Strains of ESBL Producing E. Coli | Investigation

Strains of ESBL Producing E. Coli InvestigationIntroductionBackground of StudyExtended Spectrum Beta- Lactamases (ESBL) are beta lactamases which are mainly produced by family members of Enterobacteriaceae derived from mutations of the previous broad-spectrum beta-lactamase (Sharma et al., 2010). This enzyme works by hydrolysing and destroying the - Lactam ring of all in all cephalosporins, penicillins and monobactams (Sharma et al., 2010). In recent years, the emergence of ESBL producing Escherichia coli has posed a very life-threatening problem to the management of diseases caused by this organism as only limited choice antibiotics can be given to patients. Carbapenems are the drugs of choice for the treatment of ESBL producing E.coli, however, carbapenamase apology has recently been reported (Paterson and Bonomo, 2005). Prolonged use of antibiotics was suggested as the main cause of the emergence of ESBL E.coli and the fact that the genes coding for ESBLs are easily transfer rosy-cheeked from one organism to other organism via conjugation, transduction and trans arrangement make the spread even quicker (Vaidya et al., 2011).ESBL producing organisms were first reported from a patient in Germany in 1983 and since then , several outbreaks render been reported worldwide usually one particular super strain has been involved presumably combining not only the capability to produce ESBLs tho also possessing versatile other virulence factors that contribute to their pathogenic success. (Harada et al., 2013). These pathogenic ESBL producing Escherichia coli in recent years defecate become a major reverence and their emergence is now become alarming in clinical fields and subjected to comprehensive studies worldwide.The most common infections caused by pathogenic ESBL producing E.coli are urinary nerve pathway infections (UTI), bloodstream infections, gastrointestinal infections (Fatima et al., 2012 Bekat et al., 2002). According to Petty et al., (2013), g lobally, E.coli sequence type ST131 is the multidrug resistant clone strain which is responsible for ESBL CTX-M15 bearing genes, and it is the most alarming pathogenic ESBL producing E.coli associated in causing UTIs and septicaemia in hospital community acquired infections. ? in UK or worldwide?As genes coding for ESBL in Escherichia coli are known to be transferrable this raises further fear of the spread of these genes to other strains, continuous monitoring of the predominant strains of E.coli which carry the ESBL genes is therefore important.Problem statementStudies of ESBL producing Escherichia coli in the South Manchester population develop been carried out previously. This study will investigate strains of ESBL producing E. coli currently circulating in the Stockport Population of South Manchester and compare them to those delineated in the previous studies using a molecular(a)(a) typing and pulse-field mousse electrophoresis.ObjectivesThe objectives of the project areScr een for ESBL Escherichia coli clinical isolatesIdentify strain using PFGEAssess the relatedness of the strains by PFGE analysisDetermine Escherichia coli plasmid desoxyribonucleic acid profileIdentify Escherichia coli phylotyping group1.0.4. Significance of studyFinding from this study will contribute to the existing data and the body of knowledge on the molecular relationship of predominating of E.coli isolates from South Manchester populations.1.0.5. Scope and LimitationsThere are no data on the antibiotics consumed by the patients in which the clinical isolates originates from. The availability of this data readiness assist in understanding relationship between an exposures of certain antibiotics to the emergence of ESBL producing E.coli strain.PFGE also has several limitations in which the method assess visual relatedness of an isolates and not using a phylogeny relationship which provide more accurate molecular relationship between an isolates.Escherichia coliEscherichia col i is a motile gram blackball rod, facultative anaerobe, non- spore forming bacteria taxonomically belong to the family of Enterobacteriaceae. It is considered as a normal inhabitants of gut and intestine in almost all warm blooded mammals and found as a faecal contaminant in the environment (Brennan et al., 2010 Darnton et al., 2007 Diniz et al., 2005). Most varieties of E.coli are harmless and do in the most part contribute to the normal and healthy intestine condition, go a few cause limiting abdominal cramp associated with diarrhoea. However, there are some serotypes that becoming a major threat to the human health, because they have acquired certain genetic material and virulence factors which enabling them transformed into pathogenic E.coli causing broad spectrum of disease (Clarke et al., 2003 Kaper et al., 2004). Pathotypes of E.coli are classified by item utensil in which they causing a disease, presence of certain virulence genes and their clinical manifestations (Chan g et al., 2004).Growth requirementsE.coli are non- fastidious bacteria, thus it can be cultured in artificial media with various altered physical and nutritional growth factors. It can be isolated easily from clinical samples by culturing into culture media and incubated at best temperature of 37C anaerobically or aerobically as it is a facultative organisms (Yunlin et al., 2004)Uropathogenic Escherichia coliAccording to Pitout et al., (2005) E. coli is a frequent cause of the urinary tract infections (UTIs) of a hospitalised and non- hospitalised patients. UTIs are usually self- limiting but untreated lower urinary tract infections such as simple cystitis (bladder infection) can lead to much more severe nausea known as pyelonephritis (renal infections) mainly among adult women (James et al., 2011). Infections occur by ascending movement of E. coli up the periurethral area colonising the bladder or infections by movement cumulus from the intestinal tract. Due to anatomical compl exities in women, they are more prone to be diagnosed with UTIs for at least once in their lifetime (James et al., 2001)1.3Escherichia coli typing1.3.1plasmid profilingMultidrug resistant bacteria including ESBL producing Escherichia coli acquire their resistance by various gene transfer mechanisms which include transformation, horizontal transfer either by transduction, and conjugation, jumping gene and most often, are plasmid mediated (Carattoli et al., 2005)Plasmids are an extra chromosomal fragments of self- replicating DNA present in most of the bacterial species. Plasmids contain genes that are an subjective for the replication of genes that promotes resistance to agents such as antibiotics, ultraviolet radiation, metals and bacteriophages.1.3.2Pulse-field gel electrophoresisPFGE was developed and described first by Schwartz and Cantor (1984). It is a molecular proficiency of typing a bacteria especially pathogenic Escherichia coli 0157H7, non 0157 H7, Salmonella serotypes, Shigella sonnei and Shigella flexneri. PFGE uses a gel electrophoresis- based technique that allows separation of large molecular weight DNA up to 2Mb- 10Mb using a standard PFGE method (CDC, 2013 Hansen et al., 2002 Vimonet et al., 2008)PFGE is different to conventional gel electrophoresis as the large genomic DNA is digested with restriction enzyme that recognise and cleave specific sequences of DNA known as restriction site in an organism to produce a multiple DNA fragments which differ in size of their molecular weight (Van der Ploeg et al., 1984). The fragments are then run through constant changing electric field of PFGE resulting in a formation of DNA at various discrete size bands.This typing method has also been shown to have more discriminating power and reproducibility between laboratories than the newer molecular typing method such as ribotyping and multi- locus sequence typing (MLST) which confer more on the global epidemiology and revolutionary relationship between bac terial species (Vimonet et al., 2008)1.3.3.Escherichia coli phylogenetic group2.0Materials and Methods2.0.1bacterial IsolatesBacterial isolates used in this study were Escherichia coli clinical isolates which was composed from Stepping Hill Hospital. Isolates undergo an anonymisation numbering of 1 to 20.2.0.2.Bacteriological MediaThe media used in the study were a selective differential medium for UTI Escherichia coli which is Chromogenic agar and nutrient agar which was used as a medium for growth and maintenance of isolates.2.0.3Antibiotic disksTable 1 Antibiotic disks used in this study was obtained from Oxoid.Ltd.AntibioticsAntibiotic GroupGentamicin (10g)AminoglycosidesCiprofloxacin (5g)QuinoloneAmoxicillin (25g)PenicillinCefpodozime (10g)CephalosporinMecillinam (10g)Beta lactamTrimetophrim (2.5g)BacteriostaticESBL Disk kit (Mast Diagnostics)2.0.4Buffers and solutionsTris Borate EDTA (TBE X1 and X0.5) (Sigma) pH 8.2 was used as a ladder buffer in agarose gel electrophoresis. 2.0.5Commercial kitsThe commercial kit used in this study was QIAprep Spin Miniprep kit out (Qiagen) and DNeasy Blood and Tissue Kit (Qiagen)2.1.Screening for multidrug resistance and potential ESBL producers in Escherichia coli clinical isolatesAntibiotic susceptibility of Escherichia coli to six antibiotics (Table 1) were tested using the Kirby Bauer disk airing method. A 24 hour cultures from Nutrient agar was used. Then, a single colony was taken and transferred into 5ml Mueller Hinton Broth. It was then incubated at 37C to develop a heavy suspension of overnight cultures. A sterile cotton swabs were used to streak onto the Mueller Hinton agar and the rotation were repeated for ternary times. A lowest sweep up was made around the rim of the agar. The plates were allowed to dry for several minutes. Using antibiotic dispenser, the disk that has been impregnated with a fixed antibiotic concentration was set on the draw near of the agar surface. After 24hr of an incubation p eriod, the plates were checked for the presence of inhibition zone. Each recorded inhibition zone was compared with antimicrobial susceptibility testing disc chart provided by The British Society for Antimicrobial Chemotherapy (BSAC). The inhibition zone of all(prenominal) antibiotic was reported as sensitive, intermediate or resistance. Isolates wake resistance to three or more classes of antibiotics were considered as multidrug resistance (Falagas, 2007). ESBL producers were detected by testing sensitivity of isolates against a pair discs (cefpodoxime 10g and cefepime 10g) with and without clavulanic dot placed oppositely on an agar. According to manufacturer (Mast diagnostics), isolates were considered as an ESBL if there is a presence of 5mm larger inhibition zone in disks with clavulanic acid rather than the disks without the clavulanic acid.2.2. Determination of plasmid profiles in MDR and ESBL Escherichia coli2.2.1Plasmid ExtractionPrior to Plasmid DNA extraction, a fresh overnight cultures of E.coli after an incubation at 37C in a Mueller Hinton broth were harvested. Plasmid DNA extraction was carried out using QIAprep Spin Miniprep Kit (Qiagen) following the manufacturers instructions. Extracted plasmid DNA was stored at -20C until use.2.2.2Detection of plasmid by agarose gel electrophoresisThe profiles of the plasmid DNA was determined on a 0.7% agarose gel electrophoresis which has been carried out at 70 Vcm-1 for 120 minutes. The size of DNA bands was estimated using Hyper ladder 1 (Bioline) as a reference molecular weights marker. The bands were envisioned under UV transilluminator and photographed with digital television camera connected to visualisation unit (Alpha Innotech) and the size of the plasmid were measured by visual comparison to the reference marker.2.3Escherichia coli pathotypes tendency2.3.1.Genomic DNA extractionPrimary cultures on the nutrient agar was inoculated into 3ml Mueller Hinton broth for 24 hours at 37C. The cells was then harvested by centrifugation at 12, 000 for 3 minutes. Genomic DNA extraction was carried out using DNeasy Blood and Tissue (Qiagen) kit following the manufacturers instructions. Final volume of 150l genomic DNA were collected and kept at -20C until needed.2.3.2Multiplex PCR for Escherichia coli phylotypingPCR reply combine preparation must be carried out on ice. PCR was performed in 0.2ml PCR tubes on a GeneAmp PCR carcass 9700 thermocycler (Applied Biosystems) with a total 25l of reaction volume as described in Table 2 and PCR condition according to Table 3. The negative control reaction lacking the DNA was included.Table 2PCR reaction mixComponentsRequired concentrationsVolume (l)per reactionBiomix Red2X12.5Primer (forward)chuAyjaAtspE4.c220pmol20pmol20pmol111Primer (reverse)chuAyjaAtspE4.c220pmol20pmol20pmol111DNA2Ultrapure sterile water4.5Total volume per reaction25Table 3 Conditions for PCR gene involutionGenesPrimer sequencePCR conditionchuA foregoing5-GACGAACCAAC GGTCAGGAT-3Reverse5-TGCCGCCAGTACCAAAGACA-3Initial denaturation 94C for 4 minsDenaturation 94C for 25 secsAnnealing 52C for 40 secs 30cyclesExtension 72C for 50secFinal extension 72C for 6 minsyjaAForward5-TGAAGTGTCAGGAGACGCTG-3Reverse5-ATGGAGAATCGGTTCCTCAAC-3tspE4.c2Forward5-GAGTAATGTCGGGGCATTCA-3Reverse5-CGCGCCAACAAAGTATTACG-32.3.3Detection of by agarose gel electrophoresisAfter completion of the multiplex PCR, the amplification product were separated by dry electrophoresis system. 15l of amplified product was mixed with 5 5X DNA loading buffer (Bioline) and peeved onto 2% agarose gel incorporated with SYBR green dye. After electrophoresis, the gel was visualised by exposing the gel under UV light and was photographed with a digital UV camera connected together with the visualisation unit (AlphaInnotech). The size of the amplicon were measured by visual comparison to the 1kb DNA marker (Bioline). Phylogenetic typing analysis were carried on the basis of the presence or absence of an amplicon sized 279bp, 211bp and 152bp which belong to chuaA, yjaA and tspE4.c2 genes respectively.2.4.Pulse- field gel electrophoresis (PFGE)2.4.1.DNA extractionEach isolates was inoculated into 5ml Mueller Hinton Broth and incubated overnight at 37C with gentle agitation. Cells were then harvested by placing 1ml of culture into 1.5ml microcentrifuge tube and was centrifuged at 13, 000 rpm for one minutes. The supernatant was discarded and the process was repeated until all the 5ml of culture finished. The supernatant was again discarded and dig of cells was resuspended in 500l of 0.5M EDTA buffer (see appendix) and was centrifuged at 13, 000rpm for one minutes to removes broth debris that might be interfering with the extraction processes. The washing step was repeated twice to ensure complete removal of debris.The supernatant was discarded once again and pellet was resuspended in 500l of suspension buffer.2.4.2.Preparation of low melting point (LMP) agaroseTo prepare the LMP a garose, 3g of SeaKem PFGE agarose (BioRad) were dispensed into 100ml of TE buffer (see appendix) in a normal bottle. It were then heated to dissolve. Agarose was transferred to a 56C waterbath until needed.2.4.3.Preparation of the bacterial plugsThe wells of PFGE plug molds were numbered. 3 plugs was prepared for each isolates. Then, 750l of LMP agarose was added immediately into each cell- buffer suspension and carefully mixed by pipetting up and down several times and be careful not to induce any formation of bubbles.The mixture of cells and agarose was quickly pipetted into the well of a plastic PFGE plug molds (BioRad). The wells was filled to the rim and plugs were allowed to solidify at room temperature or chilled for 5 minutes in the refrigerator.2.4.4.Lysis of the cellsThe cells were lysed by adding a mixture of 1ml of proteolysis buffer with 10l of Proteinase K stock solution (50mg/ml) (see appendix) into a 1.5ml new labelled microcentrifuge tube. The plugs were aloof fro m the plug molds by peeling the sealant tape below the wells until all tape was removed. The PFGE plastic arm was used to push the plugs out of the molds into the microcentrifuge containing the mix of proteolysis buffer-proteinase K solution. All plugs for one isolates were transferred into the same tubes. Care was taken while pushing the plugs out of the molds as not to tear the fragile plugs. Tubes was then incubated in a heating block at 50C for 24 hours for digestion to take place.2.4.5.Washing of the plugsAfter completion of an overnight incubation, the proteolysis buffer and Proteinase K activity were eliminated by carefully pipetting out the volume, care taken not to tear the plugs. The plugs were then washed with TE buffer. The washing steps was repeated three times, for every half an hour and were held at room temperature to equilibrize the plugs.2.4.6.Restriction enzyme digestionAfter completion of the washing steps, wash buffer was removed in the final wash leaving only a garose gel in the tubes. Then, 300l of 1X restriction enzyme buffer specific to the enzyme used was pipetted in each tubes containing the agarose plugs and was let to equilibrate at room temperature for 10 minutes. The restriction buffer was then discarded, taking care not to tear the plugs. Next, 300l of restriction buffer containing 50U of Xbal enzyme was added into the tubes and was incubated in an incubator for 24 hours at 37C specific to the optimal temperature for Xbal enzyme.2.4.7.Pulse- field gel electrophoresis2.4.7.1.Electrophoresis gel preparation.After incubation, restriction enzyme reaction was stopped by addition of 200l of 50mM EDTA. Plugs were cooled at 4C until needed.Then, a (1%) agarose gel was prepared by heated to dissolved 3g of PFGE coterie agarose (BioRad) into 300ml of 0.5X TBE buffer over magnetic hot plate with constant stirring or in the microwave and swirl to dissolved. The agarose was then poured into a casting tray that has been placed with PFGE comb and let to solidify at room temperature. The enzyme- buffer was aspirated and one plug of each isolates was loaded into the gel. Care was taken not to tear the plugs. Then, a thin slice high range and mid- range lambda molecular weight marker (New England Biolabs) was loaded into the wells next to each other. After all samples was loaded into wells, the wells were sealed with melted LMP agarose.2.4.7.2.Electrophoresis RunThe electrophoresis was performed by using a CHEF mapper (BioRad) which subsequently was filled with approximately 3 liters of 0.5ml TBE buffer. The running buffer was let to cool approximately at 14C before turning on the pump.The run time was set for 24 hours at 6 Vcm-1 with 120 angle using switch time of 2.16 sec to 54.17 sec.2.4.7.3.Gel stainingOnce the run was complete, the gel was stained with 3X Gel red nucleic acid stain (Biotium) with approximately 200ml distilled water and was gently agitated on rotary shaker for 20 minutes. The gel was then visualised und er UV transilluminator and a picture was taken once optimal image obtained.