The antibacterial mechanism of carvacrol and thymol against Escherichia coli

Aims:  To investigate the antibacterial mechanism of carvacrol and thymol against Escherichia coli.
Methods and Results:  The time-kill curve results showed that carvacrol and thymol at 200 mg l⁻¹ could inhibit the growth of E. coli . Flow cytometry and fluorescent dyes were used to explore the effect of two components on membrane permeability and membrane potential. In membrane permeability experiment, the mean fluorescence intensity of cells treated with 200 mg l⁻¹ carvacrol or thymol were lower than nonexposed cells. The ratio of red to green fluorescence intensity of DiOC₂(3) reflected the change of membrane potential. Carvacrol and thymol at 200 mg l⁻¹ caused the ratio of red/green decreasing from 0·42 of control to 0·08 and 0·07, respectively.
Conclusions:  Carvacrol and thymol had desired antimicrobial effect on E. coli . The antibacterial effects were attributed to their ability to permeabilize and depolarize the cytoplasmic membrane.
Significance and Impact of the Study:  This study showed the potential use of flow cytometry as a suitable method to investigate the mode of antibacterial action of essential oil components.     

Production of tylosin in solid-state fermentation by Streptomyces fradiae NRRL-2702 and its gamma-irradiated mutant (γ-1)

Aims:  To develop solid-state fermentation system (SSF) for hyper production of tylosin from a mutant γ-1 of Streptomyces fradiae NRRL-2702 and its parent strain.
Methods and Results:  Various agro-industrial wastes were screened to study their effect on tylosin production in SSF. Wheat bran as solid substrate gave the highest production of 2500 μg of tylosin g⁻¹ substrate by mutant γ-1 against parent strain (300 μg tylosin g⁻¹ substrate). The tylosin yield was further improved to 4500 μg g⁻¹ substrate [70% moisture, 10% inoculum (v/w), pH 9·2, 30°C, supplemental lactose and sodium glutamate on day 9]. Wild-type strain displayed less production of tylosin (655 μg of tylosin g⁻¹ substrate) in SSF even after optimization of process parameters.
Conclusion:  The study has shown that solid-state fermentation system significantly enhanced the tylosin yield by mutant γ-1.
Significance and Impact of the Study:  This study proved to be very useful and resulted in 6·87 ± 0·30-fold increase in tylosin yield by this mutant when compared to that of wild-type strain.     

In vivo virulence of Mycoplasma hyopneumoniae isolates does not correlate with in vitro adhesion assessed by a microtitre plate adherence assay

Aims:  Adherence of Mycoplasma hyopneumoniae to the ciliated epithelial cells of the porcine respiratory tract is considered an important first step in the pathogenesis of enzootic pneumonia. It was the aim of this study to verify the usefulness of in vitro adhesion as a virulence marker.
Methods and Results:  Adherence capacity to immobilized cilia from porcine tracheal epithelial cells of three low, two moderately and two highly virulent M. hyopneumoniae field isolates was determined by a microtitre plate adherence assay.
Conclusions:  No significant differences between the isolates were demonstrated.
Significance and Impact of the Study:  The results suggest that mechanisms other than adherence might be responsible for the observed differences in virulence of these field isolates or that the in vitro assay does not adequately reproduce in vivo adherence conditions.     

3-O-methylfunicone, a metabolite of Penicillium pinophilum, inhibits proliferation of human melanoma cells by causing G2 + M arrest and inducing apoptosis

Objectives:  Melanoma cells take advantage of impaired ability to undergo programmed cell death in response to different external stimuli and chemotherapeutic drugs; this makes prevention of tumour progression very difficult. The aim of this study was to demonstrate whether 3- O -methylfunicone (OMF), a metabolite of Penicillium pinophilum , has the ability to arrest cell population growth and to induce apoptosis in A375P (parental) and A375M (metastasis derivatived) melanoma cell lines.
Materials and methods:  Cell proliferation and apoptosis were analysed by flow cytometry, DNA fragmentation, caspase-3 and caspase-9 activation, and PARP-1 cleavage.
Results:  We demonstrated that OMF affected cell proliferation in a time- and dose-dependent manner, reaching the best effect at concentration of 80 µg/ml for 24 h. Flow cytometry revealed that OMF caused significant G₂ phase arrest, which was associated with marked decrease in cyclin B1/p34^cdc2 complex and p21 induction. OMF also induced marked decrease of survivin expression. Reduced levels of apoptosis were evident after silencing p21 expression in both cell lines. Finally, the effect exercised by OMF on hTERT and TEP-1 gene expression confirmed the ability of this molecule to interfere with replicative ability of cells.
Conclusions:  The results reported here seem to suggest that OMF as a promising molecule to include in strategies for treatment of melanoma.     

Antibacterial activity of sphagnum acid and other phenolic compounds found in Sphagnum papillosum against food-borne bacteria

Aims:  To identify the phenolic compounds in the leaves of Sphagnum papillosum and examine their antibacterial activity at pH appropriate for the undissociated forms.
Methods and Results:  Bacterial counts of overnight cultures showed that whilst growth of Staphylococcus aureus 50084 was impaired in the presence of milled leaves, the phenol-free fraction of holocellulose of S. papillosum had no bacteriostatic effect. Liquid chromatography–mass spectrometry analysis of an acetone–methanol extract of the leaves detected eight phenolic compounds. Antibacterial activity of the four dominating phenols specific to Sphagnum leaves, when assessed in vitro as minimal inhibitory concentrations (MICs), were generally >2·5 mg ml⁻¹. MIC values of the Sphagnum- specific compound 'sphagnum acid' [ p -hydroxy-β-(carboxymethyl)-cinnamic acid] were >5 mg ml⁻¹. No synergistic or antagonistic effects of the four dominating phenols were detected in plate assays.
Conclusions:  Sphagnum -derived phenolics exhibit antibacterial activity in vitro only at concentrations far in excess of those found in the leaves.
Significance and Impact of the Study:  We have both identified the phenolic compounds in S. papillosum and assessed their antibacterial activity. Our data indicate that phenolic compounds in isolation are not potent antibacterial agents and we question their potency against food-borne pathogens.     

Factors affecting the recovery of Campylobacter spp. from retail packs of raw, fresh chicken using ISO 10272-1:2006

Aims:  This study sought to determine the most effective protocol for the detection of Campylobacter spp. in retail packs of fresh, raw chicken based on ISO 10272-1:2006.
Methods and Results:  Three sample preparation protocols were studied; two based on excision and one combining excision with a rinse of the remaining sample. Enrichment cultures were incubated both in closed bottles and microaerobically, and sub-cultured at 24 and 48 h. Packs of chicken (110) were analysed and only two yielded no Campylobacter spp. Subculturing enrichment broths at 24 h gave the same prevalence as at 48 h, P  >   0·4 but microaerobic incubation yielded approximately 50% more positive samples than did incubation in closed bottles. Sampling based on excision plus rinsing gave the highest Campylobacter prevalence (92·7%).
Conclusions:  To isolate Campylobacter spp. from retail packs of chicken, enrichment cultures must be incubated in a microaerobic atmosphere and sub-cultured at 24 h and, possibly, 48 h. Sampling packs by excision plus rinsing maximized recoveries.
Significance and Impact of the Study:  ISO 10272-1:2006 permits the use of inefficient protocols which markedly underestimate the true prevalence of Campylobacter spp. in retail, fresh chicken. Equivalent results could be obtained 24 h earlier, with consequent savings. Its revision is essential.     

Investigation of functional and morphological changes in Pseudomonas aeruginosa and Staphylococcus aureus cells induced by Origanum compactum essential oil

Aims:  Evaluation of the cellular effects of Origanum compactum essential oil on Pseudomonas aeruginosa ATCC 27853 and Staphylococcus aureus ATCC 29213.
Methods and Results:  The damage induced by O. compactum essential oil on these two strains has been studied using different techniques: plate count, potassium leakage, flow cytometry (FC) and transmission electron microscopy (TEM). The results showed that oil treatment led to reduction of cells viability and dissipated potassium ion gradients. Flow cytometric analysis showed that oil treatment promoted the accumulation of bis-oxonol and the membrane-impermeable nucleic acid stain propidium iodide (PI), indicating the loss of membrane potential and permeability. The ability to reduce 5-cyano-2,3-ditolyl tetrazolium chloride was inhibited. Unlike in Ps. aeruginosa , membrane potential and membrane permeability in Staph. aureus cells were affected by oil concentration and contact time. Finally, TEM showed various structural effects. Mesosome-like structures were seen in oil-treated Staph. aureus cells whereas in Ps. aeruginosa, coagulated cytoplasmic material and liberation of membrane vesicles were observed, and intracellular material was seen in the surrounding environment. Both FC and TEM revealed that the effects in Ps. aeruginosa were greater than in Staph. aureus .
Conclusions:  Oregano essential oil induces membrane damage showed by the leakage of potassium and uptake of PI and bis-oxonol. Ultrastructural alterations and the loss of cell viability were observed.
Significance and Impact of the Study:  Understanding the mode of antibacterial effect of the oil studied is of a great interest in it further application as natural preservative in food or pharmaceutical industries.     

Sensitivity of Certain Porcine and Bovine Mycoplasmas to Antimicrobial Agents in a Liquid Medium Test Compared to a Disc Assay

The sensitivity of some porcine and bovine mycoplasmas to potent antimicrobial agents was examined. Minimal inhibitory concentration (MIC) values were estimated for M. hyosynoviae, M. hyopneumoniae, M. dispar and M. bovis against enrofloxacin, lincomycin, tetracycline, tiamulin and tylosin, in a liquid medium test and in a disc assay. All 6 examined strains of each species and the respective type strains were significantly inhibited. The greatest sensitivity was noted for tiamulin against strains of M. hyosynoviae with a final MIC50 broth value of 0.025 µg ml−1 and disc value of 0.03 µg per disc. Enrofloxacin was found very potent against M. hyopneumoniae with a final MIC50 of 0.025 µg ml−1 and 0.1 µg per disc, and for M. dispar with 0.05 µg ml−1 and 0.03 µg per disc. Most disc assay estimates in ug per disc were similar to or moderately greater than corresponding final broth figures in µg ml−1. It may be possible to convert observed disc assay values into representative final broth MIC values for use in the clinic.     

Characterization of a novel antibacterial glycopeptide produced by Penicillium sp. M03

Aims:  To isolate a novel antibiotic termed AF from fermentation broth of Penicillium sp. M03 and to examine its antimicrobial activity, biological properties and structure characteristics.
Methods and Results:  Sephadex LH-20 and HPLC were used to purify AF from fermentation broth of Penicillium sp. M03. The antimicrobial activity of AF was evaluated with the agar diffusion test. Amino acid and monosaccharide composition of AF was analysed by a HITACHI 835 detector and HPLC assay, respectively. Matrix-assisted laser desorption time of flight mass spectrometry, FT-IR and ¹H nuclear magnetic resonance spectra analyses were performed to examine the initial structure of AF. Eighty milligrams of AF was isolated as white powder from 1-l Penicillium sp. M03 fermentation broth. It consists of five amino acid and two monosaccharide residues and the molecular weight of it was 1017, and it was stable to β-lactamase, heat, acid and alkali. AF showed inhibitory activity to a wide range of bacteria, particularly to multidrug-resistant Staphylococcus aureus .
Conclusions:  AF was a novel antibacterial glycopeptide with a broad inhibitory spectrum to pathogenic bacteria including multidrug-resistant agents. Furthermore, it is difficult to generate bacteria resistant to AF.
Significance and Impact of the Study:  Characterization of AF made it a potential antibiotic to fight against antibiotic-resistant bacterial pathogens.     

Panose, a new prebiotic candidate

Aims:  To investigate the prebiotic potential of two novel candidates, sophorose and panose, with in vitro methods.
Methods and Results:  The growth of single microbial strains was first assessed for both substrates in pure cultures, and panose was further analysed in the simulated colon model with mixed human faecal culture. Quantitative PCR and flow cytometry were used to determine the microbial group and strain densities after the simulated colonic fermentation of panose, and chromatographic methods were utilized to analyse metabolite concentrations. In pure cultures, sophorose and panose were both fermented only by few beneficial strains, and in the colon simulator, panose gave a significant increase in the numbers of Bifidobacterium and Bifidobacterium lactis , concomitantly decreasing Bacteroides group. Butyrate and acetate production was significantly increased together with decreased markers of protein fermentation as a result of panose fermentation.
Conclusions:  Panose had bifidogenic activities in vitro , and these potential beneficial effects should be further assessed in vitro and in vivo .
Significance and Impact of the Study:  The current study has provided the first data on pure panose fermentation by the endogenous microbiota and extends our knowledge of the selective fermentation of oligosaccharides by the intestinal microbes.     

Use of flow cytometry for enumeration of Mycoplasma mycoides subsp. mycoides large-colony type in broth medium

Aims:  The potential of using flow cytometry (FC) in combination with a fluorescent dye (SYBR green-I) for rapidly estimating Mycoplasma mycoides subSPS. mycoides large-colony type (MmmLC) in broth culture was examined.
Methods and Results:  The FC analysis was performed by staining the MmmLC cells with a fluorescent dye, SYBR green-I (SYBR), and the results were compared with plate count method (colony forming units, – CFUs). There was a good correlation (linear regression, r ² = 0·93) between mycoplasma counts determined by FC (cells ml⁻¹) and by traditional plate count method (CFU ml⁻¹). The lowest bacterial concentration detected by FC and traditional plate count was of the order of 10⁴ cells ml⁻¹ and 10³ CFU ml⁻¹, respectively. FC method allowed results in 20–30 min, whereas at least 24 h were necessary to obtain results with the traditional plate count method (CFU).
Conclusion:  Growth rates of MmmLC in broth medium determined by FC were highly reproducible and correlated well with mycoplasma counts assessed by the plate count method.
Significance and Impact of the Study:  These findings suggest that FC could be a good alternative to replace other time-consuming techniques that are currently used to enumerate mycoplasma in broth medium, such as plate count method (CFU).     

The formation of spores in biofilms of Anoxybacillus flavithermus

Aims:  To examine the rate and the extent of spore formation in Anoxybacillus flavithermus biofilms and to test the effect of one key variable – temperature – on spore formation.
Methods and Results:  A continuous flow laboratory reactor was used to grow biofilms of the typical dairy thermophile A. flavithermus (strain CM) in skim milk. The reactor was inoculated with either a washed culture or a spore suspension of A. flavithermus CM, and was run over an 8·5 h period at three different temperatures of 48, 55 and 60°C. Change in impedance was used to determine the cell numbers in the milk and on the surface of the stainless steel reactor tubes. The biofilm developed at all three temperatures within 6–8 h. Spores formed at 55 and 60°C and amounted to approx. 10–50% of the biofilm. No spores formed at 48°C.
Conclusions:  The results suggest that both biofilm formation and spore formation of A. flavithermus can occur very rapidly and simultaneously. In addition, temperature variation has a considerable effect on the formation of spores.
Significance and Impact of the Study:  This information will provide direction for developing improved ways in which to manipulate conditions in milk powder manufacturing plants to control biofilms and spores of A. flavithermus .     

Identification and characterization of antibiotic resistance genes in Lactobacillus reuteri and Lactobacillus plantarum

Aims:  The study aimed to identify the resistance genes mediating atypical minimum inhibitory concentrations (MICs) for tetracycline, erythromycin, clindamycin and chloramphenicol within two sets of representative strains of the species Lactobacillus reuteri and Lactobacillus plantarum and to characterize identified genes by means of gene location and sequencing of flanking regions.
Methods and Results:  A tet (W) gene was found in 24 of the 28 Lact. reuteri strains with atypical MIC for tetracycline, whereas four of the six strains with atypical MIC for erythromycin were positive for erm (B) and one strain each was positive for erm (C) and erm (T). The two Lact. plantarum strains with atypical MIC for tetracycline harboured a plasmid-encoded tet (M) gene. The majority of the tet (W)-positive Lact. reuteri strains and all erm -positive Lact. reuteri strains carried the genes on plasmids, as determined by Southern blot and a real-time PCR method developed in this study.
Conclusions:  Most of the antibiotic-resistant strains of Lact. reuteri and Lact. plantarum harboured known plasmid-encoded resistance genes. Examples of putative transfer machineries adjacent to both plasmid- and chromosome-located resistance genes were also demonstrated.
Significance and Impact of the Study:  These data provide some of the knowledge required for assessing the possible risk of using Lact. reuteri and Lact. plantarum strains carrying antibiotic resistance genes as starter cultures and probiotics.     

Initial in vitro evaluations of the antibacterial activities of glucosinolate enzymatic hydrolysis products against plant pathogenic bacteria

Aims:  The aim of the study was to evaluate the in vitro antibacterial effects of glucosinolate hydrolysis products (GHP) against plant pathogenic micro-organisms namely Agrobacterium tumefaciens , Erwinia chrysanthemi , Pseudomonas cichorii , Pseudomonas tomato , Xanthomonas campestris and Xanthomonas juglandis .
Methods and Results:  Using a disc diffusion assay, seven different doses of 10 GHP were tested against each bacteria. The results showed that the isothiocyanates were potent antibacterials, whilst the other GHP were much less efficient. Moreover, the antibacterial effects were dose-dependent, increasing with the dose applied; 2-phenylethylisothiocyanate and sulforaphane showed the strongest inhibitory effects. The overall results show a great potential for using the isothiocyanates as an alternative tool to control undesired bacterial growth in plants.
Conclusions:  Glucosinolate hydrolysis products and more specifically the isothiocyanates: benzylisothiocyanate, 2-phenylethylisothiocyanate, the isothiocyanate Mix and sulforaphane, were effective phytochemicals against the in vitro growth of the phytopathogenic bacteria. The antibacterial activity exhibited by these phytochemicals reinforces their potential as alternatives to the traditional chemical control of phytopathogenic bacteria.
Significance and Impact of the Study:  This current in vitro study is the first providing comparative data on GHP as potential control agents for plant pathogenic bacteria. However, more studies are needed to determine their possible allelopathic impacts e.g. inhibition of plant growth and negative effects on beneficial soil bacteria and fungi (mycorrhizae).     

Ecology-based screen identifies new metabolites from a Cordyceps-colonizing fungus as cancer cell proliferation inhibitors and apoptosis inducers

Objectives:  This study aims to identify new anti-cancer agents from Cordyceps -colonizing fungi, using an ecology-based approach. It also aims to explore their anti-cell proliferative mechanisms, and to evaluate their anti-tumour effects in vivo .
Materials and methods:  Extracts from Cordyceps -colonizing fungi were tested on HeLa cells, and active extracts were separated to obtain anti-tumour metabolites; their structures were elucidated by mass and nuclear magnetic resonance spectroscopy. Cell cycle analysis was evaluated using flow cytometry. Tumour formation assays were performed using C57BL/6J mice.
Results:  Based on ecological considerations, the selected extracts were subjected to initial anti-tumour screening. Bioassay-guided fractionation of the active extract afforded two new epipolythiodioxopiperazines, named gliocladicillins A ( 1 ) and B ( 2 ). (A) 1 and B ( 2 ) inhibited growth of HeLa, HepG2 and MCF-7 tumour cells. Further study demonstrated that both preparations arrested the cell cycle at G₂/M phase in a dose-dependent manner, and induced apoptosis through up-regulation of expression of p53, p21, and cyclin B, and activation of caspases-8, -9 and -3. These data imply that gliocladicillins A ( 1 ) and B ( 2 ) induce tumour cell apoptosis through both extrinsic and intrinsic pathways. In addition, in vivo studies showed that they displayed significant inhibitory effects on cell population growth of melanoma B16 cells imlanted into immunodeficient mice.
Conclusions:  Gliocladicillins A ( 1 ) and B ( 2 ) are effective anti-tumour agents in vitro and in vivo and should be further evaluated for their potential in clinical use.     

Use of flow cytometry in oenology to analyse yeasts

Flow cytometry is a rapid method with many microbiological applications. This technique can be used to obtain counts of viable yeasts in 30 min, whereas a 48 h incubation is necessary with plate counts. This rapid method was tested for its suitability to analyse wine yeasts in a multicentre study in our three laboratories. The study compares measurements obtained by flow cytometry and the usual method, in order to test the reliability of the new method. The results obtained were very similar in terms of both the micro-organisms detected and the precision of measurements.     

Control of microbial contamination of Franz diffusion cell receptor phase in the development of transcutaneous breast cancer therapeutics

Aims:  To determine the micro-organism contamination of excised porcine (pig) ear, and evaluate the use of Cyclopore track-etched membranes (CTEM) for preventing ingress into Franz-type diffusion cells.
Methods:  Swabs were taken from four locations and used to inoculate Tryptone Soya Agar (TSA) and Sabouraud Dextrose Agar (SDA) plates. Diffusion cells were assembled to include porcine skin with and without CTEM, and the receptor phase sampled periodically and spread onto plates.
Results:  Five distinct colony types were isolated after incubation of all swabs on TSA plates at 37°C; on SDA plates, one fungal colony was found at 30°C and one at 37°C. The SDA agar plate incubated at 30°C resulted in the growth of a large diffused white fungal colony. No regional differences were observed. Without the CTEM, the receptor phase became contaminated within 6 h. With the CTEM present, microbial ingress was substantially retarded with visible presumptive fungal growth occurring at 24 h and detectable contamination on both microbiological media at 48 h.
Conclusions:  As expected, the native porcine ears were considerably contaminated. The ingress of contamination into the diffusion cell receptor phases can be largely, but not entirely, eliminated using CTEM. The addition of antimicrobial agents was necessary to eliminate micro-organisms that were observed at later time points.
Significance and Impact of the Study:  This article, while highlighting the presence of a high number of micro-organisms on native porcine skin, presents a practical means to reduce the risk of microbial contamination in transdermal/transcutaneous permeation studies, particularly in the study of cell cultures grown within Franz diffusion cell receptor compartments.     

Antimicrobial susceptibility of Clostridium difficile isolated from neonatal pigs with enteritis

The minimum inhibitory concentration (MIC) of eight antimicrobial agents was determined by the agar dilution method for 80 isolates of Clostridium difficile from neonatal pigs with enteritis. MICs(50) for erythromycin, tilmicosin, and tylosin were relatively low (0.25-0.50 microg/mL), but MICs(90) (64 or > or =256 microg/mL) suggest in vivo resistance of a proportion of isolates. Susceptibility to tetracycline varied widely, with MIC(50) and MIC(90) of 8 and 32 microg/mL, respectively. The MICs(90) for tiamulin (8 microg/mL) and virginiamycin (16 microg/mL) suggest moderate susceptibility. Bacitracin and ceftiofur (MICs(90) > or =256 microg/mL) have little activity against C. difficile. Tiamulin and virginiamycin may decrease fecal shedding of C. difficile by sows, and erythromycin, tetracycline, and tylosin may be useful for treatment of infected piglets.     

流式细胞术检测胞内重组耻垢分枝杆菌的活力研究

目的建立一种快速检测胞内分枝杆菌活力的方法。方法将一定量培养至对数生长期的含pMV-eis的重组耻垢分枝杆菌感染U937巨噬细胞,以含空质粒的耻垢分枝杆菌为对照,吞噬作用2 h后洗去胞外细菌,再分别培养4、12、24和48 h后收集细胞并裂解之。获得的胞内细菌用FDA荧光染料染色后用流式细胞仪检测死亡率,并与平板菌落计数法进行比较。结果流式细胞仪检测出感染12 h后重组耻垢分枝杆菌胞内死亡率较对照组均有显著下降（P〈0.05）,流式细胞仪检测法与平板菌落计数法相比差异无统计学意义（P〉0.05）。结论流式细胞术与传统的平板计数法相比具有快速、敏感、方便的特点,可用于分枝杆菌活菌快速检测。