The effect of DNasel enzyme on food pathogens subjected to different food processing treatments .

Detection of pathogens in contaminated food products by PCR can result in false-positive results due to amplification of DNA from non-viable cells. DNase treated DNA polymerase chain reaction (DTD-PCR) has been attempted to eliminate such contaminating DNAs from non­ viable cells prior to the isolation of template DNA, and to improve the overall fidelity of such detection methods. The main objective of this work was to determine the effect of different DNasel enzyme treatments on the DNA present in viable and non-viable cells that were subjected to different food processing treatments. Hypothesis was based on the use of DNasel for the differentiation of viable and non-viable cells of food borne pathogens. The effect was observed both on gram positive (Listeria monocytogenes) and gram negative cells (Escherichia coli 0157:H7) of food pathogens after subjecting the cells to a range of heat, cold and chemical treatments. The penetrability of DNasel enzyme into the cells and the subsequent digestion of DNA residing in the cell are based on the type and extent of DNasel treatment and the gram character of the cells. The results indicate the possibility of using DNasel to distinguish between viable and non-viable cells that were produced by heat treatments and some chemical treatments (ethyl alcohol and sodium hypochlorite). However, this cannot be applied for differentiation between viable and non-viable cells produced by cold treatments and sodium chloride treatments. Overall results confirmed the potential of developing DTD-PCR to differentiate the viable cells from the non-viable cells of test organisms after subjecting them to heat treatment, ethyl alcohol treatment and sodium hypochlorite treatment.


INTRODUCTION
There is a distinct need to develop rapid detection methods for food pathogens since conventional methods are cumbersome and time consuming.The most serious limitation of using standard PCR methods lies in the fact that they fail to distinguish between live and dead cells and the resultant need for an initial enrichment step.The standard PCR methods amplify both DNA sourcing from live cells and dead cells including the free extraneous DNA already released to the media due to dead and lysed cells.DNase treated DNA polymerase chain reaction (DTD-PCR) method has been attempted to eliminate such contaminating DNA from dead cells prior to the isolation of template DNA, and to improve the overall fidelity of such detection methods 1 .These methods could therefore be cheaper than the more sophisticated methods like Reverse Transcriptase Polymerase Chain Reaction (RT-PCR), nucleic acid sequence based amplification (NASBA), etc 2 .In the experiments reported in this study the cell walls of harvested bacterial cells have been exposed to crude DNasel enzyme treatment.Deoxyribonuclease (DNase) is a nuclease enzyme that catalyzes the hydrolytic cleavage of phosphodiester linkages in the DNA backbone.DNasel digestion of heterogeneous double stranded DNA (dsDNA) reported always a mixture of nucleotides with yields of dinucleotides (60%), trinucleotides (25%), and oligonucleotides 3 .The smallest substrate for DNasel is a trinucleotide.Although DNasel is commonly perceived to cleave DNA non specifically, in practice it does show some sequence preference.It acts on single stranded DNA, double stranded DNA, and chromatin 4 .Like many enzymes, DNasel activity is also affected by the composition of the reaction buffer and is not active in buffers containing Mg 2+ yet lacking Ca 2+5 .Since DNasel is a powerful research tool for DNA manipulations, it is used in a range of molecular biology applications such as: degradation of contaminating DNA after RNA Corresponding author isolation, "clean-up" of RNA prior to RT-PCR 6 , prevention of clumping when handling cultured cells, and creation of a fragmented library of DNA sequences for in vitro recombination reactions etc 7 .
In this study we report the potential of DNasel enzyme to penetrate into the cells of gram positive and negative bacteria that were subjected to different food processing and sterilization treatments.The aim was to develop an effective and low cost tool to avoid false positive results obtained in the presence of the non-viable cells by a PCR due to the presence of extraneous DNA in the sample.This was taken as a preliminary step towards the development of a PCR based method; DTD-PCR for detection of viable food borne pathogens.Activity of DNasel on the intra and extra cellular DNA was observed by means of touchdown PCR and reconfirmed by doing a second round of touchdown PCR by taking the initial PCR product as the template in the second round.

METHODS AND MATERIALS
Bacterial strains and culturing: The selected reference organisms that belong to gram positive and gram negative groups, L. monocytogenes and E. coli 0157:H7 respectively, were cultured on tryptic soy broth yeast extract (TSBYE) (Difco Laboratories, Detroit, MI, USA) agar medium (1.5% w/v) and incubated under aerobic conditions overnight at 37°C.

Preparation ofaliquots of cell suspension for treatments:
The bacterial cells in the late log phase were harvested by centrifugation at 12,000 rpm for 10 min and washed with PBS buffer (0.2 M sodium dihydrogen orthophosphate, 0.2M disodium hydrogen orthophosphate) followed by ultra pure water.Cell number was estimated by means of a haemocytometer after resuspending the cells in 250 mL of ultra pure water.Aliquots of cell suspension with a constant cell count of 5x 10 7 cells/100 pL were prepared prior to treatments.

Different food processing and sterilization treatments:
Prepared cell aliquots of gram positive and negative bacteria were subjected to various stress conditions, namely heat treatments (H1-H3), cold treatments (CI -C3) and chemical treatments (Chl-Ch3) as given in Table 2 and 3. Then the-cells were washed thoroughly with ultra pure water, to get rid of traces of salts and chemicals, prior to DNasel treatment (D1-D6).
DNasel treatment: A set of experiments were conducted to find out the penetrability of DNasel enzyme through cell walls of gram positive and negative bacteria and to find the optimum DNasel concentration and treatment time combinations required for developing DTD-PCR system.The cell number was fixed to 5xl0 7 cells/100 pL for all the treatments discussed below.Each treatment was replicated twice.DNasel (Sigma) (from bovine pancreas, minimum 2,000 Kunits/mg protein) obtained in lyophilized powder form was hydrated to a stock concentration of 5 U/pL using storage buffer (10 mM Tri-HC1 (pH 7.5), 10 mM CaCl 2 10 mM MgCl, and 50% glycerol) and an aliquot of the DNasel stock solution was diluted to a working concentration of 1 U/pL using storage buffer.The stock and working DNasel solutions were stored in 50pL aliquots at -20"C until required.DNasel treatment was carried out at a final volume of 100 pL which consisted of 20 pL of reaction buffer 5-10 uL DNasel enzyme (1 U/uL) and 50 pL cell aliquot and made up to 100 pL with ultra pure water and incubated at 37°C for one hour.Mixing was carried out at 20 min intervals using a vortex system.DNA was extracted from cells after the DNasel treatment.The DNasel activity was deactivated by boiling the reaction mixture in a constant temperature water bath for 10 min.
DNA extract preparation: DNA extracts used as templates in PCR amplifications were prepared by Triton X-100 method.Bacterial DNA extracts were prepared according to the manufacturer's instructions (Easy DNA kit, Invitrogen Co., San Diego, USA).The final DNA extract was dissolved in 50 pL of ultra pure nuclease free water (Seromed*).
Touchdown PCR: Touchdown PCR was carried out with the DNA samples extracted from the cells that were subjected to DNasel treatment using a primer pair specific to V3 region of 16S rDNA.In order to make the PCR efficiency independent of primer type, this universal primer pair with a product size of 193 bp was used in PCR amplification.This was done in order to check the effectiveness of DNasel treatment.The nucleotide sequences of the primers are as follows: forward primer (V3f): 5'-CCTACGGGAGGCAGCAG-3'; Reverse Primer (V3r)5'-ATTACC GCG GCT GCT GG-3'  C2:-10"C, 24 hours
Control: without heat, cold or chemical treatments.
transilluminator.Touchdown PCR results were reconfirmed by a second round of touchdown PCR process by taking the amplicons of first PCR assay as the template.

Effect of heat treatments
Growth was not observed in both bacteria Listeria monocytogenes and Escherichia coli 0157:H7 after being subjected to three different heat treatments (HI, H2, H3).This confirmed that the heat treatments were powerful enough to kill the cells.The DNasel treatments were found effective enough to digest all DNA present in cells subjected to H2 and H3 treatments at both concentrations of 5 U/100//Land 10U/100//Linboth species of bacterial cells.However the DNasel treatment of cells subjected to H1 treatment (for 20,40,60 min) at a concentration of 5 U/ 100 pL was not effective.The cells treated at this temperature produce PCR bands with very low intensity even after the DNasel treatment with a concentration of 5 U/100 fiL for all treatment times tested (Figure 1).However, the cells subjected to HI treatment did not yield any PCR product when treated with enzyme at a concentration of 10 U/100 fiL which showed the insufficiency of DNasel concentration in earlier treatment.
This result was common to both L. monocytogenes and E. coli 0157:H7 cells.The results obtained indicate that the heat treatments given are capable of killing cells of both bacteria tested and 10 U/100 fiL DNasel treatment for 20-60 min of incubation time is effective to remove all DNA residing in the cells of both bacteria tested.

Effect of cold treatments
In cold treatments tested, the DNasel treatments (5 U/100 fiL and 10 U/100 /.iL at all treatment times tested) were effective enough to digest all the DNA present in cells of L. monocytogenes (gel picture not given).But the cells showed normal growth after undergoing CI, C2 and C3 treatments, which proved their ability to grow after a freeze shock.The absence of PCR products indicates the possibility of penetration of the DNasel enzyme across the cell membrane of cold treated cells of L. monocytogenes.
In the case of E. coli 0157:H7 cells (Figure 2) the results were totally different.The cells subjected to the three cold treatments produced PCR products after the DNasel treatment which confirmed that the different cold shocks given are insufficient to make cell wall changes or make it permeable for DNasel in gram negative bacteria that have two layers of plasma membranes.Literature indicates that both gram positive and gram negative bacteria have been shown to be affected by cold shock 1 ' During rapid cooling, permeability changes in the cell membrane are caused by a phase transition in the cell membrane lipids from liquid crystalline to a gel state'.Slow cooling allows lateral phase separation of the lipids and proteins of the membrane, whereas rapid cooling "fixes" these components in a random, disordered state, resulting membrane leakiness 1 ".The results of this experiment with cold treatment indicate that the given treatments are capable of making permeability changes in the cell wall of L. monocytogenes but not in E. coli 0157:H7.The results also confirmed that the cold treatments tested were not powerful enough to kill the cells of both bacteria.

Effect of chemical treatments
In different chemical treatments (Figure 3), the cells (L.monocytogenes and E. coli 0157:H7) that were subjectedo Ch3 treatment grew in TSBYH and also yielded PCR products at different DNasel treatments in both bacteria.It has been reported that L. monocytogenes can survive under a variety of environmental stresses, such as 10% NaCl solutions".This bacteriumcan also be detected after 150 days in pure salt at 22"C 12 .E. a;/7 0157:H7 can survive in 10% NaCl solutions".The presence of PCR products can be due to two reasons.Firstly, the main physiological change that occurs due to salt treatment is plasmolysis, which causes cell membrane shrinkage but slight or no changes in the cell wall structure, especially permeability.Therefore, DNasel movement is not facilitated by these treatments.Secondly, as the literature says, DNasel is very sensitive to different salts present in reaction buffer".In Ch3 treatment there are chances for traces of NaCl that may remain after the washing step which could subsequently inhibit the DNasel activity.This is a practical error which was kept at minimum level by washing the cells thoroughly with ultra pure water several times to get rid of traces of salts and chemicals prior to DNasel treatment.Therefore, it was assumed that there were no traces of salts that could interrupt the activity of DNasel.
In Chi and Ch2 treatments cells (I.monocytogenes and E. coli 0157:H7) were killed after treatments and PCR products were not produced in any of the DNasel treatments.that were tested.Alcohol induced coagulation of proteins occurs al the cell wall, the cytoplasmic membrane and among the various plasma proteins 14 .Alcohols target the bacterial cell wall, with resultant lysis of the cytoplasmic membrane and release of cellular contents.Subsequently, this facilitates movement of DNasel into the cells and digestion of DNA.Protein coagulation occurs within concentration limits around an optimum alcohol level.In the absence of water, proteins are not denatured as readily as when water is present.Therefore, mixtures of alcohol with water exhibit much better efficacy than straight alcohol alone 14 .
Sodium hypochlorite forms hypochlorous acid in solution.The mechanism in which hypochlorous acid destroys microorganisms has never been demonstrated experimentally, but it has been speculated 14 that hypochlorous acid allows oxygen to emerge, which in turn supposedly combines with components of cell protoplasm, destroying the organism.The results of the experiment indicates that this could facilitate the movement of DNasel and digestion of DNA.
The overall results show that DNasel can effectively penetrate the walls of gram positive and gram negative bacterial cells that are subjected to different heat treatments.In the case of cold treatment, the cell walls seem to become permeable for DNasel after the treatment only in gram positive bacteria.In gram negative bacteria this is different and the cells produce expected PCR products even after giving DNasel treatment for one hour.This indicates that the cold treatments given are not sufficient to make permeability changes in cell walls of gram negative bacteria.In chemical treatments, Ch3 treatment was not able to make any permeability change in cells; hence all the cells subjected to salt treatments produced expected PCR products.But when the cells were subjected to Chi and Ch2 treatments the cells did not yield any PCR products.This concludes that selected Chi and Ch2 treatments are able to make permeability

3 :
PCR products, X-absence of PCR products.Control: without heat, cold or chemical treatments.L.M.N.S. Nadugala & Sudip K. Rakshit Table Effect of DNasel treatment on E. coli cells that were subjected to different food processing treatments.

Figure 3 :
Figure 3: PCR products of chemically treated cells of K. coli 0157:117 after DNasel treatment changes in the cell walls of gram positive and negative bacteria which facilitate the movement of DNasel and do DNA digestion.The results indicate that DNasel treatment could be used to destroy DNA in non-viable cells after different heat (H1-H3) treatments and some chemical (Chi and Ch2) treatments and this could be used as a tool to differentiate viable and non-viable cells.

Table 1 : Thermal cycling conditions for touchdown PCR amplification.
Taq master mixture (1.25 U Taq DNApolymerase, 1.5 mM MgCl 2 ,10 X PCR buffer, dNTPs 200 uM each), 0.25//L of each forward and reverse primers(12.5 pmole each/50 pL), lpL of template DNA and ultra pure water to bring the Journal of the National Science Foundation of Sri Lanka 35(3)final volume to 5QuL..The touchdown PCR procedure was in Table1.The PCR amplification products were visualized performed in a PCR Thermocycler (Biometra ™ Personel).in 2% agarose gels (Analytical grade, Promega) stained