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.     

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.     

Bacteriocin typing of some Lactobacillus species of the subgenus betabacterium

One hundred and forty cultures of L. fermenti, L. brevis, and L. buchneri were tested by the method of delayed antagonism for sensitivity to 39 bacteriocins produced by lactobacillus strains of different species. According to their bacteriocin sensitivity patterns, 84 L. fermenti, 43 L. brevis and 13 L. buchneri cultures were differentiated into 26, 18 and 10 bacteriocin types, respectively. Bacteriocin typing allows not only intraspecific differentiation of L. fermenti, L. brevis and L. buchneri cultures but also a subdivision of their biochemical-physiological variants.     

Response of Clostridium perfringens and its L form to bacteriocins of C. perfringens

Clostridium perfringens strain No. 28 and its penicillin-induced stable L form were treated with 10 different bacteriocins of C. perfringens. Viable count and labelled amino acid incorporation experiments revealed that the L form was sensitive to two and possibly three bacteriocins to which the bacillus was not, while both forms were commonly sensitive to two other bacteriocins and resistant to five others. Adsorption of bacteriocin, immunity factors, or perhaps uptake of bacteriocin might be proposed to explain the responses of these organisms to bacteriocins.     

Epidemiological Differentiation of Serratia marcescens: Typing by Bacteriocin Sensitivity

Strains of Serratia marcescens were compared and differentiated by a new method. Bacteriocin lysates were prepared from mitomycin-induced S. marcescens and added to lawns of test strains. From 100 bacteriocin producers, 12 were chosen with the aid of computer analysis as the most useful in differentiation. Uniform drops of the 12 standard bacteriocins were added simultaneously with a bacteriocin-bacteriophage dropper to each strain to be typed. All 93 strains of S. marcescens tested were typable and were differentiated into 79 different sensitivity patterns. One pattern had three strains, 12 patterns had two strains each, and 66 patterns had only one strain. The bacteriocins also inhibited Shigella, Klebsiella, and Enterobacter, but no other Enterobacteriaceae. Bacteriocin sensitivity was less stable as an epidemiological marker than bacteriocin production. Several colonial mutants had sensitivity patterns different from the wild types, but most mutants were identical. In three different instances when cross-infection had been shown by other methods, bacteriocin sensitivity also gave the correct epidemiological results. Until the significance and frequency of genetic variations are known, a more stable epidemiological technique should be used in conjunction with bacteriocin sensitivity.     

Epidemiological Differentiation of Serratia marcescens: Typing by Bacteriocin Production

A new method for comparing and differentiating strains of S. marcescens is described which has proved useful in determining the epidemiology of hospital infections. Strains were grown in Trypticase soy broth, and bacteriocin production was induced with mitomycin C for 5 hr. The bacteriocin lysates were then spotted onto nine standard indicator strains, which were chosen with the aid of computer analysis from the 118 indicators tested. After 24 hr at 37 C, zones of inhibition due to bacteriocins were recorded. One hundred twentynine strains were differentiated into 72 different bacteriocin production patterns, but 11 strains were nontypable. None of the 45 other strains of Enterobacteriaceae produced bacteriocins. Bacteriocin production was a stable epidemiological marker. Colonial mutants always had identical patterns, as did the same strain which has passed from patient to patient through cross-infection. The new technique does not require any specialized equipment and can be used in laboratories with limited budgets. The applications of the new method in cross-infection studies and as a supplement to serological typing are discussed.     

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.     

Divergicin 750, a novel bacteriocin produced by Carnobacterium divergens 750

Abstract Divergicin 750, a bacteriocin produced by Carnobacterium divergens 750, preferentially inhibited the growth of strains of Carnobacterium and Enterococcus . Selected strains of Listeria monocytogenes and Clostridium perfringens were also inhibited. The bacteriocin was purified to homogeneity by ammonium sulfate precipitation and sequential S-Sepharose, hydrophobic interaction and reversed phase chromatography. The complete amino acid sequence was determined by Edman degradation. The peptide consisted of 34 amino acid residues. The calculated ifM_r from the peptide sequence, 3447.7, agreed well with that obtained by mass spectrometry. Divergicin 750 did not show any sequence similarities to other known bacteriocins. The plasmid-located structural gene encoding divergicin 750 ( dvn750 ) was cloned and sequenced. The gene encoded a primary translation product of 63 amino acids with a deduced M _rmr = 6789.4 which is cleaved between amino acid residues 29 and 30 to yield the mature bacteriocin.     

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.     

New biologically active hybrid bacteriocins constructed by combining regions from various pediocin-like bacteriocins: the C-terminal region is important for determining specificity.

The pediocin-like bacteriocins, produced by lactic acid bacteria, are bactericidal polypeptides with very similar primary structures. Peptide synthesis followed by reverse-phase and ion-exchange chromatographies yielded biologically active pediocin-like bacteriocins in amounts and with a purity sufficient for characterizing their structure and mode of action. Despite similar primary structures, the pediocin-like bacteriocins, i.e., pediocin PA-1, sakacin P, curvacin A, and leucocin A, differed in their relative toxicities against various bacterial strains. On the basis of the primary structures, the polypeptides of these bacteriocins were divided into two modules: the relatively hydrophilic and well conserved N-terminal region, and the somewhat more diverse and hydrophobic C-terminal region. By peptide synthesis, four new biologically active hybrid bacteriocins were constructed by interchanging corresponding modules from various pediocin-like bacteriocins. All of the new hybrid bacteriocin constructs had bactericidal activity. The relative sensitivity of different bacterial strains to a hybrid bacteriocin was similar to that to the bacteriocin from which the C-terminal module was derived and quite different from that to the bacteriocin from which the N-terminal was derived. Thus, the C-terminal part of the pediocin-like bacteriocins is an important determinant of the target cell specificity. The synthetic bacteriocins were more stable than natural isolates, presumably as a result of the absence of contaminating proteases. However, some of the synthetic bacteriocins lost activity, but this was detectable only after months of storage. Mass spectrometry suggested that this instability was due to oxidation of methionine residues, resulting in a 10- to 100-fold reduction in activity.     

Luminescent method for the detection of antibacterial activities

A new rapid and sensitive method for the detection of antibacterial activities was based on luminescent indicator strains. Listeria innocua 8811 and Enterococcus faecalis 32 were transformed with plasmid carrying bacterial luciferase genes. Subsequent strains became capable to emit light during the exponential growth phase. The addition of bacteriocin containing culture supernatants to such cultures induced a drop of their light emission which was correlated to the combined antibacterial activity of acid stress and bacteriocin. The detection of antagonistic activity is independent of its mode of action, i.e. bactericidal or bacteriostatic. This method allowed to directly visualize the antagonistic activity of bacteriocin producer strains toward target strains in coculture experiments. However, a control co-culture with non-producing bacteriocin mutant was necessary in order to distinguish between nutrients competition and bacteriocin activity. Finally, five class IIa bacteriocins were purified from culture supernatants of eight strains detected in 3 days from a 120 lactic acid bacteria collection.     

The bacteriocin typing of Klebsiella pneumoniae strains]

Results of bacteriocin typing of 196 strains of the Klebsiella genus are presented. They are typified by their sensitivity to bacteriocins and by their production using colicinogenic and indicating strains from collection of P. Fredericq [correction of Frederick], D. G. Kudla?, N. I. Koshanova as well as klebocinogenic K-type cultures of Klebsiella previously suggested by the authors. Investigation results have shown sufficient stability of a bacteriocinotype of the cultures confirmed by the population analysis. It is concluded that bacteriocin typing may be recommended as an additional method in epidemiological labelling of Klebsiella cultures.     

Bacteriocins and temperate phage ofXanthomonas campestris pv. glycines

Sixteen strains ofXanthomonas campestris pathovar (pv.) glycines produced bacteriocins (glycinecins) on agar media. Optimal incubation conditions were for 48 h at 20°C. In addition to strains ofX. campestris pv. glycines, bacteriocins were also inhibitory towardsX. campestris pv. phaseoli andX. campestris pv. vesicatoria. All bacteriocins were susceptible to inactivation by a nonspecific protease and resistant to ribonuclease, but they differed in their sensitivity to trypsin, deoxyribonuclease, and heat treatment. Differential heat and enzyme sensitivities also indicated that some strains ofX. campestris pv. glycines produce more than one bacteriocin. Attempts to induce bacteriocin production in liquid cultures were unsuccessful. However, temperate bacteriophage were released from cultures ofX. campestris pv. glycines strains XP175, B83, 17915, and MINN after addition of mitomycin C or nalidixic acid or after exposure to UV light.Reference to brand or firm names does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Selection and fitness in bacteriocin-producing bacteria.

Bacteriocins are proteinaceous anticompetitor molecules produced by bacteria against closely related species. A number of theoretical models have been used to explain experimental data that indicate high polymorphisms among bacteriocins and a frequency-dependent nature of selection for bacteriocin-producing strains. The majority of these experimental data were, however, obtained from investigations into the colicin group of bacteriocins produced by Gram-negative bacteria. The conclusions drawn from these models have been extrapolated to other bacteriocins and allelopathic compounds in general. Examination of more recent experimental investigations into the bacteriocins of Gram-positive bacteria indicate a lower degree of polymorphism and a less frequency dependent mode of selection among these strains them among the colicin-producing strains. Here we examine these contradictions in the light of the assumptions and conclusions of the theoretical models and reported data. We show that fitness costs as indicated by decreased relative maximum growth rate associated with bacteriocin production may be much lower in many cases than is assumed in the present models. A lower fitness cost associated with bacteriocin production adequately explains the newer data from Gram-positive bacteria cited here, and indicates that extrapolation of existing models to all bacteriocins and other allelopathic compounds is not appropriate.     

An analysis of bacteriocins produced by lactic acid bacteria isolated from malted barley

AIMS: The aim of this study was to perform a detailed characterization of bacteriocins produced by lactic acid bacteria (LAB) isolated from malted barley. METHODS AND RESULTS: Bacteriocin activities produced by eight LAB, isolated from various types of malted barley, were purified to homogeneity by ammonium sulphate precipitation, cation exchange, hydrophobic interaction and reverse-phase liquid chromatography. Molecular mass analysis and N-terminal amino acid sequencing of the purified bacteriocins showed that four non-identical Lactobacillus sakei strains produced sakacin P, while four Leuconostoc mesenteroides strains were shown to produce bacteriocins highly similar or identical to leucocin A, leucocin C or mesenterocin Y105. Two of these bacteriocin-producing strains, Lb. sakei 5 and Leuc. mesenteroides 6, were shown to produce more than one bacteriocin. Lactobacillus sakei 5 produced sakacin P as well as two novel bacteriocins, which were termed sakacin 5X and sakacin 5T. The inhibitory spectrum of each purified bacteriocin was analysed and demonstrated that sakacin 5X was capable of inhibiting the widest range of beer spoilage organisms. CONCLUSION: All bacteriocins purified in this study were class II bacteriocins. Two of the bacteriocins have not been described previously in the literature while the remaining purified bacteriocins have been isolated from environments other than malted barley. SIGNIFICANCE AND IMPACT OF THE STUDY: This study represents a thorough analysis of bacteriocin-producing LAB from malt and demonstrates, for the first time, the variety of previously identified and novel inhibitory peptides produced by isolates from this environment. It also highlights the potential of these LAB cultures to be used as biological controlling agents in the brewing industry.     

Production of bacteriocins and siderophore-like activity by Azospirillum brasilense

Sixty Azospirillum strains were tested for their bacteriocin production ability; twenty-seven (45%) were able to produce bacteriocins and inhibited the growth of one or more indicator strains in solid medium. Mitomycin C treatment enhanced the proportion to 80%. Sometimes large growth inhibition zones were formed, but not when FeCl3 was added in the medium. These inhibition zones probably result from the activity of siderophores. Partially purified bacteriocins produced by four strains were inactivated at pH 4, but were very stable between pH 5 to 10; bacteriocins produced by three strains lost their activity between 55 and 80 degrees C. Loss or decrease in the bacteriocin activity was observed with pronase E treatment; trypsin, lysozyme and alpha-amylase did not have an effect on bacteriocin activity. These findings show that the antagonism among azospirilla was due principally to the bacteriocins and sometimes probably due to siderophores, but not to bacteriophages or other substances.     

Group H Streptococcal Bacteriocins Having No Relation to Bacterial Transformation

The culture filtrate of group H streptococcus strain Challis produced a competence factor (CF) for bacterial transformation as well as a bactericidal factor(s) against Wicky cells. Strain 36658, in the same streptococcal group, also produced the bactericidal factor(s) but not CF. The effect of the Challis bacteriocin was limited to strains Wicky and 58, whereas the 36658 bacteriocin affected 67% of 49 strains tested. Strain 58, one of the indicator strains, was affected by the bactericidal activity of these bacteriocins but not by CF activity, and failed to transform. No relationship between the bacteriocin-producing strains and indicator strains was observed. Both Challis and 36658 bacteriocin activities decreased markedly either when the bacteriocins were heated at 50 C for 30 min or with the addition of a protein synthesis inhibitor, but showed different sensitivities to trypsin, papain and lipase. The bacteriocins were of at least protein nature and their molecular weight was roughly estimated as 100,000 daltons by membrane filtration experiments. The 36658 bacteriocin is a new type of streptocin previously not reported. The possible absence of bacteriophage or phage-like particles in the preparations is discussed.     

Bacteriocin detection from whole bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry

Class I bacteriocins (lantibiotics) and class II bacteriocins are antimicrobial peptides secreted by gram-positive bacteria. Using two lantibiotics, lacticin 481 and nisin, and the class II bacteriocin coagulin, we showed that bacteriocins can be detected without any purification from whole producer bacteria grown on plates by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF-MS). When we compared the results of MALDI-TOF-MS performed with samples of whole cells and with samples of crude supernatants of liquid cultures, the former samples led to more efficient bacteriocin detection and required less handling. Nisin and lacticin 481 were both detected from a mixture of their producer strains, but such a mixture can yield additional signals. We used this method to determine the masses of two lacticin 481 variants, which confirmed at the peptide level the effect of mutations in the corresponding structural gene.     

Bacteriocin diversity inBacillus sphaericus

Nondenaturing polyacrylamide gel electrophoresis revealed the presence of diversity among bacteriocins produced by strains of Bacillus sphaericus. Bacteriocin bands of six strains (pathogenic and non pathogenic) were found to be located just below the stacking gel. However, in two other strains (1 pathogenic and 1 collection strain) more than one protein band with bacteriocin activity were seen in the middle of resolving gel. In bacteriocin-treated cultures, electron-microscopy studies revealed the growth of lysedswollen ghost cells, and loss of viability among sensitive strains.     

Bacteriocin Produced by Bordetella pertussis

Of the 24 strains of Bordetella pertussis examined, 2 produced bacteriocins that inhibited the growth of all but 2 other strains of this species. The two strains producing the bacteriocin and the two resistant strains were rough, whereas all susceptible strains were smooth. The bacteriocin was not active on the B. parapertussis or B. bronchiseptica strains tested. These bacteriocins appeared to be protein in nature, since they were heat-labile and partially inactivated by trypsin. They were antigenic but the neutralizing antibodies did not precipitate the antigens. Absorption of the antiserum with homologous cell suspensions removed the agglutinating, but not the neutralizing, antibody.