Further development of a bacteriocin typing system for Clostridium perfringens

The specificity of typing Clostridium perfringens with bacteriocins was improved by adding new bacteriocins and deleting others from the original typing set of ten. A total of 516 new isolates of Cl. perfringens were screened for bacteriocin production and, of these, 162 strains (31%) were found to be producers. The sensitivity patterns obtained by testing 40 bacteriocins against 200 isolates of Cl. perfringens were recorded and the data subjected to a computer analysis. A total of 18 bacteriocins capable of dividing the 200 isolates into 98 typing patterns was selected. The repro-ducibility of the new system was tested by performing three sequential typings of 60 strains of Cl. perfringens. No variation was found in 73% of the strains, while a further 16% of the strains demonstrated a change in sensitivity to only one bacteriocin. Common serological types of Cl. perfringens were divisible into subtypes based upon both their ability to produce bacteriocins and their sensitivity to bacteriocins, suggesting a useful role for bacteriocin typing in conjunction with an already well-established tool for typing Cl. perfringens.     

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak.

Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3-5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro, contained no plasmids, and was of a common bacteriocin type and serotype.     

Bacteriocin Susceptibility of Clostridium perfringens: a Provisional Typing Schema

Ninety-four strains of Clostridium perfringens were examined for bacteriocin production. Bacteriocins produced by ten of these strains were selected for typing 274 cultures of C. perfringens. The bacteriocins were prepared by growing the producer strains in broth and precipitating the active principle from the supernatant fluids of centrifuged cultures with ammonium sulfate. All bacteriocins were titrated against a common indicator strain, adjusted to equivalent titers, and spotted onto blood agar plates seeded with the test organisms. Fifty different bacteriocin sensitivity patterns were observed. These patterns were organized into seven groups bearing some relationship, and the largest number of strains falling into any one pattern did not exceed 16% of the total strains tested. Ninety-nine percent of all isolates were typable. The new method should prove useful in studies where strains must be fingerprinted.     

Multiple typing techniques applied to a Clostridium perfringens food poisoning outbreak

D.E. MAHONY, R. AHMED AND S.G. JACKSON, 1992. Twenty-one stool specimens obtained from persons implicated in two food poisoning outbreaks at the same institution in Smith Falls, Ontario, were examined for Clostridium perfringens. Ninety-two colonies of Cl. perfringens (3–5 per stool specimen) were typed with antisera, bacteriocins and by plasmid analysis. They were also tested for the in vitro production of bacteriocin and enterotoxin. Sixteen of the 21 stool specimens were tested directly for enterotoxin. This was detected in 13, five of which were from individuals listed as 'asymptomatic' food handlers. The predominant strain isolated from 15 of the 21 stool samples produced bacteriocin and enterotoxin in vitro , contained no plasmids, and was of a common bacteriocin type and serotype.     

A simple device for growing Clostridium perfringens and its application in bacteriocin studies

A simple device constructed of common laboratory material served as a minifermenter for the growth of Clostridium perfringens. A constant flow of nitrogen gas into a culture tube containing C. perfringens assured agitation of the culture and a mechanism for dispensing small volumes of liquid from the culture without disturbing the growth environment. The method was applied to examining the growth-inhibiting effect of bacteriocins of C. perfringens where a very economical use of radioactive isotopes was possible. The activity of some bacteriocins differed when compared with previous data obtained with stationary cultures. Two major categories of bacteriocin appear to exist for this species: those bacteriocins which block the incorporation of DNA, RNA, and protein precursors and those which interfere with the organism's cell wall.     

Characterization of two bacteriocins produced by Clostridium perfringens

Two types of bacteriocins were shown to be produced in succession by a strain of Clostridium perfringens SN-17. They were separated by diethylaminoethyl cellulose (DEAE) column chromatography at pH 8.5 with a linear concentration gradient of NaCl. One type of bacteriocin (named SN-a) was eluted at 0.07 M and the other type (named SN-b) was at 0.12 M. Each of these was partially purified in a series of column chromatographies: DEAE, Sephadex G-200 (or Bio Gel P-150), and hydroxyapatite. Specific activities of SN-a and SN-b after the last chromatography were at most 30- to 50-fold that of culture filtrate of the organisms. Chromatographed SN-a migrated as a single zone in polyacrylamide gel electrophoresis (PAGE) and the zone showed high biological activity. On the other hand, PAGE pattern of SN-b revealed the presence of a few contamination materials. The activity of SN-b after the last chromatography was hardly recovered from the gel but inactivated SN-b was identified in the gel by examining bacteriocin activity of the DEAE fractions recovered from the gel. The molecular weight of the SN-a and SN-b was determined to be about 70,000 and 100,000, respectively, by molecular sieve chromatography. These bacteriocins were very sensitive to protease but insensitive to DNase and RNase. Bacteriocins were both completely inactivated at 55 C and they were more stable in alkaline pH than in acidic pH. SN-a and SN-b were adsorbed in different ways on the surface of the producer and insensitive strains. Several differences and similarities between these 2 bacteriocins are discussed with special reference to the relationship between them.     

Factors affecting the adsorption of bacteriocins ST194BZ and ST23LD to Lactobacillus sakei and Enterococcus sp

Bacteriocins ST194BZ and ST23LD, produced by Lactobacillus plantarum, inhibit Gram-positive and Gram-negative bacteria. Images obtained by atomic force microscopy showed clear signs of membrane damage of Lactobacillus sakei, accompanied by the leakage of DNA and beta-galactosidase. Adsorption of the bacteriocins to cells was increased when cells were treated with buffers at pH values above neutral. An increase in bacteriocin ST194BZ adsorption to cells of Enterococcus sp. and L. sakei was observed with an increase in incubation temperatures, but at different rates for the two species. Treatment of the two species with various inorganic salts and solvents gave different results regarding the adsorption of the two bacteriocins. In general, pre-treatment of the two sensitive cells with Triton X-100, Triton X-114 and chloroform increased the adsorption of the two bacteriocins. Increased adsorption of bacteriocin ST23LD to L. sakei was recorded when the cells were pre-treated with Tris and NH4-citrate. Treatment of Enterococcus sp. and L. sakei with Na-EDTA and SDS decreased the adsorption of the two bacteriocins. Variable results were recorded with inorganic salts.     

Bacterioides fragilis: production and sensitivity to bacteriocins

Bacteriocin production and sensitivity to bacteriocins have been successfully applied as an epidemiological tool in several species of bacteria. However, little work has been carried out on the bacteriocins produced by Bacteroides fragilis, which is the most frequently isolated anaerobe species from clinical specimens. Thirty two clinical isolates of B. fragilis grown anaerobically on a 0.22 microm membrane filter spotted on an agar plate, were tested for bacteriocin production and used in a screen for bacteriocin sensitivity. Sensitivity to at least one bacteriocin was found in 94% of the strains, 62.5% were sensitive to two bacteriocins, whereas 34.4% were sensitive to three or more and finally one strain was found sensitive to 17 bacteriocins. Of the strains studied, 94% inhibited at least one strain, 66% inhibited two strains, and 30% inhibited at least three strains or more. Finally, one strain was extremely active by inhibiting the growth of 17 strains. Bacteriocin types are characterised by geographic variation, and their epidemiological investigation by a simple method could be promoted.     

Structure and Mode-of-Action of the Two-Peptide (Class-IIb) Bacteriocins

This review focuses on the structure and mode-of-action of the two-peptide (class-IIb) bacteriocins that consist of two different peptides whose genes are next to each other in the same operon. Optimal antibacterial activity requires the presence of both peptides in about equal amounts. The two peptides are synthesized as preforms that contain a 15–30 residue double-glycine-type N-terminal leader sequence that is cleaved off at the C-terminal side of two glycine residues by a dedicated ABC-transporter that concomitantly transfers the bacteriocin peptides across cell membranes. Two-peptide bacteriocins render the membrane of sensitive bacteria permeable to a selected group of ions, indicating that the bacteriocins form or induce the formation of pores that display specificity with respect to the transport of molecules. Based on structure–function studies, it has been proposed that the two peptides of two-peptide bacteriocins form a membrane-penetrating helix–helix structure involving helix–helix-interacting GxxxG-motifs that are present in all characterized two-peptide bacteriocins. It has also been suggested that the membrane-penetrating helix–helix structure interacts with an integrated membrane protein, thereby triggering a conformational alteration in the protein, which in turn causes membrane-leakage. This proposed mode-of-action is similar to the mode-of-action of the pediocin-like (class-IIa) bacteriocins and lactococcin A (a class-IId bacteriocin), which bind to a membrane-embedded part of the mannose phosphotransferase permease in a manner that causes membrane-leakage and cell death.     

Characterization of bacteriocin 28 produced by Clostridium perfringens

Bacteriocin 28, produced by Clostridium perfringens, was characterized by gel filtration and sodium dodecyl sulfate - polyacrylamide gel electrophoresis as a glycoprotein with a molecule weight of approximately 100,000. Density gradient centrifugation suggested a lower weight of 84,000. The bacteriocin bound firmly to phenyl-Sepharose CL-4B gel, indicating hydrophobic properties, and elution from this gel with ethylene glycol clearly separated bacteriocin from the alpha and theta toxins of C. perfringens, the latter of which was also hydrophobic. Bacteriocin 28 was immunogenic, inducing neutralizing and precipitating antibodies, and possessed three isoelectric points: 7.37, 7.05, and 5.4. Amino acid and carbohydrate analysis of the active material showed a composition of 15 amino acids and several carbohydrates. The molecule demonstrated instability with increasing purification, and several approaches to purification are described.     

Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A

AIMS: The aim of this research was to investigate the production of bacteriocins by Bacillus spp. isolated from native soils of south of Brazil. METHODS AND RESULTS: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified. The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited. Bacteriocin was stable at 80 degrees C, but the activity was lost when the temperature reached 87 degrees C. It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6.0-9.0. CONCLUSIONS: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food. SIGNIFICANCE AND IMPACT OF STUDY: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.     

产气荚膜梭菌实时荧光PCR方法的建立

目的:利用荧光定量PCR技术,建立快速敏感特异的检测产气荚膜梭菌的方法。方法:以产气荚膜梭菌基因为靶序列设计引物和探针,以自产气荚膜梭菌菌株中提取的DNA为模板,优化引物和探针的浓度比,同时验证方法的特异性、敏感性。结果:建立的反应体系在上游引物浓度为0.45μmol/L、下游引物浓度为0.15μmol/L、探针浓度为0.3μmol/L时,具有良好的特异性和敏感性,与创伤弧菌等12种相关细菌均无交叉反应;对纯菌检测的灵敏度低于10 CFU/反应体系。结论:建立的实时荧光PCR方法特异、灵敏、快速,能对战时气性坏疽做出快速准确的报告,实现对这种战时高发疾病的安全、快速和定量检测。     

Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.     

Mechanisms of resistance to bacteriocins targeting the mannose phosphotransferase system

The membrane proteins IIC and IID of the mannose phosphotransferase system (Man-PTS) together form a membrane-located complex that serves as a receptor for several different bacteriocins, including the pediocin-like class IIa bacteriocins and the class IIc bacteriocin lactococcin A. Bacterial strains sensitive to class IIa bacteriocins readily give rise to resistant mutants upon bacteriocin exposure. In the present study, we have therefore investigated lactococcin A-resistant mutants of Lactococcus lactis as well as natural food isolates of Listeria monocytogenes with different susceptibilities to class IIa bacteriocins. We found two major mechanisms of resistance. The first involves downregulation of Man-PTS gene expression, which takes place both in spontaneous resistant mutants and in natural resistant isolates. The second involves normal expression of the Man-PTS system, but the underlying mechanism of resistance for these cells is unknown. In some cases, the resistant phenotype was linked to a shift in the metabolism; i.e., reduced growth on glucose due to reduction in Man-PTS expression was accompanied by enhanced growth on another sugar, such as galactose. The implications of these findings in terms of metabolic heterogeneity are discussed.     

The continuing story of class IIa bacteriocins.

Many bacteria produce antimicrobial peptides, which are also referred to as peptide bacteriocins. The class IIa bacteriocins, often designated pediocin-like bacteriocins, constitute the most dominant group of antimicrobial peptides produced by lactic acid bacteria. The bacteriocins that belong to this class are structurally related and kill target cells by membrane permeabilization. Despite their structural similarity, class IIa bacteriocins display different target cell specificities. In the search for new antibiotic substances, the class IIa bacteriocins have been identified as promising new candidates and have thus received much attention. They kill some pathogenic bacteria (e.g., Listeria) with high efficiency, and they constitute a good model system for structure-function analyses of antimicrobial peptides in general. This review focuses on class IIa bacteriocins, especially on their structure, function, mode of action, biosynthesis, bacteriocin immunity, and current food applications. The genetics and biosynthesis of class IIa bacteriocins are well understood. The bacteriocins are ribosomally synthesized with an N-terminal leader sequence, which is cleaved off upon secretion. After externalization, the class IIa bacteriocins attach to potential target cells and, through electrostatic and hydrophobic interactions, subsequently permeabilize the cell membrane of sensitive cells. Recent observations suggest that a chiral interaction and possibly the presence of a mannose permease protein on the target cell surface are required for a bacteria to be sensitive to class IIa bacteriocins. There is also substantial evidence that the C-terminal half penetrates into the target cell membrane, and it plays an important role in determining the target cell specificity of these bacteriocins. Immunity proteins protect the bacteriocin producer from the bacteriocin it secretes. The three-dimensional structures of two class IIa immunity proteins have been determined, and it has been shown that the C-terminal halves of these cytosolic four-helix bundle proteins specify which class IIa bacteriocin they protect against.     

Identification of a new plasmid-encoded sec-dependent bacteriocin produced by Listeria innocua 743

Listeria innocua 743 produces an inhibitory activity demonstrating broad-spectrum inhibition of Listeria monocytogenes isolates. Gel-electrophoretic analysis of culture supernatants indicated that two inhibitors with different molecular weights were produced by this strain. Insertion of Tn917 into a 2.9 Kb plasmid (pHC743) generated mutants with either an impaired ability or a loss in ability to produce one of the inhibitors. Sequence analysis of the transposon insertion regions revealed the presence of two continuous open reading frames, the first encoding a new pediocin-like bacteriocin (lisA) and the second encoding a protein homologous with genes involved in immunity toward other bacteriocins (lisB). Translation of the bacteriocin gene (lisA) initiates from a noncanonical start codon and encodes a 71-amino-acid prebacteriocin which lacked the double glycine leader peptidase processing site common in other type II bacteriocins. Alignment of the sequence with the processed N termini of related bacteriocins suggests that the mature bacteriocin consists of 43 amino acids, with a predicted molecular mass of 4,484 Da. Mutants containing insertions into lisA were sensitive to the inhibitor, indicating that lisAB forms a single operon and that lisB represents the immunity protein. Cloning of an amplicon containing the lisAB operon into Escherichia coli resulted in expression and export of the bacteriocin. This finding confirms that the phenotype is dependent on the structural and immunity gene only and that export of this bacteriocin is sec dependent. This is the first confirmation of bacteriocin production in a Listeria spp., and it is of interest that this bacteriocin is closely related to the pediocin family of bacteriocins produced by lactic acid bacteria.     

Bacteriocin properties of Lactobacillus fermenti, Lactobacillus brevis and Lactobacillus buchneri

Four bacteriocins of L. fermenti, 3 bacteriocins of L. brevis and 1 bacteriocin of L. buchneri were studied with respect to morphology of the inhibition growth zones of the indicator strains, capacity for diffusion through cellophane, sensitivity to high temperature, bacterial proteases, trypsin, chymotrypsin, pepsin, papain, nucleases and lysozyme. According to the differences in their properties the bacteriocins were classified as belonging to 8 types, including 4 types of L. fermenti bacteriocins and 3 types of L. brevis bacteriocins.     

Molecular genetics and pathogenesis of Clostridium perfringens.

Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals. Recently significant advances have been made in the development of C. perfringens genetics. Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C. perfringens genes and the construction of shuttle plasmids. The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated. A detailed physical map of the C. perfringens chromosome has been prepared, and numerous genes have been located on that map. Reproducible transformation methods for the introduction of plasmids into C. perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned. Now that it is possible to freely move genetic information back and forth between C. perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C. perfringens infections.     

Spontaneous bacteriocin resistance in Listeria monocytogenes as a susceptibility screen for identifying different mechanisms of resistance and modes of action by bacteriocins of lactic acid bacteria

A practical system was devised for grouping bacteriocins of lactic acid bacteria (LAB) based on mode of action as determined by changes in inhibitory activity to spontaneously-acquired bacteriocin resistance (Bac^R). Wild type Listeria monocytogenes 39-2 was sensitive to five bacteriocins produced by 3 genera of LAB: pediocin PA-1 and pediocin Bac3 (Pediococcus), lacticin FS97 and lacticin FS56 (Lactococcus), and curvaticin FS47 (Lactobacillus). A spontaneous Bac^R derivative of L. monocytogenes 39-2 obtained by selective recovery against lacticin FS56 provided complete resistance to the bacteriocin made by Lactococcus lactis FS56. The lacticin FS56-resistant strain of L. monocyotgenes 39-2 was also cross-resistant to curvaticin FS47 and pediocin PA-1, but not to lacticin FS97 or pediocin Bac3. The same pattern of cross-resistance was also observed with Bac^R isolates obtained with L. monocytogenes Scott A-2. A spontaneous mutation that renders a strain cross-resistant to different bacteriocins indicates that they share a common mechanism of resistance due to similar modes of action of the bacteriocins. Spontaneous resistance was acquired to other bacteriocins (in aggregate) by following the same procedure against which the Bac^R strain was still sensitive. In subsequent challenge assays, mixtures of bacteriocins of different modes of action provided greater inhibition than mixtures of bacteriocins of the same mode of action (as determined by our screening method). This study identifies a methodical approach to classify bacteriocins into functional groups based on mechanism of resistance (i.e., mode of action) that could be used for identifying the best mixture of bacteriocins for use as biopreservatives.     

Plasmid detection in a bacteriocinogenic strain of Clostridium perfringens

Bacteriocinogenic Clostridium perfringens, strain 28, harboured plasmid DNA detectable by dye-bouyant density-gradient centrifugation. This plasmid DNA was absent from an ultraviolet light cured variant which had simultaneously lost its immunity and ability to produce bacteriocin. Agarose gel electrophoresis of the plasmid DNA revealed at least six bands but denaturation experiments suggested three plasmids occurring in more than one conformation. Electron microscopy revealed three major size distributions of circular DNA of molecular weights 1,5,6, and 7.1 megadaltons. Some evidence suggests that the 5.6 megadalton plasmid may control bacteriocin 28 production.