Identification of Potential Diagnostic Markers among Burkholderia cenocepacia and B. multivorans Supernatants

Abstract

Patients with cystic fibrosis (CF) are susceptible to chronic respiratory infections with a number of bacterial pathogens. Among them, the Burkholderia cepacia complex (Bcc) bacteria, consisting of nine related species, have emerged as problematic CF pathogens due to their antibiotic resistance, incidence of nosocomial infection, and person-to-person transmission. Bcc organisms present the clinical microbiologist with a diagnostic dilemma due to the lack of phenotypic biochemical or growth-related characterization tests that reliably distinguish among these organisms. The complex taxonomy of the Bcc species colonizing the CF respiratory tract makes accurate identification problematic. Despite the clinical implications of Bcc identification, a clinical laboratory differentiation of species within the Bcc is lacking. Additionally, no commercial assays are available to further identify the Bcc species. In the current study, secretory proteins present in the cultured supernatants of Burkholderia cenocepacia and Burkholderia multivorans were analyzed by two-dimensional gel electrophoresis (2-DE), followed by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). To assess differential expression, protein spots of B. cenocepacia and B. multivorans that were unique or displayed different intensities were chosen for MALDI-TOF MS analysis. In total, 341 protein spots were detected, of which 23 were unique to each species, demonstrating that potential diagnostic candidates between these two members of the Bcc exist.The Burkholderia cepacia complex (Bcc) consists of nine genetically distinct but phenotypically similar Gram-negative bacilli distinguished by their role as important agents of pulmonary disease in cystic fibrosis (CF) patients (12, 16). Associated with increased morbidity and mortality, Bcc bacteria make eradication very difficult due to their ability to establish infection and maintain chronic colonization in the CF lung (6,9,18,22). Furthermore, these organisms are capable of person-to-person spread, presenting the threat of nosocomial acquired infection. Highly variable and unpredictable, clinical outcomes of Bcc infection range from asymptomatic carriage to bacteremic disease and death (12). This variation is speculated to be the result of genomovar heterogeneity and differential expression of a virulence factor(s) during Bcc infection. Although all species of the Bcc have been recovered from CF patients, Burkholderia cenocepacia and Burkholderia multivorans are the most prevalent, accounting for 85% of pulmonary infections (16).Establishing accurate methods of Bcc identification is paramount for the implementation of infection control measures and appropriate treatment of infected and/or exposed CF patients. Furthermore, species identification within the Bcc is vital not only for surgical and clinical management of CF patients but also for the elucidation of the group's epidemiology. Bcc organisms present clinical microbiologists with a diagnostic dilemma due to the l...

Profiling of Burkholderia cepacia Secretome at Mid-Logarithmic and Early-Stationary Phases of Growth

“…Aside from these, 13 proteins were exclusively identified in the mid-logarithmic phase and 40 proteins were newly-released in the early-stationary phase. This was generally in slight variation with our previous report [17] and that of others [27], [29], but the differences may be attributed to the variations in the time of collection of the supernatants as well as the inherent genetic differences in the strains used in the experiments [17]. When taken together with the findings of the cytotoxicity experiments, the differential protein profiles detected in the mid-logarithmic and early-stationary growth phases of the B. cepacia secretome suggests that the proteins may have contributed to the different cytotoxic effects of the supernatants.…”

“…B. cepacia culture supernatant flagellins were also identified as potential diagnostic candidates [27]. Relatively high amounts of flagellins have been previously reported to be released by many Burkholderia species into their culture supernatants [27], [29], [34].…”

How new molecular tools can help bugbusters: a Burkholderia cepacia complex outbreak investigation

“…Multilocus sequence-based and recA gene polymerase chain reaction have emerged as effective tools for Bcc identification and classification 1 . Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) has also emerged as a potential tool for identifying species of Bcc 26 , 27 .…”

POLYCLONAL OUTBREAK OF BLOODSTREAM INFECTIONS CAUSED BY Burkholderia cepacia COMPLEX IN HEMATOLOGY AND BONE MARROW TRANSPLANT OUTPATIENT UNITS

“…Burkholderia cepacia complex (Bcc) comprises at least 15 different species or genomovars based on phenotypic and genotypic analyses 16 , 17 . Identification to species level has been troublesome in the clinical laboratory.…”

“…Automated methods seem to perform poorly in identifying species of Bcc 19 , 25 . Molecular methods are more appropriate 8 , 12 , 16 . Clinically, Bcc mainly cenocepacia and multivorans species are important colonizers and cause respiratory tract illness among cystic fibrosis patients 1 .…”