alternatus with oats resulted in slightly enhanced fruiting body production. The original form of M. alternatus showed higher production than its powder form. The solid culture medium was optimized using a response surface methodology, and the optimal medium contained the following: 8·5 g per bottle of M. alternatus and 11·5 g per bottle of oats mixed with 22·4 ml of water in a 300-ml cylindrical plastic bottle. The optimal culturing period for the fruiting body formation was 37·1 days. Under these conditions, a fruiting body dry weight of 38·0 g per bottle (actual value) was attained. The fruiting body produced using a solid culture medium based on M. alternatus had a cordycepin content of about 25 µg g⁻¹. The solid culture medium containing M. alternatus is highly efficient and eco-friendly, and its effectiveness in large-scale fruiting body production from C. militaris has been demonstrated.     

Kocuria SM1 controls vibriosis in rainbow trout (Oncorhynchus mykiss,Walbaum)

Aims: To develop probiotics for the control of vibriosis caused by Vibrio anguillarum and Vibrio ordalii in finfish. Methods and Results: Kocuria SM1, isolated from the digestive tract of rainbow trout, was administered orally to rainbow trout (Oncorhynchus mykiss) for 2 weeks at a dose equivalent to c. 10⁸ cells per g of feed and then challenged intraperitoneally with V. anguillarum and V. ordalii. Use of SM1 led to a reduction in mortalities to 15–20% compared to 74–80% mortalities in the controls. SM1 stimulated both cellular and humoral immune responses in rainbow trout, by elevation of leucocytes (5·5 ± 0·8 × 10⁶ ml^?1 from 3·7 ± 0·8 × 10⁶ ml^?1), erythrocytes (1·2 ± 0·1 × 10⁸ ml^?1 from 0·8 ± 0·1 × 10⁸ ml^?1), protein (23 ± 4·4 mg ml^?1 from 16 ± 1·3 mg ml^?1), globulin (15·7 ± 0·2 mg ml^?1 from 9·9 ± 0·1 mg ml^?1) and albumin (7·3 ± 0·2 mg ml^?1 from 6·1 ± 0·1 mg ml^?1) levels, upregulation of respiratory burst (0·05 ± 0·01 from 0·02 ± 0·01), complement (56 ± 7·2 units ml^?1 from 40 ± 8·0 units ml^?1), lysozyme (920 ± 128·8 units ml^?1 from 760 ± 115·3 units ml^?1) and bacterial killing activities. Conclusions: Kocuria SM1 successfully controlled vibriosis in rainbow trout, and the mode of action reflected stimulation of the host innate immune system. Significance and Impact of the Study: Probiotics can contribute a significant role in fish disease control strategies, and their use may replace some of the inhibitory chemicals currently used in fish farms.     

Inhibitory activity of tea polyphenol and Candida ernobii against Diplodia natalensis infections

Aims: To investigate the effect of tea polyphenol (TP) and Candida ernobii alone or in combination against postharvest disease (Diplodia natalensis) in citrus fruit and to evaluate the possible mechanisms involved. Methods and Results: TP at concentrations of 0·1%, 0·5% and 1·0% alone, or in combination with C. ernobii (1 × 10⁶ CFU ml^?1), showed a lower infection rate of stem‐end rot. TP at the concentration of 0·5% or above significantly inhibited the spore germination of D. natalensis. TP at the concentration of 1·0% showed inhibitary ability on mycelium growth of D. natalensis. The addition of TP did not affect the growth of C. ernobii in vitro and significantly increased the population of C. ernobii in vivo. Conclusions: TP exhibited an inhibitory effect against D. natalensis and improved the biocontrol efficacy of C. ernobii. It was direct because of the inhibitory effects of TP on spore germination and mycelial growth of D. natalensis in vitro and indirect because of the increased populations of C. ernobii in vivo. Significance and Impact of the Study: The results suggested that TP alone or in combination with biocontrol agents has great potential in commercial management of postharvest diseases in fruits.     

Association of Chlamydia trachomatis,Neisseria gonorrhoeae,Mycoplasma genitalium and Ureaplasma species infection and organism load with cervicitis in north Indian population

Cervicitis is predominantly caused by Neisseria gonorrhoeae and Chlamydia trachomatis, which accounts for almost half of all the cases of cervicitis. The role of newer organisms like Mycoplasma genitalium and Ureaplasma sp. and association of bacterial load with cervicitis are also not well established. So the study aimed to determine the relative frequency of these organisms and their load in association with cervicitis cases from north India. A case–control study involving 300 women was conducted using quantitative real-time PCR from endocervical swabs for identification of organisms and quantification of bacterial load. Among 150 cervicitis cases, C. trachomatis, N. gonorrhoeae, M. genitalium and Ureaplasma parvum were detected in 5 (3·3%), 10 (6·6%), 37(24·6%) and 47 (31·3%) respectively. Old age (<0·001, chi-squared test) and irregular menstrual cycles (<0·001, chi-squared test) were significantly associated with cervicitis. M genitalium was the only organism to be associated significantly with cervicitis with regard to age (<0·031) and symptoms like discharge (P < 0·033, chi-squared test) and dysuria (P < 0·044, chi-squared test) in multivariate analysis. Our finding suggests that the bacterial load of these organisms is not significantly associated with cervicitis. However, we found significant association of M. genitalium infection with clinical characteristics of cervicitis cases.     

Novel fluorine‐containing chiral hydrazide‐hydrazones: Design,synthesis, structural elucidation,antioxidant and anticholinesterase activity,and in silico studies

In this study, a series of fluorine‐containing chiral hydrazide‐hydrazone derivatives [III‐XII] from ?‐cysteine ethyl ester hydrochloride was synthesized as new antioxidant and anticholinesterase agents. The antioxidant activity of these derivatives was evaluated by ABTS^+· and DPPH^· scavenging and CUPRAC assays and the anticholinesterase activity by the Ellman method spectrophotometrically. The results of the antioxidant assay showed that compounds V , IX , and X exhibited higher activity than BHT and α‐tocopherol used as positive standards. Among the synthesized derivatives, compound IX (IC₅₀: 2.3 ± 1.6 μM) exhibited higher acetylcholinesterase inhibitory activity than galantamine (IC₅₀: 4.5 ± 0.8 μM). Compounds XI (IC₅₀: 9.6 ± 1.0 μM), IX (IC₅₀: 12.5 ± 1.6 μM), III (IC₅₀: 16.0 ± 1.6 μM), X (IC₅₀: 17.2 ± 1.8 μM), VI (IC₅₀: 20.2 ± 0.8 μM), XII (IC₅₀: 21.5 ± 1.0 μM), and VII (IC₅₀: 24.6 ± 0.6 μM) displayed better butyrylcholinesterase inhibitory activity than galantamine (IC₅₀: 46.03 ± 0.14 μM). ADME‐Tox analysis was used to probe the drug‐like properties of the compounds. Molecular docking studies were also applied to understand the interactions between compounds and targets. The docking calculations were supported by the experimental data. In particular, compound IX , having better activity than galantamine against acetylcholinesterase and butyrylcholinesterase enzymes, was visualized using molecular docking.     

Modulation of function in a minimalist heme-binding membrane protein

De novo designed heme-binding proteins have been used successfully to recapitulate features of natural hemoproteins. This approach has now been extended to membrane-soluble model proteins. Our group designed a functional hemoprotein, ME1, by engineering a bishistidine binding site into a natural membrane protein, glycophorin A (Cordova et al. in J Am Chem Soc 129:512–518, 2007). ME1 binds iron(III) protoporphyrin IX with submicromolar affinity, has a redox potential of −128 mV, and displays peroxidase activity. Here, we show the effect of aromatic residues in modulating the redox potential in the context of a membrane-soluble model system. We designed aromatic interactions with the heme through a single-point mutant, G25F, in which a phenylalanine is designed to dock against the porphyrin ring. This mutation results in roughly tenfold tighter binding to iron(III) protoporphyrin IX (K _d,app = 6.5 × 10⁻⁸ M), and lowers the redox potential of the cofactor to −172 mV. This work demonstrates that specific design features aimed at controlling the properties of bound cofactors can be introduced in a minimalist membrane hemoprotein model. The ability to modulate the redox potential of cofactors embedded in artificial membrane proteins is crucial for the design of electron transfer chains across membranes in functional photosynthetic devices.     

Control of postharvest decay on cherry tomatoes by marine yeast Rhodosporidium paludigenum and calcium chloride

Aims: In this study, the potential of calcium chloride (CaCl₂) application to improve the efficacy of the marine antagonist Rhodosporidium paludigenum in controlling postharvest diseases of cherry tomatoes was assessed. Methods and Results: CaCl₂ alone was found not to have any direct influence on the population growth of R. paludigenum in NYDB cultures or in cherry tomato wounds. However, the combined treatments with 1 × 10⁸ cells ml^?1R. paludigenum and CaCl₂ at the concentration from 0·5 to 2% showed high activities to reduce black rot caused by Alternaria alternata in cherry tomato wounds, significantly higher than those of R. paludigenum or CaCl₂ alone. Meanwhile, 0·5% CaCl₂ in combination with 1 × 10⁸ cells ml^?1R. paludigenum greatly inhibited the natural decay of cherry tomatoes in 21 days’ storage at 25°C. Conclusions: The combination of R. paludigenum and CaCl₂ enhances the inhibition of black rot and natural decay of postharvest cherry tomatoes. The results from this study provide a new way to improve the efficiency of R. paludigenum in maintaining the quality of postharvest fruits and vegetables. Significance and Impact of the Study: The marine yeast R. paludigenum combined with CaCl₂ has greatly potential use as an alternative to chemical fungicides in inhibiting postharvest decay on cherry tomatoes.     

Microbial communities in low permeability,high pH uranium mine tailings: characterization and potential effects

Aims

To describe the diversity and metabolic potential of microbial communities in uranium mine tailings characterized by high pH, high metal concentration and low permeability.

Methods and Results

To assess microbial diversity and their potential to influence the geochemistry of uranium mine tailings using aerobic and anaerobic culture‐based methods, in conjunction with next generation sequencing and clone library sequencing targeting two universal bacterial markers (the 16S rRNA and cpn60 genes). Growth assays revealed that 69% of the 59 distinct culturable isolates evaluated were multiple‐metal resistant, with 15% exhibiting dual‐metal hypertolerance. There was a moderately positive correlation coefficient (R = 0·43, P < 0·05) between multiple‐metal resistance of the isolates and their enzyme expression profile. Of the isolates tested, 17 reduced amorphous iron, 22 reduced molybdate and seven oxidized arsenite. Based on next generation sequencing, tailings depth was shown to influence bacterial community composition, with the difference in the microbial diversity of the upper (0–20 m) and middle (20–40 m) tailings zones being highly significant (P < 0·01) from the lower zone (40–60 m) and the difference in diversity of the upper and middle tailings zone being significant (P < 0·05). Phylotypes closely related to well‐known sulfate‐reducing and iron‐reducing bacteria were identified with low abundance, yet relatively high diversity.

Conclusions

The presence of a population of metabolically‐diverse, metal‐resistant micro‐organisms within the tailings environment, along with their demonstrated capacity for transforming metal elements, suggests that these organisms have the potential to influence the long‐term geochemistry of the tailings.

Significance and Impact of the study

This study is the first investigation of the diversity and functional potential of micro‐organisms present in low permeability, high pH uranium mine tailings.     

High redox potential laccases from the ligninolytic fungi Pycnoporus coccineus and Pycnoporus sanguineus suitable for white biotechnology: from gene cloning to enzyme characterization and applications

Aims: Exploitation of natural biodiversity in species Pycnoporus coccineus and Pycnoporus sanguineus to screen for a new generation of laccases with properties suitable for the lignin‐processing sector. Methods and Results: Thirty strains originating from subtropical and tropical environments, mainly isolated from fresh specimens collected in situ, were screened for laccase activity. On the basis of levels of enzyme activity and percentage of similarity between protein sequences, the laccases from strains BRFM 938, BRFM 66 and BRFM 902 were selected for purification and characterization. Each BRFM 938, BRFM 66 and BRFM 902 laccase gene encoded a predicted protein of 518 amino acids; the three deduced proteins showed 68·7–97·5% similarity with other Polyporale laccases. The three laccases (59·5–62·9 kDa with 7–10% carbohydrate content) had high redox potentials (0·72–0·75 V vs normal hydrogen electrode at pH 6), remained highly stable up to 75–78°C and at pH 5–7 mixtures, and were resistant to methyl and ethyl alcohols, acetonitrile and dimethylsulfoxide at concentrations as high as 50% (v/v). The best laccase‐1‐hydroxybenzotriazole systems permitted almost 100% of various polyphenolic dye decolourization and oxidation of adlerol and veratryl alcohol. Conclusions: The three laccases showed complementary biochemical features. BRFM 938 laccase had the highest thermo‐ and pH stability, catalytic efficiency towards 2,2′‐azino‐bis‐[3‐ethylthiazoline‐6‐sulfonate] and resistance to alcoholic solvents. BRFM 66 laccase had the highest rates of dye decolourization and oxidation of nonphenolic compounds. Significance and Impact of the Study: This study identified P. coccineus and P. sanguineus as outstanding producers of high redox potential laccases, easy to purify and scale‐up for industrial production. Three new laccases proved to be suitable models for white biotechnology processes and for further molecular breeding to create a new generation of tailor‐made enzymes.     

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.     

Evaluation of the probiotic and functional potential of Lactobacillus agilis 32 isolated from pig manure

Escherichia coli is a symbiotic bacterium in humans and animals and an important pathogen of humans and animals. Prevention and suppression of E. coli infection is of great concern. In this study, we isolated a strain of Lactobacillus agilis 32 from pig manure and evaluated its biological characteristics, and found that its bacterial survival rate was 25% after 4 h of treatment at pH 2, and under the condition of 0·5% bile concentration, its survival rate exceeds 30%. In addition, L. agilis 32 has a cell surface hydrophobicity of 77·8%, and exhibits 67·1% auto-aggregation and 63·2% aggregation with Enterotoxigenic E. coli 10 (ETEC 10). FITC fluorescence labelling showed that the fluorescence intensity of cecum was significantly higher than that of duodenum, jejunum or colon (P < 0·05), but no significant difference from ileum. Lactobacillus agilis 32 bacterial culture and CFS showed average inhibition zone diameters of 14·2 and 15·4 mm respectively. Lactobacillus agilis 32 CFS treatment can significantly reduce the pathogenicity of ETEC 10. These results show that L. agilis 32 is an active and potential probiotic, and it has a good antibacterial effect on ETEC10, which provides basic research for probiotics to prevent and treat intestinal diarrhoea pathogen infection.     

The protective effect of Bdellovibrio‐and‐like organisms (BALO) on tilapia fish fillets against Salmonella enterica ssp. enterica serovar Typhimurium

Aims: To characterize freshwater Bdellovibrio‐and‐like organisms (BALO) isolated in China and examine their potential in controlling growth of Salmonella enterica ssp. enterica serovar Typhimurium on tilapia fillets. Methods and Results: Four BALO isolates were recovered from a pond in Yanzhou of Shandong province, China, with Salm. Typhimurium as prey using double‐layer agar method. Partial 16S rDNA sequencing analysis identified BD2GL, BD5GL and BDXGL as Bdellovibrio bacteriovorus and BD2GS as a Peredibacter sp. Lysis experiments on 32 potentially pathogenic strains revealed that BALO lysis rates are in the range of 56·3–65·6%. On the five Salmonella strains tested, only BD2GS achieved 100% lysis rate. When applied on tilapia fillets against Salm. Typhimurium, BD2GS showed its growth control potential. Cell increments of Salm. Typhimurium were significantly lower (P < 0·05) in two BD2GS‐treated groups compared to control and low‐dose group (BD2GS to prey ratio, 1 : 1) was more effective than high‐dose group (BD2GS to prey ratio, 10 : 1) in controlling Salm. Typhimurium growth. Conclusions: Results of this study indicated that BD2GS could control Salm. Typhimurium growth on tilapia fillets. Significance and Impact of the Study: BALO could be used as a live protective culture in controlling bacterial growth and ensure food safety.     

Bactericidal and antioxidant effects of essential oils from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus

The extraction and characterization of the essential oils (EO) from Satureja montana L., Myristica fragrans H. and Cymbopogon flexuosus and the determination of their antibacterial and antioxidant activities were achieved. The EO were identified by gas chromatography/mass spectrometry and quantified by gas chromatography using a flame ionization detector. The antibacterial potential against Escherichia coli and Staphylococcus aureus was evaluated by cell susceptibility assays and by scanning electron microscopy. The antioxidant activity was evaluated by the 2,2-diphenyl-1-picrylhydrazyl assay, by β-carotene bleaching and by determining the reducing power. Borneol (36·18%), γ-terpineol (12·66%) and carvacrol (11·07%) were the principal components in the EO from S. montana, and sabinene (49·23%) and α-pinene (13·81%) were found in the EO from M. fragrans. Geranial (59·66%) and neral (38·98%) isomers were the only major components in the EO from C. flexuosus. The EO from S. montana was effective against E. coli, with minimum inhibitory and bactericidal concentrations (MIC and MBC) of 6·25 µl ml⁻¹, whereas bactericidal potential against both was observed for the EO from M. fragrans; MIC = 6·25 µl ml⁻¹ for S. aureus and MBC = 12·5 µl ml⁻¹ for E. coli. A significant protective role on lipid substrates in the β-carotene bleaching assay was seen for the EO from S. montana and M. fragrans. Overall, such EO can be promising agents against pathogenic bacteria and for protecting biomolecules during oxidative stress.