Detection of Escherichia coli and Associated β-Lactamases Genes from Diabetic Foot Ulcers by Multiplex PCR and Molecular Modeling and Docking of SHV-1, TEM-1, and OXA-1 β-Lactamases with Clindamycin and Piperacillin-Tazobactam

Diabetic foot ulcer (DFU) is a common and devastating complication in diabetes. Antimicrobial resistance mediated by extended-spectrum β-lactamases (ESBLs) production by bacteria is considered to be a major threat for foot amputation. The present study deals with the detection of Escherichia coli and the prevalence of bla TEM, bla SHV and bla OXA genes directly from biopsy and swab of foot ulcers of diabetic patients. In total, 116 DFU patients were screened, of which 42 suffering with severe DFUs were selected for this study. Altogether 16 E. coli strains were successfully isolated from biopsy and/or swab samples of 15 (35.71%) patients. ESBL production was noted in 12 (75%) strains. Amplification of β-lactamase genes by multiplex PCR showed the presence of bla CTX-M like genes in 10 strains, bla TEM and bla OXA in 9 strains each, and bla SHV in 8 of the total 16 strains of E. coli. Out of the ten antibiotics tested, E. coli strains were found to be resistant to ampicillin (75%), cefoxitin (56.25%), cefazolin (50%), meropenem (37.5%), cefoperazone (25%), cefepime (31.25%), ceftazidime (56.25%), and cefotaxime (68.75%) but all showed sensitivity (100%) to clindamycin and piperacillin-tazobactam. 3D models of the most prevalent variants of β-lactamases namely TEM-1, SHV-1, OXA-1, and ESBL namely CTX-M-15 were predicted and docking was performed with clindamycin and piperacillin-tazobactam to reveal the molecular basis of drug sensitivity. Docking showed the best docking score with significant interactions, forming hydrogen bond, Van der Waals and polar level interaction with active site residues. Findings of the present study may provide useful insights for the development of new antibiotic drugs and may also prevent ESBLs-mediated resistance problem in DFU. The novel multiplex PCR assay designed in this study may be routinely used in clinical diagnostics of E. coli and associated bla TEM, bla SHV, and bla OXA like genes.


Introduction
Diabetic foot infections (DFIs) are common, often resulting in potentially devastating complications in diabetic patients. DFIs are associated with high morbidity and risk of lower extremity amputation [1]. Limb amputation has a major impact on the individual, not only in distorting the body structure, but also with regard to loss of productivity, increasing dependency, and costs of treating foot ulcers if patients require inpatient care [2]. Wound infection, delay in wound healing, neuropathy and ischemia in combination with a foot ulcer are the most common causes of diabetes-related amputations [3]. Patients with diabetes are more likely to lose a limb than those without diabetes and up to eightyfive percent of lower-limb amputations in patients with diabetes are preceded by infected foot ulceration [3]. To meaningfully describe and evaluate the severity of foot ulcer, several systems of classification are currently in use [4,5]. Wagner's system of classification is routinely used in determining the surgical intervention to foot ulcer on admission. In more superficial infections which are classified according to Wagner (Wagner grades I-II), aerobic gram-positive bacteria are the predominant organisms. In deeper wounds (Wagner grades III-V), gramnegative bacteria are frequently found [6]. Escherichia coli, Proteus spp., Pseudomonas spp., Staphylococcus aureus and Enterococcus spp. are the most frequent pathogens contributing to progressive and widespread tissue destruction [7]. In the patients with DFI, there is a predominance of E. coli (24.20%) and antibiotic resistance is wide spread in this species [8]. The predominant mechanism of resistance to b-lactams in E. coli is production of extendedspectrum b-lactamases (ESBLs). Bacterial strains producing these enzymes inactivate the drugs by hydrolyzing the b-lactam ring [9]. ESBLs -producing bacteria are emerging as a worldwide clinical threat. In the early 1960s, bla TEM-1 was the first plasmid-mediated b-lactamase gene in E. coli [10]. Subsequently, another common b-lactamase gene bla SHV-1 was reported from Klebsiella pneumoniae and E. coli. Various new b-lactam antibiotics have been developed since 1960s for the treatment of patients which have resulted in emergence of other ESBL. Different types of b-lactamase have been reported during the 1990s however, TEM-and SHV-types are more common [11]. During the past decade, rapid and massive spread of CTX-M-type ESBLs have been reported. These enzymes are now the most prevalent ESBLs in Enterobacteriaceae and also occur rarely in Pseudomonas spp. and Acinetobacter baumannii [12] in Europe and in other parts of the world [13]. The list of ESBLs is increasing and the total number of well characterized ESBLs exceeds 200 [11].
The main objective of this study was to develop a simple and rapid method for the detection of E. coli isolates and associated blactamase genes (bla TEM , bla SHV , and bla OXA ) from patients suffering from DFI. Attempt was made to predict three dimensional (3D) model of TEM-1, SHV-1, OXA-1 (b-lactamases) and CTX-M-15 (ESBL). Furthermore, the identification of the amino acid residues crucial to the interaction between selected blactamases with clindamycin and piperacillin tazobactum was performed. Additionally, docking studies of TEM-1, SHV-1, OXA-1 and CTX-M-15 proteins with clindamycin and piperacillin-tazobactum were performed. It is anticipated that modeling and docking studies may be useful in developing new class of drugs to control ESBLs-mediated antibiotic resistance problem in DFUs.

Patients and Sample Collection
This study was conducted in the School of Biotechnology. Samples and details of patients were obtained from the Department of Endocrinology and Metabolism, and the Department of General Surgery, Sir Sunderlal Hospital, Institute of Medical Sciences, Banaras Hindu University, Varanasi. Approval of the institutional ethics committee of Banaras Hindu University (Ref. No. Dean/2009-10/555 dated July 11, 2009) was obtained to conduct this study. Prior written consent was also obtained from every recruited patient. In total, 116 diabetic foot patients attending to the hospital between January 2010 and October 2011 were screened and 42 suffering with severe DFIs (Wagener's grade III-V) were selected for the study. Grading of DFUs was done according to Wagner [4].
Tissue samples from infected DFUs were obtained from the ulcer using a sterilized 6 mm punch biopsy needle under local anaesthesia. Two swab and tissue samples from each patient were collected by washing the wound with sterile physiological saline. One swab and tissue sample was used for detecting E. coli through in vitro culture, the second set of sample was used for detecting E. coli by PCR.

Isolation and Identification of E. coli
A direct smear was made from each sample (swab and biopsy) and plated directly onto MacConkey agar. The inoculated plates were immediately placed in an aerobic environment and incubated at 35uC for 24 h. The plates were examined after 24h of incubation and distinct pink colonies that appeared on each plate were picked up and restreaked on respective media. Tentative identification of E. coli was made on the basis of Gram's staining, morphological characteristics, and biochemical tests namely, catalase, urease, Simmons citrate utilization and MR (methyl red) as per the standard methods. E. coli JM109 (Promega, USA) was used as reference strain.

Isolation of Genomic DNA
Genomic DNA of swab and biopsy samples was extracted using a fast tissue PCR Kit (MBI Fermentas, USA). Genomic DNA from the laboratory-grown cultures was isolated using a DNeasy tissue kit (Qiagen, Germany) according to the instructions of the manufacturer. Plasmid DNA from E. coli strains was isolated using a PureLink HiPure plasmid miniprep kit (Invitrogen, USA) according to the instructions of the manufacturer.  Primer3 (http://frodo.wi.mit.edu/) tool was used for designing E. coli gene specific primers from species-specific region (16S rRNA dimethyladenosine transferase). 16S rDNA (1467 bp) was amplified from the template DNA of the reference strain E. coli JM109, strains of E. coli isolated from DFUs, and biopsy/swab samples of DFUs. Amplification was performed in a final volume of 50 ml containing 16PCR assay buffer with 1.5 mM MgCl 2, 25 pmol of each primers (Fd.59-TGTGGGAACGGCGAGTCGGAATAC-39 and Rev 59GGGCGCAGGGGATGAAACTCAAC-39) (Integrated DNA Technologies, USA), 250 mmol each of the dNTPs, 1U Taq DNA polymerase (Bangalore Genei, Banhalore) and 100 ng of template DNA. Conditions for PCR amplification were; initial denaturation for 10 min at 94uC, 30 cycles of 40 s at 94uC, 40 s at 60uC and 1 min at 72uC followed by final extension of 7 min at 72uC. 5 ml of the amplified PCR product was electrophoresed on a 2% agarose gel in Tris-borate-EDTA buffer (TBE) containing ethidium bromide (0.5 mg/ml) and monitored in gel documentation unit (BioRad Laboratories, USA). 16S rDNA (1467 bp) amplified from E. coli (isolated from DFU) was sequenced to confirm the identity and to confirm the specificity of primers. Additionally, the specificity of primer was confirmed using template DNA from other gram-negative bacteria viz., Klebsiella spp., Enterobacter spp. Citrobacter spp, Serratia spp., and Pseudomonas spp. Based on sequence similarity, the representative isolate was identified as E. coli strain DF39TA. The sequence was submitted to NCBI database under accession number JX017293.

Phenotypic Detection of ESBL and Carbapenemases
ESBL phenotype of various isolates was determined by double disc diffusion synergy test (DDST). Briefly, equal amount of inoculum from each isolate was added to Mueller Hinton broth and grown for 24 h at 37uC. 100 ml of broth culture (approx. 10 6 cells/ml) was uniformly spread onto sterile Mueller Hinton Agar. Antibiotic discs of amoxicillin/clavulanic acid (20/10 mg) and cefotaxime (30 mg), and ceftazidime (30 mg), were placed at a distance of 15 mm apart and plates were incubated at 37uC overnight. Enhancement of zone of inhibition of any of the cephalosporins towards the amoxycillin/clavulanic acid disc was considered as ESBL producer [16]. The E. coli isolates which were found to be positive for ESBL phenotype were subjected to E-test.
E-test for confirming the ESBL phenotype was performed according to Coudron et al. [17]. ESBL results were considered positive if the isolates had an MIC (mg/ml) of $1 for ceftazidime (CAZ), $0.5 for cefotaxime (CTX), and the ratio for CAZ-CLA and CTX-CTL was more than or equal to 8 [17,18]. E. coli ATCC strain 25922 and Klebsiella pneumoniae ATCC strain 700603 (HiMedia, Mumbai, India) were used as negative and positive controls for ESBL production test respectively. E. coli strains that showed a zone diameter of 16-21 mm for meropenem were tested for carbapenemase production by Modified Hodge test (MHT) as per the CLSI recommendation [19].
Multiplex PCR and Sequencing of bla TEM , bla SHV , bla OXA, and 16S rRNA Genes of E. coli Multiplex PCR was performed in a single tube with primers of bla TEM , bla SHV , bla OXA and 16S rRNA genes. PCR assay was performed in a total volume of 50 ml which contained; 25 pmol of the primers of 16S rRNA (Fd 59-TGTGGGAACGGCGAGTCG-GAATAC-39 and Rev 59-GGGCGCAGGGGATGAAACT-CAAC-39), 10 pmol primers of each of the bla TEM , bla SHV , and bla OXA as described by Dallenne et al. [20], 200 mM each of the dNTPs, 1 U of Taq DNA polymerase, 16PCR assay buffer with 1.5 mM MgCl 2 and 100 ng of template DNA or 5 ml of macerated biopsy samples. PCR conditions were used as described by Dallenne et al. [20]. PCR was run in a PTC-100 Thermal Cycler (MJ Research, Inc., USA). 5 ml of the amplified PCR product was used for electrophoresis and visualization was made as mentioned above. Multiplex PCR was also performed separately for bla CTX-M Gp1 , bla CTX-M Gp2 and bla CTX-M Gp9 genes as described previously [20].
Amplified product of bla TEM , bla SHV , bla OXA, and bla CTX-M genes was purified by QIAquick gel extraction kit (Qiagen, Hilden, Germany) according to the instructions of the manufacturer. Sequencing and homology search of the amplified products were done on commercial basis from Chromous Biotech PVT Ltd., Bangalore, India. After complete annotation, the sequences were submitted to NCBI database (http://www.ncbi.nlm.nih.gov/) and all accession numbers are shown in Table S1.

Gene Annotation and Similarity Search
Sequences of bla TEM-1 , bla SHV-1 , bla OXA-1, and bla CTX-M-15 genes from E. coli strain DF39TA were subjected to ORF scan (http:// www.ncbi.nlm.nih.gov/gorf/gorf.html) to identify coding regions (exons). FGENESB was used to predict operons and genes in raw sequences [21]. The predicted putative protein sequences were subjected to protein functional analysis using INTERPROSCAN Table 2. Stereo-chemical properties using PDBSum.   4.4 [22]. These protein sequences were used for homology search. Similar sequences from different species were retrieved and aligned using ClustalW [23]. Phylogenetic tree was constructed using the UPGMA method and tree was inferred by bootstrap phylogenetic inference using MEGA4 [24]. The conserved motifs present in these sequences were analyzed using BLOCKS and MEME (multiple EM for motif elicitation) software version 3.5.7 [25]. For motif analysis, the selection of maximum number of motifs was set to 10 with minimum width of 10 amino acids, while for genes a maximum number of motifs were set to 20 while other factors were default selections for putative proteins.

Retrieval of the Target Protein Sequence and Template Identification
Predicted putative protein sequences of bla TEM-1 , bla SHV-1 , bla OXA-1, and bla CTX-M-15 genes were used as target for homology modeling. Discovery studio 3.1 [26,27] was used for comparative homology modeling of TEM-1, SHV-1, OXA-1, and CTX-M-15 protein using template structures. PDB advance BLAST (http:// www.rcsb.org/pdb/home/home.do) was applied for template identification to construct 3D models of the target proteins.

Active Site Prediction and Docking
After obtaining the final model, the possible binding sites of TEM-1, SHV-1, OXA-1, and CTX-M-15 proteins were searched using Q-SiteFinder (http://bmbpcu36.leeds.ac.uk/qsitefinder/). Ten binding sites were obtained for TEM-1, SHV-1, OXA-1, and CTX-M-15. These binding sites were compared to the active site of the template to determine the residues forming the binding pocket. Clindamycin (C 18

Isolation and Identification of E. coli
Sixteen strains of E. coli were successfully isolated from biopsy/ swab samples of 15 out of 42 patients (35.71%) admitted to the S.S Hospital, Varanasi. Two E. coli strains were isolated from DFU of one patient (DF30). Identity of E. coli strains was confirmed by morphological characteristics, biochemical tests and amplification of the E. coli specific 16S rDNA. Similar to our findings 32.07% of the DFU patients from south India were reported to carry infection of E. coli in DFUs [31]. Alavi et al. [32] also reported that E. coli (23.8%) was the most predominant gram-negative organisms in DFUs from patients of Iran.
One of the interesting outcomes of the study was use of species specific primers for the identification of bacteria from DFUs. All the strains of E. coli isolated from DFUs showed amplification of the desired fragment of 16S rDNA (1476 bp) with E. coli specific primers in PCR assay. Interestingly, DNA isolated from 42 biopsy/swab samples also resulted in amplification of E. coli specific amplicon in 8 biopsy and 7 swab samples. However, template DNA isolated from other gram-negative bacteria did not show amplification of E. coli specific amplicon. As the PCR-based results matched with the laboratory-grown cultures, it is concluded that direct diagnosis of E. coli and/or other species of bacteria by PCR is possible directly from biopsy/swab samples. That these strains indeed belonged to E. coli also became evident from the sequencing of amplified product from a representative strain of E. coli. The sequences showed 99% similarity with sequences available in the NCBI for E. coli.

Antibiotic Resistance Profiles
Antibiotic sensitivity test revealed that all the sixteen E. coli strains of DFUs show high percentage of resistance to a number of antibiotics. Prevalence of resistance among the isolates was; ampicillin (75%), cefoxitin (56.25%), cefazolin (50%), meropenem (37.5%), cefoperazone (25%), cefepime (31.25%) ceftazidime (56.25%), and cefotaxime (68.75%). However, all the strains showed sensitivity (100%) to clindamycin and piperacillin/ tazobactam. Similar to our findings, occurrence of multiple antibiotics resistance has been reported in several bacteria but only few reports are available for bacteria isolated from DFUs    [31][32][33]. Sensitivity of E. coli to clindamycin and piperacillin/ tazobactam has also been reported by other researchers previously [34,35]. Sana et al. [36] reported that 82.2% isolates of E. coli were susceptible to piperacillin/tazobactam. Similarly, a study conducted at Mahatma Gandhi Medical College and Research Institute, Pondicherry, reported that the members of Enterobacteriaceae were mostly susceptible to tazobactam [35].

ESBL and Carbapenemase Production in E. coli
Of the 16 E. coli isolates, 12 (75%) were ESBL producers according to the results of phenotypic tests DDST and E test (Table 1). ESBL-producing strains were found to be resistant to blactam antibiotics namely, ampicillin (83%), cefazolin (50%), cefoperazone (25%), cefepime (33.33%), cefoxitin (58.33%), ceftazidime (75%), and cefotaxime (91.66%). Additionally, six isolates (37.5%) showed resistance to meropenem which seems uncommon for E. coli species. Surprisingly, all these six isolates did not show the presence of carbapenemase by MHT. Recently, Shanmugam et al. [37] and Sahu et al. [38] have reported as high as 45.4% and 37% strains of E. coli resistant to meropenem respectively. With the available data, it is indeed hard to assign the exact mechanism of resistance to meropenem, it would be essential to confirm the presence of various types of carbapenemase genes employing PCR assay. This is the shortcoming of the present study and needs further investigation.
Available reports dealing with the prevalence of ESBL producers amongst various isolates of E. coli show marked variations [7,[33][34][35][36]. In fact, the prevalence of ESBL producing E. coli isolates show significant differences among geographical locations within India [34], and other parts of world ranging from 0% in Iceland to less than 1% in Estonia, 41% in Romania, 16.8% in Iran [35], 25.2% in Tiruchirapali, South India [39], and 31.86% in Turkey [40]. Gadepalli et al. [7] reported 54.5% E. coli isolates as ESBL producers in a tertiary care hospital in New Delhi. Detection of ESBL producing strains of E. coli is of vital importance as they are responsible for the spread of resistance among different bacteria. A combination of factors such as coselection due to MDR phenotypes, virulence factors, mobile genetic elements, clonal spread of virulent strains and the acquisition of diverse ESBL-bearing plasmids may facilitate the spread of ESBL and other resistances [41].
Occurrence of bla TEM , bla SHV , bla OXA, and bla CTX-M Genes and Comparative Analysis of the ESBL Phenotype Multiplex PCR assay was employed to detect the prevalence of bla TEM , bla SHV, and bla OXA like genes as well as E. coli in a single PCR reaction. Typical representation of multiplex PCR for simultaneous amplification of bla TEM , bla SHV , bla OXA, and 16S rRNA genes from 5 (2 biopsy and 3 swab) samples are shown in Figure 1. The amplified products were identical to those obtained by pure culture of E. coli. That the amplified products are indeed originating from strains of E. coli became evident from the fact that all the strains showed amplification of E. coli specific amplicon similar to the reference strain, E. coli (JM109) (Figure 1). bla SHV like gene was detected in 8 of the 16 E. coli positive DFUs. Further analysis of bla SHV gene revealed that four strains of E. coli possess bla SHV-1 , two bla SHV-12 , one each has bla SHV-5 and bla SHV-2 (Table 1). bla TEM was detected in 9 E. coli strains of which five isolates expressed bla TEM-1 , two bla TEM-20 and one each showed expression of bla TEM-52 and bla TEM-10 (Table 1). Interestingly, bla OXA like gene was noted in 9 strains of E. coli of which all produced bla OXA-1 (Table 1).
Multiplex PCR was also performed separately to detect the prevalence of bla CTX-M like gene. Of the 16 pure cultures of E. coli strains, presence of bla CTX-M like gene was noted in 10 strains (62.5%) ( Table 1). Further analysis revealed that bla CTX-M-15 was the most widespread among different strains of E. coli (7/16 strains, 43.47%), followed by bla CTX-M-9 , bla CTX-M-3, and bla CTXM-1 which were present in each one strain (1/16 strains, 6.25%). However, the predominance of CTX-M-15-producing E. coli in this study may be due to the virulent ST131 clone and the diverse plasmids bearing the bla CTX-M-15 gene. Further studies are needed to decipher the genetic traits responsible for showing CTX-M-15 predominance in this study. Sequences of bla TEM (TEM-1, -10, -20, and -52), bla SHV (SHV-1, -2, -5, and 12), bla OXA (OXA-1), and bla CTX-M (CTX-M-1, -3, -9, and -15) -type genes were submitted to NCBI database and accession numbers are shown in Table S1. Occurrence of several b-lactamases genes has been reported, but bla TEM , bla SHV , bla OXA, and bla CTX-M -type ESBLs genes are the most predominant [10]. Kiratisin et al. [42] screened 235 strains of ESBLs producing E. coli and reported that 87.3%, 77% and 3.8% of the strains were carriers of the bla CTX-M , bla TEM, and bla SHV genes respectively. A few strains were found to carry the bla OXA gene. The OXA b-lactamases, known as oxacillinases are equally important as they degrade isoxazolyl b-lactams such as oxacillin and methicillin. OXA enzymes belong to the Class D group of b-lactamases and are known to be present in a number of bacteria [43]. SHV, TEM, and CTX-M enzymes belong to Ambler class A and were initially reported as plasmid borne in gram-positive bacteria [44]. More than 200 types of well characterized b-lactamases enzymes have been reported [45] and several attempts have been made to categorize them since the late 1960s [46][47][48][49].

Homology Modeling of OXA-1, SHV-1, TEM-1, and CTX-M-15 Proteins
The homology modeling of OXA-1, SHV-1, TEM-1, and CTX-M-15 proteins was performed with Discovery studio 3.1 and is represented in Figure 2. Additionally, the 3D model of these proteins was constructed using the PDB BLAST for template identification. Analyses revealed that OXA-1 shares 99% similarity as well as positivity with PDB code: 1M6K of E. coli [50]. Similarly, SHV-1 showed 99% similarity and 100% positivity with the PDB code: 3D4F crystal structure of Klebsiella pneumoniae [51]. TEM-1 showed 94% similarity and 94% positivity with PDB code: 1ERM of E. coli [52]. In the case of bla CTXM-15 , there were 86% similarity and 93% positivity with PDB code: 2ZQ8 crystal structure of E. coli. Electrostatic energy of predicted OXA-1, SHV-1, TEM-1, and CTX-M-15 models were -5659.92, -7887.66, -9139.24, and -7430.49 kcal/mol respectively as per the analysis done by CHARMm force field of Discovery studio 3.1. Based on simulation study, it became evident that the predicted models are highly stable. Details of modeling and simulation results for OXA-1, SHV-1, TEM-1, and CTX-M-15 are available in Table S2.

Model Assessment (Refinement and Evaluation)
Stereochemical quality of the predicted protein structure was assessed using RAMPAGE and PDBSum. The Ramachandran plot study of OXA-1, SHV-1, TEM-1, and CTX-M-15 revealed that more than 90% of residues were in favoured regions having good stereochemical quality. Analysis of OXA-1, SHV-1, and CTX-M-15 revealed that 93.5, 98.1, and 98.9% residues occur in the favoured region respectively, with no residues present in the outlier region. TEM-1 showed 98.3% residues in favoured region and 0.4% residues in outlier region (Table S3). No residues were observed in the disallowed region. Further analysis based on PDBSum showed that the residues present in the most favoured regions for OXA-1, SHV-1, TEM-1, and CTX-M-15 were 87.7, 95.2, 95.7, and 95.3% respectively ( Table 2). ERRAT score for the models of OXA-1, SHV-1, TEM-1, and CTX-M-15 was 91.15, 82.12, 92.30, and 92.35 respectively which are well within normal range for a high quality model. The best refined and validated structures of OXA-1, SHV-1, TEM-1, and CTX-M-15 were deposited in the PMDB database with PMDB-IDs; PM0078526, PM0078524, PM0078525, and PM0078527 respectively. The weighted root mean square deviation (RMSD) of the Ca trace between the template and the final refined model of OXA-1, SHV-1, TEM-1, and CTX-M-15 showed that the target and the template structures are closely similar at the backbone and at the CA tom level, yielding a significant Z-score. Resolution of the predicted OXA-1, SHV-1, TEM-1, and CTX-M-15 structure showed significant resolution of 2.363, 1.677, 2.021, and 1.62Å respectively using RESPROX server. Structure quality estimation using PROSA showed significant Z-scores of -6.04, -5.27, -7.66, and -6.38 for OXA-1, SHV-1, TEM-1, and CTX-M-15 respectively as compared to the template Z-score. Structure quality estimation using QMEAN also resulted in significant Zscore (Table S4). The modeled structures of OXA-1, SHV-1, TEM-1, and CTX-M-15 revealed that each monomer belongs to the alpha-beta: 3-layer (aba) sandwich DD-peptidase/b-lactamase super family (3.40.710.10) ( Figure S6). Secondary element composition is also available in Table S5.

Active Site Prediction of OXA-1, SHV-1, TEM-1, and CTX-M-15 Proteins
Among the ten binding sites obtained from Q-Site finder (Figure 3), site 1 was found highly conserved in OXA-1, SHV-1, and CTX-M-15 and therefore selected as the active site for docking study with clindamycin and piperacillin-tazobactam. However, sites 3 and 7 were found highly conserved in TEM-1 and thus used for docking purpose. The results of multiple sequence alignment and active site prediction revealed that the residues in site 1 of OXA-1 (LYS 16  , and LEU 172 ) were found conserved and showed homology with the active site of TEM-1 b-lactamase from E. coli [52]. All the 10 possible active sites for OXA-1, SHV-1, TEM-1, and CTX-M-15 models are available in Table S6. All these putative predicted active sites show close similarity with the reported active sites of respective b-lactamases [51][52][53] and support our findings. With OXA-1 it was noted that clindamycin and piperacillintazobactam interacted with the major active site cavity, with site volume of 234 cubic Å . The residues TRP 18 , MET 19 , Ser 22 ,  VAL 23 , VAL 24 , SER 27 , TYR 48 , TRP 67 , LeU 68 , GLU 69 ,  ILE 74 ,GLN 79 , LYS 119 , THR 120 , GLY 121 , ALA 122 , TRP 134 , and GLU 136 were mainly involved in the interaction with clindamycin ( Figure 4a) (Figure 4b). Validity of docking is corroborated from the fact that MET 19 , MET 105 , LYS 119 , and THR 120 residues are commonly reported as prominent active site of OXA-1 protein for both, clindamycin and piperacillin-tazobactam. Our findings also show that the binding orientation of both the compounds is proper with the inhibitory active domain.
In the case of SHV-1 protein, clindamycin and piperacillintazobactam interacted with the major active site cavity with a site volume of 486 cubic Å . The residues namely, VAL 13  , and LEU 172 residues were found to be the prominent active sites.
In the case of CTX-M-15 protein, clindamycin and piperacillintazobactam showed interaction with the major active site cavity (with site volume of 255 cubic Å ). The residues namely, GLY 26  , and HIS 194 residues were found as prominent active sites.

Conclusions
The present study shows that multiplex PCR assay may be used for the simultaneous screening of E. coli and associated ESBL genes (bla TEM , bla SHV, and bla OXA ) in DFU of a large number of clinical specimens especially in laboratories/hospitals having moderate resources. Findings of this study demonstrate the high prevalence of ESBL-producing E. coli in DFUs patients. Multiplex PCR assay showed the highest occurrence of bla CTX-M like gene (62.5%) followed by bla TEM , bla OXA, and bla SHV genes among the E. coli strains isolated from DFUs patients. In view of high prevalence of bla CTX-M like gene, it is recommended that multiplex PCR may be routinely used for the screening of this gene in ESBLproducing bacteria. Our findings also showed that majority of the ESBL-producing strains were resistant to b-lactams but showed 100% sensitivity to clindamycin and piperacillin-tazobactam. Furthermore, the 3D models of most prevalent variants of blactamases viz. TEM-1, SHV-1, OXA-1, and ESBL viz. CTX-M-15 were predicted and docking studies with clindamycin and piperacillin/tazobactam were performed. The docking scores of TEM-1, SHV-1, OXA-1, and CTX-M-15 proteins with clindamycin and piperacillin-tazobactam showed significant interaction with active binding residues. The docking studies also revealed that TYR 48 , GLU 69 , SER 70 , GLN 79 , and THR 120 of OXA-1; THR 40 , ARG 212 , and ILE 214 of SHV-1; SER 56 , and LEU 172 residues of TEM-1 protein; PRO 163 residue of CTX-M-15 form hydrogen bonds with the side chain along with main chain interaction with above drugs. The results of the present study may provide useful insights for developing new antibiotic drugs to minimize ESBLs-mediated resistance problem of bacteria in DFU of diabetic patients.