Genotypic and Phenotypic Characterization of Staphylococcus aureus Isolates from the Respiratory Tract in Mechanically-Ventilated Patients

Abstract

: Staphylococcus aureus is a commensal and frequent colonizer of the upper respiratory tract. When mechanical ventilation disrupts natural defenses, S. aureus is frequently isolated from the lower airways, but distinguishing between colonization and infection is difficult. The objectives of this study were (1) to investigate the bacterial genome sequence in consecutive isolates in order to identify changes related to the pathological adaptation to the lower respiratory tract and (2) to explore the relationship between specific phenotypic and genotypic features with the patient’s study group, persistence of the clinical isolate and clinical outcome. A set of 94 clinical isolates were selected and corresponded to 34 patients that were classified as having pneumonia (10), tracheobronchitis (11) and bronchial colonization (13). Clinical strains were phenotypically characterized by conventional identification and susceptibility testing methods. Isolates underwent whole genome sequencing using Illumina HiSeq4000. Genotypic characterization was performed with an in-house pipeline (BacterialTyper). Genomic variation arising within-host was determined by comparing mapped sequences and de novo assemblies. Virulence factors important in staphylococcal colonization and infection were characterized using previously established functional assays. (1) Toxin production was assessed using a THP-1 cytotoxicity assay, which reports on the gross cytotoxicity of individual isolates. In addition, we investigated the expression of the major virulence factor, alpha-toxin (Hla) by Western blot. (2) Adhesion to the important extracellular matrix molecule, fibronectin, was determined using a standardized microtitre plate assay. Finally, invasion experiments using THP-1 and A539 cell lines and selected clinical strains were also performed. Repeated isolation of S. aureus from endotracheal aspirate usually reflects persistence of the same strain. Within-host variation is detectable in this setting, but it shows no evidence of pathological adaptation related to virulence, resistance or niche adaptations. Cytotoxicity was variable among isolates with 14 strains showing no cytotoxicity, with these latter presenting an unaltered Fn binding capacity. No changes on cytotoxicity were reported when comparing study groups. Fn binding capacity was reported for almost all strains, with the exception of two strains that presented the lowest values. Strains isolated from patients with pneumonia presented a lower capacity of adhesion in comparison to those isolated during tracheobronchitis (p = 0.002). Hla was detected in 71 strains (75.5%), with most of the producer strains in pneumonia and bronchial colonization group (p = 0.06). In our cohort, Hla expression (presence or absence) in sequential isolates was usually preserved (70%) although in seven cases the expression varied over time. No relationship was found between low cytotoxicity and intracellular persistence in invasion experiments. In our study population, persistent S aureus isolation from airways in ventilated patients does not reflect pathological adaptation. There is an important diversity of sequence types. Cytotoxicity is variable among strains, but no association with study groups was found, whereas isolates from patients with pneumonia had lower adhesion capability. Favorable clinical outcome correlated with increased bacterial adhesion in vitro. Most of the strains isolated from the lower airways were Hla producers and no correlation with an adverse outcome was reported. The identification of microbial factors that contribute to virulence is relevant to optimize patient management during lower respiratory tract infections.

Strain-specific interspecies interactions between co-isolated pairs of Staphylococcus aureus and Pseudomonas aeruginosa from patients with tracheobronchitis or bronchial colonization

“…†BC: bronchial colonization; TB: tracheobronchitis. Respiratory study groups according to the Clinical Pulmonary Infection Score (CPIS) as previously described 61 .…”

“…S. aureus and P. aeruginosa strains included were isolated simultaneously in the same respiratory sample (4 endotracheal aspirates and one sputum sample) (co-isolated) from five patients admitted to the ICU from Hospital Universitari Germans Trias i Pujol, a tertiary care hospital in Badalona (Spain) (Table 1 ). The patients were classified into respiratory study groups according to the Clinical Pulmonary Infection Score (CPIS) and as previously described 61 . At inclusion, all patients except one were under mechanical ventilation.…”

A Study on the Epidemiological-Molecular Role of Staphylococcus aureus Strains in the Development of Ventilator-Associated Pneumonia in a Tertiary Hospital in Brazil

“…Despite reports of pneumonia in community-dwelling patients, Pasquale et al [ 29 ] recently described the involvement of USA300 in nosocomial pneumonia. In these cases, participation of this strain is of great clinical importance since, in addition to the difficulty in selecting antimicrobials for treatment, the strain can carry a wide range of virulence factors that can also influence the outcome [ 3 , 9 ].…”

“…Among the virulence factors that play an important role in pneumonia, alpha-hemolysin or alpha-toxin (Hla) is one of the most prominent cytotoxins that target erythrocytes, epithelial and endothelial cells, T cells, neutrophils, monocytes, and macrophages. Alpha-toxin is an amphipathic, water-soluble, pore-forming molecule that binds primarily to lipids present on the host cell membrane [ 9 , 10 ]. The main function of alpha-toxin is the causing osmotic swelling, rupture, lysis, and subsequently, cell death.…”

Persistent Methicillin-Resistant Staphylococcus aureus Bacteremia: Host, Pathogen, and Treatment

“…Meanwhile, S. aureus must resist or avoid phagocytosis by neutrophils and the resulting oxidative and non-oxidative burst, in addition to the circulating platelet-derived antimicrobial peptides. There is significant heterogeneity in the catalog of virulence factors produced by different S. aureus clinical isolates [ 57 , 58 , 59 , 60 ], the regulators mediating virulence factor expression [ 61 , 62 , 63 , 64 ], and susceptibility to antimicrobial peptides [ 65 , 66 , 67 , 68 ]. This section discusses the key genetic and phenotypic characteristics of S. aureus that have been associated with persistent SAB.…”