Characterization and selection of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains for their effect on bile tolerance,taurocholate deconjugation and cholesterol removal

Fourteen Lactobacillus strains of six species were investigated with their characteristics of bile salt tolerance, deconjugation of sodium taurocholate and cholesterol removal in the spent broth. Meanwhile, a co-precipitation curve of cholesterol with cholic acid at concentrations ranged 0.0–6.0 μM/ml was involved in the evaluation of cholesterol removal. Results demonstrated that both co-precipitation and assimilation effects contributed to cholesterol removal during the incubation of these Lactobacillus strains. It was also indicated that the supplementation of bile salts influenced the cholesterol removal, not only as an essential factor related to co-precipitation but also a critical condition for cholesterol assimilation. Out of all strains tested, four L. plantarum strains LS12, LS31, Lp501 and Lp529 exhibited a high ability of cholesterol assimilation (maximum 20.76 μg/ml), deconjugation of sodium taurocholate (maximum 5.00 μM/ml) and bile tolerance. They could be further studied and used as potential probiotics strains to reduce serum cholesterol in humans     

Isolation and Characterization of Pediocin NV 5 Producing <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> LAB 5 from Vacuum-Packed Fermented Meat Product

A potentially novel antimicrobial compound producing Pediococcus acidilactici LAB 5 was isolated from vacuum-packed fermented meat product. This compound was found active against some species of Enterococcus, Leuconostoc, Staphylococcus and Listeria, many of which are associated with food spoilage and food related health hazards. The strain was found to be a paired cocci which can utilize a broad range of carbohydrates and produce acid identical to the P. acidilactici and P. pentoseus. Since the antimicrobial agent was sensitive to proteolytic enzymes but quite resistant to heat, it was identified as a bacteriocin and was designated as Pediocin NV 5. The molecular weight of the bacteriocin was 10.3 kDa and the bacterium possessed a 5 kbp plasmid responsible for bacteriocin production and also for vancomycin resistance phenotype.     

The probiotic properties of Lactobacillus buchneri P2

Aims: To isolate new lactobacilli strain with cholesterol‐lowering effect and analyse its probiotic properties and possible mechanisms of cholesterol removal. Methods and Results: The strain with cholesterol‐lowering effect was isolated from pickled juice. The acid and bile tolerance and antimicrobial activity were tested. The free cholalic acid, the cholesterol in supernatant fluid, washing buffer and cell extract, the cholesterol removed by growing, dead and resting cells were quantified. The isolated strain with high cholesterol‐reducing rate of 43·95% was identified as Lactobacillus buchneri (Lact. buchneri) P2. It had acid and bile tolerance and antimicrobial activity. Moreover, it could remove cholesterol via coprecipitating with deconjugated bile salts, assimilating and adsorbing by cells. And the assimilation was considered to be the main reason of cholesterol removal. Conclusions: The isolated Lact. buchneri P2 showed probiotic properties of cholesterol reduction, acid and bile tolerance and antimicrobial activity and could remove cholesterol via different ways. Significance and Impact of the Study: A new strain of Lact. buchneri P2 with efficient cholesterol‐reducing ability was isolated to provide species diversity of lactobacilli for functional dairy products. And the possible mechanism of cholesterol removal by Lact. buchneri was discussed.     

Characterization of a multifunctional feather-degrading <Emphasis Type="Italic">Bacillus subtilis</Emphasis> isolated from forest soil

In this study, we isolated and characterized a novel feather-degrading bacterium that shows keratinolytic, antifungal and plant growth-promoting activities. A bacterium S8 was isolated from forest soil and confirmed to belong to Bacillus subtilis by BIOLOG system and 16S rRNA gene analysis. The improved culture conditions for the production of keratinolytic protease were 0.1% (w/v) sorbitol, 0.3% (w/v) KNO₃, 0.1% (w/v) K₂HPO₄, 0.06% (w/v) KH₂PO₄ and 0.04% (w/v) MgCl₂·6H₂O (pH 8.0 and 30°C), respectively. In the improved medium containing 0.1% (w/v) feather, keratinolytic protease production was around 53.3 ± 0.3 U/ml at 4 day; this value was 10-fold higher than the yield in the basal feather medium (5.3 ± 0.1 U/ml). After cultivation for 5 days in the improved medium, intact feather was completely degraded. Feather degradation resulted in free –SH group, soluble protein and amino acids production. The concentration of free –SH group in the culture medium was 15.5 ± 0.2 μM at 4 days. Nineteen amino acids including all essential amino acids were produced in the culture medium; the concentration of total amino acid produced was 3360.4 μM. Proline (2809.9 μM), histidine (371.3 μM) and phenylalanine (172.0 μM) were the major amino acids released in the culture medium. B. subtilis S8 showed the properties related to plant growth promotion: hydrolytic enzymes, ammonification, indoleacetic acid (IAA), phosphate solubilization, and broad-spectrum antimicrobial activity. Interestingly, the strain S8 grown in the improved medium produced IAA and antifungal activity, indicating simultaneous production of keratinolytic and antifungal activities and IAA by B. subtilis S8. These results suggest that B. subtilis S8 could be not only used to improve the nutritional value of feather wastes but also is useful in situ biodegradation of feather wastes. Furthermore, it could also be a potential biofertilizer or biocontrol agent applicable to crop plant soil.     

<Emphasis Type="Italic">Fusarium verticillioides</Emphasis>: Evaluation of Fumonisin Production and Effect of Fungicides on In Vitro Inhibition of Mycelial Growth

The objectives of this study were to evaluate the fumonisin production by 16 F. verticillioides strains on corn cultures and the effect of quintozene and fludioxonil + metalaxyl-M fungicides on “in vitro” mycelial growth on agar. In addition, the effect of fludioxonil + metalaxyl-M on fumonisin production in defined liquid culture medium was analyzed. Fumonisin B₁ levels on corn cultures ranged from 2.41 to 3996.36 μg/g and the F. verticillioides 103F strain produced the highest level (3996.36 ± 390.49 μg/g, P < 0.05). F. verticillioides strains were inoculated in potato dextrose agar with the addition of quintozene (75 to 9,375 μg/ml) and fludioxonil + metalaxyl-M (1.5 + 0.6 to 187.5 + 75 μg/ml) in order to evaluate the effect of these fungicides on “in vitro” mycelial growth. The F. verticillioides strains showed great variability concerning ED₅₀ values, which were below the recommended application dose for quintozene, but above that for fludioxonil + metalaxyl-M. Moreover, fungicide addition to the culture medium increased mean FB₁ levels compared to the control, suggesting the importance of focusing on the effect of fungicides on mycotoxin production as well as on the phytopathogen control.     

Coexpression of bile salt hydrolase gene and catalase gene remarkably improves oxidative stress and bile salt resistance in <Emphasis Type="Italic">Lactobacillus casei</Emphasis>

Lactic acid bacteria (LAB) encounter various types of stress during industrial processes and gastrointestinal transit. Catalase (CAT) and bile salt hydrolase (BSH) can protect bacteria from oxidative stress or damage caused by bile salts by decomposing hydrogen peroxide (H₂O₂) or deconjugating the bile salts, respectively. Lactobacillus casei is a valuable probiotic strain and is often deficient in both CAT and BSH. In order to improve the resistance of L. casei to both oxidative and bile salts stress, the catalase gene katA from L. sakei and the bile salt hydrolase gene bsh1 from L. plantarum were coexpressed in L. casei HX01. The enzyme activities of CAT and BSH were 2.41 μmol H₂O₂/min/10⁸ colony-forming units (CFU) and 2.11 μmol glycine/min/ml in the recombinant L. casei CB, respectively. After incubation with 8 mM H₂O₂, survival ratio of L. casei CB was 40-fold higher than that of L. casei CK. Treatment of L. casei CB with various concentrations of sodium glycodeoxycholate (GDCA) showed that ~10⁵ CFU/ml cells survived after incubation with 0.5% GDCA, whereas almost all the L. casei CK cells were killed when treaded with 0.4% GDCA. These results indicate that the coexpression of CAT and BSH confers high-level resistance to both oxidative and bile salts stress conditions in L. casei HX01.     

Influence of aflatoxin B1 on a bacteriocinogenic strain ofSerratia marcescens

The ability of aflatoxin B₁, the most potent hepatocarcinogen known, to induce bacteriocin synthesis inSerratia marcescens strain Nima was studied. The optimal induction dose of aflatoxin B₁ was found to be 20μg/ml, which on the average increased the number of bacteriocinproducing cells by a factor of 11.5. Doses less than 5 μg or more than 40 μg per ml did not have a pronounced stimulating action. The optimum incubation time for maximum bacteriocin synthesis was found to be 1.5 h.     

Screening and molecular identification of potential probiotic lactic acid bacteria in effluents generated during ogi production

Screening and molecular identification of probiotic lactic acid bacteria (LAB) in effluents generated during the production of ogi, a fermented cereal (maize, millet, and sorghum) were done. LAB were isolated from effluents generated during the first and second fermentation stages in ogi production. Bacterial strains isolated were identified microscopically and phenotypically using standard methods. Probiotic potential properties of the isolated LAB were investigated in terms of their resistance to pH 1.5 and 0.3% bile salt concentration for 4 h. The potential LAB isolates ability to inhibit the growth of pathogenic organisms (Escherichia coli, Staphylococcus aureus, and Salmonella typhimurium) was evaluated in vitro. The pH and LAB count in the effluents ranged from 3.31 to 4.49 and 3.67 to 4.72 log cfu/ml, respectively. A total of 88 LAB isolates were obtained from the effluents and only 10 LAB isolates remained viable at pH 1.5 and 0.3% bile salt. The zones of inhibition of the LAB isolates with probiotic potential ranged from 7.00 to 24.70 mm against test organsisms. Probiotic potential LAB isolates were molecularly identified as Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus reuteri, Enterococcus faecium, Pediococcus acidilactici, Pediococcus pentosaceus, Enterococcus faecalis, and Lactobacillus brevis. Survival and proliferation of LAB isolates at low pH, 0.3% bile salt condition, and their inhibition against some test pathogens showed that these LAB isolates could be a potential probiotics for research and commercial purposes.     

Relationships Between Respiratory Function Disorders and Serum Copper Levels in Copper Mineworkers

The aim of this study was to investigate the respiratory function disorders that could be related to dust exposure during the production of copper mine in copper mineworkers (CMWs). The study included 75 male CMWs (mean age, 32.0 ± 7.1 years, 58.6% smokers) and 75 male age- and smoking status-matched healthy control subjects. Serum Cu level was significantly higher in the CMW group (0.80 ± 0.62 μg/ml) than the control group (0.60 ± 0.39 μg/ml) (p = 0.017). Significant negative correlations were found between serum Cu level and forced expiratory volume in first second (r = −0.600; p < 0.001) and between serum Cu level and forced vital capacity (r = −0.593; p = <0.001) in CMWs. Serum Cu level was significantly higher in the restrictive type pulmonary function disorders group (1.36 ± 0.62 μg/ml) than obstructive type (0.90 ± 0.55 μg/ml) and normal pulmonary function pattern group (0.53 ± 0.43 μg/ml) (p < 0.001). Patients with radiological parenchymal abnormalities had significantly higher serum copper levels than those without abnormalities (1.53 ± 0.52 vs. 0.71 ± 0.52 μg/ml, respectively; p = 0.002). In conclusion, result of the study has shown a negative association between pulmonary functions disorders and radiological abnormalities and serum Cu levels in CMWs.     

Antivibrio compounds produced by <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. W3: characterisation and assessment of their safety to shrimps

In order to explore compounds naturallly inhibitory to shrimp pathogenic vibrios, a culture filtrate of Pseudomonas sp. W3 at a pH of 2 was extracted with ethyl acetate (EtOAc) to produce 82.15 mg/l of a yellow–brown extract (EtOAc-W3) that had MIC values of 225-450 μg/ml against the growth of 18 shrimp pathogenic Vibrio harveyi strains. The MIC of EtOAc-W3 against the most pathogenic strain PSU 2015 was 450 μg/ml and this strain had the lowest LD₅₀ (50% lethal dose) to pacific white shrimp (Litopenaeus vannamei, PL 21). At this MIC value, EtOAc-W3 in artificial sea water (ASW) killed strain PSU 2015; however in natural sea water, only a partial growth inhibition was observed. The toxicity to pacific white shrimp and antivibrio activity of the EtOAc-W3 were investigated by conducting an experiment with 4 sets; native control (commercial ASW), EtOAc-W3 control (MIC/10, 45 μg/ml), challenge (inoculation 6.0 × 10⁶ c.f.u./ml PSU 2015) and treatment (6.0 × 10⁶ c.f.u./ml PSU 2015 + 45 μg/ml EtOAc-W3). The same experiment was repeated by increasing the dose of EtOAc-W3 to 90 μg/ml (MIC/5). Both concentrations of EtOAc-W3 tested had no toxicity to postlarval shrimps. A significant decrease in shrimp mortality was observed over a 72 h period as approximately 80% of the shrimps died in each challenge set but only 63 and 23% died in the presence of 45 and 90 μg/ml EtOAc-W3. The major component of EtOAc-W3 was supposed to be 2-heptyl-4-quinolone (HHQ) by FAB-MS and ¹H-NMR analyses of the purified fraction.     

Relative gene expression of bile salt hydrolase and surface proteins in two putative indigenous <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> strains under in vitro gut conditions

Probiotic bacteria must overcome the toxicity of bile salts secreted in the gut and adhere to the epithelial cells to enable their better colonization with extended transit time. Expression of bile salt hydrolase and other proteins on the surface of probiotic bacteria can help in better survivability and optimal functionality in the gut. Two putative Lactobacillus plantarum isolates i.e., Lp9 and Lp91 along with standard strain CSCC5276 were used. A battery of six housekeeping genes viz. gapB, dnaG, gyrA, ldhD, rpoD and 16S rRNA were evaluated by using geNorm 3.4 excel based application for normalizing the expression of bile salt hydrolase (bsh), mucus-binding protein (mub), mucus adhesion promoting protein (mapA), and elongation factor thermo unstable (EF-Tu) in Lp9 and Lp91. The maximal level of relative bsh gene expression was recorded in Lp91 with 2.89 ± 0.14, 4.57 ± 0.37 and 6.38 ± 0.19 fold increase at 2% bile salt concentration after 1, 2 and 3 h, respectively. Similarly, mub and mapA genes were maximally expressed in Lp9 at the level of 20.07 ± 1.28 and 30.92 ± 1.51 fold, when MRS was supplemented with 0.05% mucin and 1% each of bile and pancreatin (pH 6.5). However, in case of EF-Tu, the maximal expression of 42.84 ± 5.64 fold was recorded in Lp91 in the presence of mucin alone (0.05%). Hence, the expression of bsh, mub, mapA and EF-Tu could be considered as prospective biomarkers for screening of novel probiotic lactobacillus strains for optimal functionality in the gut.     

Functional Characteristics of <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">fermentum</Emphasis> F1

In this study, Lactobacillus fermentum (L. fermentum) F1 reduced cholesterol 48.87%. The strain was screened from cattle feces using an API 50 CHL system and the 16S rRNA sequence contrasting method. L. fermentum F1 showed acid and bile tolerance, and antimicrobial activity against pathogenic Escherichia coli and Staphylococcus aureus. L. fermentum F1 deconjugated 0.186 mM of free cholalic acid after it was incubated at 37°C in 0.20% sodium taurocholate (TCA) broth for 24 h. Heat-killed and resting cells of L. fermentum F1 showed small amounts of cholesterol removal (6.85 and 25.19 mg/g, respectively, of dry weight) compared with growing cells (33.21 mg/g of dry weight). The supernatant fluid of the broth contained 50.85% of the total cholesterol, while the washing buffer and cell extracts had 13.53 and 35.39%, respectively. These findings suggest that L. fermentum F1 may remove cholesterol by co-precipitating with deconjugated bile salt, assimilating with cells and by incorporation into cellular membranes. Cholesterol assimilated by cells held 72.0% of the reduced cholesterol, while 21.65% of the reduced cholesterol was coprecipitated with deconjugated bile salt and 5.89% was adsorbed into the surface of the cells.     

Characterization and antioxidant ability of potential probiotic lactic acid bacteria in ogi liquor and lemon juice-ogi liquor

Purpose

Ogi is an indigenous edible fermented cereal slurry but the steep liquor is usually wasted or administered as therapeutic to suppress certain illnesses. The combination of lemon juice and ogi steep liquor (OSL) is known to possess bioactive metabolites.

Method

This study evaluated potential probiotic lactic acid bacteria (LAB) in different OSL (Zea mays, Sorghum bicolor, and Pennisetum glaucum L.) and lemon juice-ogi steep liquor (LJOSL) based on low pH, bile and lysozyme tolerances, hydrophobicity and auto-aggregation, antibiotic, cholesterol removal, exopolysaccharide production, β-galactosidase, and antimicrobial and hemolytic activities using standard methods. Presumptive LAB were sequenced and assayed for radical scavenging using 2,2-diphenyl-1-picrylhydrazyl (DPPH) and lipid peroxidation inhibitory (LPI) tests.

Results

Presumptive LAB counts were higher in maize OSL (0 h:5.09 log CFU/ml) and combined cereal OSL (24–48 h:7.65 and 7.72 log CFU/ml) but decreased in all steep liquors at 72 h, except in millet OSL (7.72 log CFU/ml). A total of 120 LAB isolates were randomly selected. Based on pH and bile tolerances, 14 isolates were comparable to reference strains. All these isolates demonstrated probiotics properties except for three that did not show γ-hemolysis. Sequenced LAB isolates were identified as Lactobacillus plantarum, Lactobacillus fermentum, Pediococcus pentosaceus, and Weissella cibara. DPPH activities of LAB gradually increased during fermentation with the highest activity of DPPH (58.77%) and LPI (57.94%) activity in L. plantarum. Strong correlations were found between DPPH and LPI in all the selected isolates.

Conclusion

The antioxidant property of probiotic LAB in OSL and LJOSL could contribute to its therapeutic nature.

     

Minimum inhibitory concentration (MIC) of crude preparations of Brevibacillus laterosporus SA14 bioactive material compared to vancomycin and oxacillin, against clinical isolates of methicillin-resistant Staphylococcus aureus

Secondary metabolites, particularly bioactive compounds, from probiotic bacteria, are good candidates for replacing antibiotics to which bacteria have become resistant. In order to compare bioactive crude material from strain SA14 of Brevibacillus laterosporus with two antibiotics, the MICs of this bioactive crude and those of antibiotics vancomycin and oxacillin, against methicillin-resistant Staphylococcus aureus (MRSA), were determined. The result indicated that the MIC (3.6–19.2 μg/ml) of bioactive crude was higher than vancomycin (MIC = 1.28–2.56 μg/ml) when tested against MRSA. Interestingly, all tested strains of MRSA were susceptible to bioactive crude and were approximately 5.2-fold more potent than oxacillin (MIC > 100 μg/ml). Its activity against MRSA gives support for further evaluation, and the development of this substance for therapeutic use.     

Endogenous profiles of indoleamines: serotonin and melatonin in different tissues of Coffea canephora P ex Fr. as analyzed by HPLC and LC-MS-ESI

Endogenous indoleamine profiles in various ex vitro and in vitro tissues of commercially important Coffea canephora were analyzed by using a high performance liquid chromatography and further confirmed with electrospray ionization mass spectrometry. High content of serotonin (SER) (98.54 ± 5 μg/g) and melatonin (MEL) (115.25 ± 6 μg/g) were found in freshly harvested seeds of C. canephora followed by zygotic embryo (65.25 ± 4 and 96.54 ± 5 μg/g fresh weight) and endosperm (34.08 ± 2 and 51.08 ± 4 μg/g fresh weight) of ripened fruits. Similarly endogenous pools of SER and MEL were moderate in in vitro tissues of C. canephora, i.e. callus (25.85 ± 2 and 75.74 ± 4), somatic embryos (31.88 ± 2 and 19.30 ± 2 μg/g fresh weight) and in vitro regenerated plant stalk (15.78 ± 1 and 38.25 ± 3 μg/g fresh weight), respectively. In view of significant levels of both SER and MEL in various tissues and beans of Coffea, further investigations on their physiological role needs to be investigated.     

Effects of pH and corn steep liquor variability on mannitol production by Lactobacillus intermedius NRRL B-3693

Lactobacillus intermedius NRRL B-3693 produced mannitol, lactic acid, and acetic acid when grown on fructose at 37°C. The optimal pH for mannitol production from fructose by the heterofermentative lactic acid bacterium (LAB) in pH-controlled fermentation was at pH 5.0. It produced 160.7 ± 1.1 g mannitol in 40 h with a volumetric productivity of 4.0 g l⁻¹ h⁻¹ in a simplified medium containing 250 g fructose, 50 g corn steep liquor (CSL), and 33 mg MnSO₄ per liter. However, the mannitol production by the LAB was severely affected by the variability of CSL. The supplementation of CSL with soy peptone (5 g/l), tryptophan (50 mg/l), tryptophan (50 mg/l) plus tyrosine (50 mg/l), or commercial protease preparation (2 ml/100 g of CSL) enhanced the performance of the inferior CSL and thus helped to overcome the nutrient limitations.     

Comparison of metabolite levels in callus of <Emphasis Type="Italic">Tecoma stans</Emphasis> (L.) Juss. ex Kunth. cultured in photoperiod and darkness

Tecoma stans is a tropical plant from the Americas. Antioxidant activity and both phenolic compound and flavonoid total content were determined for callus tissue of T. stans cultured in either a set photoperiod or in darkness. Callus lines from three explant types (hypocotyls, stem, and leaf) were established on B5 culture medium supplemented with 0.5 μM 2,4-D and 5.0 μM kinetin. While leaf-derived callus grew slower under a 16-h photoperiod (specific growth rate, μ = 0.179 d⁻¹, t _D = 3.9 d) than in darkness (μ = 0.236 d⁻¹, t _D = 2.9 d), it accumulated the highest amount (p < 0.05) of both phenolics (86.6 ± 0.01 mg gallic acid equivalents/g) and flavonoids (339.6 ± 0.06 mg catechin equivalents/g). Similarly, antioxidant activity was significantly higher (p < 0.05) when callus was cultured in period light than when grown in extended darkness. Antioxidant activity measured with a 2,20-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS)-based assay was 350.5 ± 15.8 mmol Trolox/g extract for callus cultured under a defined photoperiod compared to 129.1 ± 7.5 mmol Trolox/g extract from callus cultured in darkness. Content of phenolic compounds and flavonoids was in agreement with a better antioxidant power (EC₅₀ = 450 μg extract/mg 1,1-diphenyl-2-picrylhydrazyl) and antiradical efficiency. Results of the present study show that calli of T. stans are a source of compounds with antioxidant activity that is favored by culture under a set photoperiod.     

Changes in Trace Elements during Follicular Atresia in Goat (<Emphasis Type="Italic">Capra hircus</Emphasis>) Ovary

Quantitative analysis of follicular fluid and granulosa cells from small, medium and large antral atretic follicles of goat (Capra hircus) ovaries was conducted to study the alterations in trace elements viz zinc (Zn), copper (Cu), manganese (Mn), and iron (Fe). The zinc content was lower in the follicular fluid (0.993 ± 0.001, 0.935 ± 0.002, 1.321 ± 0.001 μg/ml) and granulosa cells (0.867 ± 0.002, 0.801 ± 0.001, 1.073 ± 0.002 μg/mg) of small, medium, and large antral atretic follicles respectively than their respective controls. Copper quantity was higher in the follicular fluid (0.113 ± 0.001, 0.163±0.001 0.{163}\pm 0.00{1} , 0.224 ± 0.001 μg/ml) and granulosa cells (0.094 ± 0.001, 0.114 ± 0.001, 0.182 ± 0.001 μg/mg) from small, medium, and large antral atretic follicles respectively than their respective controls. Similarly, iron and manganese was also found higher in the follicular fluid and granulosa cells of small, medium, and large antral atretic follicles than their respective controls. The present study provides the basic data on trace elements that can be safely used as atretic marker and will find use in in vitro studies for fertility improvement plan. Thus, help in elevating the number of ovulations and screening of follicles to enhance the success rate in vivo and in vitro fertilization and embryo transfer technology.     

Identification,cloning, and functional characterization of EmrD-3, a putative multidrug efflux pump of the major facilitator superfamily from <Emphasis Type="Italic">Vibrio cholerae</Emphasis> O395

A putative multidrug efflux pump, EmrD-3, belonging to the major facilitator superfamily (MFS) of transporters and sharing homology with the Bcr/CflA subfamily, was identified in Vibrio cholerae O395. We cloned the emrD-3 gene and evaluated its role in antimicrobial efflux in a hypersensitive Escherichia coli strain. The efflux activity of this membrane protein resulted in lowering the intracellular concentration of ethidium. The recombinant plasmid carrying emrD-3 conferred enhanced resistance to several antimicrobials. Among the antimicrobials tested, the highest relative increase in minimum inhibitory concentration (MIC) of 102-fold was observed for linezolid (MIC = 256 μg/ml), followed by an 80.1-fold increase for tetraphenylphosphonium chloride (TPCL) (156.2 μg/ml), 62.5-fold for rifampin (MIC = 50 μg/ml), >30-fold for erythromycin (MIC = 50 μg/ml) and minocycline (MIC = 2 μg/ml), 20-fold for trimethoprim (MIC = 0.12 μg/ml), and 18.7-fold for chloramphenicol (MIC = 18.7 μg/ml). Among the fluorescent DNA-binding dyes, the highest relative increase in MIC of 41.7-fold was observed for ethidium bromide (125 μg/ml) followed by a 17.2-fold increase for rhodamine 6G (100 μg/ml). Thus, we demonstrate that EmrD-3 is a multidrug efflux pump of V. cholerae, the homologues of which are present in several Vibrio spp., some members of Enterobacteriaceae family, and Gram-positive Bacillus spp.     

Susceptibility of <Emphasis Type="Italic">Candida</Emphasis> biofilms to histatin 5 and fluconazole

Candida-associated denture stomatitis has a high rate of recurrence. Candida biofilms formed on denture acrylic are more resistant to antifungals than planktonic yeasts. Histatins, a family of basic peptides secreted by the major salivary glands in humans, especially histatin 5, possess significant antifungal properties. We examined antifungal activities of histatin 5 against planktonic or biofilm Candida albicans and Candida glabrata. Candida biofilms were developed on poly(methyl methacrylate) discs and treated with histatin 5 (0.01–100 μM) or fluconazole (1–200 μM). The metabolic activity of the biofilms was measured by the XTT reduction assay. The fungicidal activity of histatin 5 against planktonic Candida was tested by microdilution plate assay. Biofilm and planktonic C. albicans GDH18, UTR-14 and 6122/06 were highly susceptible to histatin 5, with 50% RMA (concentration of the agent causing 50% reduction in the metabolic activity; biofilm) of 4.6 ± 2.2, 6.9 ± 3.7 and 1.7 ± 1.5 μM, and IC₅₀ (planktonic cells) of 3.0 ± 0.5, 2.6 ± 0.1 and 4.8 ± 0.5, respectively. Biofilms of C. glabrata GDH1407 and 6115/06 were less susceptible to histatin 5, with 50% RMA of 31.2 ± 4.8 and 62.5 ± 0.7 μM, respectively. Planktonic C. glabrata was insensitive to histatin 5 (IC₅₀ > 100 μM). Biofilm-associated Candida was highly resistant to fluconazole in the range 1–200 μM; e.g. at 100 μM only ~20% inhibition was observed for C. albicans, and ~30% inhibition for C. glabrata. These results indicate that histatin 5 exhibits antifungal activity against biofilms of C. albicans and C. glabrata developed on denture acrylic. C. glabrata is significantly less sensitive to histatin 5 than C. albicans.