Association of Diverse Staphylococcus aureus Populations with Pseudomonas aeruginosa Coinfection and Inflammation in Cystic Fibrosis Airway Infection.

Staphylococcus aureus is one of the most common pathogens isolated from the airways of cystic fibrosis (CF) patients and often persists for extended periods. There is limited knowledge about the diversity of S. aureus in CF. We hypothesized that increased diversity of S. aureus would impact CF lung disease. Therefore, we conducted a 1-year observational prospective study with 14 patients with long-term S. aureus infection. From every sputum, 40 S. aureus isolates were chosen and characterized in terms of phenotypic appearance (size, hemolysis, mucoidy, and pigmentation), important virulence traits such as nuclease activity, biofilm formation, and molecular typing by spa sequence typing. Data about coinfection with Pseudomonas aeruginosa and clinical parameters such as lung function, exacerbation, and inflammatory markers in blood (C-reactive protein [CRP], interleukin 6 [IL-6], and S100A8/9 [calprotectin]) were collected. From 58 visits of 14 patients, 2,319 S. aureus isolates were distinguished into 32 phenotypes (PTs) and 50 spa types. The Simpson diversity index (SDI) was used to calculate the phenotypic and genotypic diversity, revealing a high diversity of PTs ranging from 0.19 to 0.87 among patients, while the diversity of spa types of isolates was less pronounced. The SDI of PTs was positively associated with P. aeruginosa coinfection and inflammatory parameters, with IL-6 being the most sensitive parameter. Also, coinfection with P. aeruginosa was associated with mucoid S. aureus and S. aureus with high nuclease activity. Our analyses showed that in CF patients with long-term S. aureus airway infection, a highly diverse and dynamic S. aureus population was present and associated with P. aeruginosa coinfection and inflammation. IMPORTANCE Staphylococcus aureus can persist for extended periods in the airways of people with cystic fibrosis (CF) in spite of antibiotic therapy and high numbers of neutrophils, which fail to eradicate this pathogen. Therefore, S. aureus needs to adapt to this hostile niche. There is only limited knowledge about the diversity of S. aureus in respiratory specimens. We conducted a 1-year prospective study with 14 patients with long-term S. aureus infection and investigated 40 S. aureus isolates from every sputum in terms of phenotypic appearance, nuclease activity, biofilm formation, and molecular typing. Data about coinfection with Pseudomonas aeruginosa and clinical parameters such as lung function, exacerbation, and inflammatory markers in blood were collected. Thirty-two phenotypes (PTs) and 50 spa types were distinguished. Our analyses revealed that in CF patients with long-term S. aureus airway infection, a highly diverse and dynamic S. aureus population was associated with P. aeruginosa coinfection and inflammation.

High Nuclease Activity of Long Persisting Staphylococcus aureus Isolates Within the Airways of Cystic Fibrosis Patients Protects Against NET-Mediated Killing.

Staphylococcus aureus is one of the first and most prevalent pathogens cultured from the airways of cystic fibrosis (CF) patients, which can persist there for extended periods. Airway infections in CF patients are characterized by a strong inflammatory response of highly recruited neutrophils. One killing mechanism of neutrophils is the formation of neutrophil extracellular traps (NETs), which capture and eradicate bacteria by extracellular fibers of neutrophil chromatin decorated with antimicrobial granule proteins. S. aureus secretes nuclease, which can degrade NETs. We hypothesized, that S. aureus adapts to the airways of CF patients during persistent infection by escaping from NET-mediated killing via an increase of nuclease activity. Sputum samples of CF patients with chronic S. aureus infection were visualized by confocal microscopy after immuno-fluorescence staining for NET-specific markers, S. aureus bacteria and overall DNA structures. Nuclease activity was analyzed in sequential isogenic long persisting S. aureus isolates, as confirmed by whole genome sequencing, from an individual CF patient using a FRET-based nuclease activity assay. Additionally, some of these isolates were selected and analyzed by qRT-PCR to determine the expression of nuc1 and regulators of interest. NET-killing assays were performed with clinical S. aureus isolates to evaluate killing and bacterial survival depending on nuclease activity. To confirm the role of nuclease during NET-mediated killing, a clinical isolate with low nuclease activity was transformed with a nuclease expression vector (pCM28nuc). Furthermore, two sputa from an individual CF patient were subjected to RNA-sequence analysis to evaluate the activity of nuclease in vivo. In sputa, S. aureus was associated to extracellular DNA structures. Nuclease activity in clinical S. aureus isolates increased in a time-and phenotype-dependent manner. In the clinical isolates, the expression of nuc1 was inversely correlated to the activity of agr and was independent of saeS. NET-mediated killing was significantly higher in S. aureus isolates with low compared to isolates with high nuclease activity. Importantly, transformation of the clinical isolate with low nuclease activity with pCM28nuc conferred protection against NET-mediated killing confirming the beneficial role of nuclease for protection against NETs. Also, nuclease expression in in vivo sputa was high, which underlines the important role of nuclease within the highly inflamed CF airways. In conclusion, our data show that S. aureus adapts to the neutrophil-rich environment of CF airways with increasing nuclease expression most likely to avoid NET-killing during long-term persistence.

The prevalence of Staphylococcus aureus with mucoid phenotype in the airways of patients with cystic fibrosis-A prospective study.

BACKGROUND: Staphylococcus aureus is one of the most frequently isolated pathogens in the respiratory tract of CF patients. Recently, we characterized peculiar mucoid S. aureus isolates, which are excessive biofilm formers and which carried a 5bp-deletion within the intergenic region of the ica operon. In this prospective study, we determined the prevalence of mucoid S. aureus-isolates in the airways of CF-patients during a 3-months period. METHODS: We analyzed specimens (sputa, throat swabs) from 81 CF patients who attended two CF centers in Münster, Germany. Ten S. aureus isolates were randomly picked from every S. aureus-positive airway specimen and evaluated for mucoidy using Congo Red agar and phenotypic tests. Mucoid isolates were characterized by spa sequence typing, biofilm production and sequencing of the intergenic region of the ica operon to screen for the 5bp-deletion. RESULTS: In 7 of 81 examined patients (8.6%), we detected mucoid S. aureus phenotypes (37 out of 1050 isolates; 3.5%). Twenty-five mucoid isolates carried the 5bp-deletion. Mucoid isolates produced excessive biofilm and were significantly more resistant to certain antibiotics. CONCLUSIONS: In our prospective study, mucoid S. aureus was present in 8.6% of S. aureus-positive CF-patients. In 6 of 7 patients, mucoid isolates carried the 5bp-deletion, indicating that also other so far not identified mechanisms cause excessive biofilm formation. Further studies are necessary to ascertain the clinical impact of mucoid S. aureus phenotypes on the severity of the CF disease.

Staphylococcus aureus in the airways of cystic fibrosis patients - A retrospective long-term study.

BACKGROUND: Cystic fibrosis (CF) is an autosomal recessive disease associated with chronic airway infections by Staphylococcus aureus as one of the earliest and most prevalent pathogens. We conducted a retrospective study to determine the S. aureus infection status of CF patients treated since 1994 at two certified CF-centres in Münster, Germany, to get insights into the dynamics of S. aureus airway infection and the clinical impact on lung function on a long-term perspective. MATERIALS AND METHODS: We used data from our microbiological database collected between 1994 and 2016 for patients treated at two centres in Münster, Germany, respectively, to determine the infection status for S. aureus. Furthermore, the resistance to selected antibiotics was determined for all patients' isolates and for 15 patients on a longitudinal basis. In addition, the prevalence of adaptive phenotypes such as small colony variants (SCVs) and mucoid S. aureus was assessed. RESULTS: For this study, 2867 patient years with respiratory specimens (mean of 9.3 years for every patient, range 1-22 years) were evaluated for 283 CF patients (median age of 7 years at the beginning of the observation period, range 0-57 years, 51% male). 18% of patients were rarely infected by S. aureus (≤24% of observation years), 20% of patients intermittently (25-49%) and 61% persistently (≥50% of observation period). Susceptibility testing for 12969 S. aureus isolates resulted in resistance to methicillin in 9%, trimethoprim/sulfamethoxazole in 10%, levofloxacin in 14%, gentamicin in 20%, erythromycin and/or clindamycin in 30% and penicillin in 80% of all isolates. S. aureus isolates of 15 patients revealed dynamics of resistance with increase, decrease and loss of resistant isolates to the analysed antibiotics during the study period. SCVs were isolated at least once from 42% (n = 118) of patients and mucoid isolates from 2% (n = 7) of patients. In the last study year, 89 patients were infected by S. aureus only, 44 patients by S. aureus and Pseudomonas aeruginosa and 18 by P. aeruginosa only. Patients infected by S. aureus only were younger and had better lung function compared to the other two groups. CONCLUSIONS: We determined a high percentage of patients with persistent S. aureus infection. During persistence, mostly fluctuation of resistance against various antibiotics was observed in the isolates indicating acquisition and loss of resistance genes by S. aureus. The prevalence of adaptive phenotypes during long-term persistence was high for SCVs (42% of patients), but low for mucoid isolates (2% of patients), which might be underestimated for mucoid phenotypes due to the retrospective study design and the difficulty to detect mucoid isolates in primary cultures. While patients with S. aureus only had better lung function and were younger, no difference was found between the group of P. aeruginosa and S. aureus co-infection and P. aeruginosa only with previous S. aureus infection.