Sensitive Indicator Strain Research Articles

Reuterin Demonstrates Potent Antimicrobial Activity Against a Broad Panel of Human and Poultry Meat Campylobacter spp. Isolates.

Reuterin is a broad-spectrum antimicrobial system produced by specific strains of Lactobacillus reuteri during anaerobic metabolism of glycerol. Acrolein is the main component responsible for its antimicrobial activity. Here, the sensitivity of Campylobacter jejuni (n = 51) and Campylobacter coli (n = 20) isolates from chicken meat and human stool samples to reuterin was investigated. The minimum inhibitory concentration (MIC) of C. jejuni and C. coli strains was measured between 1.5 and 3.0 µM of acrolein, below the MIC of the sensitive indicator strain Escherichia coli K12 (16.5 µM acrolein). The interaction of C. jejuni N16-1419 and the reuterin-producing L. reuteri PTA5_F13 was studied during 24 h co-cultures with or without glycerol. A high C. jejuni growth was observed in cultures without glycerol. In contrast, C. jejuni growth decreased from 7.3 ± 0.1 log CFU/mL to below detection limit (1 log CFU/mL) during co-cultures added with 28 mM glycerol. This bactericidal effect could be attributed to in situ reuterin production. The low MIC observed and the high sensitivity towards in situ produced reuterin suggests L. reuteri combined with glycerol, as a possible intervention option to reduce Campylobacter in the food chain.

POSSIBILITIES OF APPLICATION OF MALDI-TOF MASS-SPECTROMETRY FOR STUDY OF CARBOHYDRATE-SPECIFIC RECEPTORS FOR DIAGNOSTIC BACTERIOPHAGE EL TOR

Study mechanisms of interaction of diagnostic bacteriophage El Tor with sensitive strain Vibrio cholerae El Tor 18507 using direct protein profiling, identification of constant and variable proteins, taking part in interaction of the phage and cell, as well as carbohydrate-specific phage receptors. . A commercial preparation of cholera diagnostic bacteriophage El Tor, strain V. cholerae El Tor 18507 were used. Effect of carbohydrates on bacteriophage activity was determined in experiments with phage by a classic and modified by us method. Protein profiles of the studied objects were studied using MSP-analysis method. Sucrose was shown to inhibit lytic activity of bacteriophage. Proteome profiles of El Tor bacteriophage and sensitive indicator strains were studied, identification of constant and variable proteins of the studied objects by MSP Peak-list program was carried out. Analysis of changes of profiles of phage and microbial cell during interaction with sucrose gave a basis for assuming, that sucrose in the mixture of culture-phage enters interaction namely with phage protein receptors, blocking receptors specific for cholera vibrio, that subsequently manifests in a sharp decrease of phage activity against the sensitive strain.

Microbial growth inhibition caused by Zn/Ag-Y zeolite materials with different amounts of silver

Different loadings of silver exchanged on bimetallic Zn/Ag-NaY zeolite materials were studied for antimicrobial properties against four reference microorganisms. The sensitive indicator strains used were two bacteria (Escherichia coli and Bacillus subtilis) and two yeast species (Saccharomyces cerevisiae and Candida albicans). The bimetallic materials were compared with the monometallic materials prepared with the same concentrations of silver. A synergistic effect between the two metals, zinc and silver, was evidenced on the antimicrobial activity of the materials. All mono and bimetallic materials showed strong efficacy against bacteria and yeasts, although the later overall displayed lower MIC values. The results of X-ray photoelectron spectroscopy (XPS) confirm the presence of silver and zinc as ions, not homogeneously distributed throughout the zeolite framework, which implies that the metal ions are located in different sites of the faujasite structure.

In vitro inhibition of Paenibacillus larvae by different extracts and pure compounds from Flourensia spp

Paenibacillus larvae, a sporulating Gram-positive bacterium, is the etiological agent of American foulbrood disease in Apis mellifera L. Plant extracts could be a natural alternative to control this pathology. The current study assessed the anti-P. larvae effect of extracts and pure principal products from the Flourensia genus: F. riparia, F. fiebrigii and F. tortuosa. Their inhibitory effect was assayed against different P. larvae strains according to the disk diffusion technique and subsequently, the minimal inhibitory concentrations (MIC) of extracts by the agar dilution method was determined. Furthermore, toxicity of the most effective extracts against P. larvae was tested in bees. All extracts inhibited growth of the different P. larvae strains assayed. However, the magnitude of the antagonistic effect depended on the chemical nature of the extract and the P. larvae strain. Chloroform extracts (CE) and ethyl ether extracts (ETE) from F. riparia and ETE from F. fiebrigii were most active against P. larvae Azul, the most sensitive indicator strain with MIC values of 250ppm (CE) and 2000ppm (ETE) for F. riparia, and 2500ppm (ETE) for F. fiebrigii. Hexane extracts from the three species did not present any significant inhibitory effect. These results would indicate that one or some of the more polar compounds would cause inhibition of this pathogen. Toxicity assays demonstrated that even the highest concentrations assayed (125,000ppm) did not show lethal effects on exposed bees during in vitro conditions.

Preparation, characterization, and antibacterial activity of shell waste loaded with silver

A green inorganic antibacterial material was prepared using shell waste as a carrier material loaded with silver. It was characterized and analyzed by X-ray diffraction (XRD), scanning electron microscopy equipped with an energy dispersive spectrometer (EDS), X-ray photoelectronic spectroscopy (XPS) and N2 adsorption isotherms. The antibacterial activity was evaluated using Staphylococcus aureus and Escherichia coli as sensitive indicator strains. The antibacterial mechanism was probed and discussed as well. Silver carbonate was detected in the prepared material through XRD analysis. XPS measurement and EDS analysis also confirmed the loading of silver onto the carrier. The antibacterial test demonstrated that the prepared material had good antibacterial property, especially against E. coli. Based on the silver ion release and pH test, as well as comparatively analyzing the characteristics of carrier material and prepared material, we proposed that the antibacterial mechanism mainly involved the antibacterial activity of silver ion, slightly higher pH value and supplementary photocatalytic antibacterial activity of silver carbonate.

Antimicrobial activity of faujasite zeolites doped with silver

The preparation, characterization and antimicrobial activity of faujasite (FAU) zeolites (NaY and NaX) doped with silver are reported. The antimicrobial activity properties were achieved in both FAU zeolites by ion exchange method. The characterization was performed by X-ray diffraction (XRD), Scanning electron microscopy (SEM), X-ray photoelectron spectroscopy (XPS), Fourier transformed infrared spectroscopy (FTIR) and chemical analyses. The characterization results indicate that silver were interchanged in the structure of FAU zeolites. Antimicrobial potential of silver zeolites (AgY and AgX) was evaluated using bacteria (Escherichia coli and Bacillus subtilis) and yeast (Saccharomyces cerevisiae and Candida albicans) as sensitive indicator strains. The detected antimicrobial properties were generated by the silver present in the zeolite structure, as confirmed by XPS analysis and antimicrobial activity assays. Both zeolites showed good antimicrobial properties, in particular antibacterial activity, but AgY displayed lower minimum inhibitory concentration (MIC) values than AgX.

Development and in vitro evaluation of an Escherichia coli probiotic able to inhibit the growth of pathogenic Escherichia coli K881

Weaned piglets commonly suffer from gastroenteritis caused by enterotoxigenic Escherichia coli K88. Our aim was to produce E. coli strains that inhibited the growth of E. coli K88 and could be used as a probiotic against postweaning diarrhea. The inclusion criteria for the probiotics were that in addition to being able to inhibit E. coli K88, they also needed to be negative for virulence genes commonly associated with E. coli. A total of 463 E. coli isolates from the cattle rumen, cattle feces, swine feces, and soil were screened against 18 E. coli K88 clinical isolates using an agar diffusion technique. Growth inhibition of the most sensitive K88 indicator strain 2-12 occurred for 121/463 isolates: 96/358 from cattle feces, 0/33 from rumen fluid, 9/35 from swine feces, and 16/37 from soil. Of the 121 positive strains, 71/121 were negative for toxin genes (LT, STa, STb, VT1, and VT2). The 14 most inhibitory strains were screened against a range of substrates to assess the ability to utilize carbohydrates that could be included in the diet to enhance their ability to compete in the gut. Two strains, UM-2 and UM-7, were weak utilizers of starch and inulin. In vitro competition assays between the probiotic strains and E. coli K88 strain 2-12 were conducted with glucose as the only carbon source (minimal medium; MM), MM + 2% starch, or MM + 2% inulin. The UM-2 and UM-7 strains were able to outcompete strain 2-12 when glucose was the only carbon source, indicating that inhibitory activity was produced against 2-12 independent of carbon source. The UM-2 strain outcompeted strain 2-12 in assays in which potato starch or inulin was the only carbon source; the ability of 2-12 to maintain its concentrations in the culture were probably the result of cross feeding of breakdown sugars of starch and inulin that could be utilized by 2-12. In contrast, UM-7 did not grow as well as UM-2 on starch and inulin and 2-12 declined rapidly in successive cultures likely because of the lack of breakdown products of starch and inulin produced by UM-7. We conclude that probiotic E. coli without known toxins and that produce inhibitory activity against E. coli that cause postweaning diarrhea can be produced. In addition, the ability to utilize starch or inulin is an important phenotype because it likely gives the probiotic a competitive advantage in the gut.

Purification and characterization of enterocin 62-6, a two-peptide bacteriocin produced by a vaginal strain ofEnterococcus faecium: Potential significance in bacterial vaginosis

A bacteriocin produced by a vaginal isolate of Enterococcus faecium strain 62-6, designated enterocin 62-6, was characterized following purification and DNA sequence analysis and compared to previously described bacteriocins. Enterocin 62-6 was isolated from brain heart infusion (BHI) culture supernatants using ammonium sulfate precipitation followed by elution from a Sepharose cation exchange column using a continuous salt gradient (0.1-0.7 M NaCl). SDS-PAGE of an active column fraction resulted in an electrophoretically pure protein, which corresponded to the growth inhibition of the sensitive Lactobacillus indicator strain in the gel overlay assay. Purified enterocin 62-6 was shown to be heat- and pH-stable, and sensitive to the proteolytic enzymes alpha-chymotrypsin and pepsin. Results from mass spectrometry suggested that it comprised two peptides of 5206 and 5219+/-1 Da, which was confirmed by DNA sequence analysis. The characteristics of enterocin 62-6 as a small, heat- and pH-stable, cationic, hydrophobic, two-peptide, plasmid-borne bacteriocin, with an inhibitory spectrum against a broad range of Gram-positive but not Gram-negative bacteria, were consistent with its classification as a class IIc bacteriocin. Furthermore, its wide spectrum of growth inhibitory activity against Gram-positive bacteria of vaginal origin including lactobacilli, and stability under the acidic conditions of the vagina, are consistent with our hypothesis that it could have potential significance in disrupting the ecology of the vaginal tract and pave the way for the establishment of the abnormal microbiota associated with the vaginal syndrome bacterial vaginosis. This is the first class IIc bacteriocin produced by a strain of E. faecium of vaginal origin to be characterized.

A lacticin 3147 enriched food ingredient reduces Streptococcus mutans isolated from the human oral cavity in saliva

To isolate and characterise Streptococcus mutans from Irish saliva samples and to assess their sensitivity to a food-grade preparation of the lantibiotic, lacticin 3147, produced by Lactococcus lactis DPC3147. Saliva samples collected from children with varying oral health status were screened on Mitis Salivarius agar for the presence of pathogenic streptococci. Following selective plating, 16S rDNA sequencing and Pulsed Field Gel Electrophoresis (PFGE), 15 distinct strains of Strep. mutans were identified. These were grouped according to their relative sensitivity to lacticin 3147 which ranged from 0.78 to 6.25%; relative to a sensitive indicator strain, Lactococcus lactis ssp. lactis HP. Inhibition of indicator Strep. mutans strains from sensitive, intermediate and tolerant groupings were assessed in microtitre plate assays with increasing concentrations of lacticin 3147. The concentration of lacticin 3147 required to give 50% growth inhibition correlated with their relative sensitivities (as assayed by well diffusion methodology) and ranged from 1280 to 5120 AU ml(-1). Concentrated preparations of lacticin 3147 caused a rapid killing of Strep. mutans strains in broth. Moreover, in human saliva deliberately spiked with Strep. mutans, the pathogen was eliminated (initial inoculum of 10(5)) in the presence of 40,000 AU ml(-1) of lacticin 3147. Furthermore, a food-grade lacticin 3147 spray dried powder ingredient was assessed for the inhibition of Strep. mutans in human saliva, spiked with a strain of intermediate sensitivity, resulting in up to a 4-log reduction in counts after 20 min. A food grade preparation of lacticin 3147 was effective in the inhibition of oral Strep. mutans. The inhibition of oral streptococci by food grade preparations of lacticin 3147 may offer novel opportunities for the development of lacticin 3147 as an anti-cariogenic agent particularly in the area of functional foods for the improvement of oral health.

Bacteriocin-Like Substances from Tomato Race 3 Strains of Xanthomonas campestris pv. vesicatoria

ABSTRACT Tomato race 3 (T3) strains of Xanthomonas campestris pv. vesicatoria are antagonistic in vitro to tomato race 1 (T1) strains of the bacterium. All T1 strains and 11 strains of other X. campestris pathovars tested were inhibited by T3 strains. Sensitivity of tomato race 2 (T2) strains was variable. No strains from other bacterial genera tested were inhibited. Cell-free filtrates from T3 strains were inhibitory to sensitive strains. The inhibitory activity of these filtrates was lost after treatment at temperatures above 80 degrees C and with selected protease enzymes. However, treatment with trypsin or DNase had no effect on their activity. Seven cosmid clones from a genomic library of a T3 strain were selected for their ability to consistently inhibit a sensitive indicator strain in plate assays. Southern hybridization analysis placed these into three bacteriocin (BCN)-producing groups designated BCN-A, BCN-B, and BCN-C. The BCN-like groups could be differentiated by variations in inhibitory spectra and levels of activity in plate assays. Mutations that inactivated expression of each BCN group individually in a wild-type T3 strain had inhibitory activity confirming that multiple BCNs are present in the T3 strain. T3 strains were inhibitory to a sensitive indicator strain in tomato leaf tissue, but this effect was observed only when T3 strains were applied in advance of the sensitive strain. BCN-A was the major BCN-like substance involved in the suppression of the sensitive indicator strain in tomato leaf tissues.

Partial characterization of bacteriocins produced by environmental strain Enterococcus faecium EK13

The partial characterization of bacteriocins produced by an environmental strain Enterococcus faecium EK13, isolated from cattle dung water. A bacteriocin was partially purified by ammonium sulphate precipitation, followed by a SP-Sepharose column, reverse-phase chromatography and N-terminal region sequenced. The anti-microbial substance produced was found to be a heat-stable polypeptide with molecular mass 4.83 kDa, which was determined by N-terminal amino acid sequencing to be enterocin A. A second substance was specified by PCR as enterocin P. Bacteriocins were stable at 4 and -20 degrees C for long storage periods. The optimum of bacteriocin production was observed in the range of pH 5.0-6.5 at 30 and 37 degrees C. The most active substances are produced by strain EK13 in logarithmic growth phase and bacteriocins are produced after 1 h of fermentation. The highest activity detected in fermentation experiments was 51 200 AU ml(-1) and the most sensitive indicator strain was found to be Listeria innocua LMG 13568. Differences in bacteriocin activity against two indicators could be explained by more than one type of enterocin production by strain EK13, or with different mode of action or in different sensitivity of strains. Enterococcus faecium strain EK13 isolated from cattle dung water produces two bacteriocins, enterocin A and P, with an inhibitory effect against the strain of the genera Enterococcus, Leuconostoc, Lactobacillus, Streptococcus, Staphylococcus, Bacillus and Listeria (in different origin). Enterococcus faecium EK13 environmental strain is a new producer of enterocin A and P. The E. faecium EK13, isolated from cattle dung water, is presented with the further aim to utilize it for waste treatment by biotechnological processes.

Macedocin, a food-grade lantibiotic produced by Streptococcus macedonicus ACA-DC 198.

Streptococcus macedonicus ACA-DC 198, a strain isolated from Greek Kasseri cheese, produces a food-grade lantibiotic named macedocin. Macedocin has a molecular mass of 2,794.76 +/- 0.42 Da, as determined by electrospray mass spectrometry. Partial N-terminal sequence analysis revealed 22 amino acid residues that correspond with the amino acid sequence of the lantibiotics SA-FF22 and SA-M49, both of which were isolated from the pathogen Streptococcus pyogenes. Macedocin inhibits a broad spectrum of lactic acid bacteria, as well as several food spoilage and pathogenic bacteria, including Clostridium tyrobutyricum. It displays a bactericidal effect towards the most sensitive indicator strain, Lactobacillus sakei subsp. sakei LMG 13558(T), while the producer strain itself displays autoinhibition when it is grown under conditions that do not favor bacteriocin production. Macedocin is active at pHs between 4.0 and 9.0, and it retains activity even after incubation for 20 min at 121 degrees C with 1 atm of overpressure. Inhibition of macedocin by proteolytic enzymes is variable.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot)

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson's index and the Shannon-Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Characterisation of the culturable heterotrophic bacterial community in a small eutrophic lake (Priest Pot)

The community composition and structure of planktonic heterotrophic bacteria (903 isolates) sampled from a small eutrophic lake in northern England (Priest Pot) was studied with respect to season (four samples) and depth (to 3.1 m). Bacteria (887) were isolated on tryptic soy broth agar and identified to 48 genera using fatty acid methyl ester analysis. The two most abundant genera isolated were Aeromonas and Pseudomonas which, respectively, dominated the middle to bottom depths in August and all depths in February. The structure of the sampled community was described using: species richness, Simpson’s index and the Shannon–Wiener index. All three indices detected a number of significant differences with depth demonstrating stratification. The greatest stratification of the bacterial community was observed in August when bacterial counts correlated strongly and negatively with diversity. Using structural measures was found to be preferable to the use of species frequencies in the analysis of perturbation and succession in community structure. Insensitivity to one or more of eight antibiotics was observed in 71% (61/86) of the isolates tested particularly in Gram-negative genera. Bacteriocinogeny and lysogeny was observed in 36% (32/90) of isolates. Using sensitive indicator strains, two of 10 producing strains produced virus, while the others produced bacteriocins.

Bioassay for the rapid detection of bacteriocins in fermentation broth

A bioassay for the rapid detection of bacteriocins by detecting efflux of K+ ions from a bacteriocin-sensitive indicator strain was developed. There was a rapid increase in concentration of K+ in the medium from approximately 1.6 to 9.5 mM when the crude bacteriocin was added to the sensitive strain. A bacteriocin activity of 8.25 AU ml−1 produced the lowest detectable concentration of K+ (0.179 mM) and the smallest inhibition zone (0.4 mm). The smallest cell dry mass of the indicator strain capable of releasing a detectable level of K+ (0.102 mM) was 0.76 mg. Detection of the bacteriocin by measuring the efflux of K+ compares well with the conventional agar well diffusion assay. Low activities of bacteriocin and low amounts of the sensitive indicator strain biomass can be used with this bioassay.

Incidence of Inducible Bacteriophage in Lactobacillus helveticus Strains Isolated from Natural Whey Starter Cultures

We studied the incidence of inducible Lactobacillus helveticus strains among 74 isolates from natural whey starter cultures that are used for the production of Italian Grana and Provolone cheeses. Mitomycin C was used for induction of cultures. Of the 77 strains tested, 35 strains (47%) released supernatant particles in the culture that were able to inhibit up to a maximum of 47 (61%) sensitive indicator strains. The presence of bacteriophages in culture supernatants of L. helveticus strains LH38 and LH60 was verified by electron microscopy. None of the positive culture supernatants formed plaques, and inhibition zones were observed only on agar plates that had been inoculated with the indicator strains. Fourteen of the 17 positive culture supernatants were able to propagate on some indicator strains, indicating the presence of completely infective virions in liquid medium. The particles were insensitive to proteolytic enzymes, were unable to pass through a microfiltration membrane, were sensitive to heat, and were sedimented by ultracentrifugation. Some of these particles showed killer activity against nonproliferating cells of indicator strains. The lysogenic state of the L. helveticus strains was related to a stronger resistance of these strains to the products of the induction; higher prevalence of indicator strains were found among the noninducible strains.

Influences on the antimicrobial activity of surface-adsorbed nisin.

The efficacy of the antimicrobial peptide nisin was examined after adsorption to silica surfaces. Three protocols were used to evaluate nisin's activity against adhered cells of Listeria monocytogenes: bioassay using Pediococcus pentosaceous FBB 61-2 as the sensitive indicator strain; visualization and enumeration of cells by microscopic image analysis; and viability of adhered cells as determined by lodonitrotetrazolium violet uptake and crystallization. The activity of adsorbed nisin was highly dependent upon conditions of adsorption. The highest antimicrobial activity of adsorbed nisin occurred with high concentrations of nisin (1.0 mg ml-1) and brief contact times (1 h) on surfaces of low hydrophobicity. Sequential adsorption of a second protein (beta-lactoglobulin or bovine serum albumin) onto surfaces consistently resulted in decreased nisin activity. These data provide direction for the development of applications to limit microbial attachment on food contact surfaces through the use of adsorbed antimicrobial peptides.

A Milk-Based Method for Detecting Antimicrobial Substances Produced by Lactic Acid Bacteria

A new technique for the detection of antimicrobial substances produced by lactic acid bacteria has been developed. In this technique, milk agar plates were supplemented with tetrazolium chloride or tetrazolium blue dyes. Comparisons of milk agar assays with M17 agar plates indicated that, out of 30 bacterial strains, 13 strains produced bacteriocins or inhibitory substances that were detectable on milk agar plates but not on M17 agar plates. Multiple-strain lactococcal cultures are used in milk fermentations. To identify suitable strains to combine for industrial use, component strains must be tested for compatibility. The procedure described allows optimization of compatibility. The assay of putative producer and sensitive indicator strains using milk agar plates (11% nonfat dry milk plus .8% agar and .02% tetrazolium chloride or tetrazolium blue) provides an important tool to prevent allopathic interactions in mixed cultures.

Direct detection of lactococcal bacteriophages in cheese whey using DNA probes

We have designed a new method for the rapid detection of lactococcal phage directly in milk samples. The method was based on a dot blot analysis and did not require a biological assay with sensitive indicator strains. Culture media or whey permeate samples containing phage were spotted directly onto a nylon membrane and the phage were lysed in situ prior to hybridization. For skim milk, whole milk and whey samples, the samples were first treated with 50 mM EDTA, 20% Triton X-100 and heated at 60°C for 5 min, prior to spotting on the membrane. The detection limit was approximately between 10 5 and 10 7 pfu/spot. A large number of samples could be processed simultaneously and the results were obtained within 24 h.

Direct detection of lactococcal bacteriophages in cheese whey using DNA probes

We have designed a new method for the rapid detection of lactococcal phage directly in milk samples. The method was based on a dot blot analysis and did not require a biological assay with sensitive indicator strains. Culture media or whey permeate samples containing phage were spotted directly onto a nylon membrane and the phage were lysed in situ prior to hybridization. For skim milk, whole milk and whey samples, the samples were first treated with 50 mM EDTA, 20% Triton X-100 and heated at 60°C for 5 min, prior to spotting on the membrane. The detection limit was approximately between 105 and 107 pfu/spot. A large number of samples could be processed simultaneously and the results were obtained within 24 h.