Introduction
Enterobacter aerogenes is a pathogenic bacterium that causes various infections as an opportunistic pathogen. However, it is sensitive to most of the antibiotics specific to the bacterium although the sensitivity is easily lost due to its unique mechanism for resisting antibiotics particularly through the production of lactamase enzyme. The bacterium lives in soil, some chemicals, and various waste materials. In the human body, it has been found to live in the gastrointestinal tract where it does not necessarily cause diseases, especially in healthy individuals. Enterobacter aerogenes has commercial significance in the production of hydrogen during fermentation as it is one of the lactose fermenting bacteria.
Objective/hypothesis
Enterobacter aerogenes is a bacterium with the shape of a rod belonging to the wide classification of Gram-negative bacteria which have characteristic thin cell walls. The bacterium has close characteristics to Escherichia coli and Klebsiella both of which are gram-negative. Another significant characteristic of enterobacter aerogenes, as well as E. coli and Klebsiella, is the ability to grow in selective medium, particularly MacConkey agar which identifies them as lactose fermenting bacteria. One of the significant differences between these bacteria is the temperature range within which each of them grows.
Enterobacter aerogenes grows best at a temperature range of 34-40°C while E. coli grows best at a higher temperature of 44° C. similarly, enterobacter is quite similar to Klebsiella pneumonia although the two are greatly distinguished by the urease test as the enterobacter bacterium is negative for the urease test while Klebsiella gives positive results for the same.
Experimental design
Microbial culture
The first step in the identification of Enterobacter aerogenes is microbial culture using both blood agar and McConkey agar. Using a sterile loop, the inoculum in question is inoculated on both agar plates and four streak sections are made flaming the loop before each streak. The agar plates are then incubated overnight at temperatures of 37° C. Colonies are then observed and identification of lactose fermenting colonies is made. Once this is done, gram staining is conducted to classify the bacterium based on its cell wall properties.
Gram staining
Using a sterilized wire loop, the bacterial colony is picked and used to make a thin smear on a glass slide. The slide is dried by air and flame is used to fix the smear onto the slide. Crystal violet is then flooded onto the slide containing the colony smear for a few seconds and decanted. This is followed by flooding of iodine for the same period and decanted as well. Either alcohol or acetone is then flooded and washed immediately to facilitate rapid decolorization.
Finally, safranin or carbon fuchsin is added and then decanted using water. The slide is then dried using blot paper and the slide is flooded with immersion oil for observation. Due to close similarities between Enterobacter aerogenes and E. coli as well as other gram-negative lactose fermenting bacteria, it is important to conduct an indole test to distinguish enterobacter from the rest as this is the most effective test for the above-mentioned bacteria.
The indole test
This test begins with the growth of the bacterial colony in tryptophan for about a day or two. After incubation, Kovac’s reagent is added to the culture broth. For non-lactose fermenters and anaerobic bacteria, ethyl alcohol is used as a variation reagent for this test. Depending on the color change in this test, enterobacter aerogenes can now be correctly identified.
Results and discussion
The microbial culture is done on both blood agar and MacConkey ager to identify lactose fermenting bacteria. Blood agar is differentially made to distinguish normal microbes from pathogenic ones based on the sheep’s blood contained in the agar. The reaction between the sheep’s blood and the bacterium’s hemolytic enzymes results to color changes hence providing information for identity.
MacConkey on the other hand is selective for the growth of gram-negative bacteria staining them as lactose fermenters. The lactose available in the medium is utilized by these bacteria producing an acid that lowers the pH of the medium resulting in the appearance of red or pink colonies. Non-lactose fermenters are unable to utilize lactose but rather they use peptone which forms ammonia raising the pH of the medium expressed by colorless colonies. This is an indication that the bacterium in question is lactose fermenting but since many of them fall under this category including E. coli and Klebsiella, a gram stain test is carried out to further distinguish the bacterium from other lactose fermenting gram-positive bacteria.
Gram stain is used to differentiate bacteria based on the physical and chemical properties present in their cell walls. Gram-positive bacteria have characteristic thick walls consisting of peptidoglycan which stains purple for the gram stain test. Gram-negative bacteria on the other hand have cell walls with thinner layers which are identified by a pink stain for the gram stain test. A pink coloration from the gram stain test confirms that the bacterium in question is gram-negative and with the previous lactose positive result, the bacterium could be identified as either Escherichia coli or enterobacter aerogenes. As a result, an indole test is conducted to establish if the bacterium is enterobacter aerogenes.
The indole test is based on the ability of a bacterium species to deaminate tryptophan hence splitting indole from the amino acid tryptophan. This reaction is completed using intracellular enzymes resulting in the production of ammonia and pyruvic acid alongside the split of indole. A positive result gives a red or a red-purple color while a negative result gives a yellow coloration.
Conclusion
With the rising resistance of bacteria to antibiotics, proper identification of specific bacteria species is very important. However, this may pose some difficulties as many bacterial species are similar to each other in various chemical and physical properties. For a bacterial species like Enterobacter aerogenes which already has mechanisms for developing antibiotic resistance, a misidentification of the bacterium would result in further resistance which may pose health risks and undermine the body’s immune system. As a result, sensitivity tests should always be carried out not only on Enterobacter aerogenes but on all pathogenic bacterial species to identify the most effective and develop new antibiotics for specific bacteria.
A basic sensitivity test is done by inoculating bacterial culture in peptone water for 6 hours under incubation after which the inoculum is flooded on a dried agar plate. A sensitivity disk containing various antibiotics is placed on the agar plate containing the inoculum and incubated overnight. The antibiotics which are sensitive to that particular species show a clear region around them indicating that they have destroyed the bacterium.