Studies on the lactose character in Salmonella S:41:z10.
A number of plasmids carrying the Lac+ character have been reported. Lac+ character of salmonella S:41:z10:- studied for transfer of Lac+ character to standard Escherichia coli K12 Lac-F- Nalr and Escherichia coli K12 F- Lac- Rifr, failed to transfer in in vitro experiments. Similarly, identification and characterisation of plasmid DNA by agarose gel electrophoresis technique did not show specific plasmid DNA as compared to standard molecular weight plasmids. Plasmid DNA appeared to have been embedded with chromosomal DNA molecule.
Certain Salmonella serotypes viz. S. anatum, S. tennessee, S. newington, S. schwarzendgrund, S. typhimurium, S. oranienburg ferment lactose. Chander et al have attributed this atypical behaviour in Salmonella to plasmids. Numerous studies concerned with the nature of these lactose factors, their property of transfer in other Enterobacteria, their similarity with F Lac+ elements, their association with antibiotic resistance factor have been reported,,. During the period between August and October 1977, samples of frozen frog legs collected from different commercial processing centres were found to be contaminated with lactose fermenting strains of Salmonella. Transmissible extra-chromosomal element confirming the property of lactose fermentation to different species of Enterobacteria have been demonstrated. The present investigation is aimed at identifying genetic transfer of Lac + character of S:41:z10:- to standard strain of E. coli and characterising the plasmid DNA present in the system by agarose gel electrophoresis technique.
The strain of Salmonella S:41:z10: Lac+ chloramphenicol resistant (C) was isolated between months of August and October 1977. The serotypes were maintained in nutrient agar slants at refrigeration temperature and sub-cultured at frequent intervals.
1. Procedure for transfer of Lac+ character from S:41:z10:- 10ml of S:41:z10:- Lac + Cr and the recipient strains (autotrophic strains) viz. E. coli K12 F- Lac- Nallr and E. coli K12 Lac- Rifr were grown in brain heart infusion broth (Difco) BHI in late log phase. Two dilutions 1:9 and 1:1 of donor and recipient strain were mixed at 26?C. After mixing, the tubes were incubated at 37?C for 24 hrs. The culture tubes were then agitated to break the bacterial clumps and plated on MacConkey's agar plates (Difco) MC containing 40 ?g/ml of nalidixic12 acid and 50, ?g/ml of rifmapicin) to counter select the donor. After overnight incubation, single isolated lactose positive colony was picked up and inoculated into BHI broth and subequently plated out in MacConkey's agar to check the stability of lactose fermentation character and plasmids.
2. Agarose gel electrophoresis for separation of plasmid DNA Reagents:
i. Lysozyme mixture for gram negative bacteria.
ii. Lysozyme 7,500 units/ml., Ribonuclease I 0.30 units/ml, 0.05? phenol blue in Tris Borate Buffer. PH 8.2, 89 min Tris base, 12 mm diNa. EDTA and 8.9 mm Boric acid) and 20 per cent Ficoll 4,00,000 (Sigma).
iii. Ribonuclease is first dissolved in 0.4M sodium acetate buffer having pH 4.0 to achieve concentration of 10 mg/ml. and heated at 980C for min. before diluting in to the rest of the lysozyme mixture. The solution stable at room temperature for several months.
iv. Sodium dodyl sulphate (S.D.S.) mixture for Gram negative: 0.2 sodium dodyl sulphate in Tris borate buffer (89 mM Tris base, 2.5 mM EDTA and 89 mM Boric acid) in 10 per cent Ficoll 4,00,000.
v. Overlay mixture for Gram negative
vi. Same as SDS for Gram negative bacteria except it contained 5 Ficoll 4,00,000.
1. Single colony from plate (107 or 108 cell) was picked up with tooth pick and resuspended in 15 ? ml. of lysozyme mixture, left for 2 to 5 min at room temperature.
2. 30 ?ml of SDS was layered carefully on top of bacterialysozyme mixture and 2 layers were gently mixed with a tooth pick, moving them from side to side (2 to 3 times for Gram negative bacteria) complete mixing was avoided.
3. 100 ?ml of overlay mixture was layered on the top of SDS lysozyme mixture without disturbing the new viscous DNA lysate.
4. Tops were scaled. Tubes were kept in a chamber containing ELE buffer and current of 2mA was posed for 60 min followed by 40 mA for further period of 90 min.
(ELE Buffer-89mM Tris basse 2.5 mMsad EDTA and 8.9mM Boric acid, and pH 8.2)
Gels were stained with ethidium bromid (0.4 ?gm/ml) in ELE buffer for 15 mts and photographed under UV light using a short wave transilluminater type (61 from UV Products, Inc.) and poloroid type 55 or 57 film and red filter (Wratter No. 55).
Conjugation experiment to determine the transfer of lactose positive character - from S:41:z10 which could be plasmid mediated on two separate strains of Escherichia coli K12 Lac- F- Nalr and Escherichia coli K12 F- Lac- Rif failed to show the transfer on MacConkey's agar plates containing 40 ?g/ml of nahdixc acid and 50 ?g/ml of rifampicin in two separate experiments.
Determination of molecular weight of Lac+ plasmid by observing the extent of their migration in gel by agarose gel electrophoresis technique in comparison with that of standard known molecular weight plasmids showed that DNA band below the gel layer to be significant, but the plasmid band was not detected.
Biochemical tests form an important backbone for the identification of Salmonella. Even though organism of the Salmonella group have most of the tests similar to family Enterobacteraccae biochemically and antigenically, they differ in one of the important property i.e. inability of lactose fermentation. However, there are certain salmonella serotypes, which can ferment lactose and therefore, raise a possibility of wrong reporting of strains during routine laboratory analysis, which is based on lactose fermentation. This characteristics found in these Salmonellas is attributed to Lac + plasmid either as a separate entity or in conjunction with drug resistance.
The concept that extra-chromosomal genetic material, episome or plasmid expressed in one host is able to migrate to another post by conjugation, carrying specific character to recipient strain, has given a new dimension to the present knowledge of plasmid.
The Lac+ character due to transmissible plasmid have been demonstrated in S. typhimurium and S. oranienburg,. Our results showed that S:41:z10: failed to show the transfer of Lac+ character to recipient E. coli K12 strain in conjugation experiment from broth culture. Walia et al has reported low frequency transfer of Lac+ character to shigella and Salmonella strains. Plasmid band of Lac+ could not be identified in the gel electrophoresis; it may be possible that Lac+ plasmid bound within DNA molecule, did not separate during this experiment. Incidence of transferable resistance in Salmonella in nature is much less than what could be seen in laboratory experiments.
It is true that genetic transfer of different properties, are coded by plasmids of different origin. The failure to demonstrate this property may be due to the absence of induceras, plasmid coded lactose genes are barely expressed.
We are thankful to the Dean, Seth GS Medical College and King Edward Memorial Hospital, Mumbai for providing necessary facilities for conducting our research study. We thank Dr. K Prakash, Head, Microbiology, Lady Harding Medical College, Delhi and Dr. EM Lederberg, Plasmid Reference Centre, California, USA for supplying standard strains and plasmids.