INTRODUCTION:

B. cereus is one of the common soil prokaryotic [1], it contains opportunistic pathogen strains which cause food borne diseases[2] also it is considerd contamination factor in the industry food such as dairy products[2,3,4]. B. cereus produce a variety of toxins such as haemolysin, phospholipase, an emesis-inducing toxin and protease [5,6,7]. B. cereus is a gram positive aerobic, spore forming, motile, rod-shaped bacteria[5].

Plasmids are small double stranded circular extra chromosomal DNA which can replicate separately from main DNA or chromosome. they are main found in bacteria[8]. B. cereus contain one or two Plasmids which can encode for Bacteriocin and tetracyclin resistance[9].

Therefore, current study aims to detect chromate resistance gene or genes in B. cereus bacteria that isolated from soil, and try proof their location, whether it are on the plasmid itself or on the chromosome.

MATERIAL AND METHOD:

Isolation of bacteria:

Three samples of contaminated soil were collected at a depth of 10-20 cm. One gram of each soil sample was diluted with normal saline (9 ml), serially diluted in order to reduce the initial number of microorganisms. Then these dilutions were inoculated into nutrient agar media. Many different dilutions of suspension were streaked on agar plates for testing activity of the organisms. sub cultured again and streaked furthermore to get individual colonies. The isolated colonies were sub cultured. All incubations were done at 28ºC for 24-48 hours.

Stains, Reagents and media:

They were prepared according to what came in [10,11,12] It was as follows: gram s stain, sore stain, motility test, catalase test, oxidase test, blood hemolysis, O-F test, starch and gelatin hydrolysis, nitrate reductase, IMViC test, salinity tolerance on 5,7.5%NaCl, sacharides utilization and growth on 45°c.

Chemical Solutions:

Chromate was chosen as bacteria resistance marker, also to detect their ability of resisting or sensitivity to this element, it was added by progressive concentrations into two methods: first solid media from nutrient agar, then the bacteria inoculation spread on these media second, the mounting concentrations added to the bacterial broth that cultured for 24 hours and then noting the turbidity compared to negative and positive samples. The ability of bacteria to grow was tested by the presence of SDS and the knowledge of lethal concentrations in the same way as the chromate.

Induction of mutations by nitrous acid:

Method of[13] was used for mutagenesis bacterial isolates and then it was cultured on the non-lethal concentration of chromate which was added to the nutrient agar media. The treatment was carried out at four different intervals of 15,30,45 and 60 minutes.

Plasmid Extraction:

Extraction method executed in this study, according to[14] which depend on alkaline smashing for large chromosomal DNA as well as isolate small and large plasmids in the same time.

Plasmid curing:

Trevors method of plasmid curing in (1986) was followed in this study. twenty B. cereus isolates were treated by SDS concentration that take place before the minimal inhibitory concentration of SDS as (100,200,600,1000) µg/ml which viable cells count in it is similar to the zero treatment. Five ml of Bacterial suspension in nutrient broth was treated with SDS and leaving the tube without any addition of SDS as a control. Tubes were inoculated by 10³_10⁴ cell/ml and incubated at 28 c° for 18 h, then dilutions were done for treated and untreated cultures and spread 0.1 ml from it on nutrient agar media then incubated for 24 h at 28c or until bacterial growth appears. Then 100 colonies were transferred by wood sticks to the nutrient agar plus tolerance concentrations of chromate and nutrient agar alone. For recognizing curing cells, we were noting the absence of colonies on chromate media and its growth on media which lack chromate this means the bacterial cells loss chromate resistance character.

Electrophoreisis:

Agarose gel was used in electrophoresis of 1%depending on [15] .Agarose gel was prepared from melting 1g of it in 100ml of TBE (Tris-burate-EDTA) solution when the mixture was put on magnetic stirrer and the rising temperature of solution to 100°c. After cooling the mixture to (45-50) °c it was poured in the specific template. In order to get homogenous pores, a plastic comb was put on one end of the template, after solidifying the agar gel, comb was removed for avoiding any fracture in the gel layer. The gel piece was put horizontally in the tray of electrophoresis instrument after filling it with buffer solution TBE. Five microliters of loading buffer D.W.+Tris+glycerol+ bromo phenol blue was added to 100 microliters of plasmid DNA, which isolated from curing and non-curing bacteria, then the mixture was added to the pores of the gel. The small volume of try, led to use voltage 5v. agarose gel was polished by putting it in the solution of bright ethidium bromoide of the con. 2mg/L for an hour [16] then the gel was raised, it was captured under UV at a wave length 1nm, plasmid DNA bands appeared in a radiating color.

RESULTS AND DISCUSSION:

Bacillus cereus identification:

Morphological and biochemical tests were done according to Bergys Manual of Systematic Bacteriology[17]the results of these tests have written in (Table1).photos (A-k)also shows some tests which done with this bacteria.

Table 1: Results of tests that carried out on Bacillus cereus identification.

Test	Result
Gram stain	+
Cell shape	Rod
Spore shape	Ellipsoidal
Spore site	Central
Motility	+
Catalase	+
Oxidase	+
Blood haemolysis	Beta haemolysis
Indol	-
Methyl red	+
Voges-proskauer	+
Citrate utilization	+
Starch hydrolsis	+
Gelatin hydrolysis	+
Growth at 45°c	+
TSI	+
Tolerance salinity
5% NaCl	+
7.5%NaCl	+
Saccharides fermentation
Glucose	+
Fructose	+
Maltose	+
Sucrose	+

Figures A1: individuals colonies of B. cereus on nutrient agar. A2:color and morphology of B. cereus colonies on nutrient agar. B: spore position inside B. cereus. C: gram stain and cell shape of B. cereus. D: beta haemolysis of blood. E: starch hydrolysis. F: catalase test on two isolates of B. cereus. G: positive oxidase on right of isolate of B. cereus. H: positive Simmons citrate agar in the right. I: positive methyl red on the left. J: voges- proskauer test.

MIC of chromate:

The MIC of chromate has reached 50 mM because bacteria cannot grow for less than this concentration. Which was more than MIC estimated by [18] was 30 mM in the same time the MIC of chromate in recent our study lower than other studies were carried out on B cereus such as [19] its MIC was 585 Mg/ml, or on other species of Bacillus such as Bacillus sphaericus its MIC had reached to 800 Mg/ml [20]whereas MIC of chromate to Bacillus sp. Was 2500 Mg/ml [21].

These differences may be, belong to the different environments which bacteria isolated from it, and chromate pollution rate of it and consequently that affect on chromate resistance ranges among these bacteria. The high resistance of chromate in these bacteria, some papers return it to aggregate chromate molecules in plasma membrane which lead to detoxification of chromate [22]whereas others suggest resistance to chromate wasn't related to the plasma membrane, but it related only to the soluble protein molecules also [23] or by enzymatic action that works in aerobic and anaerobic conditions which reduce chromate from quaternary to ternary chromate [24].

In all chromate resistance explanation cases or any metal or antibiotic resistance proteins or enzymes there was responsible gene of resistance must code for control on its proceeding.

MIC of SDS:

The MIC of SDS has reached 30000 µg/ml the bacteria in this study, which considered very high concentration. Another study has also observed when the other isolate of B. cereus, which isolated from detergents - polluted water these bacteria could tolerate high concentrations over to 10000µg/ml of SDS [25]. Enduring B cereus to high levels of SDS may be, belong to the perfect activities of enzymes such as alkaline protease which responsibility of higher stability against most chemicals[26].

Induction of mutations by nitrous acid:

It was found that the best period for the mutagenesis is 45 min. Using nitrous acid as to get the sensitive mutants to the chromate ware observed in low concentration of chromate. While the same isolates has been able to grow on the nutrient agar media. These results indicate the loss of resistance to chromate when treated with nitrous acid that give evidence of mutation, but the location of this mutation is still unknown as to whether in the chromosomal DNA or in the plasmid. That because the nitrous acid effect is affected by three factors in a number of living cells. it decreases when: 1) pH decrease, 2) concentration of HNO₂ increase, and also if the duration of exposing to this acid increase [27] and this what had really happened in my study, because I found the 45 minutes the better period of exposing to nitrous acid .Whereas didn’t get any mutants from one hour exposing to mutagen this refer to this period represents killing dose of this organism. furthermore B. cereus is highest resistances from Pseudomonas aeruginosa which was 20 minutes just enough for killing it by nitrous acid [28].

Plasmid curing:

When the wild isolates of bacillus cereus were treated with SDS, found they lost its susceptibility to chromate resistance while they able to grow and developed on nutrient agar alone without chromate. Bacterial cells can be loss plasmids spontaneously in low frequencies this phenomenon called spontaneous curing plasmid it could be increased by exposing cells to interfering molecules such as Accridenes and other chemical's such as SDS[29].

Results from curing study in this study indicates preliminary for the probability exists of chromate resistance genes or gene on the plasmid rather than on a chromosome. that's consistent with the information we received from [30] suggested that the bacteria B. sp. becomes sensitive to heavy metals after curing the plasmid ,and the same thing happened with the spices B. cereus which lost the chromate character [31].

The feature of metals resistance is in a relation with the plasmid. It was also proved in other bacteria, such as gram positive Staphylococcus [32]. This demonstrates very concretely of ownership of the metal resistant plasmid. There is an Enzyme that responsible of mercuric reducing encoding by plasmid in E. coli [33]. Genes on plasmids or chromosome or in the transposons are cause of heavy metal's resistance in different environments [34]. Transformation was carried out between B. sp. and B subtilis also prove that heavy metal resistance genes located on a plasmid [35].

SDS was very effective in twenty isolates were tested at four concentrations that was used in this study. The results were shown the susceptibility to chromate ranged 90-100%which agree with referred by most studies on the SDS role in plasmid curing comparing with the other curing factors as ethidium bromide and Novobiocin [36].

Electrophoresis:

Genetic content of B. cereus was detected by depending on [14] method for recognizing genetic component or genetic layout in this bacteria. This method shows high ability in plasmid DNA isolation. The result of electrophoresis in agarose gel has confirmed that non-curing isolates of Bacillus cereus had a big band of plasmid numerate as (Figure K:1-7 and 9-11), whereas curing isolates of Bacillus cereus which treated with SDS had no any clear band of plasmid in (Figure K: no. 8,13,14) which observed result of electrophoresis. Encompass of Bacillus cereus of plasmid DNA was substantiated by old studies such as [9,23,37] and new studies as [38,39] and current study has also Supported this.

Figure K: Photo of electrophoresis.

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Received on 22.11.2018 Modified on 19.12.2018

Research J. Pharm. and Tech. 2019; 12(3): 1301-1306.

DOI: 10.5958/0974-360X.2019.00218.X