Biofilm is a major virulence determinant in bacterial colonization of chronic skin ulcers independently from the multidrug resistant phenotype

Bacterial biofilm is a major factor in delayed wound healing and high levels of biofilm production have been repeatedly described in multidrug resistant organisms (MDROs). Nevertheless, a quantitative correlation between biofilm production and the profile of antimicrobial drug resistance in delayed wound healing remains to be determined. Microbial identification, antibiotic susceptibility and biofilm production were assessed in 135 clinical isolates from 87 patients. Gram-negative bacteria were the most represented microorganisms (60.8%) with MDROs accounting for 31.8% of the total isolates. Assessment of biofilm production revealed that 80% of the strains were able to form biofilm. A comparable level of biofilm production was found with both MDRO and not-MDRO with no significant differences between groups. All the methicillin-resistant Staphylococcus aureus (MRSA) and 80% of Pseudomonas aeruginosa MDR strains were found as moderate/high biofilm producers. Conversely, less than 17% of Klebsiella pneumoniae extended-spectrum beta-lactamase (ESBL), Escherichia coli-ESBL and Acinetobacter baumannii were moderate/high biofilm producers. Notably, those strains classified as non-biofilm producers, were always associated with biofilm producer bacteria in polymicrobial colonization. This study shows that biofilm producers were present in all chronic skin ulcers, suggesting that biofilm represents a key virulence determinant in promoting bacterial persistence and chronicity of ulcerative lesions independently from the MDRO phenotype

Modelling co-infection of the cystic fibrosis lung by <em>Pseudomonas aeruginosa</em> and <em>Burkholderia cenocepacia</em> reveals influences on biofilm formation and host response

The Gram-negative bacteria Pseudomonas aeruginosa and Burkholderia cenocepacia are opportunistic human pathogens that are responsible for severe nosocomial infections in immunocompromised patients and those suffering from cystic fibrosis (CF). These two bacteria have been shown to form biofilms in the airways of CF patients that make such infections more difficult to treat. Only recently have scientists begun to appreciate the complicated interplay between microorganisms during polymicrobial infection of the CF airway and the implications they may have for disease prognosis and response to therapy.To gain insight into the possible role that interaction between strains of P. aeruginosa and B. cenocepacia may play during infection, we characterised co-inoculations of in vivo and in vitro infection models. Co-inoculations were examined in an in vitro biofilm model and in a murine model of chronic infection. Assessment of biofilm formation showed that B. cenocepacia positively influenced P. aeruginosa biofilm development by increasing biomass. Interestingly, co-infection experiments in the mouse model revealed that P. aeruginosa did not change its ability to establish chronic infection in the presence of B. cenocepacia but co-infection did appear to increase host inflammatory response.Taken together, these results indicate that the co-infection of P. aeruginosa and B. cenocepacia leads to increased biofilm formation and increased host inflammatory response in the mouse model of chronic infection. These observations suggest that alteration of bacterial behavior due to interspecies interactions may be important for disease progression and persistent infection

Cytokines and chemokines in lungs homogenates of C57BL/6NCrlBR mice after chronic infection.

a<p>Cytokines and chemokines are expressed as pg/ml (mean±SEM).</p>b<p>Significant difference of <i>P. aeruginosa</i> RP73 single infection <i>vs</i> co-infected mice (^#<i>P</i><0.05;^{# #}<i>P</i><0.01 Two-tailed student's t test).</p>c<p>Significant difference of <i>B. cenocepacia</i> LMG16656 single infection <i>vs</i> co-infected (^Δ<i>P</i><0.05; ^ΔΔ<i>P</i><0.01 Two-tailed student's t test).</p

<i>P. aeruginosa</i> and <i>B. cenocepacia</i> planktonic and sessile cells in single and dual cultures.

<p>Bacteria were grown overnight in 96-well polyvinyl chloride flat-bottomed microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio. CFU counts were determined at 24 h of bacterial growth in both planktonic and sessile fraction. Key: (A, left) Sessile cells of clinical pair (<i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656) in single and dual cultures; (A, right) Sessile cells of environmental pair (<i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1) in single and dual cultures; (B, left) Planktonic cells of clinical pair (<i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656) in single and dual cultures; (B, right) Planktonic cells of environmental pair (<i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1) in single and dual cultures; (C) CI and RIR mean values of sessile growth of <i>P. aeruginosa</i> versus <i>B. cenocepacia</i> (RP73 <i>versus</i> LMG16656, E5 <i>versus</i> Mex1); (D) CI and RIR of planktonic growth of <i>P. aeruginosa</i> versus <i>B. cenocepacia</i>. Each value represents the mean of RIR and CI values from three separate assays, and the bars indicate standard deviations. * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001 in Student's t test.</p

Total and differential cell counts in BAL fluid after 13 days of infection.

<p>The number of total leukocytes and in particular of neutrophils, monocytes and lymphocytes recruited in the airways were analyzed in BAL fluid (BALF) after 13 days of chronic lung infection with pairs of clinical (A) or environmental strains (B)). Values represent the mean ± SEM. The data are pooled from two or three independent experiments. Statistical significance by two tailed Student's <i>t</i>-test is indicated: * <i>P</i><0.05, ** <i>P</i><0.01.</p

Virulence of <i>P. aeruginosa</i> and <i>B. cenocepacia</i> strains alone or in co-infection in mice.

<p>C57Bl/6 mice (A and B), <i>Cftr^tm1UNCTgN(FABPCFTR)</i> (CF) and their congenic wt mice (C) were infected with <i>P. aeruginosa</i> and/or <i>B. cenocepacia</i> strains. Mortality induced by bacteremia (red) and survival (grey) were evaluated on challenged mice. Clearance (white) and capacity to establish chronic airways infection (green) after 13 days from challenge were determined on surviving mice infected with <i>P. aeruginosa</i> and <i>B. cenocepacia</i> strains alone or with pairs of clinical (A and C) or environmental (B) strains. The data are pooled from two to three independent experiments. Mortality and chronic infection are reported as median values. B6.129P2-<i>Cftr^tm1UNCTgN(FABPCFTR) Cftr</i>^+/+ and B6.129P2-<i>Cftr^tm1UNCTgN(FABPCFTR)Cftr^S489X/S489X</i> mice co-infected with RP73-LMG16656 developed a higher rate of mortality when compared with C57BL/6NCrlBR mice (<i>P</i><0.05; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052330#pone.0052330.s006" target="_blank">Table S1</a>).</p

Biofilm architecture in <i>P. aeruginosa</i> is influenced by <i>B. cenocepacia</i>.

<p>Images are of 4-day-old biofilms in flow cells in FABL medium. Key: (A) <i>P. aeruginosa</i> RP73; (B) <i>B. cenocepacia</i> LMG16656;(C) mixed culture of <i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656; (D) Quantification of biomass as determined using COMSTAT to estimate the percentage of <i>P. aeruginosa</i> cells as a function of the total biomass. For these experiments, <i>P. aeruginosa</i> was tagged with mini-Tn<i>7gfp</i>. <i>B. cenocepacia</i> was visualized with Syto62, as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052330#s4" target="_blank"><i>Materials and Methods</i></a>. Scale bars = 20 µm. Images shown are representative of 12 images from three independent experiments.</p

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

<p>The two species were individually cultured or co-cultured at a 1∶1 ratio and grown for 24 h in NB medium at 37°C with vigorous aeration. Colony-forming unit counts (CFU) were determined at 0, 2, 4, 6, 8 and 24 h of bacterial growth. The results are the mean of Log (CFU ml⁻¹) values of three separated assays. Key: (A) Growth of clinical <i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656 strains in single and dual cultures; (B) Competitive index (CI) and relative increase ratio (RIR) generated from single and dual cultures of clinical <i>P. aeruginosa</i> RP73 and <i>B. cenocepacia</i> LMG16656 strains; (C) Growth of environmental <i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1 strains in single and dual cultures; (D) Competitive index (CI) and relative increase ratio (RIR) generated from single and dual cultures of environmental <i>P. aeruginosa</i> E5 and <i>B. cenocepacia</i> Mex1 strains. CI and RIR were calculated as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052330#s4" target="_blank"><i>Materials and Methods</i></a>. Each value represents the mean of RIR and CI values from three separate assays, and the bars indicate standard deviations. * = <i>P</i><0.05, ** = <i>P</i><0.01 in the Student's t test.</p

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

<p>Bacteria were grown overnight in 96-well polyvinyl chloride flat-bottomed microtiter plates in NB medium at 37°C either individually cultured or co-cultured at a 1∶1 ratio or when individually cultured supplemented with sterile concentrated supernatant of the second organism at a final concentration of 1×. Biofilm biomass was quantified by staining with crystal violet and absorbance measurements at OD ₅₉₅. The values are means of three separated assays, and the bars indicate standard deviation. * = <i>P</i><0.05, ** = <i>P</i><0.01, *** = <i>P</i><0.001 in Student's t test. S = supernatant.</p