Fig 1.
Morphology of non-mucoid and mucoid S. aureus on Columbia blood agar and Congo red agar (CRA).
S. aureus strains were diluted in PBS and streaked on Columbia blood agar to observe single colonies. The macroscopic morphology of the non-mucoid and mucoid S. aureus isolates revealed significant differences in colony phenotypes. Whereas it was possible to isolate single colonies for the non-mucoid phenotype, the mucoid colonies stuck together, and it was almost impossible to recover bacteria from the agar. (See the movie in the supplemental data). On CRA, non-mucoid S. aureus displays smooth colonies, whereas mucoid isolates exhibit dry wrinkled colonies.
Table 1.
Prevalence and persistence of mucoid S. aureus isolates in respiratory specimens of CF patients.
Fig 2.
Biofilm formation of non-mucoid and mucoid phenotypes.
A static biofilm assay was performed after overnight culture of the non-mucoid strain 8.1 and the isogenic mucoid S. aureus strain 8.2 (Table 2) of patient 8 in BHI containing 0.25% glucose, incubation for 24 h at 37°C and staining with 1% crystal violet. Biofilms were solubilized in ethanol-acetone and the absorbance was determined at OD655nm with a microtiter plate reader. Each experiment was performed at least three times, and in each experiment the samples were prepared in eight replicate wells. To determine the nature of biofilm, detachment of the biofilm was measured after treatment of the wells, which have been cultured as described above, with sodium-metaperiodate, DNase or proteinase K before measuring the absorbance. All experiments were repeated three times. Results are shown as means and standard deviation. **** p-value ≤ 0.0001.
Table 2.
S. aureus isolates used in the studies with or without a 5 bp deletion exhibiting the non-mucoid or mucoid phenotype.
Fig 3.
Confocal microscopy of biofilms of the non-mucoid (strain 8.1) and mucoid S. aureus (strain 8.2) phenotypes.
Adherent bacteria were stained using the LIVE/DEAD BacLight kit. A). The green color indicates live and the red color dead cells. Only scant amounts of biofilm were detected for non-mucoid isolates. The average biofilm thickness was low (<10 μM at 24h). B) The mucoid strain produced a stable biofilm, which revealed the typical mushroom-shaped multicellular structure. The average height of the biofilm produced by the mucoid strain was 23 μM after 24 h.
Fig 4.
Confocal microscopy of biofilms stained with PIA/PNAG-specific antibodies.
Non-mucoid strain (A) and mucoid (B) S. aureus strains (strains 8.1 and 8.2) were grown overnight in TSB. After washing, adherent bacteria were stained using SYTO 9 (green). PIA/PNAG was detected using ALEXA-568 labeled wheat germ agglutinin (red), which is a lectin binding to the N-acetylglucosaminyl backbone of PIA/PNAG. Zoom-in shows PIA/PNAG-embedded bacteria in the mucoid strain (B), while hardly any PIA/PNAG is detected with the non-mucoid isolate. White bar = 10 μm.
Fig 5.
Opsonophagocytic killing of non-mucoid (strain 8.1) and mucoid S. aureus (strain 8.2) isolates by human neutrophils (PMNs) from healthy volunteers.
The assay contained 2.5 x 106 neutrophils, 5 x 105 CFU S. aureus, 1% guinea pig serum (as a complement source), in a total volume of 500 μl MEM-BSA. Some assays included antibodies against PIA/PNAG raised in rabbits (1% PIA/PNAG). Control samples contained PMNs and S. aureus or antiserum and S. aureus alone with no PMNs. After 2 h of incubation, samples were vortexed and diluted in sterile deionized H2O to lyse the PMNs to release internalized bacteria. The lysate was plated on TSA plates to enumerate bacterial counts. Percent killing was calculated by the reduction in CFU/ml after 2 h compared with that at time zero. A) Significantly more non-mucoid compared to mucoid bacteria were killed in the presence of neutrophils (97% versus 28%, p<0.0001). In control wells, where neutrophils were absent, only 10% of bacteria died. B) By adding antibodies against PIA/PNAG to wells with mucoid isolates and neutrophils, 93% of bacteria were killed compared to only 26% killing of mucoid isolates in the presence of neutrophils but lacking the specific antibodies (p = 0.0002). All experiments were repeated three times. Results are shown as means and standard deviation. *** p-value ≤ 0.001; **** p-value ≤ 0.0001.
Fig 6.
Mutations of non-mucoid S. aureus isolates with 5 bp deletions.
During persistence, non-mucoid S. aureus isolates with 5 bp deletions and compensatory mutations in icaA, D and C were identified. The ica-sequences of 12 non-mucoid isolates with 5 bp deletions of patients 7 (A) and 8 (B) are illustrated. The numbering of bases and amino acids (aa) follows the sequences retrieved from S. aureus strain ATCC35556 (SA113). Changes of bases, amino acids and deletions are indicated in red color. Synonymous changes are indicated in green. S. aureus strains are identified by the number of the patient's visit and the date of isolation. For patient 7, the same mutation was identified for isolates from visits 14, 15 and 16 and for patient 8 for isolates from visits 27, 43, 49 and 51, respectively. Only the mutation for the first identified isolate is shown.
Fig 7.
UPGMA-tree based on up to 1,861 cgMLST allelic profiles of S. aureus isolates of patients 7 and 8 (Table 2).
The tree on the top shows the relationship of the six isolates of patient 7 including the reference strain N315, the tree on the bottom shows the relationship of the six isolates of patient 8 including the reference strain 55/2053. The scale, i. e. distances were given in absolute number of differing alleles. The strains marked with an asterisk exhibit a mucoid phenotype. For more information about isolates see Table 2.
Fig 8.
Biofilm formation of isolates with 5 bp deletion, compensatory mutations, which were complemented with the wild-type ica genes.
Biofilm formation of the strains with compensatory mutations in icaA, icaD or icaC was abrogated. A) Isolates of patient 7. B) Isolates of patient 8. Transformation with a vector expressing the appropriate wild-type ica gene conferred biofilm formation.
Fig 9.
Competition and starvation experiments comparing non-mucoid and mucoid S. aureus isolates from patients 7 and 8.
A and C) Competition growth experiments between the non-mucoid and mucoid strain pairs of patients 7 and 8 with fitness values indicated on top of the graph. During co-culture, the non-mucoid strain of patient 7 out-competed the mucoid strain, while for the strains of patient 8 no significant difference could be observed. B and D) The same strain pairs of patients 7 and 8 were compared concerning survival under nutrient limited conditions for 48h. For both patients, the mucoid strains survived significantly better during starvation compared to the non-mucoid strains. Data were generated by performing three biological with two technical replicates. Graphs show the average of three biological replicates with error bars indicating the standard deviation. Statistical analysis of the generated data was performed using an unpaired two-tailed t-test. * p-value ≤ 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001.
Fig 10.
Growth of isolates with 5 bp deletion and compensatory mutations under nutrient limited conditions.
Under nutrient limited conditions, all tested strains with 5 bp deletions and non-mucoid phenotype were less able to survive under these conditions compared to the isogenic mucoid isolate. Data were generated by performing two technical replicates per biological replicate. Graphs show the average of three biological replicates with error bars indicating the standard deviation. Statistical analysis was performed using an unpaired two-tailed t-test. * p-value ≤ 0.05; ** p-value ≤ 0.01; *** p-value ≤ 0.001.
Table 3.
List of primer pairs used for the amplification of the four genes of the ica-operon (icaA, icaD, icaB and icaC), the upstream localized gene icaR (coding for the regulator), and the intermediate promoter region.