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MOLECULAR IDENTIFICATION OF ACINETOBACTER ISOLATED FROM EGYPTIAN DUMPSITE AS POTENTIAL BACTERIA TO DEGRADE MALATHION
Hussein H. Sabit*, Osama A.M. Said, Ali F. Shamseldin, Kholoud Elsayed College of Biotechnology, Misr University for Science and Technology, 6 October Governorate (EGYPT) *Corresponding author: h2s74@yahoo.com ABSTRACT Efficiency of Acinetobacter isolated from a dumpsite in Qaliubeyya, Egypt for Malathion degradation was investigated. It was able to utilize Malathion as a sole carbon and energy source and to degrade it. Minimal salt medium supplied with different concentrations of Malathion along with a constant amount of the bacteria were prepared and incubated for seven days at 37C. The degradation rate was obtained by using the gas chromatography/ mass spectrometry (GC/MS). The results showed an obvious degradation with a rate of 100% of the initial concentrations applied. These results indicate that Acinetobacter is an excellent candidate for the efficient and convenient way of Malathion biodegradation. 16S ribosomal DNA gene was detected and RFLP analysis was employed. Sequencing of the full length 16S rRNA was performed and obtained data were analyzed and compared to other sequences recorded in the NCBI. Key words: Molecular identification, 16S rRNA, Acinetobacter, Malathion, Biodegradation, GC. 1. INTRODUCTION
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Organophosphates are a group of highly toxic pesticides widely used for plant protection. Malathion (Diethyl [(dimethoxyphosphino-thioyl)thio]butanedioate) is still extensively used worldwide despite its high toxicity [1]. Malathion is one of the more frequently used organophosphorothioate (OPT) insecticides in the world, both in agriculture and in residential settings; in addition, it has been used in malaria eradication programs in Africa and Central America or in wide-scale pest control [2]. The reason for such widespread use lies in its relatively low toxicity to mammals and high selectivity toward insects, paralleled by a moderate persistence in the environment, when compared with other OPTs [3]. Current methods to detoxify Malathion mainly rely on chemical treatment, incineration and -landfills [4]. Chemical methods impose a risk due to production of large volume of acids and alkali that must be degraded itself. Microbial attack on wide ranges of organophosphorus insecticides have been reported [5, 6, 7, 8 and 4]. Enzymatic detoxification of organophosphorus insecticides by some bacterial species was also noted [9, 4, 10, 11, 12, and 13]. There are currently 140 Organophosphate compounds being used as pesticides and as plant growth regulators around the world [14]. In the United States of America alone, 60,000 tons/year of these types of compounds are produced [15]. Although they have very useful properties, their intensive and indiscriminate use has caused short and long term environmental hazards and health problems [16]. Synthetic organophosphates (OPs) are used widely as insecticides in agriculture. These insecticides are potent acetylcholinesterase (AchE) inhibitors, and various clinical effects can occur due to OP poisoning in humans [17 and 18]. Isolation of indigenous bacteria capable of metabolizing pesticides provides environmentally friendly means of in situ detoxification [4 and 19]. Contaminated environments have resulted through time in the evolution of autochthonous microbial populations; therefore, these sites are the most appropriate ecological niches for the isolation of strains able to degrade these compounds [20, 21, and 22]. Taxonomically, the genus Acinetobacter has a rather long and complicated history and currently comprises up to 33 (genomic) species, including seven recently described environmental species and the clinically relevant species A. parvus [23]. Acinetobacter is widespread in nature, and can be obtained from water, soil, living organisms and even from human skins. Species of Acinetobacter have been attracting increasing attention in both environmental and biotechnological applications. Some strains of this genus are known to be involved in biodegradation of a number of different pollutants such as biphenyl and chlorinated biphenyl [24]. Acinetobacter has showed that it can utilize a wide range of pollutants as a sole energy and carbon source [25 and 26]. The present study aimed at evaluates the potentiality and the efficiency of Acinetobacter to degradation Malathion and to characterize it on the molecular level. 2. MATERIALS AND METHODS

Isolation and purification: Soil samples were collected from a dumpsite in Qaliubeyya Egypt. One gram of the contaminated soil was taken in sterilized plastic bag and 10 ml of dH2O was added in a clean test tube. Sample was then used for isolation of Acinetobacter bacteria on McConkey medium. A lot of gram negative bacteria were detected on McConkey medium, but only catalase negative coccobacilli colonies were selected. The sample was tested against

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a wide range of biochemical tests including growth on different NaCl concentrations, indol, gelatinase, catalase, and oxidase. Also the ability to utilize different carbon sources (Glucose, Galactose, Fructose, etc.) was assessed according to the ordinary laboratory procedures. All these tests have been performed according to published procedures [27]. Growth and maintaining Media: LB Media was used to culture and enrich the bacteria. It was prepared according to the following constituents (g/L): NaCl, 5; Trypton, 10; and Yeast extract, 10. Bacteria were kept at 37C under the normal laboratory condition. Biodegradation media: Malathion was kindly obtained from the Faculty of Agriculture, Ain Shams University, Egypt. Mineral Salt Media was prepared for the biodegradation studies according to the following constituents (g/L): KH2PO4, 4.2; K2HPO4, 1.2; NH4NO3, 1; MgSO4.7H2O, 0.2; CaCl2, 0.04; and Fe (SO4)3, 0.01. DNA extraction and 16SrDNA gene amplification: Genomic DNA was isolated according to ordinary laboratory protocols [28]. Extracted DNA was subjected to PCR to detect and amplify the 16SrDNA gene. The reaction mixture was as follows (the total volume was 25 l): 1.5 l of genomic DNA (25 ng), 1.5 l of forward primer, 1.5 l of reverse primer, 12.5 l of Master Mix, and 8 l of d.H2O. The PCR profile was as follows: Pre-PCR 95 C for 5 min, denaturation at 94C for 45 seconds, annealing at 58C for 1 minute, extension at 72C for 2 minutes and a final extension step at 72C for 10 minutes. A 35-cycles program was performed. The PCR products (5 l) were characterized by electrophoresis on 1% agarose gels and were visualized under UV light after they were stained with a solution containing 10 pg of ethidium bromide per l. The oligonucleotide primers used in this study were purchased from LabTechnology (Promega Corp.). Primer F-5 AGA GTT TGA TCC TGG CTC AG 3 and R-5 GTA TTA CCG CGG CTG CTG 3. RFLP Analysis: The PCR product of the amplified 16SrDNA was subjected to different restriction endonuclease enzymes to fragmentize the band obtained. Three different restriction enzymes were used; BamH1, Pst1 and Hind III. The reactions were carried out according to the manufacturer instructions with minor modifications (17 l d.H2O, 5 l Buffer, 2 l enzyme and 5 l DNA. The mixture incubated for 10 min. at 37C and then placed in a water bath at 80C for 20 min. to stop the reaction). Sequencing of the 16SrDNA gene: The 16SrDNA fragment was purified from the gel using Gel Extraction kit (Promega). Using the primers used in detection and amplification of the 16SrDNA has also been used in the sequencing reactions. The sequencing reactions were carried out ABI PRISM BigDyeTM Terminator Cycle Sequencing Kits using AmpliTaq polymerase (Applied Biosystems). The procedures were as per the manufacturer instructions. Biodegradation experiments: Four concentrations of Malathion were used in this study (100, 150, 250, and 500 ppm). Five test tubes were prepared as follows: 10ml MSM, 17.5l Malathion, and 500l bacteria to obtain a final concentration of 100 ppm; 10ml MSM, 26.25l Malathion, and 500l bacteria to obtain a final concentration of 150 ppm; 10ml MSM, 35l Malathion, and 500l bacteria to obtain a final concentration of 250 ppm; 10ml MSM, 87.5l Malathion, and 500l bacteria to obtain a final concentration of 500 ppm; and the fifth tube was used as control. A standard of 50 ppm was prepared by adding 8.75l of Malathion to 10 ml of MSM. The tubes were incubated for one week at 37C. To assess the rate of biodegradation, the cells were harvested by centrifuging the tubes at 4000 rpm for 5 min. and the supernatant was used in the assessment procedures. The degradation rate was determined and estimated by the loss of Malathion in the media. The cell-free supernatants were analyzed on GC-MS. The GC protocol used was as the manufacturer instructions. 3. RESUTS AND DISCUSSION

Biochemical tests: The biochemical test results illustrated in Table (1) clarifies the biochemical test result of Acinetobacter strain. Unlike other gram negative bacteria, Acinetobacter has a high tendency to resist decolonization which could be mistaken as gram positive bacteria. The isolate was able to produce gas from glucose and also showed positive growth in 2% NaCl only. Unlike catalse, oxidase and indol test, Acinetobacter was able to liquefy gelatin by 90%. Table 1. The biochemical test results of the isolate under study
Gas from Glucose

Growth at 45C

Growth at 37C

6.5% NaCl

Cocobacilli

Gelatinase

Indol Test

2% NaCl

Catalase

Oxidase

Shape

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Carbohydrate fermentation pattern of the isolate under study was carried out. Table (2) illustrates the results. It has the same fermentation pattern with Glucose, Galactose, Lactose, Sucrose, Sorbitol, Mannose, Rhamnose and Citrate. Alternatively negative result in fermentation has been obsereved with Fructose, Mannitol and Xylose. Table 2. Carbohydrate fermentation results of the isolate under study

Rhamnose

Galactose

Mannose

Fructose

Mannitol

Glucose

Sucrose

Lactose

Sorbitol

Xylose

Citrate

The isolate under study showed alpha hemolysis on blood agar using 5% sheep blood as illustrated in Figure (1). According to the pervious data, it could that the isolate was closer to Acinetobacter haemolytics.

Fig. 1. Alpha hemolysis on blood agar of the tested isolate. 16SrDNA amplification: The genus Acinetobacter currently contains up to 33 described named and unnamed (genomic) species [29 and 30]. The 16SrDNA analysis was performed against the Acinetobacter genomic DNA using the designated primers. The results are shown in fig. 2. A fragment with molecular size of 460 bp was obtained. Other fragment sizes have been obtained by different authors [31] who obtained a 240-bp PCR product by using Ac436f/Ac676r primer set. The difference in the molecular size of the fragment we obtained may be due to the design of the primers used or the individual variation occurs between the different species. However, the 16S rRNA gene is not polymorphic enough to clearly distinguish all Acinetobacter species [32]. The currently used 16S rRNA gene sequencing determinations have failed to distinguish closely related genomic species of Acinetobacter due to its extremely low polymorphic nature. The present approach may help to elucidate the ecology and clinical significance of different species of Acinetobacter (23). 1000 bp 800 bp 460 bp 500 bp 100 bp

Fig. 2. The 16S rRNA PCR product

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RFLP analysis: The 460-bp fragment of 16S rRNA was subjected to cutting by restriction endonucleases. Three different enzymes were used (BamH1, Pst1 and Hind III). None of these three enzymes have been able to cut the fragment although the reactions were carried out according the manufacturer procedures and also some modification in the concentration of both DNA and the enzymes were employed. In the present study, we used the three mentioned restriction enzymes to find out whether they able to cut or not despite it was reported that Acinetobacter species identification method is based on PCR amplification with subsequent restriction analysis (RFLP) with HinfI and MboI enzymes [33]. This is in general may be due to the absence of restriction sites within the fragment of interest or reflect the individual variation with the species of Acinetobacter. RFLP analysis can reveal different fragmentation patterns [34] in some cases while in other cases, and depending of the RE used, no fragmentation could be obtained. Sequence analysis: With the increasing availability of sequencing facilities, sequences of specific genes may be useful for species identification. Partial [35] or nearly complete [36] sequence analyses of the 16S rRNA gene for Acinetobacter classification have been performed using ABI PRISM BigDyeTM Terminator Cycle Sequencing Kits using AmpliTaq polymerase (Applied Biosystems). The obtained data indicated that our bacterial isolate belongs to uncultured Acinetobacter sp. (Fig. 8 and 9). For the forward primer, the maximum identity was 93% with an E-value of 8e-148 for the clone number GI7-8-G13 (accession number: FJ 193823.1). For the reverse primer, the maximum identity was also 93% and an E-value of 7e -133 for the clone number GI7-8-G13 (accession number: FJ 193823.1).

Fig. 3. The main tree obtained by the forward primer

Fig. 4. The main tree obtained by the reverse primer

Biodegradation of Malathion: Organophosphorus compounds are a group of highly toxic agricultural chemicals widely used for plant protection. Up to the present these pesticides such as parathion and Malathion, are still extensively used worldwide despite their high toxicity [1]. Due to environmental concerns associated with the accumulation of these pesticides in food products and water supplies, there is a great need to develop safe, convenient and economically feasible methods for pesticide detoxification in environment [5]. Along with identifying the isolated bacteria, the biodegradation experiments were carried out on the target pollutant. Malathion was incubated for one week with the bacteria to allow the biodegradation. Four concentrations of Malathion were used (100, 150, 250, and 500 ppm). At the end of the experiment, the cells were harvested and the biodegradation rate was assessed. Gas Chromatography was used to detect the residuals of Malathion. The data obtained (Fig. 5-9) is strongly indicated the complete biodegradation of Malathion by a rate of 100% after one week of incubation; this means that the rate of degradation was 2.97 mg/ (Lx h). This might be slightly lower than the degradation rate obtained by Acinetobacter johnsonii MA19 which reached 3.5837 mg/(L x h) [37]. Meanwhile, there are other bacterial species that can degrade Malathion such as B. thuringiensis MOS-5 that can utilize it cometabolically as a sole energy and carbon source [5]. We could conclude that the strain was Aceinetobcter sp. These results have been indicated by the 16S r DNA amplification followed by sequencing of the consensus fragment of that gene. The Acinetobacter species was efficiently able to utilize Malathion as a sole carbon source and this may show a great potential for the development of a new safe, convenient and efficient way of degrading Malathion.

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