Detection of Bacteriophage Particles Containing Antibiotic Resistance Genes in the Sputum of Cystic Fibrosis Patients

Abstract

Cystic fibrosis (CF) is a chronic disease in which the bacterial colonization of the lung is linked to an excessive inflammatory response that leads to respiratory failure. The microbiology of CF is complex. Staphylococcus aureus is the first bacterium to colonize the lungs in 30% of pediatric CF patients, and 80% of adult patients develop a chronic Pseudomonas aeruginosa infection, but other microorganisms can also be found. The use of antibiotics is essential to treat the disease, but antibiotic performance is compromised by resistance mechanisms. Among various mechanisms of transfer of antibiotic resistance genes (ARGs), the recently been reported bacteriophages are the least explored in clinical settings. To determine the role of phages in CF as mobile genetic elements (MGEs) carrying ARGs, we evaluated their presence in 71 CF patients. 71 sputum samples taken from these patients were screened for eight ARGs (bla_TEM, bla_CTX-M-1-group, bla_CTX-M-9-group, bla_OXA-48, bla_VIM, mecA, qnrA, and qnrS) in the bacteriophage DNA fraction. The phages found were also purified and observed by electron microscopy. 32.4% of CF patients harbored ARGs in phage DNA. β-lactamase genes, particularly bla_VIM and bla_TEM, were the most prevalent and abundant, whereas mecA, qnrA, and qnrS were very rare. Siphoviridae phage particles capable of infecting P. aeruginosa and Klebsiella pneumoniae were detected in CF sputum. Phage particles harboring ARGs were found to be abundant in the lungs of both CF patients and healthy individuals and could contribute to the colonization of multiresistant strains.

Keywords: antibiotic resistance genes; bacteriophages; cystic fibrosis; horizontal gene transfer; sputum.

FIGURE 1

Percentage of positive samples for each antibiotic resistance gene (ARG) in phage DNA of the sputum samples considering the limit of detection (LOD) or the limit of quantification (LOQ) of the qPCR assays.

FIGURE 2

Percentage of positive samples for each ARG in phage DNA of the sputum samples of individuals aged below 18 years and above 18 years old. Lower chart shows the distribution of the number of ARGs in phage DNA detected in each individual in relation to age.

FIGURE 3

Quantification of ARGs in the phage fraction of sputum samples from cystic fibrosis patients. Values in brackets indicate the number of positive samples that were used for the calculation. In the box plot, the cross-pieces of each box represent (from Top to Bottom) the maximum, upper-quartile, median (black bar), lower-quartile and minimum values. The black diamond shows the mean value. The upper gray boxes in the box plot include samples showing values within the 75th percentile and lower white box samples show values within the 25th percentile.

FIGURE 4

Analysis of infectious phage particles isolated from pooled sputum samples of CF patients. (A) Infectivity of the phages from sputum samples onto Escherichia coli (Ec), Staphylococcus aureus (Sa), P. aeruginosa (Pa), and K. pneumoniae (Kp) hosts. Gray bands corresponding to density 1.45 g/ml in the tubes of CsCl density gradients prepared with the phages isolated from the spot test onto each strain (C) qPCR results of the ARGs present in the phage particles purified from each CsCl density band in (B). (D) Electron micrographs of phage particles purified from the CsCl bands obtained with Pa and Kp samples. Bar 100 nm.