Heavy metals found in nature and the excessive accumulations of heavy metals have an impact on humans and animals. Different effects resulted from the toxicity of heavy metal, that damage the functioning of different organs: brain, lungs, kidney and other essential organs and lowering the levels of energy. Cadmium and lead considered being toxic to organisms in a particular concentration. Patients with renal failure concerned contaminated drinking water with cadmium and lead. Biofilm produced by microbes and it is important for the remediation of pollutants. This study aimed to isolate and investigate the ability of bacterial isolates to produce a biofilm that can resistance heavy metals (cadmium chloride (CdCl2) and lead nitrate Pb (No3)2). Isolates were isolated from soil sample located at different locations from Saudi Arabia (Makkah, Taif and Jeddah). Fifty isolates have been tested for formation of biofilm by two methods. First method was Congo Red Agar CRA and the second was Tissue Culture Plate TCP. Results revealed that 3 out of 50 isolates showed high biofilm formation. The three (A2, ST and PS) isolates that form strong biofilm were screened primarily on nutrient agar plate contain 7ppm concentration of CdCl2 and Pb (NO3)2. Results indicated that all three isolates were resistance. The maximum tolerance concentration (MTC) of three (A2, ST and PS) isolates studied on nutrient agar plate supplemented with different concentrations from CdCl2 and Pb (NO3)2 respectively. Results indicated that MTC values of Pb (NO3)2 were up to (450, 350 and 500 ppm) for ST, A2 and PS isolates respectively. While in CdCl2 the MTC values were (150, 120 and 250 ppm) for ST, A2 and PS isolates respectively. The effect of CdCl2 and Pb (NO3)2 on bacterial growth using spectrophotometer, and results indicated that all three isolates (A2, ST and PS) growth decreased with the increase in concentration of Pb (NO3)2 and CdCl2. Three isolates were identified by biochemical and 16S rRNA gene. The isolates identified as B. cereus A2, B. cereus ST and P.aeruginosa PS and submitted to NCBI under accretion numbers (MK450303 and MK450304 for B. cereus A2, B. cereus ST) respectively. Plasmid curing was studied using the method of elevated temperature, and results showed that all cured B. cereus A2 and P. aeruginosa PS colonies were resistance to 7ppm of CdCl2 and Pb (NO3)2 while B. cereus ST showed different pattern of resistance after curing. B. cereus ST selected for test removal Pb (NO3)2 and CdCl2 using inductively coupled plasma optical emission spectrometry (ICP-OES). Using ICPOES showed removal lead up to 93% while in cadmium to 49 %. Antimicrobial susceptibilities patterns of identified bacteria were determined. All tested isolated strains showed resistance against to 3 or more antibiotics. Three strains B. cereus A2, B. cereus ST and P. aeruginosa PS that isolated from soil, showed the highest biofilm formation which considered important factor for heavy metals resistance. The biofilm represents a very renewable, promising, cost-effective and easy biotechnology for treatment of wide range contaminated effluents.
Heavy metals, biofilm, genomic DNA, 16S rRNA sequencing, plasmid curing, B. cereus ST, lead nitrate Pb (NO3)2 Cadmium chloride (CdCl2), phylogenetic, Gram stain.
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