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Int J Mol Sci
2017 Nov 14;1811:. doi: 10.3390/ijms18112417.
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Environmental Burkholderia cenocepacia Strain Enhances Fitness by Serial Passages during Long-Term Chronic Airways Infection in Mice.
Bragonzi A
,
Paroni M
,
Pirone L
,
Coladarci I
,
Ascenzioni F
,
Bevivino A
.
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Burkholderia cenocepacia is an important opportunistic pathogen in cystic fibrosis (CF) patients, and has also been isolated from natural environments. In previous work, we explored the virulence and pathogenic potential of environmental B. cenocepacia strains and demonstrated that they do not differ from clinical strains in some pathogenic traits. Here, we investigated the ability of the environmental B. cenocepacia Mex1 strain, isolated from the maize rhizosphere, to persist and increase its virulence after serial passages in a mouse model of chronic infection. B. cenocepacia Mex1 strain, belonging to the recA lineage IIIA, was embedded in agar beads and challenged into the lung of C57Bl/6 mice. The mice were sacrificed after 28 days from infection and their lungs were tested for bacterial loads. Agar beads containing the pool of B. cenocepacia colonies from the four sequential passages were used to infect the mice. The environmental B. cenocepacia strain showed a low incidence of chronic infection after the first passage; after the second, third and fourth passages in mice, its ability to establish chronic infection increased significantly and progressively up to 100%. Colonial morphology analysis and genetic profiling of the Mex1-derived clones recovered after the fourth passage from infected mice revealed that they were indistinguishable from the challenged strain both at phenotypic and genetic level. By testing the virulence of single clones in the Galleria mellonella infection model, we found that two Mex1-derived clones significantly increased their pathogenicity compared to the parental Mex1 strain and behaved similarly to the clinical and epidemic B. cenocepacia LMG16656T. Our findings suggest that serial passages of the environmental B. cenocepacia Mex1 strain in mice resulted in an increased ability to determine chronic lung infection and the appearance of clonal variants with increased virulence in non-vertebrate hosts.
Figure 1. Schedule of sequential chronic B. cenocepacia lung infection in mice. Two groups of C57Bl/6NCrlBR mice (n = 8–12) were inoculated with 1.5 × 107 CFU/lung of B. cenocepacia Mex1 strain embedded in agar beads for 28 days (P1). After 28 days, single colonies were recovered from two groups of infected mice and were re-grown separately; then, they were pulled for two different agar bead preparations and injected in two groups of mice (n = 8–9) (P2). The third and the fourth passages in mice (n = 8–9) (P3 and P4) were carried out as P2 for 28 days each.
Figure 3. Random amplified polymorphic DNA (RAPD) fingerprints of the B. cenocepacia Mex1 challenge and some of its persisting colonies isolated from infected mice. (A) The polymorphisms were generated using RAPD primer 270. From left to right: L123, 123-bp molecular size marker ladder; Mex1 strain; clone 410801; clone 410807; clone 410808; negative control. (B) The dendrogram showing the clonal relatedness of some persisting colonies, performed with the Unweighted Pair Group Method with Arithmetic mean (UPGMA) by using mathematic averages algorithm programs integral to the Phoretix 1D Pro software. Lanes 2–5: Mex1 strain; clone 410801; clone 410807; clone 41080.
Figure 4. Biofilm formation of Mex1 and some of its derivatives in microtiter plate assay. No significant difference among Mex1 and its derivative clones was found (p > 0.05, One-way ANOVA). The clinical LMG16656T formed a higher biofilm in comparison with Mex1 (p = 0.0311, Student’s t-test). The amount of biofilm was quantified by Crystal Violet staining. Absorbance was measured at 595 nm.
Figure 5. Colonial morphology of Mex1, its derivative (41818 clone as an example) and the clinical B. cenocepacia LMG16656T on YEM agar. Strains were grown at 37 °C for 48 h.
Figure 6. Percent survival of G. mellonella following inoculation with Mex1, Mex1-mouse persistent derivatives and the clinical LMG16656T. The B. cenocepacia strains are indicated on the right: Mex1, thick dashed line; LMG16656, thick dotted line. Y-axis, percent survival of larvae infected with the indicated bacterial strains; X-axis, time post infection (days). Mex1-mouse persistent derivatives were recovered from two groups of eight and 9 C57Bl/6 male mice, and coded as 40 (A) and 41 (B), respectively.
Figure 7. Kaplan-Meier survival plots of larvae injected with the indicated strains. (A) The killing ability of Mex1 compared with that of 41803 and 41818 Mex1-derived clones. (B) The killing ability of LMG16656 compared with that of 41803 and 41818 Mex1-derived clones. Y-axis, percent survival of larvae; X-axis, time (days) post infection. Statistical analysis was performed by log-rank test.
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