|Year : 1985 | Volume
| Issue : 1 | Page : 46-51
Bacteriocin production in Salmonella.
CV Patankar, LM Joshi
C V Patankar
|How to cite this article:|
Patankar C V, Joshi L M. Bacteriocin production in Salmonella. J Postgrad Med 1985;31:46-51
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Patankar C V, Joshi L M. Bacteriocin production in Salmonella. J Postgrad Med [serial online] 1985 [cited 2023 Oct 4 ];31:46-51
Available from: https://www.jpgmonline.com/text.asp?1985/31/1/46/5425
Bacteriocins are a class of antibiotics, like colicins which are apparently proteinic in nature and inhibit the growth of strains of same or closely related species. Bacteriocins, as a class, have a narrow spectrum of activity and they require the presence of specific receptors on the surface of sensitive cells for their action. Classification of bacteriocins involves specificity of the resistant mutant. Many chemical and physical studies have been carried out on bacteriocins of different Gram positive and Gram negative bacteria.,,,, own about bacteriocins of salmonella. In the present study, an attempt has been made to identify the bacteriocin producing strains and to study the relative properties of these bacteriocins to be compared with colicins.
MATERIAL AND METHODS
A total of 377 strains of salmonella were studied [Table 1]. These strains were obtained from different places in India and abroad and were a random collection. The indicator strain for detection of bacteriocin production was Escherichia coli K-12 Row.* Phage indicator strain used was S. gallinarum 9240. The other standard colicin producers were E. coli CA7/V, E. coli CA18/B, E. coli CA 38/E and E. coli CA 53/I.§
In all experiments, tryptic soy agar (Difco) and tryptic soy broth (Difco) were used. Soft nutrient agar contained peptone 10.0 gm, meat extract 3.0 gm, NaCl 5.0 gm, and agar-agar 10.0 gm per litre of distilled water.
Detection of bacteriocin production was done according to the method described by Fredericq. Small loopfull (1.0, mm) of overnight broth cultures of the test strains were stab-inoculated into thick, well-dried, TSA plates. In a single plate, eight inoculations could be done. The plates were incubated at 37°C for 4.8 hours. The surface growth was killed by exposing to chloroform for 30 minutes. Chloroform was evaporated and the plates were overlayered with 3-4 ml of molten soft nutrient agar containing 0.1 ml of overnight culture of the indicator strain. They were incubated overnight. Bacteriocin producing strains showed a zone of inhibition around their stab-inoculated growths. The bacteriocin producers were tested for zone production of S. gallinarum 9240. Colicin resistant mutants were picked up from the zone of inhibition produced by E. coli CA 7, E. coli CA 18, E. coli CA 38 and E. coli CA 53. After testing a number of resistant mutants, those which were resistant to only one type of bacteriocin were selected. In a similar fashion bacteriocin resistant mutants from zones of inhibition produced by salmonella bacteriocin producer strains were selected. The activity of salmonella bacteriocin producers and E. coli strains on these resistant mutants was studied to know the type of bacteriocins produced by salmonella strains. The characteristics of zones of inhibition produced by both E. coli and salmonella strains were studied. Action spectra on a set of E. coli strains were also studied. Using the cellophane paper test dialysability of colicins and salmonella bacteriocins was studied. This principally included growing the producer strains, on a piece of cellophane paper placed over a TSA plate and after removal of the paper testing for residual bacteriocin in the medium passed through the cellophane paper. Broth activity and cell bound activity were also tested. For broth activity, 10 ml aliquots of cultures of each producer strain were incubated for 24, 48 and 72 hours. They were treated with chloroform and spun at 3,000 rpm for 30 minutes. The activity of the bacteriocin in the supernatant was then determined. The supernatant was diluted two folds and a loopful was placed on the lawn of the indicator strain. The last dilution that gave a clear inhibition of growth was taken as the titre of the bacteriocin in the broth. The sediment was treated with a few drops of chloroform, shaken well and diluted and tested for the titre of bacteriocin.
The bacteriocin containing supernatants were heated in a water-bath at 60, 70, 80, 90 and 100°C for 30 minutes to test the loss of bacteriocin content in the broth.
The salmonella strains collected were distributed in all serotypes, though S. paratyphi A formed the main bulk of the collection [Table 1]. Our collection comprises 12 `O' groups and 38 serotypes. As the indicator strain used was E. coli K. 12 Row (a Colicin indicator strain), all the bacteriocins detected were necessarily related to colicins. Of the 377 strains, only five produced bacteriocin. These five strains were S. bovismorbificans, S. give, S. paratyphi B, S. saintpaul and S. wien. Group distribution of bacteriocin producers is given in [Table 2].
The characteristics of zones of inhibition produced by the bacteriocin producers, diffusibility of bacteriocin through cellophane paper, and the nature of action on bacteriophage indicator strain S. gallinarum 9240 and other E. coli strains are given in [Table 3] Identification of the type of bacteriocin produced is shown in [Table 4] The strain R/EC CA 7 was resistant to both colicins I and V while strains R/EC-CA 53 was resistant to only Colicin 1. The resistant mutants to bacteriocins produced by S. bovismorbificans, S. paratyphi B, S. saintpaul and S. wien were also resistant to colicin I of E. colt CA 53. The resistant mutant to S. give bacteriocin was resistant to action by colicin E. of E. coli CA/38. Thus 4 strains produced bacteriocins related to colicin I and S. give produced one related to colicin E. Salmonella bacteriocin producers had a narrower range of activity. [Table 5] shows the broth activity and cell-bound bacteriocin content and heat sensitivity. E. coli CA 7/V had a maximum broth activity. In salmonella strains, S. give showed maximum broth activity. All salmonella strains had greater cell-bound activity when compared with their broth activity.
The bacteriocin activity tested in the present study was essentially related to colicins. This helped in carrying out a comparative study of bacteriocin activity of the salmonella strains and colicin activity of the related colicin producers. As seen from [Table 2], only five strains out of 377 (1.04%) produced bacteriocin. This is low as compared to the varying incidences of 1-10% as reported by other authors.,,, Bacteriocin production in S. paratyphi A was completely absent. Agarwal reported that none out of the 63 strains tested of S. paratyphi A was bacteriocinogenic.
The serotype and group distribution of salmonella bacteriocin producers are not very significant though from [Table 2] it apparently seems that group B salmonella have maximum bacteriocin producing strains. The zones of inhibition produced by salmonella B-producers were comparable with those produced by colicin producers. From the mutant sensitivity to bacteriocins of salmonella and colicins it is clear that S. give produced a bacteriocin related to colicin E while other four strains produced bacteriocins related to colicin I. Incidence of colicin I production in salmonella B- producers is reported by many authors.,, Bacteriocins are usually released in the medium. Atkinson has reported that salmonella producers produce little bacteriocin in broth even after incubation of 3 days. Our findings support Atkinson's view. The low broth activity may be because of two reasons. Firstly, the strains may not be capable of producing bacteriocin at high levels. Secondly, though the bacteriocin is produced in good amount it may be that it is not released in the medium but remains adhering to the producing cell. In salmonella strains the cell bound activity is significant.
Though E. colt CA 7 and S. bovismorbificans produced a diffusible substance, the types of bacteriocin they produced were different. Their action spectra were different. S. bovismorbificans produced bacteriocin related to colicin I.
The salmonella bacteriocins were sensitive to heating for 30 minutes between 60-80°C. The activity spectrum of the salmonella and E. colt strains showed marked differences. Fredericq, opined that activity spectrum of a bacteriocin is important in classifying it into a certain group. Thus different colicins give different activity spectra. Our findings clearly indicate that the substances produced by salmonella strains are related to colicins but are not identical with them. Atkinson, in her study on salmonella bacteriocins, suggested to name these as salcols meaning salmonella colicins which are related to colicins but are not identical with them and exhibit many differences. We suggest to use the name "salcols" to designate the bacteriocins of salmonella related to colicins.
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