<i>S. aureus</i> and <i>P. aeruginosa</i> planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined in both planktonic and sessile fractions. Panel A: planktonic (left) and sessile (right) cells of <i>S. aureus</i> strain Newman in pure culture and in co-culture with <i>P. aeruginosa</i> strains PA14, AA2 and AA43. Statistically significant differences are referred to Newman in pure culture. Panel B: planktonic (left) and sessile (right) cells of <i>P. aeruginosa</i> strains PA14, AA2 and AA43 in pure culture and in co-culture with <i>S. aureus</i> strain Newman. The values represent the means of three independent experiments, and the bars indicate standard deviation. Statistically significant differences in non-parametric Mann–Whitney test are indicated by symbols when present: **: p<0.01; ***: p<0.001.</p

Competition between <i>P. aeruginosa</i> and <i>S. aureus</i> strains in a murine model of pneumoniae.

<p>Planktonic <i>S. aureus</i> strain Newman and <i>P. aeruginosa</i> clinical isolates AA2 and AA43 and reference strain PA14 were used to infect C57BL/6NCrlBR mice at a ratio of 1∶1. After 18 hours of acute infection lungs homogenates were plated on selective plates to determine <i>S. aureus</i> and <i>P. aeruginosa</i> CFU. Each circle represents the CI for a single animal in each group. A CI value equal to 1 indicates equal competition of the two species; a CI value significantly <1 indicates a competitive advantage of <i>S. aureus</i> that outcompetes <i>P. aeruginosa</i>; a CI value significantly >1 indicates a competitive advantage of <i>P. aeruginosa</i> that outcompetes <i>S. aureus</i>. Wilcoxon signed rank test of the null hypothesis that the distribution of CI is symmetric about 1 was performed. Statistically significant differences are indicated by symbols when present: *: p<0.05; **: p<0.01. The data are pooled from two or three independent experiments.</p

Biofilm formation by <i>S. aureus</i> and <i>P. aeruginosa</i> strains in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. Biofilm biomass was quantified by staining with crystal violet and absorbance measurements at OD 595 nm. The values represent the means of three independent experiments, and the bars indicate standard deviation. Statistically significant differences in Student's t test are indicated by symbols when present: **: p<0.01; ***: p<0.001.</p

Factors Contributing to Epidemic MRSA Clones Replacement in a Hospital Setting

<div><p>The mechanisms governing the epidemiology dynamics and success determinants of a specific healthcare-associated methicillin-resistant <em>S. aureus</em> (HA-MRSA) clone in hospital settings are still unclear. Important epidemiological changes have occurred in Europe since 2000 that have been related to the appearance of the ST22-IV clone. Between 2006 and 2010, we observed the establishment of the ST22-IV clone displacing the predominant Italian clone, ST228-I, in a large Italian university hospital. To investigate the factors associated with a successful spread of epidemic MRSA clones we studied the biofilm production, the competitive behavior in co-culture, the capacity of invasion of the A549 cells, and the susceptibility to infection in a murine model of acute pneumonia of the two major HA-MRSA clones, ST22-IV and ST228-I. We showed that persistence of ST22-IV is associated with its increased biofilm production and capacity to inhibit the growth of ST228-I in co-culture. Compared to ST228-I, ST22-IV had a significantly higher capacity to invade the A549 cells and a higher virulence in a murine model of acute lung infection causing severe inflammation and determining death in all the mice within 60 hours. On the contrary, ST228-I was associated with mice survival and clearance of the infection. ST22-IV, compared with ST228-I, caused a higher number of persistent, long lasting bacteremia. These data suggest that ST22-IV could have exploited its capacity to i) increase its biofilm production over time, ii) maintain its growth kinetics in the presence of a competitor and iii) be particularly invasive and virulent both <em>in vitro</em> and <em>in vivo,</em> to replace other well-established MRSA clones, becoming the predominant European clone.</p> </div

Single and dual species batch growth curves and competition index values.

<p><i>S. aureus</i> strain (Newman) and <i>P. aeruginosa</i> strains (PA14 and two clinical early and late isolates from a CF patient AA2 and AA43) were grown for 24 hours in BHI in single culture and in co-culture after inoculation at equal ratio from mid-exponential phase pure cultures. Growth rate was monitored by colony count after plating on selective media for both species. Results are represented as the mean of values obtained from three independent experiments. The error bars indicate the standard deviations. A nonlinear mixed-effect model was fitted, using a four-parameters logistic regression function. Panel A: growth curves of Newman in pure culture and in co-culture with PA14; Panel B: Competition index (CI) and Relative Increase Ratio (RIR) calculated from single and dual cultures of Newman and PA14; Panel C: growth curves of Newman in pure culture and in co-culture with AA2; Panel D: CI and RIR calculated from single and dual cultures of Newman and AA2; Panel E: growth curves of Newman in pure culture and in co-culture with AA43; Panel F: CI and RIR calculated from single and dual cultures of Newman and AA43. Each value represents the mean of CI and RIR values from three independent experiments and the bars indicate standard deviation. Statistically significant differences in Student's t test and in nonlinear mixed-effect model are indicated by symbols when present: *: p<0.05; **: p<0.01; ***: p<0.001.</p

<i>In vitro</i> growth inhibition of <i>S. aureus</i> and <i>P. aeruginosa</i>.

<p>Twenty-four <i>P. aeruginosa</i> isolates were collected from eight individuals with CF (SG, NN, BT, AA, TR, MF, KK, BST) at the onset of chronic colonization (numbered 1-2) or after 4.5–16.3 years of colonization (numbered 43-83). PAO1 and PA14 were included as reference strains. 5 µl spots of <i>P. aeruginosa</i> overnight cultures, normalized to 0.5 OD, were added to <i>S. aureus</i> lawn (normalized to 0.5 OD) on Mueller-Hinton agar and incubated overnight at 37°C. The table summarizes the results obtained: “weak inhibition” indicates an inhibition halo ≤15 mm; “strong inhibition” indicates an inhibition halo >15 mm and ≤25 mm; “very strong inhibition” indicates an inhibition halo >25 mm; “no inhibition“ indicates absence of inhibition halo (9 mm is the diameter of the <i>P. aeruginosa</i> spot).</p><p>* Indicates mucoid phenotype.</p>#<p>Indicates hypermutable phenotype. For statistical analysis see “<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0089614#s2" target="_blank">Results</a>”.</p

Clinical characteristics of patients with persistent MRSA infection identified between 2009 and 2010.

a<p>years;</p>b<p>broncho-alveolar lavage;</p>c<p>bronchial aspirate;</p>*<p>patient transferred to another hospital; - not present.</p

Histopathology in murine lung infected with MRSA clones ST22-IV (variant A) and ST228-I (variant B5).

<p>C57Bl/6NCrl were infected intratracheally with 5*10⁸ cfu per mouse of either the variant A of the clone ST22-IV (strain 54) or the variant B5 of the clone ST228-I (strain 67). Mice were sacrificed between 3 and 7 days from infection with ST22-IV (left panel) and ST228-I (right panel), respectively. Lungs were removed en bloc, fixed in 4% paraformaldehyde/PBS and processed for paraffin embedding. Longitudinal sections were stained with hematoxylin and eosin. Upper panel: 2,5× magnification; lower panel: 10× magnification.</p

<i>In vitro</i> competitive growth cultures.

<p>The MRSA clones ST22-IV (variant A, strain 54 isolated from sputum) and ST228-I (variant B5, strain 67 isolated from blood) were grown in pure culture (A) and in co-culture after inoculation in BHI broth at equal ratio (B and C). Growth rate was monitored for 24 hours by colony count after plating and normalizing the output value obtained at each time (t) to the input value at time 0 (t0). CFU: colony forming unit; statistically significant differences are indicated by symbols when present: * indicates a p value <0,05; **: p<0,01; ***: p<0,001. The competition index for mixed culture (D) was calculated at the different time points as the ST22-IV/ST228-I CFU ratio for the output (time t) divided by the corresponding ratio for the input (inoculum at time t = 0).</p

Percentage of planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well flat-bottom microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined in both planktonic and sessile fractions and the percentage of S. aureus and P. aeruginosa in the two fractions of single and dual cultures was calculated. Panel A: percentages of planktonic and sessile cells of Newman in single culture (first histogram), PA14 in single culture (second histogram), Newman and PA14 in ideal co-culture if the 2 species would not interfere each other (third histogram, percentages have been calculated considering the values of the first and second histograms), and Newman and PA14 in co-culture (fourth histogram). Panel B: percentages of planktonic and sessile cells of Newman in single culture (first histogram), AA2 in single culture (second histogram), Newman and AA2 in ideal co-culture if the 2 species would not interfere each other (third histogram, percentages have been calculated considering the values of the first and second histograms), and Newman and AA2 in co-culture (fourth histogram). Panel C: percentages of planktonic and sessile cells of Newman in single culture (first histogram), AA43 in single culture (second histogram), Newman and AA43 in ideal co-culture if the 2 species would not interfere each other (third histogram, percentages have been calculated considering the values of the first and second histograms), and Newman and AA43 in co-culture (fourth histogram). SA: S. aureus; PA: P. aeruginosa.</p