**Usefulness of Pulsed Field Gel Electrophoresis Assay in the Molecular Epidemiological Study of Extended Spectrum Beta Lactamase Producers**

Patrick Eberechi Akpaka1 and Padman Jayaratne2

*1Department of Para-Clinical Sciences, The University of the West Indies, St. Augustine 2Department of Pathology & Molecular Medicine McMaster University, Hamilton, Ontario 1Trinidad & Tobago 2Canada* 

#### **1. Introduction**

192 Gel Electrophoresis – Advanced Techniques

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> A major problem in several health institutions, countries and regions is to categorically define or delineate the source or index case of any microbial organism/s during an outbreak of an infection. Understanding bacterial distribution and their relatedness is essential for determining the epidemiology of nosocomial infections and aiding in the design of rational pathogen control methods. The role of bacterial typing is to determine if epidemiologically identical or related isolates are also genetically related **[Singh A et al, 2006].** Based on phenotypic and genotypic typing methods, multi-drug resistant bacteria organisms such as extended spectrum beta lactamase (ESBL) enzyme producing pathogens e.g. *Escherichia coli* and *Klebsiella pneumoniae* can be traced to have been transferred from one hospital to another, from one country or region to another. Such information and knowledge have greatly assisted clinicians and health care policy makers to determine the best approach of stopping or eliminating such spreads and transfers of the pathogenic organisms involved in the infection.

> As noted in the reviews by Singh A et al, the use of molecular methods for typing of nosocomial pathogen has assisted in efforts to obtain a more fundamental assessment of strain interrelationship **[Singh A et al, 2006].** Establishing clonality of pathogens can aid in the identication of the source (environmental or personnel) of organisms, distinguish infectious from non infectious strains, and distinguish relapse from reinfection. Many of the species that are key hospital-acquired causes of infection are also common commensal organisms, and therefore it is important to be able to determine whether the isolate recovered from the patient is a pathogenic strain that caused the infection or a commensal contaminant that likely is not the source of the infection. Likewise, it is important to know whether a second infection in a patient is due to reinfection by a strain distinct from that causing the initial infection or whether the infection is likely a relapse of the original infection. If the infection is due to relapse, this may be an indication that the initial

Usefulness of Pulsed Field Gel Electrophoresis Assay in the Molecular

**2. Materials & methods** 

complications.

below.

**2.1 Confirmation of ESBL phenotypes** 

**2.2 Multiplex PCR amplification** 

control) and *K. pneumoniae* ATCC 700603 (ESBL positive).

5′-TGC TTT GTT ATT CGG GCC AA-3′

5′-ACG CTC ACC GGC TCC AGA TTT AT-3′

separation PCR amplicons by agarose gel electrophoresis.

5′- TGG GTR AAR TAR GTS ACC AGA AYCAGC GG-3′

Epidemiological Study of Extended Spectrum Beta Lactamase Producers 195

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

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

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

Gene Primer bp Sizes Reference *bla*SHV 5′-ATG CGT TAT ATT CGC CTG TG-3′ 747-bp Paterson DL *et al*

*bla*TEM 5′-TCG CCG CAT ACA CTA TTC TCA GAA TGA-3′ 445-bp Boyd DA *et al*

*bla*CTX-M 5′-ATG TGC AGY ACC AGT AAR GTK ATG GC-3′ 593-bp Boyd DA *et al*

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

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

treatment regimen was not effective, and alternative therapy may be required **[Singh A et al, 2006].**

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 & Cantor CR, 1984]**

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

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 period of the outbreak

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 the isolates have been recovered or encountered.
