Isolation and Molecular Identification of Bacterial Strains to Study Biofilm Formation and Heavy Metals Resistance in Saudi Arabia

© The Author(s) 2019. Open Access. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License which permits unrestricted use, sharing, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. Makki et al. J Pure Appl Microbiol, 13(1), 419-432 | March 2019 Article 5512 | https://dx.doi.org/10.22207/JPAM.13.1.46 Print ISSN: 0973-7510; E-ISSN: 2581-690X


INTRODUCTION
The heavy metals are non-degradable, toxic, tend to accumulate in organisms and undergo food chain amplification, persistent nature and effects on the local users, so considered the most dangerous groups (Rajeev Kumar et al., 2014). Badr et al., (2008) revealed that, the activities in the coastal area of Saudi Arabia by industries and human have increased through three decades, and resulted in heavy metals pollution. There are three main categories of metals: non-toxic and essential as Calcium (Ca) and Magnesium (Mg), only toxic at high concentrations (typically, Iron (Fe) Manganese (Mn), Zinc (Zn), Copper (Cu), Cobalt (Co), Nickel (Ni) and Molybdenum (Mo), and Mercury (Hg) or Cadmium (Cd) were toxic (Valls and de Lorenzo, 2002). Air, soil, and water are contaminated with toxic heavy metals that effect on human (McDonald and Grandt, 1981; Alloway, 1995). Pandey and Jain, (2002) reported that the pollution with chemical and environmental problems has brought the possibility of longterm environmental disasters into the public conscience. Heavy metals including lead, mercury, cadmium and arsenic effects in human metabolism due to their persistence in the environment and documented potential for serious health consequences. Microorganisms can be used for the clean-up of environmental pollutants, this is known as bioremediation. Paul et al., (2005) reported that treating toxic effluents with the biological processes are the best efficiency and economy from other methods as (chemical and physical), and the potential of biofilm communities for bioremediation processes has been realized. Bioremediation with biofilm is safer because the cells in a biofilm possess good chance of adaptation and survival (during periods of stress), also as they are protected within the matrix so it is alternative to bioremediation with planktonic microorganisms (Decho, 2000). A biofilm includes one or more bacterial strains disposed in a matrix containing extracellular polymeric substances such as DNA, protein or carbohydrates (Post et al., 2013). Extracellular polymeric substances (EPS) are biosynthetic polymers produced by micro-organisms from (prokaryotic and eukaryotic) (Wingender et al., 1999).
Different factors effect on EPS production by bacterial strains in (culture or aggregates) depends on the microbial species, phases of growth, nutritional status and the environmental conditions (Sheng et al., 2006). Cell adhesion, formation of microbial aggregates (biofilms, flocs, sludges and bio-granules), depend on bacterial EPS (Sutherland, 2001;Tay et al., 2001;Comte et al., 2006) and also protect cells from hostile environments. EPS also involved in the degradation of particulate substances sorption of dissolved materials including heavy metals (Gutnick and Bach, 2000).
Different resistance mechanisms to counteract stress heavy metal have developed in bacteria, as the formation and sequestration of heavy metals in complexes, reduction of metal to a fewer toxic species, and direct efflux of a metal out of the cell (Nies, 1999), Environmentally significant microbe such as P.aeruginosa is a ubiquitous, possess many mechanisms of resistance, such as the mer operon that reduces toxic Hg 2 to volatile Hg O , which then diffuses out of the cell (Outten et al., 2000). Morillo et al., (2006) reported that EPS produced from Paenibacillus jamilae strain capable of absorbing heavy metals from a multi-metal sorption system: Pb, Cd, Cu, Zn, Ni, Co when grown in aqueous extracts of two-phase olive mill waste.
Identification of bacteria from environmental or clinical specimens by sequencing of 16S rRNA gene that determine bacterial phylogenetic relationships (Vandamme et al., 1996;Gomila et al., 2004).
The present study investigated the (1) ability of some bacterial isolates that isolated from soils to produce a biofilm that can detoxify heavy metals (2) identified the bacterial strains by biochemical and 16S rRNA gene, (3) test removal Pb (NO 3 ) 2 and CdCl 2 , plasmid curing and antibiotic resistance.

MATERIALS AND METHODS Soil sample collection and bacterial isolations
All samples taken from the soil located at different locations from Saudi Arabia (Makkah, Taif and Jeddah). These samples were taken in sterilized polyethylene bags using the sterilized spatula and stored at 4°C until the examination. One gram of soil was suspended in 9 ml of sterilized distilled water, and serial dilutions up to 10 7 were prepared, 0.1ml suspension of each 10 -5 and 10 -7 dilutions spread on nutrient agar medium then plates incubated at 30°C for 24 hrs. Several colonies of bacteria were selected and isolated.

Biofilm Formation detection by Congo Red Agar method (CRA)
This medium prepared with brain heart infusion broth, sucrose, agar and Congo Red indicator. The stain of Congo Red was prepared separately as a concentrated aqueous solution and autoclaved. Then it was added to the autoclaved brain heart infusion agar with sucrose. Inoculate the plates of CRA with test bacterial isolates and incubated at 37 o C for 24 hrs. Aerobically, black colonies indicated production biofilm (Freeman et al., 19 89).

Biofilm Formation detection using Tissue Culture Plate method (TCP)
This assay is considered as a standard test for the detection the formation of biofilm. The overnight cultures grown in NB were diluted at 10 -3 and inoculated into six individual wells of a Tissue Culture Plate Method (150µl per well). Then the plates were incubated for 24 hrs. at 30 o C. Bacterial isolates were screened for their ability to form biofilm by the TCP method with a modification of (Christensen et al., 1985) according to (O'Toole and Kolter, 1998).

Heavy Metal Resistance Screening
The isolated bacterial colonies were screened for resistance to Cadmium Chloride (CdCl 2 ) and Lead Nitrate (Pb (NO 3 ) 2 by adding 7ppm of CdCl 2 and Pb (NO 3 ) 2 , respectively, to sterilized nutrient agar medium. The isolated bacterial colonies were spot inoculated onto the plates of nutrient agar, then incubated for 24 hrs. at 37°C (Margeay et al., 1985).

Maximum Tolerable Concentration of Bacterial Isolates (MTC)
The maximum tolerable concentration is the highest concentration of the heavy metals that growth at 37°C. Isolated bacterial colonies that grew initially on the nutrient agar supplemented with the heavy metals CdCl 2 and Pb (NO 3 ) 2 were exposed to increasing heavy metals concentrations (7ppm -500ppm). Testing for tolerance of the microorganisms ended when complete inhibition of the growth was observed on the nutrient agar with metal supplementation according to (Mulik and Bhadekar, 2017).

Growth Study of Metal Resistant Isolates
The capacity bacterial strains for tolerance of heavy metals were tested, cells were grown in 50 ml LB medium supplemented with different concentrations of the heavy metals, cadmium and lead, incubated for 24, 48 and 72 hrs. at 37°C on a rotary shaker (150 rpm) of selected heavy metal (cadmium (CdCl 2 ), lead (Pb (NO 3 ) 2 ). Growth was monitored as a function of biomass by measuring absorbance at 600 nm using a spectrophotometer. The growth of the isolates on LB with no metal supplementation (control) and with metal supplementation (test) were performed and compared by plotting the optical density at 600 nm (OD 600nm ) to time in hours. According to (Chien et al., 2013).

Morphological Characterization and Biochemical test
B a c t e r i a l i s o l a t e s w e re t e s t e d for morphology on nutrient agar medium, microbiological tests such as Gram staining followed by biochemical identification tests like catalase, citrate oxidase, indole production.

Isolation of Genomic DNA
Bacterial colonies isolated from the nutrient agar with metal supplementations were characterized molecularly using the 16S rRNA sequencing. The genomic DNA was extracted using a GeneJET Genomic DNA extraction kit according to the manufacturer's instructions.

Amplification of 16S rRNA
Amplification of 16S rRNA from extracted DNA has used as a template for PCR to amplify the 16S rRNA gene. The forward primer 27F 5' (AGA GTT TGA TCM TGG CTC AG) 3 and reverse primer 1492R 5' (TAC GGY TAC CTT GTT ACG ACT T) 3' were used to amplify the 16s rRNA gene. PCR was performed with a one gel electrophoresis in 1 x TAE buffer with ethidium bromide (0.5 µg/ml) using the Mupid-One.

Sequencing of Amplified Fragments of Isolate 16S rRNA Genes
Samples for sequencing sent to MACROGEN, Korea. Sequences have compared with the available sequences against the 16S rRNA sequences database using NCBI's BlastN. Sequences were aligned using the ClustalW program in Mega 6.0. Similarity index was generated and compared with known sequences. Plasmid Curing using elevated Temperature for Bacterial Isolates Ten ml of NB was inoculated by single colony, incubated for 24 hrs. at 37°C then 0.2 ml of bacterial culture was transferred to 10 ml of fresh NB and incubated at 45°C (an elevated temperature) for 24 hrs., with shaking at 100 rpm. Several dilutions up to 10 -7 were prepared, then 0.1 ml of the last three dilutions were spread on plates of nutrient agar which supplemented with 7 ppm with CdCl 2 and Pb (NO 3 ) 2 and incubated for 24 hrs. (37°C) (Kheder, 2002).

Gel Electrophoresis
According to (Sambrook et al, 1989), a plasmid was characterized by agarose gel electrophoresis through a gel of 1% agarose submerged in 100 ml 1X TBE supplemented with 2µl of ethidium bromide running buffer at 120 V for 1 hr. 7µl from sample with 1µl from loading buffer dye. DNA bands were visualized on UV. The plasmids MW was compared and determined by using DNA ladder.

Measurements of CdCl 2 and Pb (NO 3 ) 2 Removal by Bacterial Isolate
B. cereus ST, was grown in LB medium supplemented with different concentrations ranged from (7-150 ppm) of CdCl 2 and (20-450 ppm) for Pb(NO 3 ) 2 individually. After 24, 48 and 72 hrs. of incubation, centrifuge at 10,000 rpm for 10 min. to cells separate the cells. The CdCl 2 and Pb (NO 3 ) 2 removal properties were estimated by measuring metals depletion in culture supernatants by inductively coupled plasma-optical emission spectroscopy (ICP-OES). The ICP-OES system was calibrated by serial dilution of each metals standard (Mulik and Bhadekari, 2017).

Samples collection and bacterial isolation
A total of fifty bacterial isolates were selected and storage at 4 o C until test their ability for biofilm formation. The biofilm formation was verified using two techniques, one qualitative and the other quantitative. The qualitative technique of biofilm formation for fifty isolates was assessed by congo red agar. The black colors were observed for the biofilm production. Results indicated that several isolates were positive for biofilm formation. The results in (Fig. 1 and Fig. 2) showed the strong three isolates (A2, ST and PS) give positive black color colonies on Congo red agar plate while negative isolates give pink color indicating non-biofilm formation.

Determination of the Effect of Pb (NO 3 ) 2 and CdCl 2 on Bacterial Growth
Measuring absorbance at 600 nm using the spectrophotometer, to test tolerance of the bacterial isolates to heavy metals, and then function of biomass. Growth of the isolates on LB with no metal supplementation (control) and with metal supplementation (test). Results showed that growth curves of isolates (A2, PS and ST) in presence of different Pb (NO 3 ) 2 and CdCl 2 concentrations ranged from (7-550 ppm) are shown in (Fig. 4-11). Results indicated that growth of all isolates decreased with the increase in concentration of Pb (NO 3 ) 2 and CdCl 2 compared with control.

Morphological and Biochemical Molecular Identification
The morphological and biochemical characteristics of the bacterial (A2, ST and PS) isolates were tested on the nutrient agar. On the basis of the morphological and biochemical study, A2 and ST isolates were identified as Bacillus cereus, and the isolate PS identified as Pseudomonas aeruginosa. The bacterial isolates were positive for (catalase and citrate) while negative for (indole and oxidase) test.
16S rRNA of two strains was sequenced and used to create phylogenetic development tree. Comparative analyses of the sequences from the NCBI databases showed that the strains were closed to the members of the genera Bacillus ( Table 4). The highest sequence similarities of the group are ST, B. cereus (99% similarity to B. cereus strain ST; A2, B. cereus (99% similarity to B. cereus strain A2;. Phylogenetic tree of B. cereus ST, A2 shown in (Fig. 12). The sequences were submitted to the NCBI Gene Bank (www.ncbi.nlm. nih.gov)          (Fig. 13 and Table 6) showed 9 cured B. cereus ST colonies picked up and tested on nutrient agar medium supplemented with 7ppm CdCl 2 revealed that all cured B. cereus ST colonies lost resistance to CdCl 2 . Nine colonies from cured B. cereus ST were selected for plasmid isolation compared with control.

Isolation of plasmid from cured B. cereus st strain
Results in (Table 7) indicated that wild B. cereus ST strain had 2 plasmids (23kb and 564 bp). After plasmid curing all colonies of cured B. cereus ST lost two plasmids while colonies (3, 4, and 5) had one plasmid (23kb).

Antibiotic resistances
Resistance to antibiotics was determined on Mueller Hinton agar plates. Results in (Table  8) showed the effect of ten antibiotics on the B. cereus indicate that B. cereus A2 was resistance to 5 antibiotic; Metronidazole, Taxo, Tobramycin, Ceftriaxone and Aztreonam while sensitive to  (15 mm) and were no effect for Metronidazole, Taxo, Aztreonam and Ceftriaxone     Three bacterial strains (A2, PS and ST) were selected as the most formed biofilms to study of their resistance to the two heavy metals Pb (NO 3 ) 2 and CdCl 2 : The resistance of the isolates of CdCl 2 and Pb (NO 3 ) 2 were studied on nutrient agar plates supplemented primary with 7 ppm   (Priyalaxmi et al., 2014). Raja et al., (2006) reported that P. aeruginosa (MTC) value for Cd ranged from 100-500 and 100-800 ppm for Pb.

Effect of heavy metals on bacterial growth
Using a spectrophotometer with control samples to study the effect of CdCl 2 and Pb (NO 3 ) 2 on bacterial growth studied on LB media containing different concentrations (7, 50, 100, 150ppm) and (20, 50, 200, 450ppm) respectively , however the higher the concentration of CdCl 2 and Pb (NO 3 ) 2 have the less growth due to the toxic effect on bacterial growth. Khatun et al., (2012) studied the effect of two heavy metals, CdCl 2 and Pb (NO 3 ) 2 on Bacillus cereus growth and showed different growth patterns in the presence of different heavy metals like (Cd and Pb). Rajbanshi, (2008) reported the decrease of the microbial (2012) reported that P. aeruginosa was highly sensitive to Levofloxacin. However, Salih et al., (2011) reported that P. aeruginosa resistant to Doxycycline during sensitive Tobramycin. Several 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. Results revealed that 3 out of 50 isolates showed high biofilm formation, these three (A2, ST and PS) isolates were tested primary for CdCl2 and Pb (NO 3 ) 2 resistances at 7ppm concentration results revealed that isolates were resistance. The maximum tolerance concentration (MTC) of three (A2, ST and PS) isolates studied with different concentrations for CdCl 2 and Pb (NO 3 ) 2 respectively. Results indicated that MTC values of Pb (NO 3 ) 2 were up to (450, 350 and 500 ppm) for ST, A2 and PS isolates respectively. While in CdCl 2 the MTC (150, 120 and 250 ppm) for ST, A2 and PS isolates respectively.

CONCLUSION
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.