CHAPTER III

METHODOLOGY

Research Design
The proponents will use a posttest only design, wherein bla OXA23 and blaNDM genes will be
detected from imipenem and meropenem resistant Pseudomonas aeruginosa and Acinetobacter
baumanii isolates.

Respondents of the Study

The population of interest are patients from selected tertiary hospitals in Metro Manila
that will test positive for the infection of Pseudomonas aeruginosa and/or Acinetobacter
baumanii. Isolates must be both resistant to imipenem and meropenem. No specific collection
site will be indicated. A minimum of 30 strains that will be identified over a period of 5 months
will be collected from the selected tertiary hospitals. Patients name will remain de-identified
except for some demographic date such as: age, gender, ward, the source of specimens, and the
diagnosis if available. A simple random sampling technique will be followed.

Research Instruments Used

Polymerase Chain Reaction will be used for the amplification of the genes of the clinical
isolates stated above.

Data Collection Procedures and Management

Antibiotic Susceptibility Test
The Kirby Bauer method will be utilized to confirm susceptibility of isolates to imipenem
and meropenem.
Etest
Etest will be applied to test for the minimum inhibitory concentration of imipenem and
meropenem to each bacterial isolate. Mueller-Hinton agar will be used, and results will be
analyzed using the Clinical and Laboratory Standards Institute guidelines.
Polymerase Chain Reaction
Molecular testing will be done through the application of the Polymerase Chain Reaction.
This is to amplify the DNA of the bacterial isolates and target the genes bla OXA23 and blaNDM that
may be present in collected strains of imipenem and meropenem resistant Pseudomonas
aeruginosa and Acinetobacter baumanii.

Data Analysis Plan

Antibiotic resistance profile will be grouped into two namely producing and nonproducing pertaining to blaOXA23 and blaNDM specifically. All resistance and susceptibility result
will be recorded. Statistical treatment will be done to all antibiotics used to treat Pseudomonas
aeruginosa and Acinetobacter baumanii, and p-value will be solved for to see the significant
effect and impact of the genes present.