Continued Surveillance of Serratia marcescens Infections by Bacteriocin Typing: Investigation of Two Outbreaks of Cross-Infection in an Intensive Care Unit

During an 8.5-month period, 198 additional isolates of Serratia marcescens were typed by bacteriocin sensitivity; 154 isolates were typable and were categorized according to our current system of 54 provisional bacteriocin sensitivity patterns. Two outbreaks of nosocomial infection due to S. marcescens occurred in our intensive care unit, involving two and five patients, respectively. The latter outbreak was caused by a strain of S. marcescens which was not sensitive to any of the 10 bacteriocins normally used. Therefore we developed a supplementary procedure based on bacteriocin production rather than bacteriocin sensitivity. Bacteriocin production was induced with mitomycin C, and the crude lysates were applied to 15 provisional bacteriocin indicator strains. The reverse typing procedure was necessary to determine the spread and ultimate subsidence of this particular outbreak of cross-infection.     

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.     

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.     

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.     

Bacteriocins of phytopathogenic Corynebacterium species.

The majority (85% of all strains tested) of 12 phytopathogenic Corynebacterium species produced bacteriocin(s) on nutrient broth--yeast extract (NBY) medium. All C. nebraskense, C. michiganense, C. insidiosum, C. oortii, and C. iranicum strains produced bacteriocin(s). The optimal conditions for production of 23 distinct bacteriocins by eight species of Corynebacterium generally were 20 degrees C and 4 days of incubation on NBY or on modified Burkholder's agar that lacked peptone (MBAL). Production in liquid was marginal and not augmented by adding mitomycin C. Bacteriocins generally had little effect on other strains within a species but were inhibitory to other species. Most bacteriocins appeared to be bactericidal proteins resistant to heat (75 to 80 degrees C, 30 min) but sensitive to proteolytic enzymes. Some strains of C. nebraskense, C. michiganense, C. insidiosum, and C. flaccumfaciens produced two bacteriocins which were clearly differentiated by varying or testing one or more of the following: conditions for production, the indicator, heat stability, and susceptibility to proteolysis. Within certain limitations, a convenient and reproducible typing scheme was devised for strain and species differentiation of most phytopathogenic corynebacteria.     

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.     

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.     

Distinct groups of plasmids correlated with bacteriocin production in Staphylococcus aureus

The genetic basis of bacteriocin (Bac) production by six strains of Staphylococcus aureus was examined. Gene transfer experiments (in which the plasmids were tagged with the erythromycin resistance transposon Tn551) and plasmid-elimination experiments by growth at 43 degrees C associated bacteriocin production with a particular plasmid in each strain. The Bac plasmids could be separated into two distinct groups: the first comprised plasmids larger than 40 kb, which did not specify immunity to bacteriocins; the second comprised small plasmids (8.0-10.4 kb) which also specified immunity to bacteriocins. The sequence relations among the small plasmids (pRJ6, pRJ9, pRJ10 and pRJ11) were investigated by comparing restriction enzyme digest patterns and by hybridization. Plasmids pRJ10 and pRJ11 were indistinguishable and very closely related to plasmid pRJ9. Plasmid pRJ6, although different from the others, shared regions of sequence homology with them. No homology was found between plasmids pRJ6 or pRJ9 and the large Bac plasmids.     

Native and heterologous production of bacteriocins from gram-positive microorganisms

In nature, microorganisms can present several mechanisms for setting intercommunication and defense. One of these mechanisms is related to the production of bacteriocins, which are peptides with antimicrobial activity. Bacteriocins can be found in Gram-positive and Gram-negative bacteria. Nevertheless, bacteriocins produced by Gram-positive bacteria are of particular interest due to the industrial use of several strains that belong to this group, especially lactic acid bacteria (LAB), which have the status of generally recognized as safe (GRAS) microorganisms. In this work, we will review recent tendencies in the field of invention and state of art related to bacteriocin production by Gram-positive microorganism. Hundred-eight patents related to Gram-positive bacteriocin producers have been disclosed since 1965, from which 57% are related bacteriocins derived from Lactococcus, Lactobacillus, Streptococcus, and Pediococcus strains. Surprisingly, patents regarding heterologous bacteriocins production were mainly presented just in the last decade. Although the major application of bacteriocins is concerned to food industry to control spoilage and foodborne bacteria, during the last years bacteriocin applications have been displacing to the diagnosis and treatment of cancer, and plant disease resistance and growth promotion.     

The cost and benefit of quorum sensing‐controlled bacteriocin production in Lactobacillus plantarum

Bacteria eliminate competitors via ‘chemical warfare’ with bacteriocins. Some species appear to adjust bacteriocin production conditionally in response to the social environment. We tested whether variation in the cost and benefit of producing bacteriocins could explain such conditional behaviour, in the bacteria Lactobacillus plantarum. We found that: (a) bacterial bacteriocin production could be upregulated by either the addition of a synthetic autoinducer peptide (PLNC8IF; signalling molecule), or by a plasmid which constitutively encodes for the production of this peptide; (b) bacteriocin production is costly, leading to reduced growth when grown in poor and, to a lesser extent, in rich media; (c) bacteriocin production provides a fitness advantage, when grown in competition with sensitive strains; and (d) the fitness benefits provided by bacteriocin production are greater at higher cell densities. These results show how the costs and benefits of upregulating bacteriocin production can depend upon abiotic and biotic conditions.     

Lack of Heterogeneity in Bacteriocin Production Across a Selection of Commercial Probiotic Products

Probiotics are live microorganisms that, when administered in adequate amounts, confer a health benefit to the host. Bacteriocin production has often been mooted as a desirable probiotic trait and, in specific cases, has been shown to promote probiotic survival within the gastrointestinal tract, contribute to the control of pathogens and even influence host gene expression in the gut. However, it is not clear what proportion of probiotic strains routinely found in commercial products produces bacteriocins, and additionally, it is not known which bacteriocins are produced most frequently. To address this, we conducted a culture-based assessment of the bacteriocinogenic ability of bacterial strains found in a variety of commercially available probiotic products. We detected eight bacteriocin-producing isolates from 16 tested products. Interestingly, in all cases, the isolates were Lactobacillus acidophilus, and the bacteriocin produced was identified as the narrow spectrum class II bacteriocin, lactacin B. The apparent absence of other bacteriocin-producing strains from across these products suggests a lack of heterogeneity in bacteriocin production within probiotic products and suggests that bacteriocin production is not being optimally harnessed as a probiotic trait.     

Bacteriocin detection by liquid chromatography/mass spectrometry for rapid identification

Aims: To establish a new system to detect and identify bacteriocins in the early stage of screening for novel bacteriocins. Methods and Results: Liquid chromatography/mass spectrometry (LC/MS) was employed for development of a new system for rapid detection and identification of bacteriocins. The system detected and identified bacteriocins such as nisin and lacticin 481 from 25 μl of culture supernatants of their producing strains by accurate mass determination coupled with simultaneous impurity removal within 40 min. Especially, the system clearly distinguished three nisin variants (A, Z, Q) in culture supernatants of their producing strains, although they have similar structures and molecular masses. Each one‐step pretreatment by cell adsorption–desorption or acetone precipitation improved bacteriocin detection dramatically, especially for mundticin KS. This system could be applied for detection and molecular mass determination of novel bacteriocins by extracting bacteriocin‐related ions. Conclusions: The developed system could detect and identify some kinds of bacteriocin from culture supernatants or pretreated samples. Significance and Impact of the Study: The developed system helps us to identify bacteriocins in the early stage of screening without any or with one‐step pretreatment. This system is effective on not only detection of known bacteriocins but also identification of novel bacteriocins. Consequently, this system will accelerate discovery of novel bacteriocins.     

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.     

Bacteriocin-like substances produced by Rhizobium japonicum and other slow-growing rhizobia

Bacteriocin-like substances were commonly produced by slow-growing Rhizobium japonicum and cowpea rhizobia on an L-arabinose medium. Antagonism between strains of R. japonicum was not detected in vitro; however, such strains were often sensitive to some bacteriocins produced by cowpea rhizobia. Inhibitory zones (2 to 8 mm from colony margins), produced by 58 of 66 R. japonicum test strains, were reproducibly detected with Corynebacterium nebraskense as an indicator. Quantitative production was not related to symbiotic properties of effective strains, since nine noninfective strains and one ineffective strain produced bacteriocin. Eight R. japonicum strains that did not produce bacteriocin nevertheless formed effective nodules on soybeans. R. japonicum strains that produced bacteriocin in vitro had no antagonistic effect on nonproducer strains during soybean nodulation. Under controlled conditions, a nonproducer (3I1b135) predominated over a bacteriocin producer (3I1b6) when inoculated at 1:1 and 1:9 ratios. Depending on the particular ratio, up to 38% of the total nodules formed were infected with mixed combinations. The bacteriocin(s) had a restricted host range and antibiotic-like properties which included the ability to be dialyzed and resistance to heat (75 to 80 degrees C, 30 min), Pronase, proteinase K, trypsin, ribonuclease, and deoxyribonuclease. R. japonicum strains representing genetic, serological, cultural, and geographic diversity were differentiated into three groups on the basis of bacteriocin production.     

Bacteriocin Production and Different Strategies for Their Recovery and Purification

Bacteriocins from lactic acid bacteria (LAB) are a diverse group of antimicrobial proteins/peptides, offering potential as biopreservatives, and exhibit a broad spectrum of antimicrobial activity at low concentrations along with thermal as well as pH stability in foods. High bacteriocin production usually occurs in complex media. However, such media are expensive for an economical production process. For effective use of bacteriocins as food biopreservatives, there is a need to have heat-stable wide spectrum bacteriocins produced with high-specific activity in food-grade medium. The main hurdles concerning the application of bacteriocins as food biopreservatives is their low yield in food-grade medium and time-consuming, expensive purification processes, which are suitable at laboratory scale but not at industrial scale. So, the present review focuses on the bacteriocins production using complex and food-grade media, which mainly emphasizes on the bacteriocin producer strains, media used, different production systems used and effect of different fermentation conditions on the bacteriocin production. In addition, this review emphasizes the purification processes designed for efficient recovery of bacteriocins at small and large scale.     

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.     

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.     

Development and application of a multiple typing system for Clostridium difficile

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

Development and application of a multiple typing system for Clostridium difficile.

A combination of bacteriocin, bacteriophage, and plasmid typing techniques was used to differentiate strains of Clostridium difficile. A typing set of 20 bacteriocin-producing strains was established after 400 isolates of C. difficile were screened for the ability to produce bacteriocin. These strains were used to type a collection of 114 isolates of C. difficile. Forty-six (40%) of the 114 isolates were typeable, and 31 typing patterns were distinguishable. Plasmid typing of the same 114 isolates of C. difficile showed that 67 (59%) of the isolates carried up to four plasmids ranging from 7 to 60 kb in size, although most strains contained only one or two plasmids. Twenty different plasmid typing patterns were observed among the isolates. A combination of bacteriocin and plasmid typing provided 77% typeability. Fifteen (13%) of the 114 strains were typeable with five bacteriophages isolated in our laboratory, but the increase in typeability of strains over that obtainable by plasmid and bacteriocin typing was only 1.8%. Isolates that were nontypeable by bacteriocins, plasmids, or phages could be divided into two groups on the basis of positive or negative cytotoxin production. This further division of strains would increase the typeability potential by 7%; i.e., the ability to differentiate strains would rise from 77 to 84%, or perhaps 86%, if phage typing were included. We conclude that more than one of the techniques reported in this paper must be used to achieve an acceptable level of typeability of this species.     

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.