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Co-occurrence of functionally diverse bacterial community as biofilm on the root surface of Eichhornia crassipes (Mart.) Solms-Laub
Science of the Total Environment 714 (2020) 136683
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Short Communication
Co-occurrence of functionally diverse bacterial community as biofilm on the root surface of Eichhornia crassipes (Mart.) Solms-Laub Duraivadivel P., Gowtham H.G., Hariprasad P. ⁎ Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, Delhi, India
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Eichhornia crassipes is a better substrate for the bacterial colonization. • Biofilm supports the coexistence of multiple bacterial community. • Bacteria on the rhizoplane of E. crassipes are diverse in their function. • Beneficial and deleterious characters are coexistence in these bacteria.
a r t i c l e
i n f o
Article history: Received 29 November 2019 Received in revised form 12 January 2020 Accepted 12 January 2020 Available online 15 January 2020 Editor: Frederic Coulon Keywords: Antibiotic Heavy metal Indole acetic acid Water hyacinth Yamuna
a b s t r a c t The current study investigates the functional diversity of bacterial community existing as a biofilm on the root surface of water hyacinth (Eichhornia crassipes (Mart.) Solms-Laub.) grown in Yamuna river, Delhi, India. Forty-nine bacterial isolates recorded a diverse pattern of susceptibility/resistance to 23 antibiotics tested. Most of the bacterial isolates were susceptible to Ofloxacin, Ciprofloxacin, Ceftriaxone, Gentamicin, and Cefepime and resistant to Ceftazidime, Nitrofurantoin, Ampicillin, and Nalidixic acid. Isolate RB33-V recorded resistant against 11 antibiotics tested, and RB42-V was found susceptible to most of the antibiotics tested. Among the seven heavy metals tested, the highest of 39 bacteria showed resistance to zinc, and least of 9 bacteria recorded resistance against cadmium. Isolate RB20-III was susceptible to all heavy metals tested, and RB23-III was found resistance for six heavy metals tested. A higher correlation was observed with zinc and multiple antibiotic resistance, and Ceftazidime resistance was most frequently associated with all the heavy metals tested. These bacteria grow optimally under neutral-alkali conditions and susceptible to acidic conditions, and they can withstand a broad range of temperatures and salt concentrations. They are very poor in phosphate solubilization. Further, the bacteria recorded varied results for beneficial traits, hemolytic, and DNase activity. The results of bacterial characterization indicated that this bacterial community is of multi-origin in nature and are assisting the hostplant in withstanding the adverse and fluctuating conditions of the Yamuna river by reducing the toxic effect of heavy metals, antibiotics and other xenobiotics. © 2020 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: #387, Block-III, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, Delhi, India. E-mail address: [email protected] (H. P.).
https://doi.org/10.1016/j.scitotenv.2020.136683 0048-9697/© 2020 Elsevier B.V. All rights reserved.
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1. Introduction Bacteria are ubiquitous in nature and evolved to survive in extreme habitats. The capability of withstanding adverse conditions stems from its quick adaptability to the changing environment at the genetic level through SOS response (Foster, 2007; Michel, 2005; Sutton et al., 2000). This adaptability has enabled pathogenic bacteria to develop resistance against multiple antibiotics (ABs), becoming a threat to animals and humans. Until recently, these bacteria identified as hospital strains were restricted to the hospital environment. However, nowadays, their existence is reported in water, sediments, rhizosphere, endophytes, manure, etc. (Hao and Xiao, 2017; Hassard et al., 2016; Karmakar et al., 2019). A 65% (21.1 to 34.8 billion defined daily doses) increase in the worldwide consumption of antibiotics between 2000 and 2015 was reported by Klein et al. (2018) with an estimate of 200% increase in 2030. Most of the ABs are water-soluble, and therefore, a significant amount is excreted out of the human and animal body in its original or metabolized form (Kemper, 2008; Kumar et al., 2005). Adding to this, AB's usage in the agriculture and livestock production sector is also increasing (Manyi-Loh et al., 2018). In agriculture fields, these ABs further spread by leaching into nearby water bodies or groundwater (Blackwell et al., 2009). Depending on its nature, some ABs may degrade very fast, and some may be retained in the environment for prolonged period imposing its toxic effect on non-target organisms (Kim et al., 2007; Thomulka et al., 1993). Though ABs are primarily designed to kill pathogenic microbes. However, its residue in the environment works non-specifically against non-pathogenic microorganisms. Further, the lower concentration of ABs in nature imposes selective stress on the bacterial community, which triggers the survival strategy by inducing resistance against ABs. Antibiotic resistance from non-pathogenic bacteria may transfer horizontally to pathogenic bacteria, and vice versa (Kim et al., 2010). On the other hand, human and animal pathogenic bacteria are released continuously into the environment primarily through wastewater discharge from hospitals. Many of these organisms harbor AB-resistance genes, eventually inserted into mobile genetic platforms (plasmids, transposons, integrons) able to spread among water, soil, and plant bacterial communities (Alonso et al., 2001; Karmakar et al., 2019). Eichhornia crassipes (Mart.) Solms-Laub. (Water hyacinth; Pontederiaceae), is a free-floating, perennial weed, form dense rafts in the water and mud. Although troublesome aquatic weed, it attracted the attention of scientists and environmentalists as it can thrive in highly polluted water, withstand variable environmental conditions, removes nutrients and heavy metals (HMs) faster than any other plants studied (Carvalho Dos Santos and Lenzi, 2000; Soltan and Rashed, 2003). Therefore, this plant is considered as the right choice for phytoremediation (Odjegba and Fasidi, 2007). Being a free-floating aquatic plant, the extensive root system of E. crassipes with large root surface area provides an attractive biological substrate for the microbial colonization. Some of these microbial communities are influenced by root exudates as well as nutrients and pollutants (ABs, HMs, solvents, chemicals, etc.). Additionally, these bacteria may be native or introduced in their origin (Karmakar et al., 2019). On the root surface of E. crassipes, these bacteria exist as biofilm embedded within a matrix of extracellular polysaccharide, thereby protecting themselves from toxic pollutants (Denkhaus et al., 2007; Teitzel and Parsek, 2003; Williams et al., 1997). To the best of our knowledge, most of the previous studies are limited to the bioremediation properties of bacteria associated with aquatic plants. Here, we hypothesize that bacteria colonized on the root surface of E. crassipes are of multiple origins, and they survive as a community under a common biofilm. Further, their long term exposure to the surrounding environment converged their functionality and enhanced their survival chances. These biological substrates may also act as a
site of inoculum of AB resistance pathogenic bacteria that can spread to a wide area in both lotic and lentic ecosystem for prolong period. 2. Materials and methods 2.1. Biological samples Six different sites on the riverbanks of Yamuna, starting from Wazirabad to Okhla barrage located in Delhi (India), were visited during April 2016. Six well-grown Eichhornia plants were randomly collected from each site without disturbing the root system. Roots were rinsed with distilled water to remove debris, pollutants, and other nonadherent microbes. Bacteria adhered to the root surface in the form of biofilm were collected using a sterile cotton swab. For each plant, around 10 cotton swabs were used and stored in 0.85% saline. Immediately the samples were brought to the laboratory and processed. 2.2. Isolation and maintenance of bacteria Cotton swabs in saline with bacteria were vortexed at high speed for 5 min to remove adherent bacteria. Saline was collected, and the above steps were repeated thrice with fresh saline. Saline from all batches was pooled and centrifuged at 8000 rpm for 15 min at 4 °C. The obtained pellet was dissolved in 1 mL of saline and used as stock for serial dilution. A 50 μL of different dilutions (up to 10−4) was spread plated on to Nutrient Agar (NA) media (Composition g/L: Peptone 5; NaCl 5; Beef extract 1.5; Yeast extract 1.5; Agar 15). The plates were maintained at 35 ± 1 °C for 36 h. Subsequently, morphologically different bacteria were pure cultured. All bacterial isolates were routinely maintained on NA unless specified otherwise. Long-term storage of bacterial culture was done in 40% glycerol at −86 °C. 2.3. Characterization of bacteria 2.3.1. Antibiotic resistance/susceptibility test Twenty-three different ABs (Hexa discs, Himedia, India) with different concentrations were used to study the bacterial resistance/susceptibility (Fig. 1). The bacteria were grown in 10 mL of Nutrient Broth (NB) for 16–18 h on a rotary shaker (200 rpm) at 35 ± 1 °C. The absorbance of bacterial growth was measured at 610 nm using a double beam spectrophotometer. The bacterial inoculum was prepared by adjusting optical density (OD) to 0.5 using Phosphate Buffer Saline (PBS). Bacterial inoculum (50 μL) was spread plated on the NA media, and the plates were allowed to dry for 3–5 min aseptically in a biosafety cabinet. Hexa discs containing set of 6 ABs were placed aseptically on the NA media previously spread plated with test bacteria. The plates were incubated at 35 ± 1 °C for 36 h. The plates were visually observed for bacterial growth, and the zone of inhibition was measured in millimeters (mm). The bacteria were divided into 5 groups based on the zone of inhibition (0 mm = Resistant; N0–5 mm = near resistant; N5–10 mm = moderately resistance; N10–15 mm = near susceptible; N15 mm = susceptible). 2.3.2. Heavy metal tolerance test Tolerance to HMs such as mercury (HgCl2), copper (CuSO4), silver (AgNO 3 ), zinc (ZnCl 2 ), cadmium (CdCl2 ), potassium dichromate (K2Cr2O7) and lead ((PbC2H3O2)2) was determined by an agar dilution method (Washington and Sutter, 1980). The concentrations of the HMs tested were as follows, mercury: 12.5, 25, 50, 100 and 150 μg mL −1 ; cadmium, lead, zinc and copper: 50, 100, 200, 400, 800 and 1600 μg mL −1; chromium: 3.12, 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 μg mL−1; silver: 4, 8, 16, 32, 64, 128, 256 and 512 μg mL−1. The plates were point inoculated with test bacteria and incubated at 35 ± 1 °C for 48 h. The plate containing media with no added HMs was taken as control. After the incubation period, the plates were observed visually for bacterial growth. As there were
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no clear-cut breakpoint concentration designated for HMs, the following concentrations, N512 μg mL−1 for Ag, N12.5 μg mL−1 for Hg, N100 μg mL −1 for Cd, Cr, Pb, and Zn, and N200 μg mL −1 for Cu
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(Akinbowale et al., 2007; Malik and Aleem, 2011; Sinegani and Younessi, 2017; Sütterlin et al., 2014) were considered to differentiate resistance or susceptibility of bacteria.
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2.3.3. Tolerance to abiotic stress Each well of the microtiter plate was filled with sterilized NB (250 μL) under aseptic conditions. To this 10 μL bacterial inoculum was added which was prepared as explained earlier. The inoculated plates were incubated at different temperatures such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C for 24 h. Bacterial growth was measured at 0 and 24 h of incubation by reading the absorbance at 610 nm using a microtiter plate reader (EPOCH Biotek Microplate reader). Experiments at 50, 55, and 60 °C were performed in test tubes containing 10 mL NB, and 250 μL test bacteria as inoculum. Each well of the microtiter plate was filled with sterilized NB (250 μL) with different pH ranging from 3, 4, 5, 6, 7, 8, 9, and 10 (pH was adjusted using 0.1 N HCl and 0.1 N NaOH) under aseptic conditions. To this 10 μL bacterial inoculum was added which was prepared as explained earlier. The inoculated plates were incubated at 35 ± 1 °C for 24 h. Bacterial growth was measured by reading at 610 nm using a microtiter plate reader. Each well of the microtiter plate was filled with sterilized NB (250 μL) amended with different concentrations of NaCl ranging from 0, 1, 2, 3, 4, 5, 7.5, 10, 12.5, and 15% under aseptic conditions. To this 10 μL bacterial inoculum was added which was prepared as explained earlier. The inoculated plates were incubated at 35 ± 1 °C for 24 h. Bacterial growth was measured at 0 and 24 h of incubation by reading the absorbance at 610 nm using a microtiter plate reader. In all the abovesaid experiments, bacterial growth was measured in triplicates and the experiment was repeated thrice, and average data were tabulated. In the aforementioned experiments, the growth of bacteria was considered positive only if they achieve ≥50% growth compared to the maximum growth achieved by same bacteria at optimal conditions. 2.4. Functional characterization of bacteria Biofilm production by bacteria grown in NB was determined on a 96 well microtiter plate (Merritt et al., 2006). Each well was loaded with 250 μL of sterile NB followed by 10 μL inoculation of each bacterium. The plates were maintained under static conditions for 36 h at 35 ± 1 °C. After the incubation period, the culture was removed, and the wells were washed thrice with 250 μL of phosphate buffer saline (PBS) to remove the non-adherent cells. The wells were dried aseptically in laminar airflow for 15 min. Adherent biofilm was stained with 100 μL of crystal violet (1%) and incubated for 15 min at room temperature. The surplus stain was washed three times with sterile distilled water (SDW). Finally, 200 μL of ethanol was added to each well to dissolve the stain. The development of light-dark blue coloration indicates the formation of biofilm. Other beneficial traits such as the production of indole acetic acid (IAA), Hydrogen cyanide (HCN) and siderophore, solubilization of phosphate, ACC deaminase activity was also monitored. In vitro antifungal assay against Aspergillus flavus and Naphthalene degradation were performed by following standard procedures (Abarian et al., 2018; Hariprasad et al., 2014; Tiwari et al., 2018). 2.5. Hemolysis and DNase activity The hemolytic ability of the isolated bacteria was analyzed by point, inoculating them on sheep blood agar (Himedia). The plates were incubated at 35 ± 1 °C for 48 h. Towards the end of the incubation period, the bacteria were categorized into α, β, and γ hemolytic as per the manufacturer's instruction. The ability of bacteria to degrade Deoxyribonucleic acid (DNA) by producing DNase was tested on the DNase test
agar medium (Himedia). Test bacteria were point inoculated on defined agar and incubated at 35 ± 1 °C for 48 h. The DNase activity was interpreted following the manufacturer's recommendations. 2.6. Data analysis All ABs and HMs resistance experiments with bacteria were repeated thrice, and the average value was tabulated. Zone of inhibition (mm) was used to generate a heat map using an online source (Babicki et al., 2016). Venn diagram for HM resistance was generated by considering resistance as “1” and susceptibility as “0” using an online tool (app.displayer.com). Radar chart for the correlation between ABs and HMs resistance was generated using Microsoft Excel (Version 2016). Bacterial characterization was performed in triplicates and repeated thrice, and the data was represented as a histogram and bar graph using Microsoft Excel. 3. Results and discussion In the present study, we analyzed the functional diversity of various bacteria existent on the rhizoplane layer of E. crassipes as a biofilm. The rhizosphere is defined as the zone surrounding the root, which is physically, chemically, and biologically influenced by root exudates (Walker et al., 2003). The functional diversity of the bacteria growing in the rhizoplane layer facilitates them to sustain the root exudates and the fluctuating aquatic environment of the Yamuna river. Depending on the plant type, 20–50% of photosynthate is allocated to the root (Kuzyakov and Domanski, 2000) that are released as exudates. These primarily contain sugars, amino acids, organic acids, enzymes, root cell lysates, and other substances (Huang et al., 2014; Koo et al., 2005). In addition, the root exudate is known to favor the growth of certain microbes and suppress others specifically. However, in the case of aquatic plants, it is challenging to define the rhizosphere as the water movement always disturbs it. In the aquatic environment, attachment of rhizobacteria on the root surface is a strategy adopted to stay with the host plant. As the municipal wastewater is rich in nutrients, the rhizoplane bacteria may not depend on root exudate to survive. Additionally, bacteria attached to the root surface may not be selected by the host plant. Raising concern with the increasing amount of ABs in the Yamuna is the development of antibiotic resistance in both pathogenic and nonpathogenic bacteria (Karmakar et al., 2019; Lamba et al., 2017; Mutiyar and Mittal, 2014). Similar concern on the development of AB resistance in pathogenic and non-pathogenic bacteria were reported in Zenne river (Belgium) (Proia et al., 2018), Semenyih river (Peninsular Malaysia) (Al-Badaii and Shuhaimi-Othman, 2015), Ba river in Xi'an (China) (Guan et al., 2018), Karoon river, Khuzestan (Iran) (Besharati et al., 2018), etc. Several factors determined the existence of AB resistance in bacteria in the natural ecosystem. These bacteria may be introduced along with the AB resistance gene or native bacteria, which acquired AB resistance. In line with the previous studies, our results revealed the differential sensitivity of bacteria for different ABs. It may be because of the concentration and time they exposed to a group of ABs in their natural habitat. Also, the existence of the AB gene is favored by other factors like HMs. Here, from E. crassipes collected from 6 sites of Yamuna river, 49 rhizoplane colonizing bacteria were isolated and cultured on the NA medium (Fig. S1). As shown in Fig. 1, the rhizoplane bacteria recorded a distinct pattern of AB resistance/susceptibility against 23 ABs tested. Most of the bacteria were found susceptible to Ofloxacin (OF),
Fig. 1. Heat map depicting resistance/susceptibility of rhizoplane bacteria against 23 antibiotics tested. The Roman letter followed by bacterial code indicates the site of collection. Clustering of bacteria/antibiotics is based on the zone of inhibition recorded by bacteria against antibiotics tested and is represented in mm. P: Penicillin G (10 units); OX: Oxacillin (1 μg); CEP: Cephalothin (30 μg); CD: Clindamycin (2 μg); E: Erythromycin (15 μg); AMC: Amoxyclav (30 μg); GEN: Gentamicin (10 μg); VA: Vancomycin (30 μg); FC: Fusidic acid (10 μg); C: Chloramphenicol (30 μg); MET: Methicilin (5 μg); CPM: Cefepime (30 μg); OF: Ofloxacin (5 μg); TEI: Teicoplanin (30 μg); CAZ: Ceftazidime (30 μg); CX: Cefoxitin (30 μg); AMP: Ampicillin (10 μg); CTR: Ceftriaxone (30 μg); CIP: Ciprofloxacin (5 μg); AK: Amikacin (30 μg); NIT: Nitrofurantoin (300 μg); NET: Netillin (30 μg); NA: Nalidixic acid (30 μg).
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Ciprofloxacin (CIP), Ceftriaxone (CTR), Gentamicin (GEN), and Cefepime (CPM). None were found resistant to OF, GEN, Amikacin (AK), and CTR. However, most of the bacteria were found resistant or near resistant to Ceftazidime (CAZ), Nitrofurantoin (NIT), Ampicillin (AMP), and Nalidixic acid (NA). Isolate RB33-V recorded resistance to 11 ABs, and near resistance 3 ABs tested. Similarly, RB23-III, RB41-VI, and RB19-III were also found resistant to 05, 04, and 06 ABs, respectively. There was no significant correlation observed between the site of isolation and the AB resistance pattern. Only isolate RB29 and RB30 from site IV, RB36, and RB40 from site V, and RB43 and RB48 from site VI recorded a similar pattern of AB resistance. In these cases, the possibility of duplicates was ruled out as they were morphologically different (Data not showed). The occurrence of HM tolerance bacteria in river Yamuna is frequently reported. The existence of wide variation in HM levels varying from high concentration during summer and low concentration during the winter season in the Yamuna river was reported by Kaur and Mehra (2012). The concentration of HM in the sediment of Yamuna was 22.2 μg Cu, 60.3 μg Pb, 9.5 μg Cd and 59.5 μg Zn g−1 sediments (Jain, 2004). According to them, the average metal concentration of copper and zinc is lower. In contrast, lead and cadmium are higher than the respective average shale value. Dubey et al. (2012) revealed that samples from the Yamuna flood plain showed arsenic contamination beyond the WHO limit of 10 ppb. Several recent research works expressed concern over the existence of HM resistant bacteria in sewage treatment plants, Yamuna river water and associated agriculture fields in Delhi (Azam et al., 2018; Bhardwaj et al., 2018; Karmakar et al., 2019; Rahman and Singh, 2019). Similarly, in this study, varying sensitivity among the bacterial isolates to different HMs was recorded. 100% and 98% of the isolates found tolerant to lead, and zinc, respectively, and 32.6% of the isolates were tolerant for all the HMs tested. The resistance/susceptible pattern of isolates to seven HMs are shown in Fig. 2. Among 49 isolates studied, all were found growing on the medium amended with lower concentrations of HMs. Isolate
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RB20-III was found susceptible to all HMs tested, and RB23-III recorded resistance against 6 HMs tested. 6% of bacteria recorded resistance against 5 HMs tested, 39 bacteria recorded resistance to zinc, and only nine were found resistant to cadmium. Multiple HM resistance pattern is shown in Fig. 2. None of the bacteria were found resistant to chromate and cadmium or chromate and mercury. We observed a correlation between HM tolerance and AB resistance in the bacteria. The correlation exists because of the higher likelihood of the resistant genes (ABs and HMs) to be located closely together on the same plasmid in bacteria and are thus more likely to be transmitted together in the environment (Calomiris et al., 1984). Similar results were reported by Ramteke (1997), where among 448 coliforms studied, 90% of them showed resistance to one or more than one ABs and tolerance to multiple HMs. Akinbowale et al. (2007) reported the presence of AB and HM resistant bacterial strains of Pseudomonas and Aeromonas spp. from fish and sediments from aquaculture in Australia. Through this, HM may indirectly favor the existence of AB resistance genes in a wide range of bacteria. Here, a higher correlation was recorded between zinc and CAZ, which was observed in 25 bacteria tested. Coexistence of zinc and NIT, Ampicillin (AMP), NA, Fusidic acid (FC), Penicillin G (P), Cephalothin (CEP), Amoxyclav (AMC), Vancomycin (VA), Oxacillin (OX), Erythromycin (E), Clindamycin (CD), and Methicilin (MET) was also recorded in multiple bacteria. Least correlation was observed between AB and cadmium, mercury, and chromate (Fig. 3). Further, a strong correlation between 7 HMs and 13 ABs was observed. Similarly, surrounded by many industries, the Yamuna river is frequently reported for phenol naphthalene, polychlorinated biphenyls, and organo-pesticides and other contaminants (Kumar et al., 2012; Nomani et al., 1996). The bacteria isolated from Yamuna river water were reported for their AB and HM resistant, xenobiotic degrading or detoxification, etc. (Lee et al., 2009; Najiah et al., 2009; Verma et al., 2011). Among 49 bacteria, 20 were found Gram +ve, and 29 were Gram −ve. When these rhizoplane bacteria were analyzed for the beneficial
Fig. 2. Venn diagram showing rhizoplane bacterial resistance to seven heavy metals tested.
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a.
b.
c.
d.
e.
f.
g.
a. b. c. d. e. f. g.
Lead Copper Chromate Silver Mercury Cadmium Zinc
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Fig. 4. Characterization of Rhizoplane bacterial isolates for adaptive traits. BF: Biofilm, IAA: Indole acetic acid, PS: Phosphate solubilization, ACC: ACC deaminase activity, SID: Siderophore, HCN: Hydrogen Cyanide, ND: Naphthalene Degradation, DNase: DNase activity, HL: Hemolytic activity, ANT: Antifungal.
traits which help in improving plant health, it was found that 83.6% bacteria were able to form biofilm in a microtiter plate, which indicated its ability to colonize plant surface. 63.26% produced IAA in Luria-Bertani (LB) broth to a various extent. These bacteria also showed HCN (14.2%), siderophore (48.9%), ACC deaminase (22.4%), and antagonism activity (8.1%) against A. flavus. However, only 4.0% of bacteria were able to solubilize calcium phosphate on Pikovskaya's medium. 69.3% of bacteria were found growing on the media amended with Naphthalene (0.02%, v/v), indicating their ability to degrade xenobiotics. Further, the test conducted to analyze the pathogenicity of these bacteria revealed that 42.8% of bacteria were able to show DNase activity, and 46.9% recorded hemolytic activity. For all the tests conducted, few bacteria showed variable results, which were categorized as a separate group (Fig. 4). Among 49 bacteria, both Gram +ve and Gram −ve bacteria had similar opportunity to colonize the rhizoplane of E. crassipes. Most of the bacteria isolated were positive for biofilm formation, which indicates their tendency to attach to the surface of the root. Although some bacteria are not or weak in biofilm formation, they could colonize the rhizoplane by embedding in biofilm formed by the other bacterial community. According to Rickard et al. (2003), different bacterial species are interact with each other by aggregating and are mediated by specific growth phase-dependent adhesion-receptor interactions. The presence of bacteria as a community in the biofilm gives them an advantage by protecting them from ABs, HMs, and other toxic pollutants (Ceri et al., 1999; Williams et al., 1997). Further, their survival chances are enhanced as the biofilm provides a better platform to exchange their genetic material both vertically and horizontally (Christensen et al., 1998; Hausner and Wuertz, 1999; Roberts et al., 1999). Most of the rhizobacteria are well known for their symbiotic association with the plant. Indole acetic acid production and phosphate solubilization are the two essential traits of beneficial rhizobacteria through which they improve plant health (Hariprasad et al., 2009). Here, only 4% of bacteria were found solubilizing calcium phosphate. Usually, domestic sewage is always polluted with excess phosphorus and nitrogen. In the Yamuna river, phosphate level varied from 0.029–0.245 mg L−1 during summer and 0.038–0.256 mg L−1 in monsoon (Kaur and Singh,
2012). As these bacteria are well adapted to the eutrophic condition, they were not found solubilizing calcium phosphate, which is a complex form. Similarly, ACC deaminase is reported to reduce abiotic stress by reducing ethylene level in host plant (Tiwari et al., 2018), other essential characters like siderophore production is involved in iron sequestration, HCN and secondary metabolites in suppressing other microbial growth, are discussed earlier (Hariprasad et al., 2014; Laville et al., 1992; Schippers et al., 1990). As these characters are inducible, their expression in bacteria depends on local environmental conditions. DNase and hemolytic activity recorded by these bacteria revealed their identity as possible human and animal pathogens, indicating their different origins. Most of the bacterial isolates were found optimally growing between pH 6–9; none of them were able to grow above pH 10 and below pH 4 (Fig. 5a). 35–45 °C was found optimum for the growth of most of these bacteria. But, their growth range-extended between 15 and 60 °C. 25 and 14 bacteria recorded the tendency to grow at 15 °C and 60 °C, respectively (Fig. 5b). Most of the bacteria recorded optimal growth between 0 and 3% salt concentrations. Their growth range extended from 0 to 12.5% salt concentrations. Twelve and 2 bacteria were showed a tendency to grow in as high as 10 and 12.5% salt concentrations, respectively (Fig. 5c). In the present study, for most of the bacteria, the optimum growth was observed in neutral-alkali conditions (6–9), and they were susceptible to the acidic condition. Similarly, these bacteria showed growth in a broad range of temperatures and salt concentrations. Under extreme conditions studied, certain bacteria failed to reach half of its maximum growth (achieved in optimal conditions). However, they could survive by compromising with their growth and regain their maximum growth under optimal conditions. The pH of the sediment of the Yamuna river varied from 8.01 to 8.45, indicating its alkali nature. The organic content of the sediment was of the order of 1.2% at most of the sites (Jain, 2004). Water quality analysis results done by Kaur et al. (2013) revealed the dissolved oxygen (6.0–7.5 mg L−1), biological oxygen demand (3.3–38 mg L−1), total solids (430–1268 mg L−1) and chemical oxygen demand (28–136 mg L−1) was high, which indicates the poor quality of water. Also, Delhi experiences two extreme ends of the season, in
Fig. 3. Radar chart representing the correlation between antibiotics and heavy metal resistance. The graph represents the number of bacteria resistant for both antibiotics (named across the outer circle) and different heavy metals.
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withstanding wide range of abiotic stress, degradation of xenobiotics, tolerance to heavy metals, etc. these bacterial consortia are better source of inoculum to use as industrial bioconversion or waste water treatment in biofilm bioreactors (Qureshi et al., 2005; Van Loosdrecht and Heijnen, 1993). Our earlier studies on endophytic bacteria associated with green leafy vegetables grown in the riverbank of Yamuna, Delhi (Karmakar et al., 2019) strongly support the hypothesis of convergent evolution in these microbes. Irrespective of their origin, these bacteria were able to survive by adopting several characters beneficial to host plants and resistance to ABs and HMs. Furthermore, these bacteria were found assisting the host plants in withstanding abiotic stress. The rhizoplane bacteria on E. crassipes studied in our study was also observed following the same strategy. Irrespective of their origin, they coexist in biofilm, protecting themselves and host plants from adverse conditions of the Yamuna river. CRediT authorship contribution statement P. Duraivadivel:Investigation, Data curation.H.G. Gowtham:Resources, Formal analysis.P. Hariprasad:Conceptualization, Visualization, Supervision, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful to the Director, Indian Institute of Technology Delhi, New Delhi, India for providing research space and necessary facilities. Appendix A. Supplementary data
Fig. 5. Histogram representing the ability of rhizoplane bacterial ability to tolerate abiotic stress, (a) sodium chloride, (b) pH and (c) temperature. *Number of bacteria showing ≥50% growth compared to the maximum growth achieved by same bacteria at optimal conditions.
winter, the temperature goes as low as ≈4 °C and in summer as high as ≈45 °C. As test bacteria were continuously exposed to fluctuating environmental conditions of the Yamuna river, they must adapt to these conditions to survive. 4. Conclusion The bacteria colonizing the rhizosphere also influence the host plant negatively or positively. As observed by Kirzhner et al. (2009), bacterial biofilm formation on the root surface of E. crassipes reduced its ability to bioremediation. However, selective inoculation of HM resistant bacteria improved the HM uptake and accumulation ability of E. crassipes (AbouShanab et al., 2007). Though in the present study, we have not analyzed the influence of rhizoplane bacteria on E. crassipes bioremediation potential. From the bacterial characterization, it can be postulated that these bacteria may be assisting the host plant in withstanding the adverse and fluctuating conditions of the Yamuna river. These bacteria may reduce the toxicity of HM, thereby helping the host plant to accumulate more HMs. Additionally, they may also reduce the adverse effect of ABs on the host plant (Karmakar et al., 2019). By recognizing the bacterial characters, such as biofilm formation and their coexistence,
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Short Communication
Co-occurrence of functionally diverse bacterial community as biofilm on the root surface of Eichhornia crassipes (Mart.) Solms-Laub Duraivadivel P., Gowtham H.G., Hariprasad P. ⁎ Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, Delhi, India
H I G H L I G H T S
G R A P H I C A L
A B S T R A C T
• Eichhornia crassipes is a better substrate for the bacterial colonization. • Biofilm supports the coexistence of multiple bacterial community. • Bacteria on the rhizoplane of E. crassipes are diverse in their function. • Beneficial and deleterious characters are coexistence in these bacteria.
a r t i c l e
i n f o
Article history: Received 29 November 2019 Received in revised form 12 January 2020 Accepted 12 January 2020 Available online 15 January 2020 Editor: Frederic Coulon Keywords: Antibiotic Heavy metal Indole acetic acid Water hyacinth Yamuna
a b s t r a c t The current study investigates the functional diversity of bacterial community existing as a biofilm on the root surface of water hyacinth (Eichhornia crassipes (Mart.) Solms-Laub.) grown in Yamuna river, Delhi, India. Forty-nine bacterial isolates recorded a diverse pattern of susceptibility/resistance to 23 antibiotics tested. Most of the bacterial isolates were susceptible to Ofloxacin, Ciprofloxacin, Ceftriaxone, Gentamicin, and Cefepime and resistant to Ceftazidime, Nitrofurantoin, Ampicillin, and Nalidixic acid. Isolate RB33-V recorded resistant against 11 antibiotics tested, and RB42-V was found susceptible to most of the antibiotics tested. Among the seven heavy metals tested, the highest of 39 bacteria showed resistance to zinc, and least of 9 bacteria recorded resistance against cadmium. Isolate RB20-III was susceptible to all heavy metals tested, and RB23-III was found resistance for six heavy metals tested. A higher correlation was observed with zinc and multiple antibiotic resistance, and Ceftazidime resistance was most frequently associated with all the heavy metals tested. These bacteria grow optimally under neutral-alkali conditions and susceptible to acidic conditions, and they can withstand a broad range of temperatures and salt concentrations. They are very poor in phosphate solubilization. Further, the bacteria recorded varied results for beneficial traits, hemolytic, and DNase activity. The results of bacterial characterization indicated that this bacterial community is of multi-origin in nature and are assisting the hostplant in withstanding the adverse and fluctuating conditions of the Yamuna river by reducing the toxic effect of heavy metals, antibiotics and other xenobiotics. © 2020 Elsevier B.V. All rights reserved.
⁎ Corresponding author at: #387, Block-III, Centre for Rural Development and Technology, Indian Institute of Technology Delhi, Hauz Khas, New Delhi 110016, Delhi, India. E-mail address: [email protected] (H. P.).
https://doi.org/10.1016/j.scitotenv.2020.136683 0048-9697/© 2020 Elsevier B.V. All rights reserved.
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1. Introduction Bacteria are ubiquitous in nature and evolved to survive in extreme habitats. The capability of withstanding adverse conditions stems from its quick adaptability to the changing environment at the genetic level through SOS response (Foster, 2007; Michel, 2005; Sutton et al., 2000). This adaptability has enabled pathogenic bacteria to develop resistance against multiple antibiotics (ABs), becoming a threat to animals and humans. Until recently, these bacteria identified as hospital strains were restricted to the hospital environment. However, nowadays, their existence is reported in water, sediments, rhizosphere, endophytes, manure, etc. (Hao and Xiao, 2017; Hassard et al., 2016; Karmakar et al., 2019). A 65% (21.1 to 34.8 billion defined daily doses) increase in the worldwide consumption of antibiotics between 2000 and 2015 was reported by Klein et al. (2018) with an estimate of 200% increase in 2030. Most of the ABs are water-soluble, and therefore, a significant amount is excreted out of the human and animal body in its original or metabolized form (Kemper, 2008; Kumar et al., 2005). Adding to this, AB's usage in the agriculture and livestock production sector is also increasing (Manyi-Loh et al., 2018). In agriculture fields, these ABs further spread by leaching into nearby water bodies or groundwater (Blackwell et al., 2009). Depending on its nature, some ABs may degrade very fast, and some may be retained in the environment for prolonged period imposing its toxic effect on non-target organisms (Kim et al., 2007; Thomulka et al., 1993). Though ABs are primarily designed to kill pathogenic microbes. However, its residue in the environment works non-specifically against non-pathogenic microorganisms. Further, the lower concentration of ABs in nature imposes selective stress on the bacterial community, which triggers the survival strategy by inducing resistance against ABs. Antibiotic resistance from non-pathogenic bacteria may transfer horizontally to pathogenic bacteria, and vice versa (Kim et al., 2010). On the other hand, human and animal pathogenic bacteria are released continuously into the environment primarily through wastewater discharge from hospitals. Many of these organisms harbor AB-resistance genes, eventually inserted into mobile genetic platforms (plasmids, transposons, integrons) able to spread among water, soil, and plant bacterial communities (Alonso et al., 2001; Karmakar et al., 2019). Eichhornia crassipes (Mart.) Solms-Laub. (Water hyacinth; Pontederiaceae), is a free-floating, perennial weed, form dense rafts in the water and mud. Although troublesome aquatic weed, it attracted the attention of scientists and environmentalists as it can thrive in highly polluted water, withstand variable environmental conditions, removes nutrients and heavy metals (HMs) faster than any other plants studied (Carvalho Dos Santos and Lenzi, 2000; Soltan and Rashed, 2003). Therefore, this plant is considered as the right choice for phytoremediation (Odjegba and Fasidi, 2007). Being a free-floating aquatic plant, the extensive root system of E. crassipes with large root surface area provides an attractive biological substrate for the microbial colonization. Some of these microbial communities are influenced by root exudates as well as nutrients and pollutants (ABs, HMs, solvents, chemicals, etc.). Additionally, these bacteria may be native or introduced in their origin (Karmakar et al., 2019). On the root surface of E. crassipes, these bacteria exist as biofilm embedded within a matrix of extracellular polysaccharide, thereby protecting themselves from toxic pollutants (Denkhaus et al., 2007; Teitzel and Parsek, 2003; Williams et al., 1997). To the best of our knowledge, most of the previous studies are limited to the bioremediation properties of bacteria associated with aquatic plants. Here, we hypothesize that bacteria colonized on the root surface of E. crassipes are of multiple origins, and they survive as a community under a common biofilm. Further, their long term exposure to the surrounding environment converged their functionality and enhanced their survival chances. These biological substrates may also act as a
site of inoculum of AB resistance pathogenic bacteria that can spread to a wide area in both lotic and lentic ecosystem for prolong period. 2. Materials and methods 2.1. Biological samples Six different sites on the riverbanks of Yamuna, starting from Wazirabad to Okhla barrage located in Delhi (India), were visited during April 2016. Six well-grown Eichhornia plants were randomly collected from each site without disturbing the root system. Roots were rinsed with distilled water to remove debris, pollutants, and other nonadherent microbes. Bacteria adhered to the root surface in the form of biofilm were collected using a sterile cotton swab. For each plant, around 10 cotton swabs were used and stored in 0.85% saline. Immediately the samples were brought to the laboratory and processed. 2.2. Isolation and maintenance of bacteria Cotton swabs in saline with bacteria were vortexed at high speed for 5 min to remove adherent bacteria. Saline was collected, and the above steps were repeated thrice with fresh saline. Saline from all batches was pooled and centrifuged at 8000 rpm for 15 min at 4 °C. The obtained pellet was dissolved in 1 mL of saline and used as stock for serial dilution. A 50 μL of different dilutions (up to 10−4) was spread plated on to Nutrient Agar (NA) media (Composition g/L: Peptone 5; NaCl 5; Beef extract 1.5; Yeast extract 1.5; Agar 15). The plates were maintained at 35 ± 1 °C for 36 h. Subsequently, morphologically different bacteria were pure cultured. All bacterial isolates were routinely maintained on NA unless specified otherwise. Long-term storage of bacterial culture was done in 40% glycerol at −86 °C. 2.3. Characterization of bacteria 2.3.1. Antibiotic resistance/susceptibility test Twenty-three different ABs (Hexa discs, Himedia, India) with different concentrations were used to study the bacterial resistance/susceptibility (Fig. 1). The bacteria were grown in 10 mL of Nutrient Broth (NB) for 16–18 h on a rotary shaker (200 rpm) at 35 ± 1 °C. The absorbance of bacterial growth was measured at 610 nm using a double beam spectrophotometer. The bacterial inoculum was prepared by adjusting optical density (OD) to 0.5 using Phosphate Buffer Saline (PBS). Bacterial inoculum (50 μL) was spread plated on the NA media, and the plates were allowed to dry for 3–5 min aseptically in a biosafety cabinet. Hexa discs containing set of 6 ABs were placed aseptically on the NA media previously spread plated with test bacteria. The plates were incubated at 35 ± 1 °C for 36 h. The plates were visually observed for bacterial growth, and the zone of inhibition was measured in millimeters (mm). The bacteria were divided into 5 groups based on the zone of inhibition (0 mm = Resistant; N0–5 mm = near resistant; N5–10 mm = moderately resistance; N10–15 mm = near susceptible; N15 mm = susceptible). 2.3.2. Heavy metal tolerance test Tolerance to HMs such as mercury (HgCl2), copper (CuSO4), silver (AgNO 3 ), zinc (ZnCl 2 ), cadmium (CdCl2 ), potassium dichromate (K2Cr2O7) and lead ((PbC2H3O2)2) was determined by an agar dilution method (Washington and Sutter, 1980). The concentrations of the HMs tested were as follows, mercury: 12.5, 25, 50, 100 and 150 μg mL −1 ; cadmium, lead, zinc and copper: 50, 100, 200, 400, 800 and 1600 μg mL −1; chromium: 3.12, 6.25, 12.5, 25, 50, 100, 200, 400, 800, 1600 and 3200 μg mL−1; silver: 4, 8, 16, 32, 64, 128, 256 and 512 μg mL−1. The plates were point inoculated with test bacteria and incubated at 35 ± 1 °C for 48 h. The plate containing media with no added HMs was taken as control. After the incubation period, the plates were observed visually for bacterial growth. As there were
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no clear-cut breakpoint concentration designated for HMs, the following concentrations, N512 μg mL−1 for Ag, N12.5 μg mL−1 for Hg, N100 μg mL −1 for Cd, Cr, Pb, and Zn, and N200 μg mL −1 for Cu
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(Akinbowale et al., 2007; Malik and Aleem, 2011; Sinegani and Younessi, 2017; Sütterlin et al., 2014) were considered to differentiate resistance or susceptibility of bacteria.
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2.3.3. Tolerance to abiotic stress Each well of the microtiter plate was filled with sterilized NB (250 μL) under aseptic conditions. To this 10 μL bacterial inoculum was added which was prepared as explained earlier. The inoculated plates were incubated at different temperatures such as 10, 15, 20, 25, 30, 35, 40, 45, 50, 55 and 60 °C for 24 h. Bacterial growth was measured at 0 and 24 h of incubation by reading the absorbance at 610 nm using a microtiter plate reader (EPOCH Biotek Microplate reader). Experiments at 50, 55, and 60 °C were performed in test tubes containing 10 mL NB, and 250 μL test bacteria as inoculum. Each well of the microtiter plate was filled with sterilized NB (250 μL) with different pH ranging from 3, 4, 5, 6, 7, 8, 9, and 10 (pH was adjusted using 0.1 N HCl and 0.1 N NaOH) under aseptic conditions. To this 10 μL bacterial inoculum was added which was prepared as explained earlier. The inoculated plates were incubated at 35 ± 1 °C for 24 h. Bacterial growth was measured by reading at 610 nm using a microtiter plate reader. Each well of the microtiter plate was filled with sterilized NB (250 μL) amended with different concentrations of NaCl ranging from 0, 1, 2, 3, 4, 5, 7.5, 10, 12.5, and 15% under aseptic conditions. To this 10 μL bacterial inoculum was added which was prepared as explained earlier. The inoculated plates were incubated at 35 ± 1 °C for 24 h. Bacterial growth was measured at 0 and 24 h of incubation by reading the absorbance at 610 nm using a microtiter plate reader. In all the abovesaid experiments, bacterial growth was measured in triplicates and the experiment was repeated thrice, and average data were tabulated. In the aforementioned experiments, the growth of bacteria was considered positive only if they achieve ≥50% growth compared to the maximum growth achieved by same bacteria at optimal conditions. 2.4. Functional characterization of bacteria Biofilm production by bacteria grown in NB was determined on a 96 well microtiter plate (Merritt et al., 2006). Each well was loaded with 250 μL of sterile NB followed by 10 μL inoculation of each bacterium. The plates were maintained under static conditions for 36 h at 35 ± 1 °C. After the incubation period, the culture was removed, and the wells were washed thrice with 250 μL of phosphate buffer saline (PBS) to remove the non-adherent cells. The wells were dried aseptically in laminar airflow for 15 min. Adherent biofilm was stained with 100 μL of crystal violet (1%) and incubated for 15 min at room temperature. The surplus stain was washed three times with sterile distilled water (SDW). Finally, 200 μL of ethanol was added to each well to dissolve the stain. The development of light-dark blue coloration indicates the formation of biofilm. Other beneficial traits such as the production of indole acetic acid (IAA), Hydrogen cyanide (HCN) and siderophore, solubilization of phosphate, ACC deaminase activity was also monitored. In vitro antifungal assay against Aspergillus flavus and Naphthalene degradation were performed by following standard procedures (Abarian et al., 2018; Hariprasad et al., 2014; Tiwari et al., 2018). 2.5. Hemolysis and DNase activity The hemolytic ability of the isolated bacteria was analyzed by point, inoculating them on sheep blood agar (Himedia). The plates were incubated at 35 ± 1 °C for 48 h. Towards the end of the incubation period, the bacteria were categorized into α, β, and γ hemolytic as per the manufacturer's instruction. The ability of bacteria to degrade Deoxyribonucleic acid (DNA) by producing DNase was tested on the DNase test
agar medium (Himedia). Test bacteria were point inoculated on defined agar and incubated at 35 ± 1 °C for 48 h. The DNase activity was interpreted following the manufacturer's recommendations. 2.6. Data analysis All ABs and HMs resistance experiments with bacteria were repeated thrice, and the average value was tabulated. Zone of inhibition (mm) was used to generate a heat map using an online source (Babicki et al., 2016). Venn diagram for HM resistance was generated by considering resistance as “1” and susceptibility as “0” using an online tool (app.displayer.com). Radar chart for the correlation between ABs and HMs resistance was generated using Microsoft Excel (Version 2016). Bacterial characterization was performed in triplicates and repeated thrice, and the data was represented as a histogram and bar graph using Microsoft Excel. 3. Results and discussion In the present study, we analyzed the functional diversity of various bacteria existent on the rhizoplane layer of E. crassipes as a biofilm. The rhizosphere is defined as the zone surrounding the root, which is physically, chemically, and biologically influenced by root exudates (Walker et al., 2003). The functional diversity of the bacteria growing in the rhizoplane layer facilitates them to sustain the root exudates and the fluctuating aquatic environment of the Yamuna river. Depending on the plant type, 20–50% of photosynthate is allocated to the root (Kuzyakov and Domanski, 2000) that are released as exudates. These primarily contain sugars, amino acids, organic acids, enzymes, root cell lysates, and other substances (Huang et al., 2014; Koo et al., 2005). In addition, the root exudate is known to favor the growth of certain microbes and suppress others specifically. However, in the case of aquatic plants, it is challenging to define the rhizosphere as the water movement always disturbs it. In the aquatic environment, attachment of rhizobacteria on the root surface is a strategy adopted to stay with the host plant. As the municipal wastewater is rich in nutrients, the rhizoplane bacteria may not depend on root exudate to survive. Additionally, bacteria attached to the root surface may not be selected by the host plant. Raising concern with the increasing amount of ABs in the Yamuna is the development of antibiotic resistance in both pathogenic and nonpathogenic bacteria (Karmakar et al., 2019; Lamba et al., 2017; Mutiyar and Mittal, 2014). Similar concern on the development of AB resistance in pathogenic and non-pathogenic bacteria were reported in Zenne river (Belgium) (Proia et al., 2018), Semenyih river (Peninsular Malaysia) (Al-Badaii and Shuhaimi-Othman, 2015), Ba river in Xi'an (China) (Guan et al., 2018), Karoon river, Khuzestan (Iran) (Besharati et al., 2018), etc. Several factors determined the existence of AB resistance in bacteria in the natural ecosystem. These bacteria may be introduced along with the AB resistance gene or native bacteria, which acquired AB resistance. In line with the previous studies, our results revealed the differential sensitivity of bacteria for different ABs. It may be because of the concentration and time they exposed to a group of ABs in their natural habitat. Also, the existence of the AB gene is favored by other factors like HMs. Here, from E. crassipes collected from 6 sites of Yamuna river, 49 rhizoplane colonizing bacteria were isolated and cultured on the NA medium (Fig. S1). As shown in Fig. 1, the rhizoplane bacteria recorded a distinct pattern of AB resistance/susceptibility against 23 ABs tested. Most of the bacteria were found susceptible to Ofloxacin (OF),
Fig. 1. Heat map depicting resistance/susceptibility of rhizoplane bacteria against 23 antibiotics tested. The Roman letter followed by bacterial code indicates the site of collection. Clustering of bacteria/antibiotics is based on the zone of inhibition recorded by bacteria against antibiotics tested and is represented in mm. P: Penicillin G (10 units); OX: Oxacillin (1 μg); CEP: Cephalothin (30 μg); CD: Clindamycin (2 μg); E: Erythromycin (15 μg); AMC: Amoxyclav (30 μg); GEN: Gentamicin (10 μg); VA: Vancomycin (30 μg); FC: Fusidic acid (10 μg); C: Chloramphenicol (30 μg); MET: Methicilin (5 μg); CPM: Cefepime (30 μg); OF: Ofloxacin (5 μg); TEI: Teicoplanin (30 μg); CAZ: Ceftazidime (30 μg); CX: Cefoxitin (30 μg); AMP: Ampicillin (10 μg); CTR: Ceftriaxone (30 μg); CIP: Ciprofloxacin (5 μg); AK: Amikacin (30 μg); NIT: Nitrofurantoin (300 μg); NET: Netillin (30 μg); NA: Nalidixic acid (30 μg).
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Ciprofloxacin (CIP), Ceftriaxone (CTR), Gentamicin (GEN), and Cefepime (CPM). None were found resistant to OF, GEN, Amikacin (AK), and CTR. However, most of the bacteria were found resistant or near resistant to Ceftazidime (CAZ), Nitrofurantoin (NIT), Ampicillin (AMP), and Nalidixic acid (NA). Isolate RB33-V recorded resistance to 11 ABs, and near resistance 3 ABs tested. Similarly, RB23-III, RB41-VI, and RB19-III were also found resistant to 05, 04, and 06 ABs, respectively. There was no significant correlation observed between the site of isolation and the AB resistance pattern. Only isolate RB29 and RB30 from site IV, RB36, and RB40 from site V, and RB43 and RB48 from site VI recorded a similar pattern of AB resistance. In these cases, the possibility of duplicates was ruled out as they were morphologically different (Data not showed). The occurrence of HM tolerance bacteria in river Yamuna is frequently reported. The existence of wide variation in HM levels varying from high concentration during summer and low concentration during the winter season in the Yamuna river was reported by Kaur and Mehra (2012). The concentration of HM in the sediment of Yamuna was 22.2 μg Cu, 60.3 μg Pb, 9.5 μg Cd and 59.5 μg Zn g−1 sediments (Jain, 2004). According to them, the average metal concentration of copper and zinc is lower. In contrast, lead and cadmium are higher than the respective average shale value. Dubey et al. (2012) revealed that samples from the Yamuna flood plain showed arsenic contamination beyond the WHO limit of 10 ppb. Several recent research works expressed concern over the existence of HM resistant bacteria in sewage treatment plants, Yamuna river water and associated agriculture fields in Delhi (Azam et al., 2018; Bhardwaj et al., 2018; Karmakar et al., 2019; Rahman and Singh, 2019). Similarly, in this study, varying sensitivity among the bacterial isolates to different HMs was recorded. 100% and 98% of the isolates found tolerant to lead, and zinc, respectively, and 32.6% of the isolates were tolerant for all the HMs tested. The resistance/susceptible pattern of isolates to seven HMs are shown in Fig. 2. Among 49 isolates studied, all were found growing on the medium amended with lower concentrations of HMs. Isolate
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RB20-III was found susceptible to all HMs tested, and RB23-III recorded resistance against 6 HMs tested. 6% of bacteria recorded resistance against 5 HMs tested, 39 bacteria recorded resistance to zinc, and only nine were found resistant to cadmium. Multiple HM resistance pattern is shown in Fig. 2. None of the bacteria were found resistant to chromate and cadmium or chromate and mercury. We observed a correlation between HM tolerance and AB resistance in the bacteria. The correlation exists because of the higher likelihood of the resistant genes (ABs and HMs) to be located closely together on the same plasmid in bacteria and are thus more likely to be transmitted together in the environment (Calomiris et al., 1984). Similar results were reported by Ramteke (1997), where among 448 coliforms studied, 90% of them showed resistance to one or more than one ABs and tolerance to multiple HMs. Akinbowale et al. (2007) reported the presence of AB and HM resistant bacterial strains of Pseudomonas and Aeromonas spp. from fish and sediments from aquaculture in Australia. Through this, HM may indirectly favor the existence of AB resistance genes in a wide range of bacteria. Here, a higher correlation was recorded between zinc and CAZ, which was observed in 25 bacteria tested. Coexistence of zinc and NIT, Ampicillin (AMP), NA, Fusidic acid (FC), Penicillin G (P), Cephalothin (CEP), Amoxyclav (AMC), Vancomycin (VA), Oxacillin (OX), Erythromycin (E), Clindamycin (CD), and Methicilin (MET) was also recorded in multiple bacteria. Least correlation was observed between AB and cadmium, mercury, and chromate (Fig. 3). Further, a strong correlation between 7 HMs and 13 ABs was observed. Similarly, surrounded by many industries, the Yamuna river is frequently reported for phenol naphthalene, polychlorinated biphenyls, and organo-pesticides and other contaminants (Kumar et al., 2012; Nomani et al., 1996). The bacteria isolated from Yamuna river water were reported for their AB and HM resistant, xenobiotic degrading or detoxification, etc. (Lee et al., 2009; Najiah et al., 2009; Verma et al., 2011). Among 49 bacteria, 20 were found Gram +ve, and 29 were Gram −ve. When these rhizoplane bacteria were analyzed for the beneficial
Fig. 2. Venn diagram showing rhizoplane bacterial resistance to seven heavy metals tested.
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b.
c.
d.
e.
f.
g.
a. b. c. d. e. f. g.
Lead Copper Chromate Silver Mercury Cadmium Zinc
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Fig. 4. Characterization of Rhizoplane bacterial isolates for adaptive traits. BF: Biofilm, IAA: Indole acetic acid, PS: Phosphate solubilization, ACC: ACC deaminase activity, SID: Siderophore, HCN: Hydrogen Cyanide, ND: Naphthalene Degradation, DNase: DNase activity, HL: Hemolytic activity, ANT: Antifungal.
traits which help in improving plant health, it was found that 83.6% bacteria were able to form biofilm in a microtiter plate, which indicated its ability to colonize plant surface. 63.26% produced IAA in Luria-Bertani (LB) broth to a various extent. These bacteria also showed HCN (14.2%), siderophore (48.9%), ACC deaminase (22.4%), and antagonism activity (8.1%) against A. flavus. However, only 4.0% of bacteria were able to solubilize calcium phosphate on Pikovskaya's medium. 69.3% of bacteria were found growing on the media amended with Naphthalene (0.02%, v/v), indicating their ability to degrade xenobiotics. Further, the test conducted to analyze the pathogenicity of these bacteria revealed that 42.8% of bacteria were able to show DNase activity, and 46.9% recorded hemolytic activity. For all the tests conducted, few bacteria showed variable results, which were categorized as a separate group (Fig. 4). Among 49 bacteria, both Gram +ve and Gram −ve bacteria had similar opportunity to colonize the rhizoplane of E. crassipes. Most of the bacteria isolated were positive for biofilm formation, which indicates their tendency to attach to the surface of the root. Although some bacteria are not or weak in biofilm formation, they could colonize the rhizoplane by embedding in biofilm formed by the other bacterial community. According to Rickard et al. (2003), different bacterial species are interact with each other by aggregating and are mediated by specific growth phase-dependent adhesion-receptor interactions. The presence of bacteria as a community in the biofilm gives them an advantage by protecting them from ABs, HMs, and other toxic pollutants (Ceri et al., 1999; Williams et al., 1997). Further, their survival chances are enhanced as the biofilm provides a better platform to exchange their genetic material both vertically and horizontally (Christensen et al., 1998; Hausner and Wuertz, 1999; Roberts et al., 1999). Most of the rhizobacteria are well known for their symbiotic association with the plant. Indole acetic acid production and phosphate solubilization are the two essential traits of beneficial rhizobacteria through which they improve plant health (Hariprasad et al., 2009). Here, only 4% of bacteria were found solubilizing calcium phosphate. Usually, domestic sewage is always polluted with excess phosphorus and nitrogen. In the Yamuna river, phosphate level varied from 0.029–0.245 mg L−1 during summer and 0.038–0.256 mg L−1 in monsoon (Kaur and Singh,
2012). As these bacteria are well adapted to the eutrophic condition, they were not found solubilizing calcium phosphate, which is a complex form. Similarly, ACC deaminase is reported to reduce abiotic stress by reducing ethylene level in host plant (Tiwari et al., 2018), other essential characters like siderophore production is involved in iron sequestration, HCN and secondary metabolites in suppressing other microbial growth, are discussed earlier (Hariprasad et al., 2014; Laville et al., 1992; Schippers et al., 1990). As these characters are inducible, their expression in bacteria depends on local environmental conditions. DNase and hemolytic activity recorded by these bacteria revealed their identity as possible human and animal pathogens, indicating their different origins. Most of the bacterial isolates were found optimally growing between pH 6–9; none of them were able to grow above pH 10 and below pH 4 (Fig. 5a). 35–45 °C was found optimum for the growth of most of these bacteria. But, their growth range-extended between 15 and 60 °C. 25 and 14 bacteria recorded the tendency to grow at 15 °C and 60 °C, respectively (Fig. 5b). Most of the bacteria recorded optimal growth between 0 and 3% salt concentrations. Their growth range extended from 0 to 12.5% salt concentrations. Twelve and 2 bacteria were showed a tendency to grow in as high as 10 and 12.5% salt concentrations, respectively (Fig. 5c). In the present study, for most of the bacteria, the optimum growth was observed in neutral-alkali conditions (6–9), and they were susceptible to the acidic condition. Similarly, these bacteria showed growth in a broad range of temperatures and salt concentrations. Under extreme conditions studied, certain bacteria failed to reach half of its maximum growth (achieved in optimal conditions). However, they could survive by compromising with their growth and regain their maximum growth under optimal conditions. The pH of the sediment of the Yamuna river varied from 8.01 to 8.45, indicating its alkali nature. The organic content of the sediment was of the order of 1.2% at most of the sites (Jain, 2004). Water quality analysis results done by Kaur et al. (2013) revealed the dissolved oxygen (6.0–7.5 mg L−1), biological oxygen demand (3.3–38 mg L−1), total solids (430–1268 mg L−1) and chemical oxygen demand (28–136 mg L−1) was high, which indicates the poor quality of water. Also, Delhi experiences two extreme ends of the season, in
Fig. 3. Radar chart representing the correlation between antibiotics and heavy metal resistance. The graph represents the number of bacteria resistant for both antibiotics (named across the outer circle) and different heavy metals.
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withstanding wide range of abiotic stress, degradation of xenobiotics, tolerance to heavy metals, etc. these bacterial consortia are better source of inoculum to use as industrial bioconversion or waste water treatment in biofilm bioreactors (Qureshi et al., 2005; Van Loosdrecht and Heijnen, 1993). Our earlier studies on endophytic bacteria associated with green leafy vegetables grown in the riverbank of Yamuna, Delhi (Karmakar et al., 2019) strongly support the hypothesis of convergent evolution in these microbes. Irrespective of their origin, these bacteria were able to survive by adopting several characters beneficial to host plants and resistance to ABs and HMs. Furthermore, these bacteria were found assisting the host plants in withstanding abiotic stress. The rhizoplane bacteria on E. crassipes studied in our study was also observed following the same strategy. Irrespective of their origin, they coexist in biofilm, protecting themselves and host plants from adverse conditions of the Yamuna river. CRediT authorship contribution statement P. Duraivadivel:Investigation, Data curation.H.G. Gowtham:Resources, Formal analysis.P. Hariprasad:Conceptualization, Visualization, Supervision, Writing - review & editing. Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgments The authors are grateful to the Director, Indian Institute of Technology Delhi, New Delhi, India for providing research space and necessary facilities. Appendix A. Supplementary data
Fig. 5. Histogram representing the ability of rhizoplane bacterial ability to tolerate abiotic stress, (a) sodium chloride, (b) pH and (c) temperature. *Number of bacteria showing ≥50% growth compared to the maximum growth achieved by same bacteria at optimal conditions.
winter, the temperature goes as low as ≈4 °C and in summer as high as ≈45 °C. As test bacteria were continuously exposed to fluctuating environmental conditions of the Yamuna river, they must adapt to these conditions to survive. 4. Conclusion The bacteria colonizing the rhizosphere also influence the host plant negatively or positively. As observed by Kirzhner et al. (2009), bacterial biofilm formation on the root surface of E. crassipes reduced its ability to bioremediation. However, selective inoculation of HM resistant bacteria improved the HM uptake and accumulation ability of E. crassipes (AbouShanab et al., 2007). Though in the present study, we have not analyzed the influence of rhizoplane bacteria on E. crassipes bioremediation potential. From the bacterial characterization, it can be postulated that these bacteria may be assisting the host plant in withstanding the adverse and fluctuating conditions of the Yamuna river. These bacteria may reduce the toxicity of HM, thereby helping the host plant to accumulate more HMs. Additionally, they may also reduce the adverse effect of ABs on the host plant (Karmakar et al., 2019). By recognizing the bacterial characters, such as biofilm formation and their coexistence,
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