Strains of ESBL Producing E. Coli | Investigation

Introduction

  1. Background of Study

Extended 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 β- Lactam ring of all cephalosporins, penicillins and monobactams (Sharma et al., 2010). In recent years, the emergence of ESBL producing Escherichia coli has posed a very serious 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 resistance 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 transferred from one organism to another organism via conjugation, transduction and transformation 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 have been reported worldwide usually one particular “super” strain has been involved presumably combining not only the capability to produce ESBLs but also possessing various other virulence factors that contribute to their pathogenic success. (Harada et al., 2013). These pathogenic ESBL producing Escherichia coli in recent years have become a major concern 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 tract infections (UTI), bloodstream infections, gastrointestinal infections (Fatima et al., 2012; Bekat et al., 2002). According to Petty et al., (2013), globally, 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 transferable 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.

  1. Problem statement

Studies of ESBL producing Escherichia coli in the South Manchester population have 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 typing and pulse-field gel electrophoresis.

  1. Objectives

The objectives of the project are:

  1. Screen for ESBL Escherichia coli clinical isolates
  2. Identify strain using PFGE
  3. Assess the relatedness of the strains by PFGE analysis
  4. Determine Escherichia coli plasmid profile
  5. Identify Escherichia coli phylotyping group

1.0.4. Significance of study

Finding 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 Limitations

There are no data on the antibiotics consumed by the patients in which the clinical isolates originates from. The availability of this data might help 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.

  1. Escherichia coli

Escherichia coli is a motile gram negative 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, while 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 specific mechanism in which they causing a disease, presence of certain virulence genes and their clinical manifestations (Chang et al., 2004).

  1. Growth requirements

E.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 optimum temperature of 37ºC anaerobically or aerobically as it is a facultative organisms (Yunlin et al., 2004)

  1. Uropathogenic Escherichia coli

According 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 illness 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 down from the intestinal tract. Due to anatomical complexities in women, they are more prone to be diagnosed with UTIs for at least once in their lifetime (James et al., 2001)

1.3 Escherichia coli typing

1.3.1 Plasmid profiling

Multidrug 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, transposon 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 essential for the replication of genes that promotes resistance to agents such as antibiotics, ultraviolet radiation, metals and bacteriophages.

1.3.2 Pulse-field gel electrophoresis

PFGE was developed and described first by Schwartz and Cantor (1984). It is a molecular technique of typing a bacteria especially pathogenic Escherichia coli 0157:H7, 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 bacterial species (Vimonet et al., 2008)

1.3.3. Escherichia coli phylogenetic group

2.0 Materials and Methods

2.0.1 Bacterial Isolates

Bacterial isolates used in this study were Escherichia coli clinical isolates which was collected from Stepping Hill Hospital. Isolates undergo an anonymisation numbering of 1 to 20.

2.0.2. Bacteriological Media

The 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.

2.0.3 Antibiotic disks

Table 1: Antibiotic disks used in this study was obtained from Oxoid.Ltd.

Antibiotics

Antibiotic Group

Gentamicin (10µg)

Aminoglycosides

Ciprofloxacin (5µg)

Quinolone

Amoxicillin (25µg)

Penicillin

Cefpodozime (10µg)

Cephalosporin

Mecillinam (10µg)

Beta lactam

Trimetophrim (2.5µg)

Bacteriostatic

ESBL Disk kit (Mast Diagnostics)

 

2.0.4 Buffers and solutions

Tris Borate EDTA (TBE X1 and X0.5) (Sigma) pH 8.2 was used as a running buffer in agarose gel electrophoresis.

2.0.5 Commercial kits

The commercial kit used in this study was QIAprep Spin Miniprep Kit (Qiagen) and DNeasy Blood and Tissue Kit (Qiagen)

2.1. Screening for multidrug resistance and potential ESBL producers in Escherichia coli clinical isolates

Antibiotic susceptibility of Escherichia coli to six antibiotics (Table 1) were tested using the Kirby Bauer disk diffusion 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 37°C 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 three times. A final sweep 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 placed on the surface of the agar surface. After 24hr of an incubation period, 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 each antibiotic was reported as ‘sensitive’, ‘intermediate’ or ‘resistance’. Isolates showing 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 10µg and cefepime 10µg) with and without clavulanic acid 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 coli

2.2.1 Plasmid Extraction

Prior to Plasmid DNA extraction, a fresh overnight cultures of E.coli after an incubation at 37ºC in a Mueller Hinton broth were harvested. Plasmid DNA extraction was carried out using QIAprep Spin Miniprep Kit (Qiagen) following the manufacturer’s instructions. Extracted plasmid DNA was stored at -20ºC until use.

2.2.2 Detection of plasmid by agarose gel electrophoresis

The 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 visualized under UV transilluminator and photographed with digital camera connected to visualisation unit (Alpha Innotech) and the size of the plasmid were measured by visual comparison to the reference marker.

2.3 Escherichia coli pathotypes determination

2.3.1. Genomic DNA extraction

Primary cultures on the nutrient agar was inoculated into 3ml Mueller Hinton broth for 24 hours at 37ºC. 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 manufacturer’s instructions. Final volume of 150µl genomic DNA were collected and kept at -20ºC until needed.

2.3.2 Multiplex PCR for Escherichia coli phylotyping

PCR reaction mix preparation must be carried out on ice. PCR was performed in 0.2ml PCR tubes on a GeneAmp PCR System 9700 thermocycler (Applied Biosystems®) with a total 25µl 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 2: PCR reaction mix

Components

Required concentrations

Volume (µl)

per reaction

Biomix Red

2X

12.5

Primer (forward)

chuA

yjaA

tspE4.c2

20pmol

20pmol

20pmol

1

1

1

Primer (reverse)

chuA

yjaA

tspE4.c2

20pmol

20pmol

20pmol

1

1

1

DNA

 

2

Ultrapure sterile water

 

4.5

Total volume per reaction

 

25

Table 3: Conditions for PCR gene amplification

Genes

Primer sequence

PCR condition

chuA

Forward

5’-GACGAACCAACGGTCAGGAT-3’

Reverse

5’-TGCCGCCAGTACCAAAGACA-3’

Initial denaturation: 94°C for 4 mins

Denaturation: 94°C for 25 secs

Annealing: 52°C for 40 secs 30

cycles

Extension: 72°C for 50sec

Final extension: 72°C for 6 mins

yjaA

Forward

5’-TGAAGTGTCAGGAGACGCTG-3’

Reverse

5’-ATGGAGAATCGGTTCCTCAAC-3’

tspE4.c2

Forward

5’-GAGTAATGTCGGGGCATTCA-3’

Reverse

5’-CGCGCCAACAAAGTATTACG-3’

2.3.3 Detection of by agarose gel electrophoresis

After completion of the multiplex PCR, the amplification product were separated by dry electrophoresis system. 15µl of amplified product was mixed with 5µ 5X DNA loading buffer (Bioline) and loaded 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 extraction

Each isolates was inoculated into 5ml Mueller Hinton Broth and incubated overnight at 37ºC 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 pellet of cells was resuspended in 500µl 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 500µl of suspension buffer.

2.4.2. Preparation of low melting point (LMP) agarose

To prepare the LMP agarose, 3g of SeaKem PFGE agarose (BioRad) were dispensed into 100ml of TE buffer (see appendix) in a universal bottle. It were then heated to dissolve. Agarose was transferred to a 56ºC waterbath until needed.

2.4.3. Preparation of the bacterial plugs

The wells of PFGE plug molds were numbered. 3 plugs was prepared for each isolates. Then, 750µl 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 cells

The cells were lysed by adding a mixture of 1ml of proteolysis buffer with 10µl of Proteinase K stock solution (50mg/ml) (see appendix) into a 1.5ml new labelled microcentrifuge tube. The plugs were removed from 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 50ºC for 24 hours for digestion to take place.

2.4.5. Washing of the plugs

After 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 equilibrate the plugs.

2.4.6. Restriction enzyme digestion

After completion of the washing steps, wash buffer was removed in the final wash leaving only agarose gel in the tubes. Then, 300µl 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, 300µl of restriction buffer containing 50U of Xbal enzyme was added into the tubes and was incubated in an incubator for 24 hours at 37ºC specific to the optimal temperature for Xbal enzyme.

2.4.7. Pulse- field gel electrophoresis

2.4.7.1. Electrophoresis gel preparation.

After incubation, restriction enzyme reaction was stopped by addition of 200µl of 50mM EDTA. Plugs were cooled at 4ºC until needed.

Then, a (1%) agarose gel was prepared by heated to dissolved 3g of PFGE grade 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 Run

The 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 14ºC before turning on the pump.

The run time was set for 24 hours at 6 Vcm-1 with 120º angle using switch time of 2.16 sec to 54.17 sec.

2.4.7.3. Gel staining

Once 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 under UV transilluminator and a picture was taken once optimal image obtained.

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