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

Maryury Brown-Jaque,Joan Jofre,Maria T Martín-Gómez,Silvia Gartner,Juan J González-López,Lirain Rodriguez Oyarzun,Thais Cornejo-Sánchez,Sandra Rovira,Javier De Gracia,Antonio Alvarez,Maite Muniesa

doi:10.3389/fmicb.2018.00856

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 (blaTEM, blaCTX-M-1-group, blaCTX-M-9-group, blaOXA-48, blaVIM, 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 blaVIM and blaTEM, 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.

Highlights

Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population (Panitch and Rubenstein, 2010)
Recovery of Stx phage 933W spiked in the samples ranged from 96.0 to 99.8% in five CF sputum samples, suggesting that the purification protocol efficiently recovered viral particles inoculated in the samples
The limit of quantification (LOQ) is defined as the last valid Ct in the standard curve that is consistent in the diverse replicates and is used to calculate the efficiency of each qPCR assay

Summary

Introduction

Cystic fibrosis (CF) is the most common autosomal recessive disease in the Caucasian population (Panitch and Rubenstein, 2010). The important morbidity and mortality of this disease are related to pulmonary affectation and its complications, which are responsible for up to 85% of the deaths of patients suffering from CF, including children and adults (Panitch and Rubenstein, 2010; Elborn, 2016). Chronic pulmonary infection with Pseudomonas aeruginosa is developed, which remains in up to 80% of adult CF patients and is capable of growing in biofilms in the lungs, thereby greatly complicating antibiotic treatment of the disease. Other opportunistic pathogens have been isolated from CF patients, including Burkholderia cepacia and Haemophilus influenza Additional opportunistic pathogens such as Stenotrophomonas maltophilia, Achromobacter xylosoxidans, and nontuberculous Mycobacterium are being recovered from adult patients with increasing frequency (Bittar et al, 2008; Lipuma, 2010)

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