oa Sampling of Qatari Sand Microbes: Serious about B.cereus

Objective: A shortage of studies regarding the ecology of Qatar led to the sampling sand to determine the composition of microbes within the sand (below a certain size). This sampling began with a sand sample obtained from Al Rayyan, Doha Qatar. The sample was purified, and sequenced after isolating the DNA. A comprehensive analysis of the local Qatar sand sample shows a range of various microbes (which are smaller than 0.2 micrometer), which exist in the sand. Among these organisms was Bacillus cereus a bacterium from the Bacillus family that is most commonly responsible for mild food poisoning. The genes are being studied further to understand better what the sand microbial composition is like. Furthermore, it will help us better understand the genomes of these microbes and possibly how the proteins allow them to survive within the sand. Method: After obtaining the sequenced DNA, a Short Oligonucleotide Analysis Package- Denovo (SOAP-Denovo) system that helps assemble the genetic information into short reads called contigs was used. In addition to this, another program called Velvet was also used in conjunction to ensure the best results were obtained using the optimal k-mer, providing the longest contig length. The longest length obtained was a contig of 12,380 at a k-mer of 59. Subsequently, the results of the assembly program were assembled into nucleotide and protein sequences using the gene prediction software, GeneMarkS available online. Each of the genes from the contigs were then searched against the National Centre for Biotechnology Information (NCBI's) online database using Basic Local Alignment Search Tool (BLASTp) which utilizes certain algorithms to compare the protein input sequences in FASTA format to the entire non redundant protein database of available sequenced genes. This tool provided us with a possible list of the gene's (percentage) identity, along with the maximum and total scores, the query cover (percentage of sequence that matches that available for a particular organism's gene) and the E-value (the smaller the value the less the probability of the similarity occurring due to random chance) in order to assess the probability that the results are accurate. All this data was collected are currently being analyzed further. Results: It was found that the microbe majorly contributing to presence in the sand was Bacillus thuringensis with 50%, closely followed by Bacillus cereus with 40%, 5% Methylobacterium, 3% Enterobacteriophages, 2% other forms of Bacillus species and finally some traces of Streptococcus pneumonia as well as some other organisms. Bacillus thuringiensis or Bt is a common biological pesticide used in agriculture and is safe for human consumption; this bacterium is a common soil dwelling organism. Bacillus cereus on the other hand is less safe for human consumption as it produces harmful toxins that cause mild food poisoning and it is similarly a soil-dwelling microbe. Methylobacterium, are bacteria that are usually found in soil and are the natural flora found on the ground. Enterobacteriophages are bacteriophages that commonly infect E.coli. And finally Streptococcus pneumoniae are pathogenic bacteria that exist in the body but are the main causative agents of pneumonia in humans when they exceed a certain threshold or if the immune system is vulnerable. Discussion: The results showed that the microbes did not share 100% identity with their respective matches. There is a high chance that the small percentage differences (which ranged from 1-6%) could be due to mutations that enable the microbes to adapt to their environment's temperature. For example, some of the B.cereus genes that have undergone mutation are those that code for polysaccharide synthase (94% identity), histidinol dehydrogenase (99%), a capsular polysaccharide biosynthesis protein (99%), or a sodium/hydrogen exchanger (also 99%). Future work: In order to verify the presence of the organism in the sand, there will need to be more samples, and a repeat of the experiments. With repeated experiments, the organisms will not be filtered; the threshold for the size of microbes will be increased, allowing for a greater view of the composition of the sand samples in Qatar. This will allow us to gain a better picture of microbial life in the ecology of Qatar; especially in terms of the presence of pathogenic microbes.

The sand samples will be cultured and grown on blood agar plates and the microbes would be identified through morphological characteristics. A repeat of the DNA isolation and sequencing will be carried out to confirm the presence of the microbes such as B.cereus. Furthermore, PCR tests will have to be conducted using universal primers for 16sRNA of certain bacteria as a control, to check against the DNA extracted from the sand sample during the initial round of DNA sequencing. If any of the genes are the same as the B.cereus from the first time, it would be confirming its presence in the sand.

It would be possible also to design primers specific for B. cereus in order to develop a convenient method of testing for such organisms. This could be extremely useful for test in food since food poisoning is commonly caused by these easily detectable and curable microbe infections. This could help reduce the level of food poisoning in the country, which is quite a common occurrence in Qatar.

In future studies, more samples could be taken from various parts in Qatar, as well as at a range of different depths within the sand, to further increase our knowledge of the types of microbes present in the ecology.