Flu season begins in August and continues well into the new year. During this extended period of exposure, influenza spreads easily. It is a particularly risky time for patients with cystic fibrosis (CF). The Cystic Fibrosis Foundation strongly recommends that CF patients be vaccinated against influenza each year and also that labs that run IVD tests of CF-patient isolates use agar-based media to measure the antibiotic susceptibility of some of the most common and dangerous infections that plague CF patients.
The cause of cystic fibrosis is a mutation to the cystic fibrosis transmembrane conductance regulator (CFTR) gene. The specific mutation disrupts ion transport in the epithelial cells lining the passageways of the lungs, digestive system, pancreas, and many other organs. In the lungs, this leads to the buildup of thick mucus that fosters infections.
This recurring buildup of sticky mucus in the lungs gives rise to dangerous lung infections, the most dangerous of which is Pseudomonas aeruginosa. This bacterium is particularly dangerous to CF patients because it releases toxins that damage the lungs and is capable of adapting a mucoid phenotype, which makes it highly resistant not only to antibiotics but to innate immunological defenses as well.
Amikacin MIC of mucoid Pseudomonas aeruginosa isolate
The CF Foundation recommends that all CF patients be tested for respiratory infections every three months, even very young patients who have yet to develop serious symptoms. Young CF patients and infants who seem perfectly healthy often are, in fact, colonized with Staphylococcus aureus and Haemophilus influenza, among other dangerous pathogens. These microbes, dangerous in their own right, also can cause epithelial cell damage within the lung, which can increase attachment points for more microbes and eventually create ideal conditions for P. aeruginosa.
Agar-based methods
There are 110 labs in the United States accredited by the CF Foundation to test CF isolates. The Foundation guidelines recommend that labs use agar-based methods such as disk-diffusion or agar gradient diffusion when testing respiratory samples from CF patients because agar-based media can best replicate the growing conditions within the CF lung and therefore provide the most accurate diagnosis of infection and the best possible antibiotic susceptibility data.
The primary advantage of agar-based systems is the growth modality of CF isolates on solid medium versus in broth microwells. Growth on a solid medium provides an environment more similar to the CF lung as compared to broth-based systems. For example, growth on agar is better suited to approximate the growth of P. aeruginosa as a biofilm. Because of reduced diffusion of antibiotics and the reduced rate of metabolism in a biofilm, isolates growing as a biofilm are more resistant to antibiotics than planktonically growing P. aeruginosa.
To measure the susceptibility of a P. aeruginosa isolate taken from a CF specimen, the growth of the microbe as a biofilm is ideal, because the biofilm morphology will give the most accurate susceptibility data.
The importance of looking specifically at the full range of morphologies in CF patients should not be underestimated. Mucoid P. aeruginosa variants are typically resistant. So if a patient has three non-mucoid and one mucoid variant, the patient probably has some early resistance. If this ratio flips as the patient ages—one non-mucoid and three mucoid variants—this can help a clinician see that patient’s condition is worsening.
The atypical variations of microbial strains seen in the CF lung are a function of the long co-existence of the microbe with the patient. Frequent exposure to antibiotics produces mutations that can reduce the growth rate of the bacteria within the patient’s lungs. We know that sulfamethoxazole/trimethoprim therapy can give rise to small colony variants (SCVs) of S. aureus in the CF patient. P. aeruginosa that grows as a biofilm within the CF lung tends to be resistant because this variant is “selected” over time by recurring antibiotic therapy.1,2
Automated AST systems
All of this is critical to a microbiology lab that is certified to run CF samples because the wrong culture methodology can create a misleading false negative or underestimate the degree of resistance. For example, automated Antibiotic Susceptibility Testing (AST) systems are based on interval testing to access growth rate. If this rate is under a certain threshold, an automated AST system could conclude that the microbe is susceptible when, in fact, it could be a highly resistant, slow-growing variant being measured in an automated system designed for P. aeruginosa strains with standard morphology.
An automated system that looks at growth rate intervals is going to have a blind spot when it comes to looking for AST data in CF patients. These systems tend to underestimate resistance, which is simply not acceptable in CF cases.
Of the CF Foundation-recommended agar-based AST methods, a case can be made for the superiority of agar gradient diffusion over standard disk diffusion. Unlike standard disk-diffusion systems, the agar gradient diffusion test provides a minimum inhibitory concentration (MIC) of the antimicrobial being tested. In a standard disk diffusion system, the lab must make an interpretation based on the diameter of the zone of inhibition around the antibiotic disk. The diameter of this zone indicates if an organism is “susceptible,” “intermediate,” or “resistant” (SIR) to the antibiotic. However, this is far from precise. Measuring the zones can be difficult and can introduce human error when the difference between S, I, and R can be a fraction of an inch.
The MIC on an agar gradient diffusion system is easily read at the point where the zone of inhibition intersects the test strip. Even with slow growing or scantly growing isolates, one can make a determination of zone edge intersection with the gradient test strip.
MIC information is vital to physicians treating CF patients. CF patients often receive inhalation therapy, whereas the categorical susceptibility or resistance reported refers to serum-achievable antibiotic concentrations. MIC information gives pulmonologists and pharmacists more flexibility in choosing antibiotics and designing effective therapies.
Frequent testing and susceptibility measurement is vital to the care of the cystic fibrosis patient, particularly as flu season begins. Managing CF patients effectively requires a constant stream of information between the microbiology lab and the treating physicians, as well as cutting-edge microbiology lab tests, including the agar gradient diffusion test for measuring susceptibility and providing MIC.
References
- Looney WJ. Small-colony variants of Staphylococcus aureus. Br J Biomed Sci. 2000;57(4):317-322.
- Kahl B, Herrmann M, Everding AS, et al. Persistent infection with small colony variant strains of Staphylococcus aureus in patients with cystic fibrosis. J Infect Dis. 1998;177:1023-1029.
Greg Porter, PhD, is Clinical Manager, Etest, for bioMerieux, Inc. Dr. Porter earned his doctorate in Biochemistry at the University of Illinois at Urbana-Champaign and has worked with companies in the laboratory automation, health care, and forensic DNA identity fields.
