Inactivation of Cronobacter spp. (Enterobacter sakazakii) in infant formula using lactic acid,copper sulfate and monolaurin

Aims: To investigate the effect of lactic acid (LA), copper (II), and monolaurin as natural antimicrobials against Cronobacter in infant formula. Methods and Results: The effect of LA (0·1, 0·2 and 0·3% v/v), copper (II) (10, 50 and 100 μg ml^?1) and monolaurin (1000, 2000, and 3000 μg ml^?1) suspended into tween‐80? or dissolved in ethanol against Cronobacter in infant formula was investigated. Reconstituted infant formula and powdered infant formula were inoculated with five strains of Cronobacter spp. at the levels of c. 1 × 10⁶ CFU ml^?1 and 1 × 10³ CFU g^?1, respectively. LA at 0·2% v/v had a bacteriostatic effect on Cronobacter growth, whereas 0·3% v/v LA resulted in c. 3 log₁₀ reduction. Copper (II) at the levels of 50 μg ml^?1 and 100 μg ml^?1 elicited c. 1 and 2 log₁₀ reductions, respectively. The combination of 0·2% LA and 50 μg ml^?1 copper (II) resulted in a complete elimination of the organism. Monolaurin exhibited a slight inhibitory activity against Cronobacter (c. 1·5 log₁₀ difference) compared to the control when ethanol was used to deliver monolaurin. Conclusions: A complete elimination of Cronobacter was obtained when a combination of sublethal concentrations of LA (0·2%) and copper (II) (50 μg ml^?1) was used. Significance and Impact of the Study: The use of the synergistic interactive combination of LA and copper (II) could be beneficial to control Cronobacter in the infant formula industry.     

Postharvest biological control of Penicillium digitatum decay on citrus fruit by Bacillus pumilus

A new antagonist/pathogen combination is reported, with Bacillus pumilus showing strong antagonistic activity against Penicillium digitatum. B. pumilus (1.6 × 10¹⁰ to 1.6 × 10¹²*cfu ml^-1) gave significant control of P. digitatum infection in Valencia orange, which was as effective as imazalil (500 μg ml^-1) and was significantly better than benomyl treatment (500 μg ml^-1). When lower concentrations of B. pumilus (1.9 × 10⁷ to 1.9 × 10⁹ cfu ml^-1) were tested on Washington Navel orange and Lisbon lemon fruit, the antagonist caused significant control of P. digitatum infection at two inoculum levels (6.5 × 10⁴ and 6.5 × 10⁵ spores ml^-5). Concentrations of both the pathogen and the antagonist affected the biocontrol effect.     

Content of cysteine, reduced and oxidized glutathione in spermatozoa of representatives of Acipenseriformes (Acipenser baerii and A. ruthenus) as well as teleosts (Perca fluviatilis and Sander lucioperca)

Glutathione belongs to a vital intra‐ and extra‐cellular protective antioxidant and is found almost exclusively in its reduced form. The ratio between its reduced and oxidized within cells is often used as a marker of cellular toxicity. The objectives of the study were to (i) determine both the reduced (GSH) and oxidized glutathione (GSSG) and cysteine (Cys) in the sperm of the Acipenser baerii and Acipenser ruthenus, as well as in Perca fluviatilis and Sander lucioperca, and (ii) to demonstrate the differences in concentration levels between representatives of acipenseriform and teleost species. High performance liquid chromatography with electrochemical detection was employed. The average content of the thiols determined in the sperm samples were as follows: Acipenser baerii (cysteine 55 ± 8 μg ml^?1; GSH 126 ± 19 μg ml^?1; GSSG 49 ± 7 μg ml^?1), Acipenser ruthenus (cysteine 62 ± 9 μg ml^?1; GSH 768 ± 115 μg ml^?1; GSSG 180 ± 16 μg ml^?1), Sander lucioperca (cysteine 251 ± 38 μg ml^?1; GSH 185 ± 28 μg ml^?1; GSSG 93 ± 14 μg ml^?1), Perca fluviatilis (cysteine 281 ± 42 μg ml^?1; GSH 496 ± 74 μg ml^?1; GSSG 138 ± 21 μg ml^?1). Based on the results obtained it can be concluded that this method is sensitive and selective for the determination of these compounds in real samples. Results revealed differences in cysteine content between species of the two systematic categories but also showed that ratios between GSH and GSSG can vary between species while potentially predict oxidative stress in fish sperm.     

Antifungal effects of the flavonoids kaempferol and quercetin: a possible alternative for the control of fungal biofilms

This study aimed to determine the minimum inhibitory concentration (MIC) of kaempferol and quercetin against planktonic and biofilm forms of the Candida parapsilosis complex. Initially, nine C. parapsilosis sensu stricto, nine C. orthopsilosis and nine C. metapsilosis strains were used. Planktonic susceptibility to kaempferol and quercetin was assessed. Growing and mature biofilms were then exposed to the flavonoids at MIC or 10xMIC, respectively, and theywere also analyzed by confocal laser scanning microscopy. The MIC ranges were 32-128 µg ml^?1 for kaempferol and 0.5-16 µg ml^?1 for quercetin. Kaempferol and quercetin decreased (P?<?0.05) the metabolic activity and biomass of growing biofilms of the C. parapsilosis complex. As for mature biofilms, the metabolic effects of the flavonoids varied, according to the cryptic species, but kaempferol caused an overall reduction in biofilm biomass. Microscopic analyses showed restructuring of biofilms after flavonoid exposure. These results highlight the potential use of these compounds as sustainable resources for the control of fungal biofilms.     

Bioactive 3,8‐Epoxy Iridoids from Valeriana jatamansi

Twelve 3,8‐epoxy iridoids, including four new compounds, jatamanins R–U ( 1 – 4 ), and eight known compounds ( 5 – 12 ), were obtained from the roots and rhizomes of Valeriana jatamansi. The structures were elucidated from analysis of spectroscopic data. The absolute configurations of 1 – 4 were determined by comparison of experimental and literature ECD spectra. Moreover, the compounds were evaluated for cytotoxic effects against glioma stem cells, inhibition of NO production, activity against influenza A virus and reversal of multidrug resistance of HepG2/ADR cells. Compounds 9 and 12 showed significant cytotoxic potency against GSC‐18# (IC₅₀=1.351 and 4.439 μg ml^?1, respectively) and GSC‐3# (IC₅₀=10.88 and 6.348 μg ml^?1, respectively) glioma stem cells, while compound 12 was also slightly less potent against GSC‐12# (IC₅₀=13.45 μg ml^?1) glioma stem cell growth. In addition, compounds 9 and 12 displayed obvious inhibition of NO production (IC₅₀=4.6 and 15.8 μm , respectively).     

Investigation of the antibacterial activity of 3-O-octanoyl-(-)-epicatechin

Aims: To measure antibacterial activity of the semi-synthetic flavonoid 3-O-octanoyl-(–)-epicatechin and investigate the mechanism of action. Methods and Results: MICs determined by the broth microdilution method were 50 μg ml⁻¹ for β-lactam sensitive and resistant Staphylococcus aureus, and 100 μg ml⁻¹ for vancomycin sensitive and resistant enterococci. In time-kill studies, 100 μg ml⁻¹ 3-O-octanoyl-(–)-epicatechin reduced colony forming unit numbers of antibiotic sensitive and methicillin-resistant Staph. aureus below detectable levels within 120 min. Bacterial aggregation was not observed when cells exposed to 3-O-octanoyl-(–)-epicatechin were examined by light microscopy. It was also shown that 50 μg ml⁻¹ 3-O-octanoyl-(–)-epicatechin is capable of reducing colony forming unit numbers of high cell density Staph. aureus populations by 80-fold within 60 min incubation, and inducing leakage of 50% of their internal potassium within just 10 min. Conclusions: 3-O-Octanoyl-(–)-epicatechin is active against Gram-positive bacteria, has bactericidal activity against both antibiotic sensitive and resistant strains, and is likely to exert its primary antibacterial effect by damaging the cytoplasmic membrane. Significance and Impact of the Study: 3-O-Octanoyl-(–)-epicatechin has significant antibacterial activity and additional structural modification and/or formulation studies may allow this to be potentiated.     

Effects of All-trans retinoic acid on germ cell development of embryos and larvae of the giant freshwater prawn, Macrobrachium rosenbergii

Embryos of the giant freshwater prawn, Macrobrachium rosenbergii, were treated with 1, 10 or 50 μg ml⁻¹ all-trans retinoic acid (AtRA) for 2 days. Survival and hatching rates were not affected. However, an increase in the number of primordial germ cells (PGCs), progenitors of gametes, and a slightly more advanced stage of the gonads were found in those treated with 10 or 50 μg ml⁻¹ AtRA. Newly hatched larvae were treated with 0.1, 0.5 or 1 μg ml⁻¹ AtRA for 2 days. Survival rates were lower in those treated with 0.5 or 1 μg ml⁻¹ AtRA; nevertheless, the gonads were slightly more developed. The results indicated that AtRA, an active metabolite of vitamin A, affected germ cell and gonad development of embryos and the larvae of giant freshwater prawn.     

Enhancement of anaphylactic mediator release from guinea-pig perfused lungs by fatty acid hydroperoxides

Fragments of chopped lung from indomethacin treated guinea-pigs had an anti-aggregating effect when added to human platelet rich plasma (PRP), probably due to the production of prostacyclin (PGI₂) since the effect was inhibited by 15-hydroperoxy arachidonic acid (15-HPAA, 10 μg ml^?1). Both 15-HPAA (1–20 μg ml^?1 min^?1) and 13-hydroperoxy linoleic acid (13-HPLA, 20 μg ml^?1 min^?1) caused a marked enhancement of the anaphylactic release of histamine, slow-reacting substance of anaphylaxis (SRS-A) and rabbit aorta contracting substance (RCS) from guinea-pig isolated perfused lungs. This enhancement was not reversed by the concomitant infusion of either PGI₂ (5 μg ml^?1 min^?1) or 6-oxo-prostaglandin F_1α (6-oxo-PGF_1α, 5 μg ml^?1 min^?1). Anaphylactic release of histamine and SRS-A from guinea-pig perfused lungs was not inhibited by PGI₂ (10 ng - 10 μg ml^?1 min^?1) but was inhibited by PGE₂ (5 and 10 μg ml^?1 min^?1). Antiserum raised to 5,6-dihydro prostacyclin (PGI₁) in rabbits, which also binds PGI₂, had no effect on the release of anaphylactic mediators. The fatty acid hydroperoxides may enhance mediator release either indirectly by augmenting thromboxane production or by a direct effect on sensitized cells. Further experiments to distinguish between these alternatives are described in the accompanying paper (27).     

Activities of muscadine grape skin and quercetin against Helicobacter pylori infection in mice

Aims: To explore the preventative potential of muscadine grape skin (MGS) and the single flavonoid, quercetin, as an alternative means for ameliorating Helicobacter pylori infection and/or the H. pylori‐induced inflammatory response in mice. Methods and Results: The antimicrobial and anti‐inflammatory properties of MGS and quercetin, a major phenolic constituent, were evaluated against H. pylori in vitro and in vivo. The antimicrobial activity of quercetin was evaluated against 11 H. pylori strains in vitro with inhibition of all strains at 128–64 μg ml^?1. In vivo studies showed a moderate reduction in H. pylori counts following treatment with 5 and 10% MGS or quercetin (25 mg kg^?1 body weight) in addition to significantly reduced inflammatory cytokines (TNF‐α, IL‐1β and IFN‐γ) when compared with untreated mice. Conclusions: MGS and quercetin did not significantly reduce H. pylori growth in a mouse model. However, these products were effective in regulating the inflammatory response to H. pylori infection. Significance and Impact of the Study: Our results suggest that H. pylori infection may be reduced or prevented via the consumption of fruits rich in certain phenolic compounds (e.g. quercetin) such as muscadine grapes.     

Development of a modified selective medium to enhance the recovery rate of Brachyspira hyodysenteriae and other porcine intestinal spirochaetes from faeces

Aims: The aim of this study was to develop a modified selective medium to improve the recovery rate of Brachyspira hyodysenteriae and other clinically significant intestinal spirochaetes from porcine faeces. Methods and Results: The susceptibility of five Brachyspira spp. type strains and five Thai field isolates of B. hyodysenteriae to the antimicrobials halquinol and flavomycin was determined by in vitro susceptibility tests in the agar dilution method, and optimal incorporation rates were confirmed by broth dilution. All the spirochaetes were susceptible to halquinol at ≤1 μg ml^?1, while 16 μg ml^?1 of flavomycin (F) allowed their growth, and therefore, only the latter was selected for further use. F and different combinations of colistin (C), spectinomycin (S) and rifampacin (R) were incorporated into pre‐enrichment broths and/or agar plates, and growth of the spirochaetes from seeded faeces was determined. Two solid media were selected for further testing using faeces from 90 finishing pigs on 10 farms. A previously recommended method of pre‐enrichment did not increase the recovery rate. The use of blood agar modified medium (BAM) containing F (16 μg ml^?1), S (400 μg ml^?1), R (30 μg ml^?1) and colistin (C, 100 U ml^?1) (assigning as BAM‐CSRF) reduced the growth of contaminating intestinal microbiota and resulted in a significantly higher rate of spirochaete recovery than the previous recommended medium. Conclusion: BAM‐CSRF is a useful new selective medium for the isolation of B. hyodysenteriae and other intestinal spirochaetes from pig faeces. Significance and Impact of the Study: The new selective medium for isolating B. hyodysenteriae and other Brachyspira spp. from pig faeces will improve their recovery and subsequent disease diagnosis.     

Lysozyme activities and immunoglobulin concentrations in seminal plasma and spermatozoa of different teleost species and indications on its significance for sperm function

The occurrence of lysozyme and immunoglobulin (Ig) in semen of different teleost species (brown trout—Salmo trutta, perch—Perca fluviatilis, burbot—Lota lota) was studied. In all investigated species lysozyme activities (1.13-1.45 U ml⁻¹) and Ig concentrations (T-Ig: 1.11-1.61 μg ml⁻¹, IgG [measured only in brown trout]: 1.49 μg ml⁻¹) were detected in seminal plasma. Ig was also found in spermatozoa (T-Ig: 0.234-0.357 μg/g protein, IgG: 0.198 μg ml⁻¹) while spermatozoal lysozyme activities were low and fluctuating (0.093-0.164 U/g protein). In Salmo trutta lysozyme activities and immunoglobulin levels were compared between semen samples with high and low sperm motility as motility is an indicator for sperm fertility. Lysozyme activities were higher in seminal plasma of samples with high motility than in those with low motility while seminal plasma and spermatozoal immunoglobulin concentrations (T-Ig, IgG) were increased in samples with low motility in comparison to samples with high motility. Seminal plasma and spermatozoal IgG concentrations and seminal plasma lysozyme activities showed significant correlations with the sperm motility rate and swimming velocity. Moreover, lysozyme improved the viability of spermatozoa in in vitro experiments. Possible physiological meanings of these results are discussed.     

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 butenolide on behavioral and morphological changes in two marine fouling species,the barnacle Balanus amphitrite and the bryozoan Bugula neritina

Butenolide [5-octylfuran-2(5H)-one] is a very promising antifouling compound. Here, the effects of butenolide on larval behavior and histology are compared in two major fouling organisms, viz. cypris larvae of Balanus amphitrite and swimming larvae of Bugula neritina. Butenolide diminished the positive phototactic behavior of B. amphitrite (EC₅₀ = 0.82 μg ml^?1) and B. neritina (EC₅₀ = 3 μg ml^?1). Its effect on the attachment of cyprids of B. amphitrite was influenced by temperature, and butenolide increased attachment of larvae of B. neritina to the bottom of the experimental wells. At concentrations of 4 μg ml^?1 and 10 μg ml^?1, butenolide decreased attachment of B. amphitrite and B. neritina, respectively, but the effects were reversible within a certain treatment time. Morphologically, butenolide inhibited the swelling of secretory granules and altered the rough endoplasmic reticulum (RER) in the cement gland of B. amphitrite cyprids. In B. neritina swimming larvae, butenolide reduced the number of secretory granules in the pyriform-glandular complex.     

The phytotoxicity of selected mycotoxins on mature,germinatingZea mays embryos

Mature maize (Zea mays) embryos were exposed to 5, 10 and 25 µg ml^–1 of deoxynivalenol (DON), zearalenone (ZEA), ochratoxin A (OA) and a mixture of zearalenone and deoxynivalenol (ZEA/DON) for 9 days. DON and the ZEA/DON combination were consistently more inhibitory of the measured parameters than either ZEA or OA. Based on the predicted additive values, it would appear that, in combination, ZEA and DON act synergistically to inhibit root and shoot growth. For ZEA alone, a concentration of 5 µg ml^–1 ZEA was generally inhibitory of root and shoot elongation and fresh mass accumulation, while at 10 and 25 µg ml^–1, this toxin had a stimulatory effect on these parameters. For OA, the measured effects on root and shoot growth at 5 and 25 µg ml^–1 were stimulatory, while at 10 µg ml^–1 OA, an inhibitory effect was observed. For all toxins, inhibitory/stimulatory effects were generally more marked for root parameters than for shoot elongation or mass.Abbreviations ADON acetyldeoxynivalenol - AFB₁ aflatoxin B₁ - DAS diacetyoxyscirpenol - DON deoxynivalenol - FB₁ fumonisin B₁ - FHB Fusaium head blight - MON moniliformin - NIV nivalenol - OA ochratoxin A - ZEA zearalenone     

The effect of triadimenol on the cytology and growth of sensitive and resistant strains of Ustilago avenue

Two triazole resistant mutants of Ustilago avenae and a wild type strain were treated with different concentrations of the fungicide triadimenol in liquid cultures. Growth rates were measured and the morphology of the sporidia examined by light and electron microscopy. After treatment with 1 – 10 μg ml^-1 for 4 days wild type sporidia increased in growth rate, changed from budding to filamentous growth and had considerably thicker cell walls. Their cytoplasm also showed strong degeneration and damage to the cell organelles. The resistant strains were not influenced under these conditions but under longer exposure (14 days) to higher fungicide concentrations (50 to 200 μg ml^-1) similar changes to growth rates, morphology and cell wall thickness also occurred. However, cell disruption only occurred at 200 to 250 μg triadimenol ml^-1 and there were differences in cell wall thickening. One resistant strain (r13) never showed thickening, while in the other (r8), the wall consisted of two distinct layers. The implications of these results are discussed in relation to possible resistance mechanisms.     

Effects of the nematicide oxamyl on life cycle stages of Globodera rostochiensis

Hatched second stage juveniles of Globodera rostochiensis were found to be more sensitive than either unhatched juveniles or adult males to low concentrations of oxamyl (EC₅₀ value for activity 0.5 μg oxamyl ml^-1). Development of juveniles in roots was significantly inhibited by 2.0 μg oxamyl ml^-1 probably due to impairment of normal feeding activity. The behavioural events necessary for orientation of juveniles towards roots for invasion and orientation of males towards females for fertilisation were found to be impaired by 0.5 and 1.0 μg oxamyl ml^-1 respectively. A comparison with previous work on Meloidogyne incognita showed G. rostochiensis juveniles to be more sensitive to oxamyl.     

Effect of garlic and allium-derived products on the growth and metabolism of Spironucleus vortens

Spironucleus is a genus of small, flagellated parasites, many of which can infect a wide range of vertebrates and are a significant problem in aquaculture. Following the ban on the use of metronidazole in food fish due to toxicity problems, no satisfactory chemotherapies for the treatment of spironucleosis are currently available. Using membrane inlet mass spectrometry and automated optical density monitoring of growth, we investigated in vitro the effect of Allium sativum (garlic), a herbal remedy known for its antimicrobial properties, on the growth and metabolism of Spironucleus vortens, a parasite of tropical fish and putative agent of hole-in-the-head disease. The allium-derived thiosulfinate compounds allicin and ajoene, as well as an ajoene-free mixture of thiosulfinates and vinyl-dithiins were also tested. Whole, freeze-dried garlic and allium-derived compounds had an inhibitory effect on gas metabolism, exponential growth rate and final growth yield of S. vortens in Keister’s modified, TY-I-S33 culture medium. Of all the allium-derived compounds tested, the ajoene-free mixture of dithiins and thiosulfinates was the most effective with a minimum inhibitory concentration (MIC) of 107 μg ml⁻¹ and an inhibitory concentration at 50% (IC_50%) of 58 μg ml⁻¹. It was followed by ajoene (MIC = 83 μg ml⁻¹, IC_50% = 56 μg ml⁻¹) and raw garlic (MIC >20 mg ml⁻¹, IC_50% = 7.9 mg ml⁻¹); allicin being significantly less potent with an MIC and IC_50% above 160 μg ml⁻¹. All these concentrations are much higher than those reported to be required for the inhibition of most bacteria, protozoa and fungi previously investigated, indicating an unusual level of tolerance for allium-derived products in S. vortens. However, chemically synthesized derivatives of garlic constituents might prove a useful avenue for future research.