**4. Discussions**

The chromosomal DNA is the most fundamental component of identity of the cell and therefore represents a preferred method to assess the relatedness of the strains. And the PFGE method is the gold standard for now in assessing this property of the microbial agents

One hundred and ninety-eight bacterial isolates comprising 120 *K. pneumoniae* and 78 *E. coli* isolates from patients with ages ranging from 2 days old to 85 years had higher MIC values from the E-test assays and thus fulfilled the criteria for further molecular characterization. More than 70% of the isolates were recovered from female patients. The isolates involved in urinary tract infections were 60%. Skin and soft tissue isolates and infections contributed 30% of the isolates and the rest of the isolates were either from respiratory tract system (5%), blood streams (4%) or central nervous systems (1%) respectively. Most of these ESBL isolates were recovered from adult patients admitted into the medical (48%) and surgical (35%) facilities of the hospital. The rest were from patients seen in the paediatrics wards

The multiplex PCR assay detected the 100% *bla*TEM genes, 25% *bla* SHV and 52% *bla* CTX-M genes among the *E. coli* isolates. Similarly, 94% *bla*TEM, 42% *bla*SHV and 70% *bla*CTX-M genes in the *K. pneumoniae* isolates were detected. All CTX-M genes were identified as alleles

The PFGE picture of all the isolates used for this study is partly represented in Figure 3 below. As depicted in the figure, the PFGE typing of the ESBL-producing isolates revealed various different and diverse DNA banding profiles among the *E. coli* and *K. pneumoniae*  isolates. Bacteriophage lambda ladder PFGE marker (New England Biolabs) is all depicted on the lanes λ. The *E. coli* isolates are demonstrated on lanes 1 – 6, 7 – 12 while lanes 13 – 18 and 19 – 24 shows the *K. pneumonia* isolates. Note that except for lanes 4 and 7, all the lanes containing the isolates have significantly divergent banding patterns. From these results therefore, one would interpret the data as stating that all the *E. coli* or *K. pneumoniae* isolates

are distinguishable by the PFGE and divergent from each other (>7 band difference).

with only the two isolates the bands were fully separated.

**4. Discussions** 

There was no major clonal similarity or relatedness of either the *K. pneumoniae* or *E. coli* producing ESBL isolates regardless of which hospital facility the patient was admitted to or specimen the bacterial pathogen was recovered from. In addition, one could notice that from the Figure 2, the bands of the DNA were not separated (i.e. no resolution) for isolates in lanes 4 and 7. This phenomenon is called smearing and occurs when there is a contamination of the nucleases (agarose plug, buffer or reagents), or use of abnormal temperature and concentration of the buffers or wrong conditions which may all affect the enzyme. All these were the case with these isolates because when the tests were repeated

The chromosomal DNA is the most fundamental component of identity of the cell and therefore represents a preferred method to assess the relatedness of the strains. And the PFGE method is the gold standard for now in assessing this property of the microbial agents

(9%), Obstetrics and gynaecology ward (5%), and intensive care units (3%).

**3. Results** 

**3.1 Bacterial isolates** 

**3.2 Multiplex PCR gene detection** 

belonging to the phylogenetic group I.

**3.3 Pulsed-field gel electrophoresis** 

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

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

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 and is a method of choice for many epidemiologic evaluations.

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

Usefulness of Pulsed Field Gel Electrophoresis Assay in the Molecular

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be long and this can also be a point against the use of PFGE. But despite the longer turnaround time PFGE method in performing molecular epidemiology of bacterial isolates still remains a gold standard for now.

Using the questionnaire to retrospectively review the laboratory and medical records, it was observed that these isolates did not significantly share the same patient demographics and occurrence periods. Despite being isolated mostly from urine of patients admitted in the medical and surgical facilities of the hospitals sharing significant patient demographics and isolate characteristics yet the ESBL enzymes differed. This clearly indicated that most ESBLproducing isolates were not sporadic but that multiple clones were widespread in the hospitals. The occurrence of these ESBL producing pathogens were definitely not from spread from one patient to another or from one ward to another. It must probably therefore be as a result of antibiotic use pressure stemming from the use and overuse of antibiotics such as third generation cephalosporins in these facilities and hospitals as already has been reported in the country **[Pinto Pereira LM et al, 2004; Akpaka PE et al, 2010].** This therefore calls for a need for continuous and active surveillance measures; and effective infection controls practices, most especially antibiotic stewardship which is nonexistent in these hospitals.

This is the first study to report bla*TEM*, bla*SHV* and bla*CTX-M* in the country that reveals that phylogenetic group 1 is the predominant CTX-M types prevalent in the hospitals. This study clearly indicated that CTX-M, mainly CTX-M-1 for ESBL-producing *E. coli* and *K. pneumoniae* was highly prevalent and probably endemic in Trinidad & Tobago. Most ESBL producers were resistant to oxyiminocephalosporins and other non-beta-lactam agents at high levels and exhibited a high rate of the MDR phenotype. The spread of ESBL-producing bacteria appeared to be polyclonal, and none of the major epidemic strains were identified.
