| Case A | Case A results |
Background: Approximately one out of 20 hospitalized patients acquires an infection during their hospital stay; such infections are known as nosocomial infections. Oftentimes, the microorganisms that cause such infections are transmitted accidentally by personnel working at the health care facility who have not followed Safety Universal Precautions such as changing their gloves often and washing their hands carefully. The most common nosocomial pathogens include Klebsiella pneumoniae, Pseudomonas aeruginosa, Enterococcus spp, Clostridium difficile, and Staphylococcus aureus.
Antibiotic-resistant microbes may develop due to genetic mutations or genetic transformations. In the latter case, an antibiotic-sensitive bacterium acquires DNA containing genes that confer antibiotic resistance either from another bacterium (horizontal transfer) or as a result of transfection from a bacterial-infecting virus (a bacteriophage or phage). Spontaneous genetic mutations may also happen in antibiotic-sensitive bacteria rendering them resistant to certain drugs that normally prevent them from growing. The drug-resistant bacteria will then be able to grow and replicate even in the presence of the antibiotic, giving rise to the spread of antibiotic-resistant strains.
The selective drug pressure induced by antibiotic abuse or misuse usually causes the genetic mutations associated with antibiotic resistance. Examples of such misuse include: using antibiotics without medical prescription; not finishing the full doses of a prescribed antibiotic; or not complying with the instructions given by the physician or pharmacist.
Staphylococcus aureus is one of the most problematic nosocomial pathogens because certain strains of the bacterium are resistant to multiple antibiotics. One such strain is Methicillin-resistant Staphylococcus aureus (MRSA), which is resistant not only to Methicillin but also to penicillin and many other penicillin derivatives known as Beta lactams S. aureus is commonly found on the human skin in areas of the body such as the underarms, the groin, and the perianal area, but the bacterium tends to concentrate in higher amounts in the nostrils. It can cause infections of the respiratory tract and bones and joints, endocarditis, septicemia, and toxic shock syndrome. However, the most common infection caused by MRSA is the necrotizing infection of soft tissues that may lead to amputations.
There are also antibiotic-resistant strains of bacteria that can cause infections outside of hospitals and healthcare facilities. These community-associated (CA) resistant strains can be transmitted easily from one person to another and are often present in crowded places such as schools, childcare centers, eldercare nursing facilities, jails, and gyms. A person carrying CA-MRSA may not develop an infection right away, but any trauma of the skin such as a cut or bruise could allow the bacteria to penetrate the skin and establish an infection. A bacterial-culture medium named Chromagar can be used to discriminate between MRSA and antibiotic-sensitive strains of S. aureus. However, the only way to differentiate between hospital-acquired (HA) and community-associated (CA) MRSA is by molecular diagnostic procedures that rely on DNA analysis. CA-MRSA strains often harbor a prophage encoding the pvl toxin gene that is not found in HA-MRSA (see diagram comparing HA and CA MRSA).
Case A. Nelson was a very talented boy who grew up in the mountains of Utuado, Puerto Rico. At twelve years of age, Nelson sang at the Fiestas Patronales where he shared the stage with the famous Puerto Rican singer Ednita Nazario, who had just returned to the island after her Broadway debut in Paul Simon’s The Cave Man. Ednita was ecstatic with Nelson’s talent and said to him “keep up the good work, because you are Broadway material”. Her words still resounded in Nelson’s mind when at age 22, he received a call confirming an audition to enter the American Music and Dramatic Academy (AMDA) in New York City.
Just two months from graduating with a double major in Dramatic Arts and Music from the University of Puerto Rico, he was confident that the AMDA was going to be his way to finally make it to Broadway. There was only one detail that he thought could become an obstacle to his triumph, and that was his nose. Nelson had inherited the distinctive big nose of the Castillo family and he was terrified when he learned that the nose and ears are the two parts of the human body that continue to grow as we get older. It was then that he decided to undergo cosmetic surgery to reduce and re-shape his nose before going to New York.
“What’s wrong with our nose?” his sister Daliana asked. “It’s just like Barbra Streisand’s and it certainly was never an obstacle to her success in showbiz.” But Nelson had made up his mind. He gathered all of his savings and convinced his sister to put down her signature for a $15,000 loan. With that amount of money, he would be able to schedule an abdominal-sculpting surgery as well. The abs-sculpting surgery went perfectly well and three weeks later, Nelson was ready for the nose job.
A week after the second surgery, Nelson’s nose and face were quite swollen, bruised, and aching, and his body temperature was 99.5°F. “There must be something wrong,” said Daliana, who was taking care of her brother during his convalescence. “You were not this sick a week after the abs surgery.” Nelson reminded Daliana that the surgeon warned him about the swelling and pain because some bone had been shaved to fix his nose. The surgeon also mentioned that fever was one of the potential side effects of beta-lactam antibiotics and he was on amoxicillin to prevent Staph infections. “I know you very well, Nelson,” Daliana said. “You are willing to risk your health in order to get what you want. I’m taking you to the emergency room even if I have to carry you.” Reluctantly, Nelson agreed to go.
The physician at the ER confirmed Daliana’s fears. The swelling and fever where not just normal post-surgical side effects, and the bruises where actually necrotic lesions. Nelson was suffering an infection, probably a Staph infection. As they waited for the lab results, Nelson did not understand how he could develop an infection since he never missed a single dose of his antibiotic and he had been very careful in complying with the surgeon’s post-surgical instructions. Daliana, on the other hand, was looking into the possibility of filing a lawsuit against the hospital and the surgeon on the basis that her brother acquired a nosocomial infection during the surgery.
DNA has been isolated from the following bacteria samples:
- culture of bacteria from Nelson’s lesions
- an antibiotic-sensitive S. aureus
- a hospital-acquired methicillin-resistant S. aureus
- a community-acquired methicillin-resistant S. aureus
PCR primers are available for the following (individually or all together in one primers file):
- glp, a housekeeping gene found in all strains of S. aureus (fragment size = 843 bp)
- pvl, a toxin gene from a prophage common in community-acquired MRSA in this region (fragment size = 433 bp)
- mec, part of the methicillin-resistance cassette found in most samples of MRSA (fragment size = 393 bp)
Procedure: Use these primers to run PCR and gel electrophoresis on the DNA samples, to see if the corresponding sequences are detected. What do the results tell you about Nelson’s infection?
Results of PCR and gel electrophoresis analysis for Case A
PCR and gel electrophoresis analysis for additional controls
Bioinformatics: Compare the glp PCR products using the sequence alignment/tree building feature. Do the tree results support your initial conclusions about the source of Nelson’s infection?
Results of bioinformatics analysis
- Is Nelson suffering a Staphylococcal infection? If so, with what strain of S. aureus is Nelson infected?
- Does Daliana have grounds to file a lawsuit against the hospital? According to the DNA analysis, do you think that Nelson developed a nosocomial infection or could he have picked up the bacteria before going to the hospital?
- What treatment options are there for Nelson?
- Explain the difference between HA-MRSA and CA-MRSA at the molecular level.
- How does antibiotic resistance happen in bacteria?
- How could Nelson get a Staph infection while taking antibiotics as prescribed by his doctor?
- How is MRSA diagnosed? Compare and contrast the culture-based methods vs. the molecular methods of diagnosis in terms of turn-around time and specificity.
- What do you think of Nelson’s decision to undergo cosmetic surgery?
- Is the risk of developing an infection greater for cosmetic surgery than for other kinds of surgeries?
- What are Safety Universal Precautions? How could antibiotic-resistant bacteria be prevented?
a
a
a
a
a
a
a
a
a
a