Case Presentation: Bacterial Infection in a Cystic Fibrosis Patient
by Dr. Pradeep Singh

Attachment A:  Case study, questions and discussion materials

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Part I:  Presentation

MZ is a 34-year-old male with cystic fibrosis.

His medical history begins at age 15 months of age when his mother took him to be evaluated by a pediatrician with complaints of cough, wheezing, and shortness of breath. He had been sick on and off in his first months, catching what she thought were common colds. Unlike the illnesses his siblings sometimes experienced, MZ's colds would often progress to prolonged respiratory illnesses that he "can't shake like his brother and sister can."

There is no one who smoked in the house, and the family history was negative for asthma, allergies, or other respiratory problems. In close questioning his mother did report that one of her siblings had died at a very early age of "weak lungs".

The physical examination was unremarkable except for low body weight (15^th percentile for age) and wheezing in the lungs.

Given this history, the pediatrician was concerned that the patient had cystic fibrosis (CF). Diagnostic tests were performed which revealed normal lung function tests. The patient's sweat was collected to determine the concentration of chloride present and an elevated level of 128 was found (normal = 0 - 60). DNA from the patient's blood was analyzed and mutations were found in both copies of the gene responsible for cystic fibrosis.

Part I:  Questions

What do you suppose the family meant when they described the mother's sibling as having died of "weak lungs"? What sorts of clinical conditions might be described by lay people as "weak lungs"?

What symptoms might have led the physician to suspect cystic fibrosis as a possible cause of this child's frequent infections?

Mothers often contribute to the diagnosis of CF by reporting to doctors that their babies taste salty (as experienced while giving hugs and kisses). Why is this a useful clinical sign?

What is the inheritance of cystic fibrosis?

How common is this condition (you might check http://ncbi.nlm.nih.gov/Omim/)?

Part I:  Discussion

Cystic fibrosis is the most common inherited disease of Caucasians. It is caused by defects in the gene known as the cystic fibrosis transmembrane conductance regulator (CFTR) located on chromosome 7. The CFTR protein plays a key role in the regulation and transport of chloride and sodium ions in and out of epithelial cells. Epithelial cells line many mucus membranes in the body including those of the respiratory tract. They act as a barrier to the external environment, help regulate the composition of secretions, and play an important role in the defense against infectious agents.

Patients with CF have abnormal secretions in many organs including the sweat glands, pancreas, liver, intestines, and testes. The main clinical problem in CF, however, is progressive lung disease. At birth, the lungs of CF patients are histologically normal. Early in life, however, lung infections develop and over time damage the lungs. Interestingly, it is only the surface of the conducting airways (the passages that carry air to the air sacs or alveoli) that become infected. CF patients do not develop infections at other sites in the lungs, or elsewhere in the body.

Part II:   Presentation

Over the next three or four years MZ was sick with bacterial infections of the airways. He would frequently present with cough, increased production of sputum, and wheezing. Bacterial culture of his expectorated sputum revealed the presence of multiple organisms including Haemophilus influenzae, and Staphylococcus aureus. These infections were treated with oral antibiotics and when particularly severe, MZ was admitted to the hospital for intravenous antibiotics. Most of the time these treatments would result in reduced concentrations of bacteria in the sputum.

Part II:  Questions

What is the relationship between the genetic defect in CF and the microbiological complications associated with the disease? Why are CF patients predisposed to pulmonary infections?

The child experienced recurrent infections of Haemophilus influenzae and Staphylococcus aureus. Where could these pathogens have come from?

How do clinicians determine which antibiotic to use and the concentration of antibiotic to be administered for bacterial infections? What is a culture and sensitivity? What is an MIC?

Part II:  Discussion

One of the most vexing questions in medicine today is how a defect in an epithelial cell chloride channel results in airway infections in CF. The nature of the genetic defect and the function of the CFTR protein have been known for many years. Despite this molecular understanding, there is as yet no definitive explanation as to how these abnormalities result in the clinical disease. Many theories abound.

One leading hypothesis (known as the "high salt hypothesis") postulates that the defective CFTR chloride channel causes abnormalities in the thin layer of fluid that lines human airways known as airway surface liquid. Cells and glands lining the airway secrete natural antibiotic factors into this fluid layer. Research has shown that there are multiple factors in this fluid (at last count approximately 10) that have a wide spectrum of activity and that some factors have synergistic activity in combination. Together, these antimicrobial factors create a chemical shield that provides a first line of defense against multitudes of bacteria to which we are commonly exposed. Interestingly, the antimicrobial activity of most all factors is inhibited by high salt concentrations. According to the "high salt" hypothesis, the defective chloride channel in CF results in high salt concentrations in the airway surface liquid. This inactivates the antimicrobial activity, and makes the airways susceptible to infection.

Part III:   Presentation

At age 6, MZ again presented to the clinic with increasing respiratory symptoms. This time the opportunistic bacteria Pseudomonas aeruginosa was isolated from his sputum. He was admitted to the hospital and treated with 3 weeks of intravenous antibiotics, which resulted in a resolution of the symptoms. Follow up sputum cultures revealed that the P. aeruginosa had been eradicated. This pattern repeated 2 more times in the next 14 months — both times P. aeruginosa infections developed and were cleared with antibiotic therapy.

At age 8 he again became infected with P. aeruginosa. Laboratory tests revealed that the infecting strain was sensitive to all commonly used anti-Pseudomonal antibiotics. This time, however, two 3-week courses of intravenous therapy with multiple antibiotics failed to eradicate the infection. Follow up cultures 3, 6 and 12 months later revealed continued presence of P. aeurginosa in the sputum.

Part III:  Questions

Something changed during this phase of the patient's disease. Describe in your own words the change in the clinical course of the infection.

Propose hypotheses to account for the odd observation that at age 8, the P. aeruginosa strain causing the boy's infection was sensitive to all commonly used anti-Pseudomonal antibiotics, but that extensive antibiotic therapy failed to eradicate the infection.

Part III:  Discussion

The airway infections of cystic fibrosis characteristically occur in two clinically distinct stages. The first stage consists of intermittent infections. During this phase (as occurred in our patient MZ) patients will have periodic respiratory infections usually caused by S. aureus, H. influenzae, and also, later P. aeruginosa. These infections are similar to bronchitis (although more severe than a non-CF patient might experience). Patients will have cough, increased sputum production, and shortness of breath. These symptoms and often the bacterial infection can be eradicated with a course of antibiotics. In between infections, lung secretions are sterile, and the patient is often without respiratory symptoms. Studies using DNA fingerprinting techniques (similar to those used in forensics) have revealed that these intermittent infections are caused by different strains of bacteria. This suggests that each infection is completely eliminated and that subsequent infections represent the acquisition of a new strain from the environment.

In the second stage of CF airway infections the opportunistic P. aeruginosa establishes permanent colonization in the lungs. Once this occurs, P. aeruginosa is present in the patient's airway for life. Unlike in the intermittent phase (where serial infections are often caused by different strains of bacteria), in permanent colonization DNA fingerprinting studies have shown that the same strain can persist for years or even decades. Most commonly, the persistent strain remains sensitive to antibiotics until very late in the course of the disease. Why the permanent infections cannot be eradicated is of great interest to researchers.

Part IV:  Presentation

Since the onset of the permanent colonization MZ's respiratory symptoms have worsened. He is now bothered by daily cough and produces up to one cup of sputum per day. His lung function studies begin to show decline and he is admitted to the hospital 3 or 4 times per year for intravenous antibiotics. Cultures always show P. aeruginosa.

During one admission, MZ volunteers for a clinical research study to investigate the reasons antibiotics cannot eliminate established CF infections. At the start of this study the bacteria in his sputum are isolated and tested in the laboratory against tobramycin, one of the most potent antibiotics available against this organism. In these tests, the bacteria are grown in increasing concentrations of tobramycin to determine the level of drug which kills the bacteria. These studies revealed that tobramycin at a concentration of 2 µg/ml completely killed the bacteria. This concentration is known as the minimum inhibitory concentration, or MIC.

In the next stage of this study, MZ is placed on intravenous tobramycin. Over the next 3 weeks his sputum is periodically collected and the concentration of tobramycin in the sputum is measured (Figure 1). These results are plotted on the left Y-axis in the accompanying graph. The tobramycin concentration steadily increased throughout the treatment period and at the end of 3 weeks the concentration reached almost 100 µg/ml (50 times the concentration required to kill these bacteria in a test tube). Simultaneously the researchers measured the number of bacteria present in the sputum. As expected, at the beginning of the study MZ had a high concentration of P. aeruginosa in the sputum (approximately 10⁸ bacteria/ml). This decreased during the treatment period, however, after three weeks treatment 1 x 10⁴ bacteria were still present. This despite the fact that the sputum contained tobramycin at concentrations that greatly exceeded the laboratory MIC of the bacteria for the majority of the treatment period.

Figure 1. Density of P. aeruginosa (▲) and tobramycin concentrations in sputum of 10 patients with cystic fibrosis during hospitalization. Tobramycin was quantitated by radioenzymatic (●) assay. (Reprinted with permission, American Review of Respiratory Disease; 1985; 132:763 Figure 1 modified for this exercise.)

Part IV:  Questions

Why do concentrations of tobramycin that eliminate P. aeruginosa from the sputum (50 times MIC) fail to control the infection?

What aspects of this patients disease are probably responsible for his declining lung function?

At the end of the experiment described, there were still 1 x 10⁴ bacteria in this patient's sputum although the sensitivity testing suggests that these bacteria are sensitive to the concentrations of tobramycin being used. Where are they coming from?

Part IV:  Discussion

Why are very high concentrations of antibiotics not killing bacteria in MZ's sputum? One hypothesis for the remarkable persistence of this bacterium in the CF lung is that they grow as a biofilm. In biofilms bacteria live in structured communities rather than as free-swimming individual cells. Biofilms are a protective mode of growth that allows bacteria to survive in hostile environments and represents a very different bacterial lifestyle. The formation of biofilms is a multi-step process. First, bacterial cells attach to a surface, and then clusters known as microcolonies form. Finally, the bacteria form well-organized structures shaped like towers or mushrooms separated by well-defined spaces known as water channels (Figure 2). One of the most intriguing properties of biofilm bacteria is that formation of these structures requires cell to cell communication between the bacteria. In bacteria genetically engineered to be missing one of these signals, normal biofilm formation was inhibited (Figure 3).

Figure 2. Illustration of the structure of a biofilm including water channels. (© Center for Biofilm Engineering, reprinted with permission.)


Figure 3. Epifluorescence and scanning confocal photomicrographs of the wild type and the mutant P. aeruginosa biofilms which lack cell to cell communication capability. Reprinted (abstracted/excerpted) with permission from Science 280:295-298. © 1998 AAAS¹

The hypothesis that bacteria form biofilms in a CF lung has been difficult to test rigorously because there are no specific markers that identify biofilm growth. Electron microscopy of CF sputum reveals that in CF sputum P. aeruginosa are clustered together, encased in a densely stained matrix (Figure 4). This morphology is quite consistent with biofilm growth, however microscopic appearance alone is not sufficient to prove this.

Figure 4. Transmission electron microscope images of P. aeruginosa
in sputum. Low magnification. Scale bars, 1µm.
© Nature, reprinted with permission. Nature 407:762

A recent study adds new, physiologic evidence that bacterial biofilms form in the lungs of CF patients. As mentioned above, P. aeruginosa makes two cell-to-cell communication signals that control the expression of dozens of genes. These two signal compounds are called 3OC12-HSL (N-(3oxododecanoyl)-L-homoserine lactone) and C4-HSL (N-buytryl-L-homoserine lactone. Researchers recently developed a radiometric technique that allows measurement of the relative synthesis rates of these two signals in biological samples. Using this technique it was found that some P. aeruginosa strains reversed the ratio of the two signals when growing in biofilms as compared to the free-swimming state. In these strains, 3OC12-HSL predominates in the free swimming or planktonic state and C4-HSL predominates in the biofilm. The presence of this measurable change in signal ratios raised the possibility that the signal ratios could be used as an indicator of biofilm growth. To test this hypothesis, researchers collected sputum from CF patients colonized with P. aeruginosa. In the sputum P. aeruginosa made the signal ratio characteristic of biofilm bacteria. The ratio of signals reversed when the strains were grown in the free-swimming state (Figure 5).

Figure 5. Quantities of communication signals made byP. aeruginosa in broth cultures and CF sputum. Examples of P. aeruginosa (top) and sputum (bottom). The size of the peak represents the amount of signal synthesized. ©Nature, modified figure reprinted with permission. Nature 2000; 407:763

This finding and previous studies provide strong evidence that P. aeruginosa form biofilms in CF lungs. The presence of biofilms may explain the failure of current antibiotic therapies to eradicate these infections. While traditional antibiotics work very well against a rapidly dividing, free-swimming bacteria, they have minimal activity against bacteria living in biofilms. Development of new antimicrobial agents specifically targeted against biofilm growth may provide hope for patients with cystic fibrosis and other chronic bacterial infections thought to be formed by biofilms.

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¹ AAAS material may be used for non-commercial, classroom purposes only. Any other uses require the prior written permission of the copyright holder.

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