CORRELATION STUDIES OF INDIGENOUS BIOFILM FORMING BACTERIA FOR RESISTANCE AGAINST SELECTED METALS, ANTIBIOTICS AND DYES DEGRADATION

This study determines correlation of biofilm-forming bacteria for selected metals, antibiotics and dyes. Total 12 strains having biofilm production potential were targeted in this study. Majority of them were Gram negative excluding MB377 and MB378, and facultative anaerobes. Isolates were mesophilic and exhibited growth capacity over a wide variety of pHs. Almost all isolates conferred resistance capacity against a wide range of multiple antibiotics having 0.0063 MAR for bacitracin, ampicillin, clindamycin, amoxicillin and sulphamethoxazole. Bacterial isolates displayed tolerance to diverse variety of metals i.e. cadmium, lead and iron 1000 mg L-1, copper 600-1000 mg L-1, chromium 700-1000 mg L-1 and nickel 200-600 mg L-1. Maximum heavy metal tolerance of 0.033 was observed for all metals excluding nickel. Correlation among metal tolerance and antibiotic resistance was extremely significant for chromium and bacitracin 0.862. Furthermore, moderate relationship (r= 0.682) between copper resistance and Basic Green 4 dye degradation was observed. Correlation coefficient for cadmium and Acid Red 2 dye was recorded as 0.512, while that for bacitracin and Basic Green 4 was noted as 0.665. These correlations proposed that resistance conferring genes might be present on same locus, genetic elements, chromosome or on a plasmid. 16S rRNA sequenced isolates were identified to be Klebsiella pneumoniae MB375, Staphylococcus sp. MB377, Klebsiella oxytoca MB381, Klebsiella pneumoniae MB394 and Klebsiella pneumoniae MB398. Potential of these biofilm forming strains to thrive under stressed environmental conditions proves them good entrants for being used in bioremoval of polluted sites after further studies on their metabolic pathways.


Site selection, sample collection, isolation and purification of bacteria from biofilm samples
Different samples in the form of biofilms (i.e. slimy layers) to obtain biofilm forming bacteria were collected from multiple sites of Pakistan [including Flour Mill (Hassan Ibdaal), Ayub Park Lake (Rawalpindi), Plywood Industry (Golrah Mor), Tops Food Industry (Hattar) and Nullah at the back side of EPA (Environmental Protection Agency) dumping site for waste from flour mills, marble factories and laboratory waste (Rawalpindi)]. These layers were scrapped from the particular sites by disinfected spatula, placed in disinfected plastic bags and taken to laboratory for bacterial isolation. Slimy layers were resuspended in autoclaved distilled water by vortexing, and then spread (via spread plate method) on to the nutrient agar plates maintaining the temperature at 37 °C for 24 and 48 hours. Colonies with distinct phenotypic features were then selected and further purified by streaking single colony.

Congo red agar (CRA) method
Screening CRA method was used for evaluating the capability of bacteria to form biofilm, and appearance of black bacterial colonies on plate after incubation (24-48 hrs) at 37°C showed the production of slime (Freeman et al., 1989).

Ring test/Tube test assay
This screening system was carried out to evaluate the amount of slime produced in broth [tryptic soy broth (TSB)] containing carbon source as 1% glucose. In autoclaved medium, 0.04% Congo red dye was added along with inoculum of overnight grown bacterial culture. Incubation was performed at temperature 37°C (100 rpm). The appearance of black color with the formation of rings was observed after 24 and 48 hours (Atshan and Shamsudin, 2011).

Microtiter plate assay
The assay was used to assess the extent of biofilm formation by the bacterial isolates. It was performed via methodology of Cerca et al. (

2007) and
Stepanovic et al. (2007) using few amendments. Firstly, a sterile loop was used to inoculate the bacterial culture in a separate glass tube which contained growth medium (nutrient broth) and incubation for 24 hours was performed at 37°C to get primary/mother culture. Optical density was adjusted to 0.2 OD at 600 nm in order to get appropriate colony forming units i.e. 10 5 -10 8 CFU.ml -1 . In this assay, 96 well plates were used to evaluate biofilm production. For this purpose, bacterial cultures (20 µl) were vortexed and added into 80 µl of autoclaved nutrient broth. The broth (devoid of bacteria) was represented as a control/blank in the assay. Cultures were incubated for 24 and 48 hrs at 37 °C and then the bacterial suspension was discarded from respective wells by moderate tapping. Washing was done to remove non-adherent bacterial cells from microtiter plates by using 200 µl volumes of saline solution (0.85% NaCl). After washing, the plates were dried for about 10 minutes and remaining bacterial cells in the microtiter plates were fixed with fixative solution [methanol (200 µl)] for 15-20 minutes. In the last step, attached bacterial cells were stained using 200 µl of 0.1% aqueous crystal violet (CV) stain for 10 minutes. To remove unnecessary CV stain, each well was washed three times with saline solution and 200 µl glacial acetic acid (35%) was dispensed in individual well for quantifying bound bacterial cells using the microtiter plate reader system (Microplate Reader BioRad680XR) at 578 nm. All experimentations were done twice.

Phenotypic and biochemical characterization
Isolates were studied for distinct phenotypic and biochemical characteristics through standard procedures (Gerhardt et al., 1994). Effects of different pHs and temperature ranges on bacterial growth was analyzed using UV-VIS spectroscopy. Overnight grown bacterial inoculum was dispensed in test tubes containing nutrient broth and incubated at a range of temperatures (30, 37 and 45°C). Similar experiments were performed with pH (5,6,7,8,9,10 and 11) at 37°C under shaking conditions. After 24hrs, UV/VIS spectrophotometer at OD600 was used to observe the effects on physiological activity of isolates.

Evaluation on the basis of HMT and MAR index
According to source of isolation, bacteria were grouped into five categories for the analysis of resistance and tolerance against various metal salts and antibiotics. Following formula was employed for calculation of HMT (heavy metal tolerance) index of bacterial cells against each metal salt.

Correlation among metals tolerance and antibiotics resistance
The correlation between metals and different antibiotics was determined through Pearson's correlation coefficient using SPSS statistics software (version 24). Perfect positive correlation was interpreted as +1, strong positive +0.70, moderate positive +0.50, weak positive +0.30, no linear correlation 0, weak negative correlation -0.30, moderate negative -0.50, strong negative -0.70 and perfectly negative was inferred as -1.

Assessment of similarity between isolates
Similarities between the isolates on basis of staining, physiological and biochemical features, motility patterns, metal and antibiotic resistances were assessed via Jaccard coefficient and un-weighted average clustering (UPGMA) algorithm. Positive results and negative results were assigned binary codes as 1 and 0. Paleontological statistics software (PAST3.08, Hammer et al., 2001) was employed for estimating similarities dendogram.

Evaluation of color removal proficiency of isolates for dyes
Different dyes including C.I. Acid Red 2, C.I. Solvent Yellow 2, C.I. Acid Orange 52, C.I. Mordant Black 11, C.I. Basic Green 4, C.I. Basic violet 3 and C.I. Direct Red 28 were used to assess decolorization potential in the current study by employing streak plate procedure (Saranraj and Sivasakthivelan, 2014). M9 medium (Tahir and Yasmin, 2019) augmented with variable concentrations (50-1000 mg.L -1 ) of dye stocks was used without any carbon source (glucose) for detection of dye decolorization activity of bacteria. Clear zones around biofilm forming bacterial colonies showed the decolorization ability of bacteria. This method was performed three times.

Association of antibiotics resistance with dye degradation capability
The association of antibiotics resistance with dye degradation ability was evaluated by Pearson's correlation coefficient method through SPSS statistics software (24 version). The results for correlation were analyzed as described above.

Correlation among metal tolerance and dye degradation capability
For evaluating the association between metal tolerance and ability of bacteria to degrade dyes, Pearson's correlation coefficient method (SPSS statistics software24 version) was used. Results were inferred as described above.

Molecular characterization of selected bacterial isolates
Pure bacterial cultures for further identification at molecular level were submitted to Macrogen (UK). 16S rRNA gene sequences obtained from Macrogen was submitted to NCBI database in GenBank. Accessions numbers of these sequences were given by GenBank and investigated using BLAST search tool and MEGA6 software (Tamura et al., 2013).

RESULTS AND DISCUSSION
In the current study, bacterial isolates were evaluated for their biofilm formation capability isolated from various polluted sites. These microbes were collected on the basis of phenotypic variations among them and then purified as a single colony by using nutrient agar medium.

Screening for biofilm formation
Different screening methods like Congo red agar method, ring test and microtiter plate assay were used to assess the biofilm forming capacity of isolates.

CRA assay
Around 75%, 9/12 of isolates showed black colonies on plate while two bacterial isolates including MB386 and MB387 showed colonies in reddish black color.

Ring test/tube test assay
All of the bacterial isolates produced black colored pigmentation in addition to formation of rings around the walls of flasks, thus conferred positive potential for biofilm formation, expect MB386 and MB387 as they appeared non-biofilm formers.

Microtiter plate assay
The bacterial isolates displayed variations in biofilm forming ability over a period of 24-48 hrs. Despite of this variability all isolates showed an escalation towards biofilm formation potential with lapse of time ( Figure S1 Supplementary Data). MB394, MB396 and MB398 displayed highest extent of biofilm formation with OD values 1.241, 2.035 and 1.094, respectively, indicating their ability to survive by forming biofilms in diverse environments. Pui et al. (2017) reported Leptospira biofilm producers P38 obtained from rats, soil and water samples displaying biofilm formation at 16.700±0.265 OD600 on 8th day, P18 with OD value 21.760±0.332 on 7th day and P22 with OD 19.793±0.144 on 7th day of incubation.
(2011) and Liaqat and Sabri (2010) documented almost similar findings about the role of optimum temperature on growth of bacteria. Though variable range of pHs favored the growth of bacterial isolates, but it declined at extreme pHs (pH 5 and 11). Around seven isolates (MB375, MB378, MB381, MB387, MB394, MB396 and MB398) were acidophilic in nature as they displayed optimal growth towards acidic pH (5 and 6), along with good potential to grow at alkaline pH up to 10. At pH 7 and 8, MB391 and MB392 revealed maximum growth, while MB377 showed optimal growth towards neutral pH (neutrophile), MB393 grew at pH 8, and MB386 towards pH 9, thus categorized as moderate alkaliphiles ( Figure 2).

Metal resistance
A large proportion of bacteria (MB377, MB378, MB386, MB387 and MB392) possessed the ability to grow and resist o chromium metal (1000 mg.L -1-). MB381 exhibited strong growth up to 600 mg.L -1 , however very weak growth patterns were observed at 700 mg.L -1 of chromium. Isolates MB391 and MB396 exhibited almost the same pattern for chromium metal at 800 and 900 mg.L -1 . MB381 exhibited a color change of growth medium to pale yellow. All isolates exhibited good resistance capability in presence of iron up to 1000 mg.L -1 . Likewise, all presented good resistance potential against lead metal up to 1000 mg.L -1 (Table  1). All isolates grew optimally in medium containing cadmium (up to 1000 mg.L -1 ), whereas, MB377 and MB378 displayed good resistance at lower concentrations (200 and 100 mg.L -1 ). Most of them showed strong resistance potentials towards copper 1000 mg.L -1 excluding four isolates (MB377, MB378, MB386 and MB387) they showed fluctuations in their resistance potentials. MB386 and MB387 appeared more sensitive towards higher concentrations of copper (tolerated up to 600 mg.L -1 ). MB378 displayed strong growth at lower concentration of copper (500 mg.L -1 ). Variability in growth patterns of bacterial isolates was noted in presence of nickel metal (up to 600 mg.L -1 ). Four isolates including MB386, MB387, MB394 and MB398 showed good resistance at 400 mg.L -1 of nickel, whereas, MB375, MB377, MB378 and MB396 grew strongly at 300 mg.L -1 . Only one isolate MB391 didn't show any growth in medium containing nickel. These results indicated competence of biofilm forming bacteria to survive and resist different metals. Bacteria have the ability to tolerate or detoxify metals by different mechanisms including segregation, attachment at protoplast and to different surfaces present on living cell surfaces. It also involves the formation of biofilms and mechanism like bioaccumulation process and precipitation. Bacteria has the ability to synthesize proteins that can trap heavy metals e.g. metallothionein and then convert them into volatile and unstable compounds. Furthermore, the negative charges on bacterial surfaces and presence of different anionic structures lead to the complexation of metals ions (iron, copper, manganese) by providing active sites for metal adsorption (Selenskapobell and Merroun, 2010; Tripathi et al., 2011).

Pearson's relation between metal tolerance and multiple antibiotics resistance
According to analysis by Pearson's correlation coefficient, the relation between copper and antibiotic bacitracin tolerance was very significant (r = 0.862). A moderate relation was found amongst copper metal tolerance and resistance against antibiotic amoxicillin (r = 0.668), while, the relationship among copper tolerance and sulphamethoxazole was detected to be moderate (r = 0.521). Similarly, cadmium resistance showed moderate correlation with that of amoxicillin (r = 0.631). The correlation coefficient was lowest at (r = 0.343) in case of nickel metal tolerance with clindamycin resistance. Whereas, in case of chromium metal and rifampicin resistance, the correlation was detected to be minimum (r = 0.302) and the antibiotic chloramphenicol conferred strongly negative correlation with cadmium i.e. r = -0.720. The resistance against other antibiotics was not related to metals tolerance. All these findings proposed that resistance conferring genes for both metals and antibiotics might be present on same locus, genetic elements, chromosome or on a plasmid. Moreover, strongly positive correlations were detected among various metallic salts and targeted antibiotics (Table 2), indicating the probability of stimulation/induction of same resistance mechanisms for different metal salts and antibiotics due to their coexistence within the environmental compartments.

Legends:
Correlation between metals tolerance, Correlation between antibiotics resistance, Positive association between metals and antibiotics resistance, Negative association between metals and antibiotics resistance, * = Significant correlation, + = Positive correlation, -= Negative correlation

Similarity assessment among isolates
Bacterial isolates were grouped into three clusters based on phenotypic, functional (physiological) and biochemical features (Figure 3). First group (Cluster A) contained isolates which had the ability to grow in acidic environments. Bacterial isolates in cluster A varied from second group (Cluster B) by the presence of capsule structure and formation of resistant spores, diverse patterns in motility, enzymatic activities (decarboxylase, deaminase and gelatinase), production of chemicals (indole, acetoin), utilization of citrate molecules, capability to ferment various sugar derivatives e.g. melibiose rhamnose, amygdalin and sorbitol, and growth on brilliant green bile and MacConkey's agar media, and resistance potential against various antibiotics (clindamycin, erythromycin, bacitracin, ampicillin, sulphamethoxazole, rifampicin, amoxicillin, and chloramphenicol). The third group (Cluster C) was further divided into two sub-groups, C1 comprised of neutrophilic isolates while C2 involved acidophilic bacterial isolates.

Figure 3
Similarity indices of biofilm forming bacteria via clustering through Jaccard's similarity coefficient

Evaluation of decolorization potential by biofilm forming bacteria
To evaluate the bacterial aptitude towards various dyes decolorization, C.I. Acid Red 2 dye concentration was slowly increased from 25 mg.L -1 to 500 mg.L -1 . Information regarding isolates' growth and percentage of decolorization capacity was mentioned in Table 1. Only one isolate MB394 had the ability to decolorize C.I. Solvent Yellow 2 dye up to 500 mg.L -1 concentration whereas all other isolates presented decolorization potential around 200 mg.L -1 . MB381 decolorized the dye supplemented in solid medium in addition to yellow pigment production. All isolates decolorized C.I. Acid Orange 52 dye in the range of 100-1000 mg.L -1 . Almost all the isolates excluding MB377, MB381 and MB396 decolorized up to 1000 mg.L -1 of C.I. Mordant Black 11. All the isolates showed the capability of growing and decolorizing C.I. Direct Red 28 dye at various concentrations 100-1000 mg.L -1 . At low concentrations of dye, bacterial isolates revealed different trends of decolorization for dye molecules, whereas, upon increasing the concentrations, bacterial growth/cells transformed in to dark red color indicating the elimination of respective dye via adsorption. Decolorization of C.I. Basic violet 3 dye was also observed at 400 mg.L -1 concentration by four isolates (MB377, MB391, MB394 and MB398), and C.I. Basic Green 4 dye was decolorized by all isolates (except MB386) at 300-1000 mg.L -1 concentration.

Association of multiple antibiotics resistance with dye degradation capability
Moderately significant correlation was noticed between bacitracin and C.I. Basic Green 4 dye degradation ability. Results of correlation coefficient analysis are given in Table 3. All these findings indicated the possible reasons for coexistence of resistance conferring genes as mentioned above regarding the correlation for both metals and antibiotics.

Association of metal tolerance with dye degradation capability
Interpreted data of correlation coefficient analysis is presented in Table 4. Results revealed moderate relationship (r= 0.682) between copper resistance and C.I. Basic Green 4 dye degradation capability. Correlation coefficient for cadmium and C.I. Acid Red 2 dye was recorded as 0.512. All these findings indicated the possible reasons for coexistence of resistance conferring genes as mentioned above regarding the correlation for both metals and antibiotics.

CONCLUSION
Current findings showed greater diversity among the bacterial strains based on the phenotypic, functional, biochemical and physiological characteristics. Furthermore, these biofilm producing bacteria possessed the potential to survive and thrive in stressed environments by showing the ability to resist various groups of antibiotics as well as heavy metal salts. In addition to toxic metals metabolism these biofilm forming strains revealed the potential to remove and degrade diverse dyes through different methods involving adsorption as well as enzymatic pathways. These diversified features specified the capabilities of bacteria of naturally adapting to withstand and grow in the existence of hazardous compounds and exploit these compounds by transforming them into different metabolite. Moreover, detailed studies about bacterial genetics and metabolic pathways will be greatly helpful in determining their potential roles in pollution abatement.

Figure S1
Biofilm formation proficiency of bacteria over time (24-48 hours)