The Lab Chronicles: My Experience in the Microbiology Laboratory – Week 3

During my third week in Microbiology, I again alternated between the primary and secondary benches. One of the main tests I performed throughout the week was the β-lactamase test for Staphylococcus aureus. S. aureus is a common bacteria found in many types of infections. Unfortunately, it is also known to be resistant to a multitude of antibiotics. The β-lactamase test is performed to see if the bacteria contains the enzyme, β-lactamase. This enzyme breaks down the β-lactam ring that certain antibiotics (such as penicillin) contain. This test is performed using a small disk that is placed on a slide, which is wetted with sterile water, and inoculated with the bacteria. The testing for the specimen is performed alongside quality control specimens that are known to be positive and negative. If the control organisms do not give the expected results, the test is invalid. A positive result appears as a pink color on the disk, whereas a negative result displays no color change.

Beta-lactamase test. Positive test (right) showing pink color and negative test (left) showing no color change.

Beta-lactamase test. Positive test (right) showing pink color and negative test (left) showing no color change.

Throughout the week, I also processed many samples for MRSA screening. MRSA stands for Methicillin Resistant Staphylococcus Aureus, which is a common antibiotic resistant bacteria, especially in hospitals. These bacteria contain the mecA gene and have developed resistance towards the beta-lactam group of antibiotics, which include the penicillins, cephalosporins, cephamycins, carbapenems, and monobactams, as well as beta-lactam and beta-lactamase inhibitor combinations. Bacteria can become resistant to antibiotics naturally over time, or through the act of conjugation. Conjugation is the exchange of genetic information between bacteria with the use of a pilus. The pilus acts as a bridge in which genes found in plasmids (circular DNA) are exchanged. At the hospital, MRSA is detected using specific agar plates known as Brilliance MRSA Agar. This type of agar is chromogenic and the MRSA are detected as pink colonies. This color is produced because the chromogen on the agar is paired with a phosphate substrate. MRSA contains the enzyme phosphatase, which breaks apart the chromogen from the substrate, releasing the pink color. Anytime a positive MRSA is identified, the patient’s nurse has to be called immediately. Afterwards, the sample is frozen for future examination.

MRSA agar demonstrating pink colonies

MRSA agar demonstrating pink MRSA colonies

Another antibiotic resistant bacteria that is screened within the hospital is Vancomycin Resistant Enterococci (VRE). This bacteria contains either the vanA, vanB or vanC genes that make it resistant to Vancomycin. Bacteria containing the vanA gene are of the most concern, because their resistance is inducible and transferred between other bacteria through plasmids. The bacteria that contain this gene include Enterococcus faecium and Enterococcus faecalis and they are a large infection control risk. The vanB gene is also inducible, however it displays a lower level of resistance in E. faecium strains. Finally, bacteria containing the vanC gene are not infection control issues as the gene is found in their chromosomes and is not easily transferred between bacteria (as opposed to genes present on plasmids). These bacteria include Enterococcus gallinarum and Enterococcus casseliflavus. Once a VRE is identified, further testing should be performed to determine whether the enterococcus is an infection control risk. VREs are also detected using chromogenic agar known as Brilliance VRE Agar. This type of media produces blue colonies in the presence of VRE. Unfortunately, there is a lot of breakthrough within these plates, where bacteria that are not VRE grow as blue colonies. Therefore, a wet prep needs to be performed in order to determine if the colonies are cocci or bacilli as there have been many instances in which bacilli grow on these VRE plates.

VRE agar demonstrating blue VRE colonies

VRE agar demonstrating blue VRE colonies

Towards the end of the week, my clinical coordinator gave me the topic of my clinical project. I am very excited to say that I will be validating the recently purchased plastic blood culture for transport within the pneumatic tube system (PTS). Previously, blood cultures at the hospital were collected using glass bottles and transported to the lab by Medical Laboratory Assistants (MLA). Blood cultures are clinically significant specimens as they are ordered by the physician whenever bacteremia is suspected. Bacteremia is whenever bacteria are present within the blood, and is a critical infection since blood is normally a sterile site. Therefore, timely delivery to the lab and diagnosis of septicemia is important. A second component to my clinical project will also be to validate the use of smudge plates in the earlier diagnosis of septicemia, as well as any effects made by the PTS. Smudge plates are made using a concentrated amount of bacteria. The bacteria is concentrated with the use of centrifugation. Then, a drop of centrifuged blood is cross-streaked in three directions. This creates an inoculum that can be used to inoculate the Vitek Mass Spectrometer in a few hours so that an identification can be performed and results can be released earlier to the physician.

Smudge plate on Chocolate agar

Smudge plate on Chocolate agar