Deciphering the Multi-Dimensional Abilities of Indigenous Bacteria Enterobacter Cloacae Isolated from Arsenic Contaminated Industrial Sites


 Arsenic (As) is a quintessential toxic metalloid and it has been classified as Group 1 human carcinogen. The evolution of arsenic defense mechanisms due to the omnipresent nature of arsenic has resulted in its alteration to less toxic forms. The present study deals with the isolation of arsenic remediating microbial strains from soil samples and their integration into bioremediation strategy. From the metal contaminated site, 118 different bacterial strains were isolated from heavy metal contaminated site. Twenty-five strains were tolerant to arsenic and one bacterial strain Enterobacter cloacae (RSC3) demonstrated maximum growth at high concentration of arsenate (6000ppm). The cell growth kinetics of RSC3revealed the specific growth rate (µ) to be 0.55 h-1. The The bacteria hosts arsC gene in the genome involved in the reduction of arsenate to arsenite. AAS, SEM, TEM and EDX studies confirmed the arsenate transportation and efflux of arsenic by the bacteria. Furthermore, the strain showed multi-resistance to other heavy metals like zinc, cadmium, selenium and nickel and several antibiotics indicating its application for facilitating bioremediation of toxic metal contaminated sites.


Introduction
Last few decades have seen tremendous rise in the environmental pollution in all forms. The contamination of soil and water resources by toxic metals/heavy metals is a major cause of health and environmental concern worldwide. The heavy metals include cadmium, arsenic, mercury, zinc, copper and selenium. Among these, arsenic (As) is one of the most prominent pollutant and is a persistent Group 1 human carcinogen (Kaur et al. 2011). The crisis involving the presence of arsenic in water and soil is rampant in parts of China, USA, Europe and Southeast Asia including parts of India. Arsenic contamination in India has been reported from Chhattisgarh, Bihar, Assam, West Bengal and Uttar Pradesh (Shrivastava et al. 2015;Satyapal et al. 2018). Natural processes as well as anthropogenic activities are accountable for the release of arsenic into the environment. Primarily human activities such as usage of insecticides and fertilizers, fossil fuel combustion, mining etc contributed to elevated levels of arsenic in the environment (Srivastava and Sharma 2013).
The maximum arsenic concentration limit recommended by World Health Organization (WHO) in drinking water and groundwater are 0.01 mg/L and 10 parts per billion (ppb) or 10 µg/L respectively (Graham 1999). But the concentration of arsenic in water and soil is way higher than the permissible limit. The arsenic presence in groundwater of Bengal Delta Plain (West Bengal, India and Bangladesh) ranges from 50 to 3200 µg/L (Bachate et al. 2009). In the Upper/Trans-Ganges Plains covering Punjab in northwestern India, arsenic concentration of surface soils has reportedly varied from 1.09 to 2.48 mg As kg − 1 . While the mean arsenic contents in soil of central India have been found to be higher than soil of West Bengal and Bangladesh. The lowest level of arsenic in the soil of this region is 3.7 mg/kg (Patel et al. 2005).
The arsenic accumulated in soil have the tendency to leach into water bodies and this could result in detrimental effects on human health through ingestion of contaminated water (Nithya et al. 2011).
Arsenic associated malignancies include skin lesions, hypertension, ischemia, some endemic peripheral vascular disorders, severe arteriosclerosis, neuropathies and noticeably, many types of cancer. The toxicity of arsenite lies in its ability to bind to sulfhydryl groups of cysteine residues in proteins and to deactivate them (Paul et al. 2015).
Studies involving the toxic metal-microbe interactions has gained wide attention. These interactions could result in the biogeochemical cycling of metals and in detoxi cation of metal-contaminated sites (Abbas et al. 2014). Further the correlation between tolerance to heavy metals and antibiotic resistance is another major cause of concern (Wright et al. 2006; Thomas et al. 2020).
Though in the environment, arsenic is present in many organic and inorganic forms, the most prevailing forms found in soil and aquatic surroundings are inorganic arsenate As(V) and arsenite As(III). The widespread occurrence of arsenic in the ecosystem has led to the evolvement of arsenic detoxi cation system in myriad of organisms (Yamamura and Amachi 2014). The ars operon consisting of three to ve genes, i.e., arsRBC or arsRDABC located on plasmids/chromosomes of prokaryotes is well speci ed and is known to involve in arsenic resistance mechanism (Dunivin et al. 2019). The microbial arsenic detoxi cation entails the reduction of arsenate to arsenite via a cytoplasmic arsenate reductase (arsC). In majority of bacterial groups, the arsenite is extruded by a membrane-associated arsB e ux pump encoded by three-gene arsRBC operons while others employ ArsAB pump encoded by ve-gene arsRDABC operons (Mukhopadhyay et al. 2002 The situation demands for sustainable and biogenic option for bioremediation of metal contaminated sites. The present study describes the isolation of arsenic resistant bacteria from a heavy metal e uent contaminated soil and its subsequent characterization. The capability of the isolate to withstand high concentration of arsenic was also determined. For getting better insight into the underlying mechanism of arsenic transformation the gene involved was also studied. In this regard, the isolated strain can be envisioned as a promising and sustainable biogenic bioremediation tool for mitigating As toxicity.
Materials And Methods

Isolation and screening of bacteria
Soil sample was collected from heavy metal contaminated industrial area (pesticide, herbicide industry) in Delhi NCR. Samples were collected from 1-15 cm depth in a sterilized and sealed polyethylene bags and were preserved at 4 C till further use.
The isolation of cultures was done at different concentrations (500 − 10,000 ppm) of sodium arsenate using the modi ed method of Saxena and Singh 2011. The strain which survived at the highest concentration of sodium arsenate was maintained by subculturing. Pure cultures were used for further studies.

Screening of transforming property of the isolate
The ability of the selected bacterial isolate to oxidize and reduce arsenic was tested using the modi ed method of Dey et al. 2016.

Physicochemical characterization
The selected pure culture of arsenic resistant isolate was initially characterized by microscopic examination, biochemical and molecular characterization.

Molecular characterization a) Genomic and Plasmid DNA isolation
The genomic DNA was isolated using the standard phenol-chloroform extraction method while plasmid DNA was isolated according to Anderson and Mckay 1983. The isolated DNA was run on 0.8% agarose gel and documented subsequently. b) PCR ampli cation of 16S rDNA Bacterial isolate with high arsenic tolerance capacity was selected for molecular identi cation by 16S rDNA sequencing. PCR ampli cation of 16S rDNA gene of bacterial genome was performed using speci c forward (5' -AGAGTTTGATCMTGGCTCAG -3') and reverse (5' -TACGGYTACCTTGTTACGACTT -3') primers. The ampli cation reaction was carried out using the procedure of Sreedharan et al. 2019.

c) Gene sequencing and strain identi cation
The gel eluted ampli ed DNA fragment was sequenced bi-directionally in ABI3500 Genetic Analyzer. Accomplishment of sequencing reaction was done using Big Dye Terminator version 3.1 following the manufacturer's protocol. The procured 16S rDNA sequence was submitted to nucleotide blast (blastn) at NCBI (http://blast.ncbi.nlm.nih.gov/Blast.cgi) averse to the nucleotide database to recover homologous sequences to recognize the strain to the generic level.
For phylogenetical analysis, the bacterial 16S rRNA gene sequences from this work and other sequences retrieved from database were aligned using System Software aligner. The phylogenetic analysis was made using the neighbor method using Jukes-Cantor Correction.

d) PCR and sequencing ofarsCgene
The arsC gene ampli cation was performed using the speci c degenerated forward (5' -ATGAGCAACATYACCATTTATCACAAC-3') and reverse (5'-MTTCAYRCGVTTACCTTWWTCATCAAC -3') primers from genomic DNA of strain RSC3. The ampli cation reaction was done as previously described by Porwal and Singh 2015. The gel eluted ampli ed DNA fragment was sequenced bi-directionally in ABI3500 Genetic Analyzer. Accomplishment of sequencing reaction was done using Big Dye Terminator version 3.1 following the manufacturer's protocol. The gene sequence thus obtained was analyzed based on homology analysis using NCBI BLASTn and was submitted to Genebank database.

Evaluation of minimum inhibitory concentration (MIC)
To determine the level of arsenic resistance, MIC of the selected isolate was determined. The MIC value of the selected isolate for sodium arsenate was assessed according to the clinical and laboratory standards institute (CLSI) protocol. Bacterial inoculum was prepared using Mueller-Hinton broth. The turbidity of the bacterial inoculum was adjusted to 0.5 McFarland turbidity standards (approx. 10 8 CFU/mL). Two-fold serial dilutions of the sodium arsenate in the range of 500-6000ppm were prepared and inoculated with standardized inoculum. Control tubes were maintained without arsenic. The MIC was determined after 24h of incubation at 30 C by observing the visible turbidity. Optical density was measured spectrophotometrically at a wavelength of 600nm. All the experiments were carried out in triplicates. The MIC was de ned as the lowest concentration of arsenate that suppressed visible growth of bacteria.

Cell growth rate kinetics
To study the effect of arsenic on bacterial growth and dry weight, inoculum (1 mL) from an exponentially growing culture (mother culture) was added to 100 mL Nutrient broth supplemented with 500, 1000, 1500 ppm of sodium arsenate. The nutrient broth without arsenic acted as the control. The culture was incubated at 37°C at 120rpm up to 96h. At every 4h time interval samples were withdrawn from each conical ask and the growth rate was measured as absorbance at 600 nm using UV-Vis Spectrophotometer. For plate count results, after every 12h, 100µL of the culture from each broth was serially diluted up to 10 − 8 and was spread on the nutrient agar plates for CFU counting. The growth of the bacterial isolate was monitored by measuring the CFU.
The growth absorbance of Enterobacter cloacae (RSC3) was converted into dry biomass using linear coe cient derived from growth absorbance vs. dry biomass (X), the cell growth was follows rst-order kinetics.
dX/X = µ dt (2) when integrating Eq. (2) with limit t 0 (initial time) to t (a time when maximum biomass reached), it becomes lnX -lnX0 = µ(t 0 -t) Where X0 -initial biomass, X -biomass at time t, and Eq. (3) can be rewritten as The speci c growth rate (µ) was calculated from the slope of a semi-logarithmic plot of dry biomass ln(X/X 0 ) vs. time (Duraisamy et al. 2020).

Analysis of resistance to other heavy metals
The arsenic resistant bacterial isolate was analyzed for resistance to various other heavy metals which includes Lead, Nickel, Mercury, Cadmium, Selenium, Zinc, Tin, Antimony, Arsenate and arsenite of 1000ppm concentration standard solution. Overnight culture from nutrient agar was inoculated in nutrient broth containing different concentrations of heavy metals (10 to 60ppm), separately. The minimum inhibitory concentration (MIC) of the culture was determined after 24h of incubation at 37 C by observing the visible turbidity. The MIC was de ned as the lowest concentration of heavy metals that suppressed the visible growth of bacteria.
1.6 Deciphering the mechanism of arsenic e ux: To study about the mechanism of arsenic e ux, bacteria was inoculated (approx. 10 8 CFU/mL) in nutrient broth with 1500 ppm of sodium arsenate. The nutrient broth without arsenic acted as the control. The culture was incubated at 37°C, 120rpm for 24h. The sample were withdrawn after 8h and16h for further assay.

Transmission Electron Microscopy Analysis (TEM)
In order to con rm the intracellular arsenic accumulation, the arsenic treated as well as untreated cells were analyzed using high resolution transmission electron microscope (HRTEM) (Philips, CM-10 model).

Atomic absorption spectroscopy (AAS) analysis
AAS was performed in order to estimate the concentration of arsenic in the selected isolate. The actively grown cells were inoculated in the medium containing 500 ppm arsenic and also in medium without arsenic serving as control. The asks were then kept for shaking at 37°C at 120rpm. The cells were drawn at different time interval of 0, 4, 8, 12, 16, 20 and 24h for centrifugation at 8000rpm for 20 min at 4 C. After centrifugation, supernatant and pellet were separated, and the pellet was allowed to sonicate. The supernatant and pellet samples were then analyzed for the presence of arsenic by ame atomic absorption spectrophotometer.

Characterization and identi cation of the strain
From the metal contaminated site, 118 different bacterial strains were isolated from heavy metal contaminated site. A total of 25 different arsenic tolerant bacterial strains were isolated using enrichment experiment. Out of the 25 isolates, only 3 isolates were able to survive in nutrient agar amended with 6000ppm of sodium arsenate. Among the 3 isolates, one isolate RSC3 that showed the maximum growth at high concentration of arsenate was chosen for the further studies.
The selected isolate RSC3 was screened for its arsenic transforming property and the results showed that it possessed the ability to reduce As(V) as observed by the silver nitrate test (Fig. 1). The arsenic transforming ability of the isolate resulted in the transformation of media color to yellow due to the reduction of arsenate to arsenite. However, the isolate lacks the ability to oxidize arsenite to arsenate. The morphological and microscopic studies revealed it to be Gram negative non-sporulating bacilli. The biochemical results indicated that it belongs to Enterobacteriaceae family (Supplementary Table 1).
Taxonomic identi cation of the isolated strain (RSC3) was determined by analyzing the 16S rDNA gene sequence data with NCBI BLAST database. Blast search using the 16S rDNA gene sequence revealed its a liation to Enterobacter cloacae (Fig. 2). The 16S rDNA nucleotide sequence of the isolate was submitted to NCBI database under the GeneBank accession number (MN904978.1). The strain RSC3 exhibited 99.44% similarity with Enterobacter hormaechei strain Y2152 plasmid pIHI2-2152, complete sequence. The taxonomic assignment was in accordance with the phenotypic and biochemical analysis.
PCR and sequencing of arsC gene The property of reduction of arsenate to arsenite by the isolated strain revealed presence of the gene in the strain. The gene responsible for reduction was screened in the isolated genomic DNA as no plasmid was isolated from the selected strain. Primer for the ars gene retrieved amplicon of 364bp from the isolated genomic DNA (Fig. 3).
The obtained sequence was compared with the available database using BLAST n search, which revealed that the amplicon contains a partial arsC gene sequence.

Evaluation of minimum inhibitory concentration (MIC)
The resistance to arsenate was tested to determine the potential of the isolated bacteria for bioremediation of arsenic. Microbial resistance to arsenate was determined by visible growth after 24 hours in Mueller-Hinton broth amended with varying concentrations of sodium arsenate.
From the recorded optical density, it was observed that the growth of the isolate decreased with increasing arsenate concentration up to 5000 ppm and ultimately stopped growing at 6000 ppm of arsenate. The minimum inhibitory concentration (MIC) of the isolate was 6000 ppm for arsenate.

Evaluation of Growth kinetics
Time course assay of Enterobacter cloacae (RSC3) revealed that the growth in presence of arsenic at concentration range from 500-1500 ppm was found to be 50 mg L − 1 of biomass around 24 h (Fig. 4). The biomass at different arsenic concentration measured every 4h at OD 600 nm was converted into dry biomass (mg L − 1 ) to calculate growth rate (Fig. 5). The speci c growth rate (µ) was calculated as 0.55 h − 1 .
The growth response of RSC3 in the presence of As(V) ions was de ned in terms of colony forming unit (CFU) on nutrient agar plates. The presence of the arsenic ions resulted in the lengthening of the log phase as evident from Fig. 6.
The growth of the isolate RSC3 decreased with the increase in concentration of arsenate. The shift in the concentration of arsenate from 500ppm to 1000 ppm resulted in 1.5 log reduction during maximum bacterial growth. Irrespective of arsenic concentration, the strain exhibited maximum growth around 50-55h of incubation but after that its growth decreased and it entered the decline phase.

Determination of antibiotic sensitivity
The Sensitivity and/or resistance to a particular antibiotic was determined by growth tests on solid medium plates containing disc of antibiotic. The results revealed that the isolate RSC3 was resistant to all the protein synthesis inhibitor antibiotics used in the experiment except Gentamycin  2.6 Deciphering the mechanism of arsenic e ux:

Scanning electron microcopy (SEM) analysis and Energy dispersive x-ray spectroscopy (EDX) analysis
The Scanning electron microscopic (SEM) studies were performed on the bacterial isolate grown in the presence and absence of arsenic. SEM images are shown in Fig. 8. The images illustrated minor changes in cell morphology and size in terms of reduction in cell size with aggregation of cells when the isolate was grown in the presence of arsenic.
When the cells were exposed to arsenic some morphological features were evident from SEM micrograph. The cells were observed as a uniform mixture of spherical and elongated cells in packed aggregation as well as individuals. The arsenic which entered the cell was e uxed out of the cell due to the presence of active e ux mechanism of resistance in the isolate as con rmed by AAS analysis.
EDX analysis was used to characterize their elemental composition. The presence of arsenic was also con rmed by EDX analysis. The EDX analysis exhibited an EDS signal corresponding to arsenic peak which was perceived in the presence of arsenate treated cell (Fig. 9), however no such peak was observed in case of control.

Transmission Electron Microscopy Analysis (TEM)
In order to con rm the intracellular arsenic accumulation, the arsenic treated as well as untreated cells were observed by transmission electron microscope (TEM). The presence of electron dense layer in the center of the cell after 8h of incubation (Fig. 10a) and its migration towards the walls of the bacterial cell after 16h of incubation (Fig. 10b) was noticed in cells treated with arsenic. Moreover, we also detected insoluble precipitates of arsenic in both TEM analysis ( Fig. 10a and 10b).

Atomic absorption spectroscopy (AAS) analysis
The AAS results revealed higher concentration of arsenic in the pellet of selected isolate at the start of log phase which then decreased towards the end of log phase while trend reversal was detected for the concentration of arsenic in the supernatant, i.e. there was a lower concentration of metal in the initial phase and then it improved with time illustrating the e ux of metal out of the cell (Fig. 11). This was in concurrence with the SEM results which indicated that there was minor change in the cell morphology of the isolate, indicating towards no accumulation of the metal and hence supporting the e ux mechanism of the isolate RSC3.

Discussion
Microbe-mediated transformation of arsenic has tremendous potential in bioremediation of contaminated soil and aquifers. In microbial communities, the implication of arsenic presence has catalyzed the development of survival instinct in the form of detoxi cation mechanisms. In this study, the multi-dimensional abilities of indigenous bacteria (RSC3) isolated from arsenic contaminated industrial sites has been deciphered.
The arsenate reducing RSC3 was characterized to be Enterobacter cloacae using 16S rDNA gene sequence. Prior studies by Anderson and Cook (2004)  The detection of partial arsC gene sequence in the bacterial isolate has demonstrated its potential to reduce arsenate to arsenite. Bachate et al. 2009 have also ampli ed A 275 bp fragment of putative arsC gene from Bacillus sp. Rice C. The presence of arsBC gene pair is reported in the chromosomes of gram negative bacteria. In case of bacteria such as Haemophilus in uenzae, Neisseria gonorrhoeae and Pseudomonas aeruginosa arsC genes was not found to be associated with arsB genes. While on the other hand, P. aeruginosa reportedly had a second arsC gene apart from the one existing within the arsRBC operon (Mukhopadhyay et al. 2002).
The property of selected isolate RSC3 to reduce As(V) was in concurrence with the already reported paper which has also described Enterobacter cloacae as an arsenate reducing bacteria (Selvi, et al. 2014 The possibility of the isolate harboring antibiotic resistance was analyzed and results obtained from this study indicating the resistance of Enterobacter cloacae towards ampicillin and amoxyclav is consistent with previous reports (Selvi et al. 2014). The isolated strain RSC3 was also found to be resistant to chloramphenicol while most isolates of the E. cloacae complex are susceptible to uoroquinolones, trimethoprim/sulfamethoxazole, chloramphenicol, aminoglycosides, tetracyclines, piperacillintazobactam and carbapenems (Mezzatesta et al. 2012). This indicated that may be Enterobacter cloacae RSC3 have also acquired resistance for chloramphenicol. These results could be interpreted as that resistance can be conferred by a plasmid or chromosome-encoded resistance and/or by a system not yet described. In this study as no plasmid was isolated, it indicated the possibility of having coexistence of both types of determinants for antibiotic and heavy metals in the same genetic element (chromosome) which may allow antibiotic resistance to be selected upon heavy metal selective pressure in the contaminated environment (Farias et al. 2015).
The MIC value for arsenate was 6000 ppm for our isolate which has been comparable to Dey et al. 2016 who isolated Bacillus sp. KM02 and Aneurini bacillus aneurinilyticus that could tolerate 4500ppm of arsenate. Moreover, an isolate of Providencia rettgeri has also been reported that can tolerate As (V) upto 10,000µg/mL (Kale et al. 2015). Higher tolerance towards arsenic apt its possible use in reclaiming the different contaminated sites.
The growth pattern of Enterobacter cloacae RSC3 in the presence of arsenic demonstrates that it could reproduce and survive despite the metal stress. In this study, we are reporting the strain is able to grow comfortably at 1500 ppm upto 60h indicating this is one of the effective arsenic tolerating strain reported so far. The increase in the duration of lag phase may be due to the toxic effect of arsenic on the functionality of bacteria leading to the arduous task of repairing and recti cation of the processes affected by the metalloid presence. Simultaneously, the duration of log phase observed in this study was also quite lengthy. It could be due to the interference in phosphate transport system for the uptake of arsenic leading to the extension of logarithmic growth. Paul et al., also reported a growth response of KUMAs15 at different concentrations of arsenate and arsenite with lengthened lag phase but the maximum growth obtained for KUMAs15 was after 28-30h of incubation (Paul et al. 2018).
In addition to arsenic resistance, multi-resistance to zinc, cadmium, selenium and nickel was observed for Enterobacter cloacae RSC3. These results remain in agreement with those reported previously by Selvi et al. 2014. Structural modi cations of arsenic exposed Enterobacter cloacae RSC3 observed by TEM examination were in accordance with the reports by Pandey and Bhatt 2015 who reported that increased arsenic accumulation in cells. Further they reportedly found a fourfold increase in bacterial cell volume when grown in the presence of arsenic. The EDX analysis exhibited an EDS signal corresponding to arsenic peak which was perceived in the presence of arsenate treated cell which is contributing to the hypothesis of entry and exit of arsenic in a modi ed form from the cell. The result of AAS analysis revealed that arsenic which entered the cell was emitted out of the cell due to the presence of active e ux mechanism of resistance in the isolate. Similar results were reported in a study Saluja et al. 2011, in which the 2 strains (AG24 and AGM13) exhibited a similar pattern of e ux of metal from the cell. Altogether these ndings emphasized the presence of arsenic e ux system in the isolated strain. Figure 12 depicts the schematic representation of arsenic uptake and e ux of our Enterobacter cloacae strain. The arsenic enters inside the bacterial cell along with the phosphate using phosphate transferase system (pst ABC).
The arsC gene responsible for production of arsenate reductase reduces As (V) to As(III). This As (III), through a series of reaction, is e uxed out through the cell using other ars operons.

Conclusions
Cost effective and biogenic remedy tools to circumvent the environmental arsenic pollution in both lands as well in water is the need of the hour. Indigenous arsenic resistant microbes isolated from the contaminated sources are really cherished in this aspect. The arsenic resistant bacteria isolated in this study was Enterobacter cloacae based on phylogenetic analysis of 16S rDNA sequence.
This current work demonstrates the capabilities of the isolated strain to withstand concentrations of upto 6000ppm of arsenic. The strain possessed an active e ux system which was con rmed with SEM-EDX, TEM and AAS analysis. Further multi-resistance to several heavy metals and antibiotics enhances the desirability factor of the isolate for judicial application in developing an in situ bioremediation technology.

Declarations
Ethics approval and consent to participate: Not applicable Phylogenetic tree based on 16S rRNA gene sequences depicting relationship between RSC3 and related bacteria. Biomass of RSC3 at a various arsenic concentration.

Figure 5
Relationship between speci c growth rate and initial arsenic concentration