Negative Bacteria Using API 20e, Research Paper Example

Abstract

There is a variation of different types of bacteria found in every habitat and can be associate with various diseases and illnesses.  In order to find out what bacteria is present and the course of action in regard to diagnosis and plan of care; there have been tests known as “API” developed in order to get an accurate and fast reading. API Rapid 20NE test was used in order to identify the type of Enterobacteriaceae. The resulting numbers were tabluted on the point system and it was indicated as a final seven digit code “5215773”.  The code was referenced to the API profile index as “Klebsiella pneumoniae”.  The results of this study were successful in that an Enteriobacteria was determined using the API profile index.

Introduction 

Antibiotics are a type of antimicrobial agent that is developed from microorganisms, such as bacteria and fungi. Antibiotics were developed in order to fight bacteria and are used in clinical medicine, as well as in agriculture.  There are many different types of bacteria and not every antibiotic can fight against every bacteria.  Antibiotics are characterized according to their molecular make-up, type of microbial action, and spectrum of antimicrobial action.  In fact, according to Madian et al., (2012), there are more than 10,000 metric tons of different types of antimicrobial drugs developed and used per year.   There is a variation of different types of bacteria found in every habitat and can be associate with various diseases and illnesses.  In order to find out what bacteria is present and the course of action in regard to diagnosis and plan of care; there have been tests known as “API” developed in order to get an accurate and fast reading.

The API-20E test strip is a type of API test that was developed in order to identify enteric gram negative rods.  The test contains 20 test compartments, in which some of the compartments will change color with pH differences when samples of bacterial suspension are added and other compartments will produce end products that need to be identified using different reagents.  After results are recorded, a code is determined from the amount of + and – tests results.  The code is compared to a codebook, which determines the bacterial species.    (Reynolds, 2009)  Researchers have investigated the effectiveness of the API 20E kit in order to determine how effective the use of these tests are in identifying different strains of Enterobacteriaceae.  Results from a study conducted by Bartoli et al. (1972) found that the API test was able to correctly identify 671 strains of Enterobacteriaceae, including Klebsiella, Enterobacter Protues, and Providencia; however, the tests also included conventional methods.

The purpose of the current paper was to investigate the use of API tests in identifying gram negative rods.  In microbiology, the first step to classifying and characterizing bacteria is usually the gram-stain; however, these tests can directly identify gram negative rods to species level.  The goal, therefore, of this lab is determine the gram negative rods to species level.

Materials and Methods

API Rapid 20NE test was used in order to identify the type of Enterobacteriaceae.  An oxidase test was performed on the unknown bacteria in order to determine that it was an oxidase-negative bacterium. Mueller-Hinton agar plates were given with an unknown Enterobacteriaceae.  A microorganism suspension was created using the bacteria from the agar plates in order to test using the API Rapid 20NE test strips.  An API tray was labeled with students name and 5 ml of tap water was added to the bottom of the tray.  The saline suspension containing the organisms was dispensed into cupules in 20 compartments of the tray.  The API strip was then taken and held at a slight angle from the table.  The bacteria from the agar plates were inoculated into each well using a sterile pipette.  The end of the pipette was moved in order to allow for capillary action of the fluid, as well as to remove any bubbles.  Each of the wells was filled to the neck of the strip.  In addition, the API tray was evaluated in order to make sure the suspension was homogenous and without clumps of floating bacteria.  A McFarland barium sulfate standard #4 was used to quantitate the suspension.    The strips were incubated for 24 hours and the results were analyzed.  Spontaneous reaction for the GLU, ADH, URE, ESC, GEL and PNPG were recorded.  Other results were determined through a color reactions and the use of reagents, such as Kovac’s reagent.  Results were recorded using a point system, such as 1, 2, or 4 points for positive reactions for the first, second and third tube in the triad and zero points for the negative test.  (Figure 1)  The type of bacteria present was identified using a seven-digit code, or codebook provided by the manufacturer.

Results

The results of the experiment are illustrated in Table 1.  The Table shows which wells were positive and negative. As seen in the Oxidase column, the result was positive indicating that the test did contain an Enteriobacteria, and therefore the API test could be performed.  Table 2 illustrates examples of wells in which are noted as “positive” or “negative” for specific bacteria.  The resulting numbers were tabluted on the point system and it was indicated as a final seven digit code “5215773”.  The code was referenced to the API profile index as “Klebsiella pneumoniae”.

Discussion

The API 20E test resulted in the name of the unknown bacteria to be, “Klebsiella pneumoniae”.  The results of this study were successful in that an Enteriobacteria was determined using the API profile index.  The fact that the profile number that was derived from the results of the incubated strains, indicates that there was a correct identification and that the taxon correlated with the identity of the strain.  Klebsiella is a Gram-negative bacterium that is associated with different illnesses, such as pneumonia, meningitis, bloodstream infections or surgical infections.  The Klebsiella bacteria are found in the human intestinal tract and feces as normal fauna; however, infections of Klebsiella are often found in patients undergoing insertion of catheters, use of ventilators and patients undergoing long term antibiotic treatments.  In order to treat Klebsiella infections, microbiology tests need to be run in order to determine which antibiotics are most effective.  Therefore, it is important that tests, such as the API 20E test are conducted in order to correctly identify the bacteria and provide the best course of care.

The API test did result in the identification of a species from the API profiling index; however, there is a possibility of error due to the identifying of the different results in the wells.  For instance, determining positive and negative results could have been incorrectly reported.  In order to determine if the results were correct, a larger sample size using two additional tests should be performed to verify results.

Table 1. Results recorded after 24 hr.  Positive and negative reactions; points, and seven digit code. 

Table 2.  Examples of characteristic results for Bacteria using API test.  http://www.biotech.univ.gda.pl/odl/biochem/api.html

Figure 1.  Example of API test results. 

References

Analytical Profile Index.  Retrieved from: http://www.biotech.univ.gda.pl/odl/biochem/api.html

Bartoli, M., Chouteau, J., Ettori, D., Bonnet, D., and Payen, G. (1972). Utilisation en practique journaliere d’une nouvelle galerie d’identification des enterobacteries. A propos de 671 souches. Lyon Pharmaceutique, 23, 269-275.

Madigan, M.T., Martinko, J.M., Stahl, D.A., Clark, D.P.  (2012).  Madian et al., 2012 Biologyof

Microorganisms.  Benjamin Cummings Publsihing, San Francisco CA.  1004pp.

Reynolds, J.  (2009).  API 20E. Richland College.  Retrived from: http://delrio.dcccd.edu/jreynolds/microbiology/2421/lab_manual/API.pdf

Analytical Profile Index.  Retrieved from: http://www.biotech.univ.gda.pl/odl/biochem/api.html