Whole-Genome Sequencing of Three Clonal Clinical Isolates of B. cenocepacia from a Patient with Cystic Fibrosis

Abstract

Burkholderia cepacia complex bacteria are amongst the most feared of pathogens in cystic fibrosis (CF). The BCC comprises at least 20 distinct species that can cause chronic and unpredictable lung infections in CF. Historically the species B. cenocepacia has been the most prevalent in CF infections and has been associated in some centers with high rates of mortality. Modeling chronic infection by B. cenocepacia in the laboratory is challenging and no models exist which effectively recapitulate CF disease caused by BCC bacteria. Therefore our understanding of factors that contribute towards the morbidity and mortality caused by this organism is limited. In this study we used whole-genome sequencing to examine the evolution of 3 clonal clinical isolates of B. cenocepacia from a patient with cystic fibrosis. The first isolate was from the beginning of infection, and the second two almost 10 years later during the final year of the patients’ life. These isolates also demonstrated phenotypic heterogeneity, with the first isolate displaying the mucoid phenotype (conferred by the overproduction of exopolysaccharide), while one of the later two was nonmucoid. In addition we also sequenced a nonmucoid derivative of the initial mucoid isolate, acquired in the laboratory by antibiotic pressure. Examination of sequence data revealed that the two late stage isolates shared 20 variant nucleotides in common compared to the early isolate. However, despite their isolation within 10 months of one another, there was also considerable variation between the late stage isolates, including 42 single nucleotide variants and three deletions. Additionally, no sequence differences were identified between the initial mucoid isolate and its laboratory acquired nonmucoid derivative, however transcript analysis indicated at least partial down regulation of genes involved in exopolysaccharide production. Our study examines the progression of B. cenocepacia throughout chronic infection, including establishment of sub-populations likely evolved from the original isolate, suggestive of parallel evolution. Additionally, the lack of sequence differences between two of the isolates with differing mucoid phenotypes suggests that other factors, such as gene regulation, come into play in establishing the mucoid phenotype.

Genome-based classification of Burkholderia cepacia complex provides new insight into its taxonomic status

“…At present, with the advent of next-generation sequencing technologies, an increasing number of complete genome sequences of the bacteria in the BCC have been published [21][22][23][24]. This provides an ideal opportunity for re-examining the taxonomy of BCC by traditional molecular methods.…”

Phenotypic diversity and genotypic flexibility of Burkholderia cenocepacia during long-term chronic infection of cystic fibrosis lungs

“…B. cenocepacia is the most prevalent Bcc species colonizing CF patients, with most isolates from the United Kingdom and Canada belonging to a single molecular type (electrophoretic type ET12 or RAPD02) that includes epidemic strain J2315 (Speert et al 2002b;McDowell et al 2004). Along with RAPD02; RAPD01, -04, and -06 comprise a distinct genomic group (previously genomovar IIIA, now called recA subgroup A), whereas other RAPD types belong to a divergent B. cenocepacia genomic group (previously genomovar IIIB, now recA subgroup B) that includes PHDC epidemic lineages HI2424 and AU1054 strains (Johnson 1994;Rozee et al 1994;Mahenthiralingam et al 1996;Speert 2002;Speert et al 2002a;Lipuma 2005;Holden et al 2009;Miller et al 2015).…”

1H-NMR-Based Endometabolome Profiles of Burkholderia cenocepacia Clonal Variants Retrieved from a Cystic Fibrosis Patient during Chronic Infection

“…However, these results should be interpreted with caution. Several recent papers are supporting the idea of the occurrence of a high Bcc phenotypic and genotypic diversity within a CF patient, where multiple lineages coexist presumably as the result of bacterial exposure to the dynamic CF lung environment during chronic infection (Coutinho et al, 2011a; Lieberman et al, 2014; Moreira et al, 2014, 2016; Miller et al, 2015). However, the B. cenocepacia clonal isolates examined in this work may not be representative of the expected population heterogeneity actually present at each isolation time in the CF airways.…”

“…Once inside the CF lung, Bcc bacteria face a changing and stressful environment resulting from the activity of the host immune system, antimicrobial therapy, fluctuating levels of nutrients, decreasing oxygen availability and the presence of other co-infecting microbes (Döring et al, 2011). As proposed for Pseudomonas aeruginosa (Smith et al, 2006; Clark et al, 2015; Marvig et al, 2015), it is believed that these challenging environmental conditions stimulate genetic alterations in the initial infecting strain, leading to the diversification of genotypes and phenotypes and to the emergence of multiple clonal variants from the underlying Bcc population (Coutinho et al, 2011a,b; Lieberman et al, 2011, 2014; Moreira et al, 2014; Zlosnik et al, 2014; Miller et al, 2015; Maldonado et al, 2016). …”

“…The genome sequences of late Burkholderia cenocepacia isolates retrieved from a CF patient after 10 years of chronic infection were compared with the initial infecting strain and mutations in genes involved in metabolism, including genes from amino acid, iron and purine metabolism, were identified (Miller et al, 2015). Another comparative genomic study focusing on B. multivorans isolates retrieved from a CF patient over 20 years of infection has shown that genes involved in gene expression regulation, cell envelope biogenesis, fatty acid and amino acid metabolism exhibit recurrent mutation patterns (Silva et al, 2016).…”