P. aeruginosa biofilms in models don’t match those in CF: Study
Researchers say new data may help with model design
Biofilms of Pseudomonas aeruginosa visualized directly in sputum (phlegm) samples collected from children with cystic fibrosis (CF) did not match biofilms when bacteria were first isolated from sputum and grown as an in vitro model, a study revealed.
In vitro (in-the-lab) model systems are typically used to study CF-related lung infections and screen for new antibiotics.
The findings may explain the “clear disconnect between the results of in vitro testing of antimicrobials against bacterial pathogens and clinical outcomes in people with cystic fibrosis,” the researchers wrote.
Correlating in vitro and in-patient model systems is “imperative for elucidating mechanisms of CF lung disease and ensuring efficacy of new (and existing) antimicrobials,” they wrote in the study, “Lack of correlation between in vitro and within patient measures of P. aeruginosa biofilms in cystic fibrosis,” which was published in Heliyon.
The hallmark accumulation of thick mucus in the lungs of CF patients increases their vulnerability to infections, such as the opportunistic bacterium Pseudomonas aeruginosa, or P. aeruginosa, a major contributor to CF lung disease.
Resistant infections
P. aeruginosa infections often resist immune responses and antibiotics because they form clusters, or so-called biofilms — layers of microorganisms that stick together on wet surfaces as a protective mechanism.
In the search for antimicrobial agents to treat such infections, decades of research have focused on developing in vitro models of P. aeruginosa biofilm-associated lung infection. With in vitro models, bacteria are first isolated from patient sputum samples, grown to form biofilms, then examined.
However, potential antibiotics developed using in vitro models have not necessarily improved clinical outcomes in CF, in part because mimicking the complexities of the CF airways is challenging.
Few studies have examined the accuracy of these models and compared them to in-patient observations of P. aeruginosa biofilm structure.
But a new method, Microbial Identification after Passive CLARITY Technique (MiPACT), can directly visualize communities of bacteria within sputum samples.
Using MiPACT, a team led by scientists at The Hospital for Sick Children in Toronto compared an in vitro P. aeruginosa biofilm model to biofilms in sputum from CF children at the time of initial infection. Directly assessing samples from patients is known as ex vivo.
First, sputum was collected from 11 CF children who had a new-onset P. aeruginosa infection before they started inhaled antibiotic treatment.
Half of the sputum samples were then subjected to MiPACT. P. aeruginosa was directly visualized using fluorescent in situ hybridization (FISH), which can detect and visualize specific bacterial DNA sequences. Psl, a key component of biofilms, was also visualized using antibody staining.
For the in vitro model, P. aeruginosa was first isolated from the remaining sputum. Then, bacteria were grown as 48-hour biofilms in standard media with and without 5% sputum supernatant, the liquid portion of sputum that remains after the solid components have been removed. Adding sputum supernatant to the growth media creates a more realistic environment for studying P. aeruginosa.
Replication is challenging
To compare the two methods, the scientists generated images of the experiments to measure the P. aeruginosa biovolume, the volume occupied by bacterial cells within the biofilm. The extent of bacterial aggregation within biofilms was measured by calculating the ratio of the biofilm surface to its biovolume.
The analysis found that the biovolume of P. aeruginosa in biofilms in sputum samples from CF children did not correlate with biovolumes from the in vitro model when bacteria were grown in media without sputum supernatant.
With sputum supernatant, there was a trend toward statistical significance in biovolume measures between the two methods, “suggesting that mimicking CF lung growth conditions in vitro results in a better correlation,” the researchers wrote.
There was no correlation between the two methods in measures of P. aeruginosa aggregation, with or without the addition of sputum supernatant.
Psl antibody binding (without sputum supernatant) significantly correlated with a strong linear relationship between the ex vivo and in vitro models. Still, the correlation was no longer significant when normalized to the biovolume.
“We highlight the challenges of replicating aspects of in-patient [Pseudomonas aeruginosa] biofilm communities in vitro,” the scientists concluded. These data can help with in vitro model design, which can “ultimately further our understanding of CF airway infections and response to antimicrobial treatment,” they wrote.
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