Inhibition and Reduction of Biofilm Production along with Their Antibiogram Pattern among Gram-Negative Clinical Isolates

Background Bacterial biofilm is a significant virulence factor threatening patients, leading to chronic infections and economic burdens. Therefore, it is crucial to identify biofilm production, its inhibition, and reduction. In this study, we investigated biofilm production among Gram-negative isolates and assessed the inhibitory and reduction potential of ethylene diamine tetra acetic acid (EDTA) and dimethyl sulfoxide (DMSO) towards them. In addition, we studied the antimicrobial resistance pattern of the Gram-negative isolates. Methods Bacterial isolation and identification was done using standard microbiological techniques, following the Clinical and Laboratory Standards Institute (CLSI) guideline, 28th edition. The Kirby–Bauer disk diffusion method was used to determine the antibiotic susceptibility pattern of the isolates, and β-lactamase production was tested via the combination disk method. Biofilm formation was detected through the tissue culture plate (TCP) method. Different concentrations of EDTA and DMSO were used to determine their inhibitory and reduction properties against the biofilm. Both inhibition and reduction by the various concentrations of EDTA and DMSO were analyzed using paired t-tests. Results Among the 110 clinical isolates, 61.8% (68) were found to be multidrug resistant (MDR). 30% (33/110) of the isolates were extended-spectrum β-lactamase (ESBL) producers, 14.5% (16/110) were metallo-β-lactamase (MBL), and 8% (9/110) were Klebsiella pneumoniae carbapenemase (KPC) producers. Biofilm formation was detected in 35.4% of the isolates. Biofilm-producing organisms showed the highest resistance to antibiotics such as cephalosporins, chloramphenicol, gentamicin, and carbapenem. The inhibition and reduction of biofilm were significantly lower (p < 0.05) for 1 mM of EDTA and 2% of DMSO. Conclusion Isolates forming biofilm had a higher resistance rate and β-lactamase production compared to biofilm nonproducers. EDTA and DMSO were found to be potential antibiofilm agents. Hence, EDTA and DMSO might be an effective antibiofilm agent to control biofilm-associated infections.


Introduction
Bacterial bioflm is the community of microbial cells that adhere to the solid surface, which can either be biotic or abiotic and remains enclosed in a self-produced polymeric matrix or slime [1].Depending on bacterial species, strain type, and environmental conditions, the bioflm matrix consists of substances of diverse chemical nature, such as exopolysaccharides, proteins, and extracellular DNA (eDNA) [2].Bioflm formation evades the host immune response, conventional antimicrobial agents, and biocides through the "bulky shields" built by extracellular polymeric substances (EPSs) [3].Te bioflm matrix acts as a barrier for difusion and fails to penetrate the antimicrobial agent; consequently, it results in diferentiation into persister cells and inactivates the action of antibiotics [4,5].Bioflms are mainly associated with the tissue or indwelling medical devices such as implants and catheters, leading to chronic or recurrent infections [6].
According to the CDC and NIH, 65-80% of all persistent infection is due to bioflm-producing organisms, leading to therapeutic failure [7].In addition, bioflm-associated pathogens possess 10-1000 times higher resistance to antibiotic treatment and are difcult to eliminate in established infections compared to planktonic cells [6].
Currently, most research is focused on developing nontoxic antibioflm agents that prevent drug resistance [8].EDTA possesses a potent activity for inhibition and reduction of bioflm.EDTA inhibits Gram-negative bacteria by its metal chelation property, as it chelates the cation which was responsible for stabilizing the negatively charged polysaccharides [9].Similarly, DMSO solubilizes the EPS matrix by forming electrostatic repulsion due to which destabilization of bioflm occurs.A study by Jain et al. [10] showed that 4 mM concentration of EDTA inhibited bioflm and 20 mM concentration reduced the bioflm membrane of MRSA.Similarly, in the context of DMSO, Guo et al. [11] reported that 2% DMSO had inhibited Pseudomonas aeruginosa bioflm.In the study of Yahya et al., 32% of DMSO was used to reduce the bioflm of Escherichia coli and Pseudomonas aeruginosa [12].
Terefore, the present study aimed to fnd the bioflm production and to determine the antibioflm activity of EDTA and DMSO against bioflm-producing Gram-negative isolates.Also, we studied the antibiogram profle and resistance pattern of bacterial isolates and compared it among the bioflmproducing and bioflm-nonproducing Gram-negative bacteria.

Materials and Methods
A cross-sectional study was conducted at Manmohan Memorial Institute of Health Sciences for six months from February 2018 to July 2018.We included Gram-negative isolates from various clinical samples such as urine, sputum, blood, pus, and body fuids (pleural fuids) collected during our study period.Informed written consent was taken from the patients before including their sample in the study.Samples were cultured in blood agar (HiMedia, India), Mac Conkey agar (HiMedia, India), and chocolate agar (HiMedia, India) and incubated at 37 °C for 24 hours.Te identifcation of signifcant isolates was performed based on standard microbiological techniques (CLSI guideline, 28 th edition), which involved the morphological appearance of the colony, gram-staining reactions, and biochemical tests.In addition, the purity plate was employed to ensure that the inoculation used for the biochemical test was pure culture.

Determination of MDR Isolates.
Te isolate resistant to at least three antimicrobial agent classes was considered as MDR isolates [14].

Detection of Beta-lactamases
2.3.1.Detection of ESBL Production.For screening of ESBL, ceftazidime (CAZ) (30 µg) and cefotaxime (CTX) (30 µg) were used (HiMedia India).Te zone of inhibition (ZOI) equal to or less than 22 mm for ceftazidime and equal to or less than 27 mm for cefotaxime were considered potential ESBL producers as recommended by the CLSI guideline, 28 th edition.Further phenotypic confrmation of ESBL production was carried out by a combined disk test (CDT).In this method, cefotaxime (30 µg) alone and cefotaxime in combination with clavulanic acid (CA) (30 µg/10 µg) were placed at 20 mm apart (center to center) on the test strain inoculated on the MHA plate.An increase in ZOI of ≥5 mm for a combined disc in comparison to cefotaxime alone confrmed ESBL production [13].

Detection for MBL and KPC Production.
All the isolates, nonsusceptible to meropenem (MRP) or imipenem (IPM), were considered potential MBL producers.Te confrmation of MBL production was done by the EDTAcombination disk test method.In this method, test isolates (comparable to 0.5Mc Farland) were inoculated on the MHA plate where two IPM discs were placed 20 mm apart from the center, one with 10 µl of 0.1 M (292 µg) anhydrous ethylene diamine tetra acetic acid (EDTA) (Sigma-Aldrich) and one with IPM alone.Te inhibition zone of the EDTA + IPM and IPM alone was compared.Te zone of inhibition of IPM + EDTA ≥5 mm than that of IPM alone confrmed the MBL production [15].
Similarly, for KPC detection, two MRP (10 µg) discs are placed 20 mm apart from the center, one with 20 µl of 3amino phenylboronic acid (3-APBA) containing 400 µg and one with MRP alone.Te inhibition zone of the APBA + MRP and MRP alone was compared.Te zone of inhibition of MRP + APBA ≥5 mm than that of MRP alone was considered positive for KPC production [16].

Detection of Bioflm Production.
Te tissue culture plate or microtiter plate technique was carried out for bioflm production.First, organisms isolated from fresh agar plates were inoculated in 2 ml of Luria-Bertani broth (HiMedia, India) with 2% glucose and incubated at 37 °C for 24 hours.Ten, the cultures were diluted at the ratio of 1 : 100 with a fresh medium.Next, 200 μl of the diluted culture of different strains were inoculated in each well of the sterile fatbottom 96-well polystyrene tissue culture plates and incubated for 24 hours at 37 °C.After incubation, the contents of each well were removed and washed with 0.2 mL of phosphate bufer saline (pH 7.2) three times.Ten, the bioflm formed by the bacteria adherent to the wells were fxed by incubating the plate at 60 °C for 1 hour and then stained by crystal violet (2%).Excess stain was removed by International Journal of Biomaterials rinsing three times using deionized water, followed by decolorization with 30% acetic acid.Te stained adherent bioflm's optical density (OD) was measured using a micro-ELISA autoreader at the wavelength 570 nm.
Uninoculated wells containing broth only were considered as a negative control.Te experiment was performed in triplicate two times.Te average optical density (OD) values of each test strain and negative control were calculated, and the fnal OD values of a test strain were obtained by subtracting the OD cutof (ODc) value of the negative control from the average OD value of the test strain.Te interpretation of bioflm production was made according to Stepanovic et al.'s criteria.Te ODc value had been specifed as three standard deviations (SDs) above the negative control [10,17,18].

Preparation of EDTA and DMSO.
A 10 mM EDTA stock solution was prepared by dissolving 3.72 g of disodium EDTA in 1000 ml of distilled water.Ten, dilution of this stock solution was made in distilled water for the preparation of 0.5 mM, 1 mM, 2 mM, 4 mM, and 5 mM of EDTA.For the preparation of DMSO solution, 99% concentrated DMSO solution was used and diluted to 1%, 2%, 4%, 8%, 16%, and 24%, respectively, in distilled water [12].

Inhibition of Bioflm.
In assessing EDTA and DMSO's capacity to inhibit the production of bioflms, organisms were separately grown overnight in LB broth with 2% glucose.Ten, an equal volume of the culture and various concentrations of inhibiting agents (EDTA and DMSO) were transferred into sterile 96-well polystyrene tissue culture plates.For about 24 hours, the plates were incubated at 37 °C, washed three times with 200 μl of sterile phosphate bufer saline (PBS), and cleaned and stained with 2% crystal violet.Te residual stain was removed by rinsing with purifed water and decolorized with 30% acetic acid.Te stained adherent bioflm's optical density (OD) was obtained using a micro-ELISA autoreader at 570 nm.Wells containing LB broth were used as a negative control [10,17,19].Inhibition data were presented in the form of magnitude.
Te magnitude of inhibition � OD before the inhibition of bioflm divided by OD after treatment with the inhibiting agent.

Reduction of Bioflm.
It was performed to evaluate the inhibiting agents EDTA and DMSO's ability to dissociate Gram-negative bioflm.From each bacterial suspension, 200 μl was inoculated in sterile 96-well polystyrene tissue culture plates and were further incubated without agitation for 24 hours at 37 °C for bioflm production.Te formed bioflm was then exposed for the next 24 hours to diferent concentrations of inhibiting compounds (EDTA and DMSO) by adding it to the corresponding microtiter wells.After that, the wells were washed three times with 200 μl of sterile phosphate bufer saline (PBS), dried, and stained with 2% crystal violet.After rinsing, the stain was decolorized by 30% acetic acid and the absorbance of the adherent bioflm was measured at 570 nm in a microplate reader [10,17,19].Reduction data were presented in the form of magnitude.
Te magnitude of reduction � OD before the reduction of bioflm divided by OD after treatment with the reducing agent.

Statistical Analysis.
Te statistical analysis was done by using SPSS version 20 (IBM Corporation, Armonk, NY, USA).Te students paired t-test was used to evaluate the mean diference between the OD value for control (without an inhibiting agent) and diferent concentrations of EDTA and DMSO used for both inhibition and reduction.

Comparison of the Antibiotics-Resistance Pattern among
Bioflm Producers and Bioflm Nonproducers.Te association of antimicrobial resistance is higher with bioflm producers.In comparison to bioflm nonproducers, bioflmproducing isolates were found to be more resistant to antibiotics such as cephalosporin, chloramphenicol, gentamicin, piperacillin-tazobactam, and carbapenems (Table 5).

Discussion
Bioflm leads to the spread of antimicrobial resistance and the generation of more virulent strains as it favors horizontal gene transfer by which resistance and virulence factors pass among bacteria [20].Te inefectiveness of oral antimicrobial agents in eradicating the bacterial cells in bioflm has led to the search for topical therapies.Terefore, developing novel agents that prevent or eliminate bioflm without involving the resistance mechanism is needed for a potential therapeutic approach to control bioflm-associated infections [21].
Among Enterobacteriaceae, 92.2% of Escherichia coli were resistant against amoxicillin, and 84.4% of Klebsiella species were resistant against third-generation cephalosporin (cefxime, cefotaxime, and ceftazidime).Tis resistance of Escherichia coli was harmonical with the study performed in Tanzania [26].In contrast to our study, Eldomany and Abdelaziz reported that resistance towards fuoroquinolones, aminoglycosides, and carbapenem was lower in Klebsiella species [27].Among nonfermenter, all the Acb complex (N � 9) were resistant to piperacillintazobactam, cephalosporin, ciprofoxacin, and gentamicin and 88.9% to carbapenems, and this fnding was similar to  International Journal of Biomaterials the study of Parajuli et al. from Nepal [28].However, this result is nearly twofold higher than that reported by Parajuli et al. [28].Increasing resistance to commonly used antibiotics is mainly due to improper use, easy access, and inadequate monitoring.Te rise in resistant isolates can signifcantly afect patient's health and increases the economic burden [25,29].Terefore, an appropriate choice of antibiotics after antibiotic susceptibility testing is needed to overcome these burdens.Beta-lactams are the drug of choice for treating infections caused by Gram-negative organisms.However, resistance to these antibiotics is increasing [30].Out of the total isolates, 30.0%were ESBL producers, the highest being in Klebsiella species (46.9%), similar to that reported in previous studies [31,32].Our study reported 14.5% of Gram-negative isolates as MBL producers, with a higher rate in the Acb complex (66.7%) followed by Klebsiella species (25%).Tis result is consistent with the study done by Chaudhary et al. [33].Similar to the study done by Robledo et al., our present study reported Acb complex and Klebsiella species as a signifcant KPC producer.Te increased prevalence of ESBL-, MBL-, and KPC-producing isolates might be due to the rational use of option drugs such as third-generation cephalosporins and carbapenems and horizontal transmission of beta-lactamase genes [34,35].
Bioflm-related infections are more troublesome and expensive to treat [7].In this study, Pseudomonas aeruginosa was found to be a potent bioflm producer, followed by the Acb complex.At the same time, minor numbers of Klebsiella species and Escherichia coli were detected as bioflm showed 37% of Gram-negative isolates as bioflm positive [36].Sanchez JR et al. reported bioflm production in 57.7% of Gram-negative isolates, with the highest rate in Pseudomonas aeruginosa, followed by Klebsiella pneumoniae, Acb complex, and Escherichia coli [37].In our study, bioflmproducing organisms had a higher degree of antimicrobial resistance compared to bioflm nonproducers, which was similar to the study conducted by Mishra et al. [17], Panda et al. [38], and Zubair et al. [39].Tis higher antibiotic resistance in bioflm producers might be due to the close contact of organisms in bioflm, activity of the exopolysaccharide matrix, growth rate alteration, pH and osmotic variation, and resistant gene or plasmid transfer among isolates within a bioflm [40].Upon comparative evaluation of the drug-resistance pattern and bioflm production among Gram-negative isolates, it was observed that 66.1% of bioflm producers were MDR, which was similar to the study of Aasti and Chaudhary [41] and Fatima et al. [22].In our study, the rate of production of ESBL among bioflm producers was lower than that reported by Neupane et al. [42] and Gawad et al. [43].Similarly, among bioflm producers, the rate of MBL production was 25.6%, which was lower than in the study by Singhai et al. [44].Among our bioflm-producing isolates, 15.4% were KPC producers, which was similar to those in the  International Journal of Biomaterials study of Hussein et al. [11].Te combination of virulence factors such as bioflm and various enzyme productions might be species specifc.
Bioflm-producing microorganisms cause multiple prosthetic device-mediated infections, leading to severe complications and ultimately resulting in high morbidity, mortality, medical costs, and hospital stay.Tere is a critical need for identifying therapeutic strategies for inhibiting bioflm formation and for efective treatment of bioflms [45].Our study used diferent concentrations of EDTA and DMSO as bioflm-inhibiting agents.We tested 0.5 mM, 1 mM, 2 mM, 4 mM, and 5 mM EDTA to evaluate their antibioflm activity.Among them, 5 mM was found to be the most efective.Te inhibitory efect of EDTA was higher for all isolates as compared to the bioflm reduction capacity.Te action of EDTA was concentration dependent and species specifc, with the highest inhibition of the Acb complex bioflm with a magnitude of 3.37 (70%) and a more signifcant reduction in Pseudomonas bioflm with a magnitude of 2.5 (60.7%).Te inhibitory efect of EDTA on the bioflm formation of Escherichia coli was 2.26 times (55%) and reduction activity (39.4%) was lower than presented by the study of Gawad et al. [43] and Singhai et al. [44].In a study by Al-Bakri et al., the percentage reduction in the viable count of the established bioflm of Pseudomonas aeruginosa after 1 hour exposure of 8 mg/ml (21 mM) of EDTA was 98.98% and Escherichia coli was 53.18%, which was similar to our study that demonstrated a higher activity of EDTA towards Pseudomonas aeruginosa as compared to that of Escherichia coli [9].
Furthermore, various DMSO concentrations were used as the antibioflm agent.Te increasing concentration showed a better efect in bioflm inhibition with the highest magnitude in the Acb complex at 3.70, Pseudomonas at 3.0, Klebsiella species at 2.29, and Escherichia coli at 2.23.However, the reduction rate was lower compared to the inhibitory efect, with magnitude between 1 and 2 for all the isolates.Te magnitude of inhibition by 2% DMSO against Pseudomonas aeruginosa bioflm was similar to the study of Guo et al. [11].Te reduction activity of 24% DMSO for Escherichia coli showed a magnitude of 1.69 (40.8%) and in the case of Pseudomonas aeruginosa (42.8%).In contrast, the study of Yahya et al. reported 38.6% reduction in Escherichia coli and 60.7% in Pseudomonas aeruginosa by 32% DMSO [12].
Although our present study has provided antibioflm activity of the EDTA and DMSO, this study should be considered with some limitations.Our study was based on the phenotypic method of bioflm detection as molecular methods and sophisticated microscopy techniques are constrained in our country.We only performed the susceptibility pattern with the commercially available antibiotic concentration in this study, and it does not provide information on the minimum inhibitory concentration of any antibiotics.Further studies with a large sample size should be considered to establish bioflm production, inhibition, and reduction potential.

Conclusion
Tis study demonstrated a high level of bioflm production among Gram-negative isolates.Consequently, bioflm producers had a higher rate of antimicrobial resistance than bioflm nonproducers.EDTA and DMSO were found to be potent bioflm inhibitors.EDTA and DMSO signifcantly inhibited and reduced the bioflm formation in a dosedependent manner and were species specifc.Tus, our study recommends that EDTA and DMSO are potentially benefcial for bioflm-related infections.Tese fndings would help to establish antibioflm activity for the bioflmproducing Gram-negative bacteria in the future.

Table 1 :
Distribution of organisms in the various clinical samples.

Table 3 :
Incidence of MDR and beta-lactamases production.

Table 4 :
Distribution of bioflm production among the Gram-negative isolates.

Table 5 :
Comparison of the antibiotics-resistance pattern among bioflm producer and bioflm nonproducer.