PMC3417363_01
Characterization of the CF subpopulation that has been monitored at the major Portuguese CF Treatment Center at Santa Maria Hospital, in Lisbon, for the past 16 years. The population was characterized in terms of gender, age, type of colonization, and clinical outcome. Among the deceased patients, at least five had developed the cepacia syndrome at the time of death (each patient marked as *). Based on the information published in Richau et al., Cunha et al., and on other recent unpublished data.
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F1
When analyzing the clinical outcome of both the adult and pediatric subpopulations of chronically infected patients (Figure 1), it is clear that the worst outcomes lye in the female subpopulation.
Profiling of the Bcc-infected CF subpopulation. (A) Distribution of the Bcc clinical isolates into the corresponding Bcc species; (B) Chronological distribution of the different Bcc species isolates; (C) Number of patients and date of first Bcc positive isolation (pediatric - white bars and adults - black bars); (D) Number of patients infected with the different species, including co-infection (cen - B. cenocepacia; cep - B. cepacia; stab - B. stabilis; mult - B. multivorans; cont - B. contaminans; dol - B. dolosa); (E) Period of duration of chronic infection and clinical outcome (eradication - white bars, death - black bars, * denotes a confirmed cepacia syndrome). Based on the information published in Richau et al., Cunha et al., and on other recent unpublished data.
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F2
According to this criterion, approximately 68% of the 41 Bcc-infected patients were persistently colonized (9 adults and 19 children), for time periods ranging from 7 months to almost 10 years, while the remaining 32% registered only intermittent isolation (Figure 2E).
PMC3417363_02
Characterization of the CF subpopulation that has been monitored at the major Portuguese CF Treatment Center at Santa Maria Hospital, in Lisbon, for the past 16 years. The population was characterized in terms of gender, age, type of colonization, and clinical outcome. Among the deceased patients, at least five had developed the cepacia syndrome at the time of death (each patient marked as *). Based on the information published in Richau et al., Cunha et al., and on other recent unpublished data.
fcimb-01-00012-g001.jpg
PMC3417363_02_fcimb-01-00012-g001.jpg
F1
When analyzing the clinical outcome of both the adult and pediatric subpopulations of chronically infected patients (Figure 1), it is clear that the worst outcomes lye in the female subpopulation.
Profiling of the Bcc-infected CF subpopulation. (A) Distribution of the Bcc clinical isolates into the corresponding Bcc species; (B) Chronological distribution of the different Bcc species isolates; (C) Number of patients and date of first Bcc positive isolation (pediatric - white bars and adults - black bars); (D) Number of patients infected with the different species, including co-infection (cen - B. cenocepacia; cep - B. cepacia; stab - B. stabilis; mult - B. multivorans; cont - B. contaminans; dol - B. dolosa); (E) Period of duration of chronic infection and clinical outcome (eradication - white bars, death - black bars, * denotes a confirmed cepacia syndrome). Based on the information published in Richau et al., Cunha et al., and on other recent unpublished data.
fcimb-01-00012-g002.jpg
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F2
According to this criterion, approximately 68% of the 41 Bcc-infected patients were persistently colonized (9 adults and 19 children), for time periods ranging from 7 months to almost 10 years, while the remaining 32% registered only intermittent isolation (Figure 2E).
Phenotypic characteristics of the B. cenocepacia clonal variants IST439 and IST4113, retrieved from the same CF patient sputum during long-term residence in the lung and whose genomic expression was compared by quantitative proteomic and transcriptomic analyses. (A) Minimum inhibitory concentration (MIC) values; (B) Growth of IST439 and IST4113 under limited or load iron conditions; (C) Fatty acid saturation of the clonal variants obtained during the 41-months of chronic colonization; (D) Colony morphology; (E) Amount of biofilms formed based on crystal violet method; (F) Swarming and swimming motilities; (G) Zeta potential; (H) Cell size; (I) Exopolysaccharide produced; (J) Fraction of cells at the interface of water and n-hexadecane. Based on results from Coutinho et al. and Madeira et al..
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F3
In this context, the genomic expression of 2 of the 11 B. cenocepacia sequential clonal variants from patient J were compared in our laboratory: IST439, the first isolate that was collected from the patient and is thought to have initiated the infection, and IST4113, an isolate that was recovered almost 3 years later after a period of exacerbated infection and intravenous therapy with gentamicin and ceftazidime (IST4113; Figure 3; Cunha et al.,; Coutinho et al.,; Madeira et al.,; Mira et al.,). Among other phenotypic differences, the IST4113 isolate was found to be much more resistant than IST439 to all the tested antibiotics belonging to different classes and having distinct biological targets (Figure 3A; Coutinho et al.,; Madeira et al.,). Several of the observations that emerged from the transcriptomic and proteomic analyses are consistent with the previously carried out phenotypic characterization of the two isolates (Figure 3; Coutinho et al.,), and are suggestive of genetic adaptation from isolate IST439 to IST4113. For example, the up-regulation of proteins and genes associated with iron binding and transport in IST4113 cells (Madeira et al.,; Mira et al.,) is in line with the higher efficiency that these cells exhibit to recover iron from an iron-limited environment, when compared to IST439 cells (Figure 3B; Coutinho et al.,; Madeira et al.,). The up-regulation of many proteins/genes related with protein synthesis, translation, and protein folding in the IST4113 isolate might also contribute to the higher resistance exhibited by IST4113 against antimicrobials targeting protein synthesis (Figure 3A; Leitao et al.,; Coutinho et al.,; Madeira et al.,), which include the aminoglycosides gentamicin and tobramycin (Jana and Deb,). Alterations observed at the level of LPS structure and/or content, cell wall, and outer membrane biogenesis are also consistent with the highly antibiotic-resistant phenotype of IST4113 cells (Figure 3A). O-antigen has also been associated with a rough colony morphotype (Saldias et al.,), an hypothesis that is consistent with the rough colonies formed by IST4113 whereas IST439 cells possess a smooth colony morphotype (Figure 3D; Coutinho et al.,; Madeira et al.,). Our results also suggest that the content of several outer membrane proteins is lower in IST4113 than in IST439, including several porines that are significantly down-regulated (Madeira et al.,; Mira et al.,).
Proposed mechanistic model of the adaptive responses that might occur during long-term residence of B. cenocepacia in the stressing environment of the CF lung under antimicrobial therapy. The model is based on the results obtained from the comparison of transcriptomic, proteomic, and phenotypic profiling of sequential clinical isolates collected from the same chronically colonized CF patient.
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F4
A working model summarizing the most significant findings suggested by our studies and described in this review is proposed in Figure 4.