Antimicrobial Activity of Bacteria Isolated From the Rhizosphere and Phyllosphere of Avena Fatua and Brachiaria Reptans Growing in Heavy-Metal Polluted Environment


 Environmental pollution especially heavy metal contaminated soils adversely affect the microbial communities associated with the rhizosphere and phyllosphere of plants growing in these areas. In the current study, we identified and characterized the rhizospheric and phyllospheric bacterial strains from Avena fatua and Brachiaria reptans with the potential for antimicrobial activity and heavy metal resistance. A total of 18 bacterial strains from the rhizosphere and phyllosphere of A. fatua and 19 bacterial strains from the rhizosphere and phyllosphere of B. reptans were identified based on 16S rRNA sequence analysis. Bacterial genera, including Bacillus, Staphylococcus, Pseudomonas and Enterobacter were dominant in the rhizosphere and phyllosphere of A. fatua and Bacillus, Marinobacter, Pseudomonas, Enterobacter, and Kocuria were the dominating bacterial genera from the rhizosphere and phyllosphere of B. reptans. Most of the bacterial strains were resistant to heavy metals (Cd, Pb and Cr) and showed antimicrobial activity against different pathogenic bacterial strains. The whole genome sequence analysis of Pseudomonas putida BR-PH17 was performed by using Illumina sequencing approach. The BR-PH17 genome contained a chromosome with size of 5774330 bp and a plasmid DNA with 80360 bp. In this genome, about 5368 predicted protein coding sequences with 5539 total genes, 22 rRNAs and 75 tRNA genes were identified. Functional analysis of chromosomal and plasmid DNA revealed a variety of enzymes and proteins involved in antibiotic resistance and biodegradation of complex organic pollutants. These results indicated that bacterial strains identified in this study could be utilized for bioremediation of heavy metal contaminated soils and as a novel source of antimicrobial drugs.

released by these industries. Plants and animals of this area are also affected by water and air pollution.
Plants especially various grasses, such as Avena fatua, Brachiaria reptans, Cymbopogan jwarancusa, Cynodon dactylon, and Dactyloctenium aegyptium are dominant and abundantly found here (Ahmad et al. 2009). Rhizospheric soil samples were collected by gently removing the plants and obtaining the soil attached the roots. Soil and plant samples were collected from three sites that are about 1 km far from each other. At each site, approximately 1 kg soil samples were collected in black sterile polythene bags.
These samples were stored at 4 ℃ for further analysis.

Soil physicochemical parameters
Each soil sample (500 g) was thoroughly mixed and sieved through a pore size of 2 mm. Physical properties (salinity, pH, moisture content and temperature) of soil samples were determined. Soil salinity or electrical conductivity (dS/m) was measured by 1:1 (w/v) soil to water mixture at 25 ℃ (Adviento-Borbe et al. 2006), pH was measured by 1:2 (w/v) soil to water mixture, moisture (%) and texture class were measured by Anderson method (Anderson et al. 1993) and organic matter (C org ) was calculated by the Walkley-Black method (1934). The total concentration of heavy metals (Cd, Pb, and Cr) in the soils and plants were analyzed by ame atomic absorption spectrophotometry (AAS, Z-5300) by digesting 100 mg of soil in a mixture of HNO3 and HClO4 (4:1, v/v).
Isolation of bacterial strains from the rhizosphere and phyllosphere of A. fatua and B. reptans Rhizospheric samples were taken as a collective sample of soil and roots and phyllospheric samples were taken as epiphytic and endophytic shoot tissues. For the isolation of rhizospheric bacteria, the sieved soil and roots were mixed thoroughly and then one gram representative soil sample was taken. In case of phyllosphere, the shoot samples were washed with tap water for 5 min and then with distilled water for 5 min. These tissues were dried at room temperature and one gram sample was macerated using sterilized pestle and mortar. Serial dilutions (10 − 1 -10 − 10 ) were made for all samples (Somasegaran 1994). Dilutions from 10 − 4 to 10 − 6 were inoculated on Luria-Bertani agar (LB) plates for counting colony forming units (CFU) per gram of dry weight. Plates were incubated at 37 ℃ until the appearance of bacterial colonies. Bacterial colonies were counted and the number of bacteria per gram sample was calculated. The bacteria were puri ed by repeated sub-culturing of single colonies. Single colonies were selected, grown in LB broth and stored in 30% glycerol at -80 ℃ for subsequent characterization.
Sequences of 16S rRNA were compared to those sequences deposited in the GenBank nucleotide database by using the NCBI BLAST. These sequences were aligned by using Clustal W software. A neighbor-joining tree was constructed by Bootstrap test with 1000 replicates (Saitou and Nei 1987). The evolutionary distances were compared using the Maximum Composite Likelihood method in MEGA7 software (Tamura et al. 2004;Kumar et al. 2016). The 16S rRNA sequences of bacterial strains were deposited in the GenBank with accession numbers MT317180-MT317216.
Antibiotic resistance assay using disc diffusion method The antibiotic resistance pattern of bacterial strains was studied according to Kirby-Bauer disk diffusion method (Bauer et al. 1966; El-Sayed and Helal 2016). Five antibiotics; ampicillin (AMP), amoxicillin (AM), erythromycin (E), cipro oxacin (CIP), tobramycin (TOB), gentamicin (GN) and vancomycin (VA) were used to check antibiotic sensitivity of bacterial strains. Antibiotic discs were placed over freshly prepared LB medium seeded with bacterial strains under study. All antibiotic disks were placed on each of the seeded plates at appropriate distances from one another and plates were incubated at 37 ℃ for 48 h. The strains were classi ed as sensitive or susceptible if they showed a growth inhibition zone around the antibiotic disc.
Analysis of heavy metal resistance A total of 18 bacterial strains from the rhizosphere and phyllosphere of A. fatua and 19 bacterial strains from the rhizosphere and phyllosphere of B. reptans were tested for resistance of Cadmium (Cd), Lead (Pb) and Chromium (Cr). About 2, 5, 7.5, and 10 mM of each metal was used to analyze the resistance in the bacterial strains isolated using LB agar plates supplemented with these heavy metals.

Antimicrobial resistance against different pathogenic bacterial strains
On the basis of antibiotics and heavy metals resistance, 8 bacterial strains from the rhizosphere and phyllosphere of A. fatua and 10 bacterial strains from the from the rhizosphere and phyllosphere of B. reptans were tested for antimicrobial activity against six pathogenic bacterial strains. Antimicrobial activity test of bacterial strains isolated from the rhizosphere and phyllosphere of A. fatua and B. reptans was performed by using the drop test method as described by Rao et al. (2005) with little modi cations. Six pathogenic bacterial strains including Bacillus cereus (LT221128), Staphylococcus aureus (MT355444), Pseudomonas aeruginosa (LT797517), Escherichia coli (MT355445), Klebsiella oxytoca (LT221131), and Enterobacter cloacae (AM778415) were used for antimicrobial activity in this study. All the pathogenic strains were grown in 20 mL of LB broth at 37 ℃ for 24 h. The bacterial strains identi ed in this study were also grown in 50 mL of LB broth at 37 ℃ for 24 h. These cultures were centrifuged at 10,000 rpm for 15 min and the cell pellet was dissolved in 25 mL of saline water (1% NaCl). About 100 µL of a pathogenic strain was spread on LB agar plate and dried for 30 min. Then a drop (10 µL) of the target bacterial culture with about 10 10 cells mL − 1 was spotted on this plate and incubated at 37 ℃ for 48 h. The zone of inhibition around the tested strain was measured in mm (millimeter).
Genome sequencing and annotation of P. putida BR-PH17 P. putida BR-PH17 showed maximum potential for heavy metals and antimicrobial resistance. The next generation whole-genome sequencing was performed by using Illumina Hiseq 2000 platform. Paired-end genome fragments were annealed to the ow-cell surface in a cluster station (Illumina). Sequencing-bysynthesis was performed with a total 100 cycles. All reads were quality ltered and assembled using the

Physical and chemical characteristics of rhizospheric soils
The physical and chemical analysis showed that the rhizospheric soils of Avena fatua were more alkaline and saline as compared to soils Brachiaria reptans (Table 1). There is no signi cant diffference in soil moisture content and temperature in the rhizospheric soils of both plants. All the polluted soils had high concentrations of heavy metals Cd, Pb, and Zn ( Table 1)  Identi cation of bacterial strains based on 16S rRNA analysis A total of 10 bacterial strains from the rhizosphere and 8 bacterial strains from the phyllosphere of A. fatua were identi ed on the basis of 16S rRNA gene analysis. Four strains including AV-HP1, AV-HP2, AV-HP4 and AV-HP10 were identi ed as different species of Bacillus, AV-HP3 strain identi ed as Staphylococcus equorum, AV-HP5 strain as Pseudomonas plecoglossicida, AV-HP7 strain as Enterococcus durans, AV-HP8 as Nocardia farcinica, AV-HP6 and AV-HP11 as Enterobacter aerogenes from the rhizosphere of A. fatua (Table S1; Fig. 1A). While from the phyllosphere of A. fatua, three strains AV-RO1, AV-RO6 and AV-RO8 showed more than 99 similarity with Bacillus spp., AV-RO2 strain had 99% homology with Pseudomonas uorescens and bacterial strains belonging to Virgibacillus sp., Enterococcus durans, Kocuria rosea and Nocardia also identi ed in this study (Table S1; Fig. 1A). From the rhizosphere of B. reptans, 4 strains were related to Bacillus spp., 2 bacterial strains BR-PH1 and BR-PH10 were identi ed as Staphylococcus equorum, BR-PH4 strain were belonging to the Marinobacter sp. and BR-PH5 strains was identi ed as Pseudomonas uorescens (Table S2; Fig. 1B). From the phyllosphere of B. reptans, out of nine, 5 bacterial strains were identi ed as different species of Bacillus, BR-PH11 strain had more than 99% similarity with Exiguobacterium aurantiacum, BR-PH13 and BR-PH16 strains identi ed as Enterobacter aerogenes and BR-PH17 strain identi ed as P. putida (Table S2; Fig. 1B).
Antibiotic resistance pro le of bacterial strains About 50% bacterial strains showed resistance against both ampicillin and amoxicillin, 20% bacterial strains showed resistance against both erythromycin and tobramycin, 10% bacterial strains showed resistance against each cipro oxacin, gentamicin and vancomycin from the rhizosphere of A. fatua, while 55% bacterial strains showed resistance against both ampicillin, and amoxicillin, 37% bacterial strains showed resistance against cipro oxacin, 25% bacterial strains showed resistance against each erythromycin, gentamicin and vancomycin and 12.5% strains were resistant to tobramycin from the phyllosphere of A. fatua (Table 2; Fig. S1).  From the rhizosphere of B. reptans, 30% bacterial strains showed resistance against both ampicillin and vancomycin, 50% bacterial strains showed resistance against both amoxicillin and erythromycin, 40% bacterial strains showed resistance against both cipro oxacin and gentamicin and 20% bacterial strains resisted against tobramycin, while from the phyllosphere of B. reptans, maximum bacterial strains (55%) showed resistance against amoxicillin, 33% bacterial strains showed resistance against each ampicillin, erythromycin, and tobramycin, 22% bacterial strains showed resistance against both cipro oxacin and vancomycin, and 44% bacterial strains were resistant to gentamicin (  Fig. S1).

Heavy metal resistance pro le of bacterial strain
More than 80% of the rhizospheric bacterial strains and 90% of the phyllospheric bacterial strains from both plants showed Cd tolerance at a concentration of 2 mM, 71-78% bacterial strains showed Cd tolerance at a concentration of 5 mM and only few strains (0-19%) from the rhizosphere and 22-37% of bacterial strains from the phyllosphere of A. fatua and B. reptans showed Cd tolerance concentration of 10 mM (Table S3; Fig. 2A).
Similar results were obtained in case of Pb resistance. About 80-89% bacterial strains from the rhizosphere and 91-99% of bacterial strains from the phyllosphere of A. fatua and B. reptans showed Pb resistance at a concentration of 2 mM, 60% of bacterial strain from the rhizosphere and 71% bacterial strains from the phyllosphere of both plants showed Pb resistance at a concentration of 5 mM and phyllospheric bacterial strains from both plants showed Pb resistance (21-36%) as compared to rhizospheric bacterial strains with only 9-10 % Pb resistance (Table S4; Fig. 2B).
About 60-75% of rhizospheric bacterial strains and 77-87% of the phyllospheric bacterial strains from A. fatua and B. reptans showed Cr resistance at a concentration of 2 mM, 49-51% from the rhizosphere and 57-64% of bacterial strains from the phyllosphere of both plants. None of the rhizospheric bacterial strains was able to tolerate 10 mM Cr concentration while 11-24% of the phyllospheric bacterial strains from both plants showed Cr tolerance at a concentration of 10 mM (Table S5; Fig. 2C).

Antimicrobial activity of bacterial strains
From the rhizosphere and phyllosphere of A. fatua, ve strains had antimicrobial activity against E. cloacae, four strains showed antimicrobial activity against P. aeruginosa, four bacteria strains showed positive results against E. coli, four strains had antimicrobial activity against K. oxytoca two strains AV-HP5 and AV-RO7 showed antimicrobial activity against B. cereus and one strain AV-RO3 showed antimicrobial potential against S. aureus (Table 3; Fig. 3). Similar results were obtained in case of bacterial strains isolated from the rhizosphere and phyllosphere of B. reptans. Seven strains showed antimicrobial activity at least against three pathogenic strains. Six bacterial strains showed antimicrobial potential against K. oxytoca, and P. aeruginosa each, ve bacterial strains showed antimicrobial activity against E. coli and E. cloacae and three strains had antimicrobial activity against B. cereus and S. aureus (Table 3; Fig. 3). and ≤ 6 mm a weak positive (+).*Average of three replicates ± standard error of means General features of chromosomal and plasmid DNA of P. putida BR-PH17 The genome size of P. putida BR-PH17 is 5,774,421 bp with an average GC content of 61.07% (Fig. 4). A total 5,423 genes were identi ed and total coding sequences were 5,321. The protein coding CDSs were 5,241 and 102 RNA genes with 22 rRNA genes, 75 tRNA genes, and 5 non-coding RNA genes were present on the chromosome (Table 4). A total of 80 genes were predicted as pseudogenes because of missing Cor N-terminus or frameshift mutations. Pseudo genes (total) 80 The functional analysis of these genes using KEGG pathway database showed that they have an important role in various metabolic pathways including plant growth promotion, bioremediation of different toxic compounds, heavy metal and antimicrobial resistance and other abiotic stresses. Many small proteins detected were also annotated as hypothetical proteins. The functional analysis of CDSs showed that they could be classi ed into 22 general COG categories including metabolism of carbohydrates, amino acids, lipids, transcription, energy, cofactors and vitamins, inorganic ions, signal transduction and cellular processes, glycan biosynthesis and metabolism, cell motility, translation, ribosomal biogenesis, DNA replication and repair, secondary metabolites, defense mechanisms and xenobiotics biodegradation (Table S6; Fig. 5A).
Whole genome sequence analysis also showed that there was one plasmid pBR-PH17 with 80360 bp size. A total of 97 genes were encoded by plasmid pBR-PH17. Functional analysis of these showed that 16% genes involved in xenobiotics biodegradation and metabolism, 14% genes coded different proteins and enzymes which caused human diseases, 11% genes involved in genetic information processing, 9% genes coded proteins and enzymes involved in energy metabolism, 7% genes involved in carbohydrate metabolism, 6% genes involved in amino acid metabolism and 22% genes were unclassi ed (Table S7; Fig. 5B).
Heavy metal resistance and bioremediation potential of P. putida BR-PH17 Based on functional genome analysis of P. putida BR-PH17, different heavy metal determinants were identi ed. Heavy metal resistance genes such as cadmium (Cd), lead (Pb), chromium (Cr), zinc (Zn), copper (Cu), nickel (Ni), and mercury (Hg) were encoded by chromosomal DNA and lead, cadmium, copper, zinc, cobalt (Co) and manganese (Mn) resistance genes were encoded by plasmid DNA (Table S9; Fig. 6C and 6D).
Identi cation of gene clusters involved in secondary metabolism of P. putida BR-PH1 Genome annotation of P. putida BR-PH1 showed that a number of gene clusters including NAGGN (Nacetylglutaminylglutamine amide), RiPPs (ribosomally synthesized and post-translationally modi ed peptides), ranthi-peptide, phenazine, NRPS (non-ribosomal peptide synthetase) biosynthesis and redoxcofactors involved in secondary metabolism were identi ed in the genome of P. putida BR-PH1 which might be involved in plant growth improvement and biocontrol mechanisms (Fig. 7).

Discussion
In the recent years, microbial diversity analysis from the polluted environments is getting more attention From the rhizosphere and phyllosphere of both plants, more than 40% of strains showed resistance against ampicillin, amoxicillin, and cipro oxacin while more than 37% bacterial strains were resistant to amoxicillin, erythromycin, cipro oxacin, and gentamicin. A number of previous studies also reported that microbial diversity from polluted environments such as contaminated soil, water and wastewater activated sludge samples showed a variety of antibiotics resistance bacterial strains (Balcom et

Conclusion
To the best of our knowledge, the present study is the rst report about comparative analysis of microbial diversity from the rhizosphere and phyllosphere of A. fatua and B. reptans collected from polluted sites of Kala-Shah Kaku industrial area near Lahore. Bacillus, Staphylococcus, Pseudomonas and Enterobacter were the dominating bacterial genera identi ed from the rhizosphere and phyllosphere of A. fatua and Bacillus, Marinobacter, Pseudomonas, Enterobacter and Kocuria were the dominating bacterial genera identi ed from the rhizosphere and phyllosphere of B. reptans. The bacterial strains identi ed in this study also showed great potential for heavy metal resistance and antimicrobial activity against different pathogenic bacterial strains. From the results of genome sequence analysis, it was con rmed that P. putida BR-PH17 had various antimicrobial resistance genes such as, tetracycline, beta-lactam, cationic antimicrobial peptide (CAMP), aminoglycoside, vancomycin, sulfonamide, and rifampin and metal resistance genes such as, Cd, Pb, Cr, Zn, Cu, Ni, Hg, Mn and Co. Bacterial strains characterized from the rhizosphere and phyllosphere of plants growing in polluted areas can be utilized as a new source of antibiotics that might be used as promising antifungal and antibacterial drugs against different diseases.
Declarations Figure 1 Phylogenetic tree based on 16S rRNA gene sequences of bacterial isolates associated with from the rhizosphere and phyllosphere of A. fatua (A) and B. reptans (B). The percentage of replicate trees in which the associated taxa clustered together in the bootstrap test (1,000 replicates) is shown next to the branches.