**2.3 DNA Electrophoresis**

The molecular genotyping method employed to compare the DNA of the ESBL producing isolates was the PFGE. This was carried out as previously described **[Kaufmann ME, 1998]** with some modifications. Briefly, the bacterial isolate suspensions were embedded in agarose plugs. The cells were lysed and the proteins digested. The plugs were washed to remove cellular debris and they were sectioned. Restriction analysis of chromosomal DNA with *Xba*1 (New England BioLabs, Beverly, MA) was carried out, and separation of the DNA was performed using 1% pulsed-field gel agarose (Bio-Rad Laboratories, La Jolla, CA). The pulsed-field gel electrophoresis was performed using a contour-clamped homogeneous electric field apparatus set (CHEF DRIII, Bio-Rad Hercules, CA, USA) as in Figure 1 below.

The different components of the CHEF system are indicated as electrophoresis chamber, chiller, pump, and the programmed power supply in the above figure. Alternating the electric field between spatially distinct pairs of electrodes causes large and small DNA fragments to re-orient and move at different speeds through the pores in an Agarose gel

Fig. 1. Picture of CHEF DRIII, Bio-Rad Hercules, CA, USA used for the Pulsed field gel Electrophoresis for the microbial agent DNA separation technique.

The gels were stained and images captured on the Gel Doc imaging system using Quantity One software version 4.4.1 (Bio-Rad Laboratories, Hercules CA, USA), Figure 2 below. After viewing the banding patterns, the results were compared and analyzed by manual visualization from a computer monitor following previously established criteria **[Tenover FC et al, 1995]** so as to determine potential outbreak patterns or spread of the infections from one patient to another or hospital facility to another.

The established criteria or guidelines proposed by Tenover *et al*. were used for the interpretation of PFGE **[Tenover FC et al, 1995].** With these guidelines, a banding pattern difference of up to three fragments could have occurred due to a single genetic event and thus these isolates are classified as highly related, differences of four to six restriction fragments are likely due to two genetic events, and differences of equal to or greater than

The molecular genotyping method employed to compare the DNA of the ESBL producing isolates was the PFGE. This was carried out as previously described **[Kaufmann ME, 1998]** with some modifications. Briefly, the bacterial isolate suspensions were embedded in agarose plugs. The cells were lysed and the proteins digested. The plugs were washed to remove cellular debris and they were sectioned. Restriction analysis of chromosomal DNA with *Xba*1 (New England BioLabs, Beverly, MA) was carried out, and separation of the DNA was performed using 1% pulsed-field gel agarose (Bio-Rad Laboratories, La Jolla, CA). The pulsed-field gel electrophoresis was performed using a contour-clamped homogeneous electric field apparatus set (CHEF DRIII, Bio-Rad Hercules, CA, USA) as in Figure 1

The different components of the CHEF system are indicated as electrophoresis chamber, chiller, pump, and the programmed power supply in the above figure. Alternating the electric field between spatially distinct pairs of electrodes causes large and small DNA fragments to re-orient and move at different

The gels were stained and images captured on the Gel Doc imaging system using Quantity One software version 4.4.1 (Bio-Rad Laboratories, Hercules CA, USA), Figure 2 below. After viewing the banding patterns, the results were compared and analyzed by manual visualization from a computer monitor following previously established criteria **[Tenover FC et al, 1995]** so as to determine potential outbreak patterns or spread of the infections

The established criteria or guidelines proposed by Tenover *et al*. were used for the interpretation of PFGE **[Tenover FC et al, 1995].** With these guidelines, a banding pattern difference of up to three fragments could have occurred due to a single genetic event and thus these isolates are classified as highly related, differences of four to six restriction fragments are likely due to two genetic events, and differences of equal to or greater than

Fig. 1. Picture of CHEF DRIII, Bio-Rad Hercules, CA, USA used for the Pulsed field gel

Electrophoresis for the microbial agent DNA separation technique.

from one patient to another or hospital facility to another.

**2.3 DNA Electrophoresis** 

speeds through the pores in an Agarose gel

below.

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 isolates have been encountered.

The Gel Doc image system captures picture of the stained gel with the bands and this is transmitted to a computer and monitor for better visualization and analysis

Fig. 2. Picture of Gel Doc (Bio-Rad Hercules, CA, USA) imaging system used to visually analyze images captured after staining the bands formed in the gels.

Usefulness of Pulsed Field Gel Electrophoresis Assay in the Molecular

and 7 hence the DNA particles were not completely separated or resolved.

and is a method of choice for many epidemiologic evaluations.

Fig. 3. PFGE picture of *Escherichia coli* and *Klebsiella pneumoniae* ESBL producers.

Epidemiological Study of Extended Spectrum Beta Lactamase Producers 199

Figure 3 Picture depicting patterns generated by PFGE of *xba*1-digested chromosomal DNA obtained from *bla* TEM, SHV and CTX-M genes produced by *Escherichia coli* and *Klebsiella pneumoniae* isolates. Lane λ, bacteriophage lambda ladder PFGE marker (New England Biolabs), lanes 1 – 6, 7 – 12 *E. coli* isolates and lanes 13 – 18 and 19 – 24, *K. pneumonia* isolates. Smearing phenomenon occurred in lanes 4

including bacterial cells. All state public health laboratories in the USA as well as Centers for Disease Control and Prevention (CDC) perform molecular epidemiology testing using the PFGE. The PFGE assay can adequately be used to type several organisms including the ones involved in nosocomial infections or pathogens associated with food-borne diseases. PFGE is one of the most reproducible and highly discriminatory typing methods that is available

The PFGE typing method used in this study to characterize the ESBL-producing *Klebsiella pneumoniae* and *Escherichia coli* isolates showed various DNA banding profiles. These banding profiles were in no way similar or related to each other indicating their independent origin. This clonal diversity detected among these ESBL-producing isolates suggests that most of the strains have been unable to be maintained or spread in the different wards or facilities of the hospitals from where the bacterial isolates used in this present study were recovered from. This observation may challenge the many conventional thoughts about the nosocomial epidemiology of antibiotic resistance in the hospitals where the isolates were recovered in Trinidad and Tobago. But it is clearly obvious from the PFGE picture that the isolates were in no way closely related as there were different band patterns

produced after restriction by the same enzymes under the same physical conditions.

The smearing phenomenon whereby the DNA particles were not completely separated or resolved that occurred and was observed in lanes 4 and 7 in Figure 2 highlights some of the drawbacks to using the PFGE method in studying molecular studies. Once the experimental or laboratory errors are eliminated, results obtained are perfect. Again, an arguement can be made or put forward that the procedures takes several days to be completed. The PFGE process can take less than 48 hours to complete. More time is expended in recovering the bacterial isolates in pure cultures from the clinical specimen because this is the time required for incubation and identification of the bacterial isolate. Thus the turnaround time tends to
