**2. Materials & methods**

194 Gel Electrophoresis – Advanced Techniques

treatment regimen was not effective, and alternative therapy may be required **[Singh A et** 

Gel electrophoresis and in particular Pulsed-Field-Gel-Electrophoresis (PFGE) is a tool that has made genotyping of bacterial isolates possible. The PFGE is a laboratory technique used for separation of large deoxyribonucleic acid (DNA) molecules if electric current that periodically changes direction is applied to it. The PFGE is the "gold standard "technique used in this discipline of molecular epidemiological studies and it is basically the comparison of large genomic DNA fragments after digestion with a restriction enzyme that cuts infrequently. Since the bacterial chromosome is typically a circular molecule, the digestion by the enzyme yields several linear large DNA molecules. Moving these large DNA molecules posed a problem but Schwartz and Cantor in 1984 introduced a voltage gradient that gave better resolution and the ability to move large molecules **[Schwartz DC** 

Conventional agarose gel electrophoresis can only be used for the separation of DNA fragments that ranges between 20 – 25 base pair (kbp) by using specialized apparatus no matter how long it is run. The distance between DNA bands of a given length is determined by the percent agarose in the gel. The disadvantage of higher concentrations is the long run times (sometimes days). PFGE uses a special type of agarose that has a larger matrix pore sizes even at a higher percentages such as 1%. The most commonly utilized PFGE methods approaches include the contour-clamped homogenous electric field (CHEF) and field inversion gel electrophoresis (FIGE) **[Carle, G. F et al, 1986; Finney, M. 1993].** Field inversion gel electrophoresis utilizes a conventional electrophoresis chamber in which the orientation of the electric field is periodically inverted by 180o and has an upper limit of resolution about 200kbp. CHEF uses a more complex electrophoresis chamber with multiple electrodes to achieve highly efficient electric field conditions for separation; typically the electrophoresis apparatus reorients the DNA molecules by switching the electric fields at

Interpreting DNA fragment patterns generated by PFGE and relating or associating them into epidemiologically useful information for typing nosocomial pathogens, the clinical microbiologist or researcher must understand how to compare PFGE patterns and how random genetic events can alter these patterns. Ideally, the PFGE isolates representing an outbreak strain will be indistinguishable from each other and distinctly different from those of epidemiologically unrelated strains. If this occurs, the outbreak is relatively easy to identify. A random genetic activity such as mutation in a DNA can occur and when this happens, it will change the restriction fragment profile obtained during the course of the outbreak **[Hall LMC, 1994; Quintiliani R., Jr., & P. Courvalin, 1996; Thal LA et al, 1997]** These random variations in the fingerprints will depend on the organism and the time

The aim of this study is to demonstrate the usefulness of PFGE techniques as a tool to be used in identifying outbreaks of bacterial infection and hence can be used as a tool for infection control measures in a hospital. Also to determine its importance in delineating the clonal relatedness or diversity of bacterial strains isolated from several regional hospitals in Trinidad and Tobago. The PFGE has been shown to be useful in the determination of the sources, clonal relatedness and spread of bacterial isolates in hospitals and countries where

**al, 2006].**

**& Cantor CR, 1984]**

period of the outbreak

120o angles. CHEF can separate even up to 2-3 Mbp.

the isolates have been recovered or encountered.

More than 230 strains of *Klebsiella pneumoniae* and *Escherichia coli* obtained routinely from three major regional hospitals in Trinidad and Tobago were used for this study. These non consecutive bacterial isolates were identified using standard microbiological methods as had been previously reported **[Akpaka PE & Swanston WH; 2008].** The initial screening for ESBL production by these pathogens using MIC values at concentrations and breakpoints recommended by the CLSI for ESBL screening **[CLSI 2010]** were performed with the automated micro dilution MicroScan WalkAway-96 System (Siemens, USA). Structured standardized questionnaire was used to extract epidemiological information from hospital records of the patients yielding these isolates. Such information included bio data, gender, hospital facilities where the patients were attended to, clinical signs and symptoms, diagnosis, other forms of investigations and treatments, treatment failures and complications.

### **2.1 Confirmation of ESBL phenotypes**

In accordance with the protocols from the manufacturer to phenotypically determine the ESBL production by bacterial isolates, the E-test strips (AB Biodisk, Solna Sweden), a very sensitive and convenient assay to use was employed to confirm ESBL production in the isolates. The control strain for all the phenotypic testing were *E. coli* ATCC 25922 (negative control) and *K. pneumoniae* ATCC 700603 (ESBL positive).

#### **2.2 Multiplex PCR amplification**

The detection of gene sequences coding for the TEM, SHV, and CTX-M enzymes were carried out using multiplex PCR as previously described with some modifications **[Monstein HJ et al, 2007].** The cycling conditions used in the PCR assays were as previously described **[Paterson DL et al, 2003; Boyd DA et al, 2004].** The oligonucleotide primer sets specific for the SHV, TEM and CTX-M genes and the cycling conditions used in the PCR assays were as described previously and are depicted in the Table 1 below.


Table 1. Showing primers used for amplifications of the genes in ESBL producers

A Multiplex PCR method previously described [**Woodford et al, 2006]** for detection of *bla*CTX-M alleles was used to identify the CTX-M phylogenetic group of positive isolates. All PCR reactions were carried out using 2µl bacterial cell suspension (density of 70%T in Vitek Colorimeter) as the DNA template. Respective genes were detected by the size separation PCR amplicons by agarose gel electrophoresis.

Usefulness of Pulsed Field Gel Electrophoresis Assay in the Molecular

isolates have been encountered.

Epidemiological Study of Extended Spectrum Beta Lactamase Producers 197

seven restriction fragments are due to three or more genetic events. Isolates that differ by three fragments in PFGE analysis may represent epidemiologically related subtypes of the same strain. Conversely, isolates differing in the positions of more than three restriction fragments may represent a more tenuous epidemiologic relation. Some studies using PFGE and other typing methods indicate that single genetic events, such as those that may alter or create a new restriction endonuclease site or DNA insertions/deletions associated with plasmids, bacteriophages, or insertion sequences, can occur unpredictably even within the time span of a well-defined outbreak (1 to 3 months) **[Arbeit RD et al 1990; Sader HS et al 1993; Tenover FC et al 1995].** With the detection of two genetic variation events by differences in fragment patterns compared to the outbreak strain, the determination of relatedness to an outbreak falls into a gray zone. The results may indicate that these isolates are related (especially if isolates were collected over a long period of time, such as 3 to 6 months), but there is also a possibility that strains are unrelated and not part of the outbreak, hence demonstrating the usefulness of PFGE techniques as a tool in infection control measures in a hospital. PFGE results should always be considered in conjunction with the epidemiologic information and data. The bacterial isolates may also show some degree of clonal relatedness or diversity, thus helping in the determination of the sources, clonal relatedness and spread of the bacterial isolates in hospitals and countries where the

The Gel Doc image system captures picture of the stained gel with the bands and this is transmitted to a

Fig. 2. Picture of Gel Doc (Bio-Rad Hercules, CA, USA) imaging system used to visually

computer and monitor for better visualization and analysis

analyze images captured after staining the bands formed in the gels.
