African Journal of
Microbiology Research

  • Abbreviation: Afr. J. Microbiol. Res.
  • Language: English
  • ISSN: 1996-0808
  • DOI: 10.5897/AJMR
  • Start Year: 2007
  • Published Articles: 5182

Full Length Research Paper

Effect of EDTA on biofilm formation and antibiotic susceptibility of multidrug resistant uropathogenic Escherichia coli clinical isolates in Egypt

Wesam Elsayed Gawad
  • Wesam Elsayed Gawad
  • Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University for Science and Technology, Giza, Egypt.
  • Google Scholar
Omneya Mohamed Helmy
  • Omneya Mohamed Helmy
  • Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
  • Google Scholar
Wael Mostafa Tawakkol
  • Wael Mostafa Tawakkol
  • Department of Microbiology and Immunology, Faculty of Pharmacy, Misr University for Science and Technology, Giza, Egypt.
  • Google Scholar
Abdelgawad Mohamed Hashem
  • Abdelgawad Mohamed Hashem
  • Department of Microbiology and Immunology, Faculty of Pharmacy, Cairo University, Cairo, Egypt.
  • Google Scholar


  •  Received: 05 September 2017
  •  Accepted: 26 September 2017
  •  Published: 14 October 2017

 ABSTRACT

Urinary tract infections are mainly caused by uropathogenic Escherichia coli (UPEC). Biofilm-producer UPEC tends to have a high level of resistance to antibiotics and this leads to recurrent episodes of urinary tract infections. The study tested the effect of a non-antibiotic adjuvant, ethylenediaminetetraacetic acid (EDTA) on the bacterial susceptibility to antibiotics and biofilm formation by multidrug resistant (MDR) strong biofilm producer UPEC from Egypt. The ability for in vitro biofilm formation was detected in 88 MDR UPEC isolates in the absence and presence of two concentrations of EDTA (10 and 20 mM). The minimum inhibitory concentrations (MIC) of the tested antibiotics were detected in the presence and absence of sub-inhibitory concentration of EDTA (2 mM) by the two-fold broth microdilution method. The effect of polyvinylchloride gelatin-EDTA coat on biofilm formation by strong and moderate biofilm producers was tested. The addition of 2 mM EDTA to antibiotics resulted in a decrease in the antimicrobials MIC values with the highest effect recorded with Meropenem (81.6%) and Nitrofurantoin (61.4%). EDTA with concentrations (10 and 20 mM) and Gelatin-EDTA coat inhibited biofilm formation by strong and moderate biofilm producing UPEC by 45.8, 78.8, and 81.1%, respectively. The combination of Carbapenems with EDTA in parenteral preparations to treat life threatening infections could greatly improve the clinical outcome. There is a continuous need for the development of new strategies for treatment of MDR biofilm-producer UPEC.  Novel approaches to control microbial biofilm are needed.

Key words: Ethylenediaminetetraacetic acid (EDTA), Escherichia coli, biofilm, antibiotic resistance.


 INTRODUCTION

Urinary tract infection (UTI) is one of the most common infectious diseases affecting all ages (Neupane et al., 2016). Catheter associated  UTI  (CAUTI)  is  common  in patients with indwelling bladder catheter leading to an increase in the length of hospitalization and prolonging the   antibiotic   therapy   period   than    non-catheterized patients (Jacobsen et al., 2008). Uropathogenic Escherichia coli (UPEC) is responsible for more than 80% of UTI in healthy people and are the most common isolates in catheterized patients with UTI (Kumar et al., 2017). The multidrug resistant (MDR) UPEC strains are major public threat worldwide (Lee et al., 2016) and are highly prevalent in Egypt (El-Sokkary and Abdelmegeed, 2015; Abdel-Moaty et al., 2016).
 
Biofilms are the microbial communities of the surface attached to cells embedded in a self-produced extracellular polymeric matrix (Niveditha et al., 2012) and biofilm-producers show higher resistance to antimicrobial agent and this leads to recurrent episodes and persistence of UTI (Tayal et al., 2015). Biofilm-producer UPEC are also the most common cause of UTI (Bang et al., 2016), which are difficult to treat with a single antibiotic (Wu et al., 2015). Several strategies have been tested to inhibit biofilm formation on the indwelling urinary catheter (Cai et al., 2016), including coating catheters with natural products as green tea and Dandasa, fresh garlic extract, honey and Oregano essential oil (Sadekuzzaman et al., 2015); ethylenediaminetetraacetic acid (EDTA)-gallium gelatin coating (Zhu et al., 2013); and using levofloxacin and vitamin C (El-Gebaly et al., 2012). 
 
EDTA is a polyamine carboxylic acid used as a metal chelator with established anticoagulant activity (Raad et al., 2003) and in low concentrations act as a food preservative and in combination with antibiotics (Lerma et al., 2014); ZOSYN® (Wyeth Pharmaceuticals Inc.) is a commercially available antibiotic combination (Piperacillin/Tazobactam) for intravenous use that contains EDTA in the formulation. It is used intravenously, in combinations with vitamins and minerals in treatment of various diseases including atherosclerotic vascular disease and renal ischemia. EDTA is shown to be safe up to 40 mg/kg/body weight when administrated intravenously to swiss albino mice (Chaudhary et al., 2012) and can be administered with a daily dosage of 50 mg/kg of body weight in humans (ENDRATE®, Hospira inc).
 
EDTA prevents curli production and inhibits bacterial adhesion which is required for biofilm development. EDTA chelates divalent ions present in lipopolysaccharide layer of biofilm (Chaudhary et al., 2013); potentiating the antibiotic effect by enhancing the drug penetration and disrupting the lipopolysaccharide present in the outer membrane, hence increasing the porosity of membrane and increasing the drug permeability (Abd et al., 2000; Chaudhary et al., 2013).
 
This study was conducted to evaluate the effect of a non-antibiotic adjuvant EDTA on in vitro biofilm formation and  the  antibiotic  susceptibility  of  clinical  MDR  strong biofilm-producer UPEC from Egypt. 


 MATERIALS AND METHODS

Bacterial strains and identification
 
The study was performed on a total number of 88 MDR UPEC from inpatients and outpatients. Seventy seven (77) isolates were collected from Mansoura University Hospital (Dakhalia Governorate), and 11 isolates were collected from Misr University for Science and Technology (MUST) Hospital (Giza Governorate) in the period between January 2014 and December 2015. All experiments in this study were conducted in accordance with and approval of the ethical committee at Cairo University, Cairo, Egypt with approval number MI (1045).
 
Identification of the isolates was done by Gram staining and isolation on MacConkey agar (Oxoid, UK) and eosin methylene blue (Oxoid, UK) (Brenner, 1984). The molecular identification of E. coli was done by the PCR amplification of uspA gene (Chen and Griffiths, 1998). Multiplex PCR for detecting gadA, chuA, yjaA and TspE4.C2 genes was used to determine the phylogenetic groups for each UPEC isolate (Doumith et al., 2012).
 
The antibiotic susceptibility testing of 88 MDR UPEC isolates was performed by using double disk diffusion using the following antibiotics (Cockerill et al., 2012): amikacin (30 µg), amoxicillin/clavulanic acid (30 µg), ampicillin (10 µg), ampicillin/sulbactam (10/10 µg), cefixime (5 µg), cefotaxime (30 µg), ceftazidime (30 µg), cefuroxime (30 µg), ciprofloxacin (5 µg), CO-trimoxazole (25 µg), gentamicin (10 µg), levofloxacin (5 µg), nalidixic acid (30 µg), nitrofurantion (300 µg), and norfloxacin (10 µg) all were supplied from Himedia, India; aztreonam (30 µg), imipenem (10 µg), meropenem (10 µg), piperacillin/tazobactam (100/10 µg), and tetracycline (30 µg) were supplied from (Oxoid, UK). E. coli strain ATCC 25922 was used as a reference strain and the result was interpreted according to CLSI guidelines (CLSI, 2012).  The isolates were classified as MDR according to Magiorakos et al. (2012).
 
Effect of EDTA on the bacterial susceptibility to antibiotics
 
The MIC of EDTA (E. MERCK. Darmstadt, GERMANY) and the following antibiotics: ciprofloxacin, levofloxacin, amoxicillin/ clavulanic acid (Sedico Pharmaceutical Co., 6th of October city, Giza, Egypt), nalidixic acid, gentamicin (Memphis Pharmaceutical Co, Cairo, Egypt), nitrofurantoin (El-Kahera Pharmaceutical Co, Cairo, Egypt), cefotaxime and ceftazidime (EPICO, 10th of Ramadan), and meropenem (AstraZeneca Co, Cairo, Egypt) were performed using the microdilution broth method (Andrews, 2001). The antimicrobials MICs were determined in the absence of EDTA and in the presence of sub MIC of EDTA (2 mM EDTA).
 
Effect of EDTA on curli production
 
The presence of curli fibers were determined using Luria-Bertani agar (L.B.) (Difco Laboratories, U.S.A) without salts containing 40 mg/L congo red dye (Aldrich Chemical Co. Ltd. England) (Baugh et al., 2013). The effect of EDTA on curli production by curli positive strains was tested using two different concentrations of EDTA (5 and 10 mM) (Chaudhary et al., 2013).
 
Effect of EDTA on in vitro biofilm formation
 
The ability of in vitro biofilm formation was determined using the microtiter plate assay (SarojGolia et al., 2012) in a 96-well microtiter plate (Greiner Bio-one, Stuttgart, Germany), in the absence and presence of EDTA (10 and 20 mM), in triplicates. The optical density was measured at 570 nm with ELISA reader (BioTek®, MQX 200, USA) and the degree of biofilm formation was estimated (SarojGolia et al., 2012).
 
Effect of coating polyvinyl chloride microtiter plate with 50 mM EDTA on in vitro biofilm formation
 
Coating of polyvinyl chloride (PVC) microtiter plates was performed using an EDTA-gelatin coating according to Zhu et al. (2013) with some modifications. The surface coat was developed by adding 150 µl of a mixture of 0.5% gelatin and 50 mM EDTA in triplicates to each well of a 96-well microtiter plate (Greiner Bio-one, Stuttgart, Germany) and drying overnight at 40°C. After drying, 125 µl of fresh Brain Heart Infusion broth (Difco Laboratories, U.S.A) supplemented with 2% sucrose (EL Naser Chemical Co. Egypt) (BHIS) was transferred to each well. Finally, these wells were inoculated with 25 µl bacterial suspension (108 CFU/ml) and incubated at 37°C for 24 h and then washed three times with sterile phosphate buffer saline (PBS) and air dried for 45 min (Chazotte, 2012). The wells were stained with 0.1% (w/v) crystal violet (Winlab, UK) for 15 min. The excess dye was removed by washing three times with bi-distilled water and then 200 µl of 95% ethanol was added for 1 h to release the attached dye and the optical density was measured at 595 nm using ELISA reader (BioTek®, MQX 200, USA). A negative control was performed (Rukayadi and Hwang, 2006). The extent of in vitro biofilm formation was also measured in PVC microtiter plates coated with gelatin only and in the absence of an EDTA-gelatin coat for comparison (SarojGolia et al., 2012).
 
Scanning electron microscope (SEM)
 
The biofilms produced by strong biofilm-producers MDR UPEC in the absence and presence of two concentrations of EDTA (10 and 20 mM) were scanned using SEM (JSM-840 SEM, JEOLE ltd., Tokyo, Japan). The biofilm was prepared in 6-well cell culture plate (Greiner Bio-one, Stuttgart, Germany) using BHI broth containing 5% sucrose. The biofilm produced was fixed with glutaldehyde 2.5% (v/v) in Dulebecco PBS (PH 7.2) for 1.5 h, rinsed with PBS and then dehydrated through ethanol series. The sample was dried and coated with gold-platinum coat (Soboh et al., 1995).


 RESULTS

Bacterial strains, identification and antibiotic susceptibility
 
A total of 88 MDR UPEC isolates presumptively identified using the conventional culture methods and molecularly identified, were included in the study. The phylogenetic analysis of the 88 UPEC isolates revealed that a percentage of 62.5% (55/88), 18.2% (16/88), 13.6% (12/88) and 5.7% (5/88) belonged to the following phylogenetic groups B2, D, A and B1, respectively. High resistance levels were recorded with ampicillin (97%, 86/88) and cefuroxime (85.2%, 75/88), while high susceptibility was recorded with amikacin (12.5%, 11/88). Several patterns of antibiotic resistance were recorded as shown in Supplementary Table S1;  patterns C,  D and  F were recorded each in 2 isolates from Mansoura hospital and all belonged to phylogenetic group B2, while pattern G was recorded in 3 isolates; all of them were isolated from Mansoura hospital and they all belonged to phylogenetic group A.
 
 
Effect of EDTA on bacterial susceptibility to antibiotics
 
The addition of a sub-MIC (2 mM EDTA) with antibiotics resulted in a decrease in the antimicrobials MIC values. The decrease in the fold of antimicrobials MIC in the presence of sub-MIC of EDTA is shown in Supplementary Table S2. The highest inhibitory effect of EDTA was observed with meropenem and nitrofurantoin rendering 81.6 and 61.4%, respectively of resistant UPEC to sensitive as shown in Figure 1.
 
 
 
Effect of EDTA on in vitro biofilm formation and curli production
 
The degree of biofilm formation in the tested MDR UPEC clinical isolates revealed that 85.2 (75/88), 11.3 (10/88) and 3.4% (3/88) of the isolates were strong, moderate and weak biofilm producers, respectively.
 
The degree of in vitro biofilm formation was determined for strong and moderate biofilm producers (85 isolates) in the presence of two different concentrations of EDTA (10 and 20 mM). The ability of in vitro biofilm formation decreased with the increase in EDTA concentration as shown in Supplementary Table S3 and Figure 2; where 45.8 (39/85), 43.5 (37/85), 3.5 (3/85) and 7% (6/85) were rendered negative, weak, moderate and still strong (no effect) biofilm-producers, respectively after the addition of 10 mM EDTA. Also, 78.8 (67/85), 17.6 (15/85) and 3.5% (3/85) were rendered negative, weak and still strong (no effect) biofilm producers, respectively after the addition of 20 mM EDTA. 
 
 
 
 
The curli production was detected in 67% (59/88) of tested isolates; they showed bright red colonies on congo red agar plate (CRA) and were confirmed to be curli producers. The ability for curli production was tested in the presence of two different concentrations of EDTA (5 and 10 mM), where the ability of curli production decreased by increasing the concentration of EDTA; 69.4% (41/59) and 89.8% (53/59) of curli producing isolates were negative producers after the addition of 5 and 10 mM EDTA, respectively.
 
SEM analysis showed reduction in biofilm formation following treatment with EDTA at both tested concentrations, with the highest reduction following the addition of 20 mM EDTA, as shown in Figure 3.
 
 
Effect of coating polyvinyl chloride microtiter plate with 50 mM EDTA on in vitro biofilm formation
 
Gelatin coating alone had no effect on biofilm formation. EDTA at concentration 50 mM in gelatin coat effectively inhibited biofilm formation, where 81.1 (69/85), 17.6 (15/85) and 1.17% (1/85) were negative, weak and moderate biofilm-producers, respectively as shown in Figure 2.


 DISCUSSION

UTI is a major cause of morbidity and may sometimes lead to mortality (Tajbakhsh et al., 2016) and represents a major health threat due to antibiotic resistance and high recurrence rate (Ponnusamy and Nagappan, 2013). Microbial biofilms in CAUTIs play an important role in antibiotic resistance and limits the therapeutic options (Deotale et al., 2015), so the effect of a non-antibiotic adjuvant EDTA on in vitro biofilm formation and antibiotic susceptibility of MDR strong biofilm producing UPEC clinical isolates from Egypt was studied.
 
The results revealed that using EDTA with concentrations 5 and 10 mM inhibited curli production, the first step in biofilm production. A similar study in India showed that EDTA at concentrations 4 and 5 mM can inhibit curli production (Chaudhary et al., 2013). It was also shown that EDTA with concentrations of 10 and 20 mM inhibited biofilm formation in UPEC biofilm producers by 45.8 and 78.8%, respectively and Chaudhary and colaboraters (2013) showed a decrease in biofilm formation by increasing EDTA concentrations.
 
In the present study, a novel approach was used to eradicate in vitro biofilm production and further evaluated the effect of EDTA coating of PVC microtiter plates, the material is often used for medical implants such as urinary catheter, on biofilm production. The results indicated that EDTA-gelatin coat was effective in inhibiting biofilm formation in 81.1% of tested isolates. Another study in China used EDTA and gallium coat in gelatin to inhibit the bacterial biofilms (Zhu et al., 2013). Trials to sustain the release of EDTA in wound dressings and contact lenses were done, using the therapeutic polymer of chitosan-EDTA (Netsomboon et al., 2017) and polylactic-glycolic acid disc containing 10% EDTA (Nishi et al., 1996). From the results, the coating of urinary catheters using combinations of EDTA with other anti-biofilm agents could greatly improve the clinical outcome.
 
Very low concentrations of EDTA (2 mM) was found to reduce the antimicrobials’ MIC of MDR UPEC in the findings; the reduction of antimicrobials’ MIC in the presence of EDTA was highly observed with Meropenem 81.6%, Nitrofurantoin 61.4%, Levofloxacin 26.4%, Ciprofloxacin 23.2% and Nalidixic acid 20.3%, Amoxicillin Clavulanic acid 18.8%, Ceftazidime 18.7%, Cefotaxime 15.8% and Gentamycin 6.5%.
 
Carbapenems are broad spectrum antimicrobial agent used as last resort treatment for Gram-negative bacteria. Emergence of resistance to carbapenems is a major threat and started to increase in the Middle East, and in this study, 55.6% (49/88) of isolates were resistant to Meropenem. In similar studies from Egypt, 44% of tested Gram negative bacteria were Carbapenem resistant (Khalifa et al., 2017). High prevalence of Carbapenem resistance among Gram negative bacteria was recorded worldwide, where similar studies in North Lebanon recorded Carbapenem resistance among 24.4% of tested Enterobacteriacea (Christophy et al., 2017) and in Germany, 16% of Carbapenem resistance organisms were detected among MDR Gram negative organisms (Maechler et al., 2015). EDTA is an inhibitor of metallo β-lactamases (MBLs) activity (Franklin et al., 2006), and in the present study, the addition of 2 mM EDTA to meropenem rendered 82.7% of meropenem resistant isolates to completely sensitive ones. Yet, no pharmaceutical preparations are available in the market that combines carbapenems with EDTA in parenteral preparations to treat life threatening infections.


 CONCLUSIONS

The high prevalence of MDR phenotype among strong biofilm producers UPEC from Egypt is recorded and the combination  of  carbapenems  with  EDTA  in  parenteral preparations to treat life threatening infections could greatly improve the clinical outcome. There is a continuous need for the development of new strategies for treatment of biofilm-producing UPEC with MDR profile and novel approaches to control microbial biofilm are needed.


 CONFLICT OF INTERESTS

The authors have not declared any conflict of interests.



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