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Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic colonization with in vivo strain evolution over time
Diagnostic Microbiology and Infectious Disease xxx (2016) xxx–xxx
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Diagnostic Microbiology and Infectious Disease journal homepage: www.elsevier.com/locate/diagmicrobio
Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic colonization with in vivo strain evolution over time Alexander L. Greninger a,b,c, Jessica Streithorst a, Jeffrey A. Golden d, Charles Y. Chiu a,b,e, Steve Miller a,b,⁎ a
Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA c Department of Laboratory Medicine, University of Washington, Seattle, WA, USA d Department of Medicine, Division of Pulmonology, University of California San Francisco, San Francisco, CA, USA e Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, USA b
a r t i c l e
i n f o
Article history: Received 31 August 2016 Received in revised form 7 October 2016 Accepted 7 October 2016 Available online xxxx Keywords: Pandoraea apista Genome sequence Strain evolution
a b s t r a c t Pandoraea apista in the family Burkholderiaceae is an emerging opportunistic pathogen in cystic fibrosis patients. Here, we describe a case from which 3 separate isolates of P. apista were recovered over a 1-year period. Using a combination of first-, second-, and third-generation sequencing technologies, we sequenced and de novo assembled the complete genomes of these 3 P. apista isolates. The genome of P. apista TF81F4 sequenced in this study was 5.58 Mb with a GC% of 62.3%, differed in sequence from other Pandoraea species by N20%, and included a number of previously undescribed loci. Three P. apista isolates cultured over a 12-month period were N99.999% identical by nucleotide, consistent with a model of chronic colonization by a single strain. Over time, the isolates accumulated point mutations, deletions, and insertions in a stepwise fashion, indicating in vivo strain evolution within the cystic fibrosis lung niche. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Pandoraea is a genus of gram-negative nonfermenting bacteria in the family Burkholderiaceae and contains the emerging pathogens P. apista, P. pulmonicola, P. sputorum, and P. pnomenusa. All of these Pandoraea species have been associated with lung infections in cystic fibrosis patients. The genus was originally described in 2000 on the basis of polyphasic taxonomy integrating unique housekeeping gene sequence alignments and biochemical profiles of putative Burkholderia and Ralstonia spp. isolated from the sputa of cystic fibrosis patients across a broad geographic range (Coenye et al., 2000). Notably, Pandoraea species have the potential for invasion, with bloodstream infections being one of the most common sources of isolation, and have also been shown to translocate across cultures of lung epithelial cells and to induce a strong proinflammatory response (Caraher et al., 2008). The name Pandoraea was chosen to refer to Pandora's box given the surprising genetic and biochemical diversity of the members within the genus (Coenye et al., 2000). The diversity of the genus merits a genomic approach to better understand the full genetic basis of this diversity as well as evolution of the genus and contributions to pathogenicity. To date, complete genomes have been assembled for strains of P. pnomenusa, P. sputorum, P. pulmonicola, and P. apista isolated from ⁎ Corresponding author. Tel.: +1-415-353-9630. E-mail address: [email protected] (S. Miller).
human infections (Chan et al., 2014). Here, we describe a clinical case from which 3 isolates of P. apista were recovered over a 12-month interval. We leveraged a combination of Sanger (first generation), Illumina (second generation), and Oxford Nanopore (third generation) technologies to sequence the complete genome of all 3 isolates, revealing that they are nearly completely identical to each other but dissimilar from other P. apista strains within locally sequenced regions or other Pandoraea species. 2. Materials and methods 2.1. Bacterial isolates Bacterial isolates were derived from clinical sputum cultures and maintained in glycerol frozen stock suspensions. Initial identification was performed at the Burkholderia cepacia Research Laboratory and Repository, Michigan and University of Gent, Belgium (Jørgensen et al., 2003). This study was approved by the committee for human research at University of California, San Francisco. Consent for publication of this study was obtained from patient's next of kin. 2.2. Genomic sequencing and analysis More than 10 μg of DNA was extracted from pure bacterial colonies grown on blood agar plates of each of the 3 isolates using the DNA EZ1
http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013 0732-8893/© 2016 Elsevier Inc. All rights reserved.
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Tissue kit (Qiagen, Valencia, CA). For each isolate, 1 μg of genomic DNA was used for sample preparation using the Nextera mate-pair kit or Oxford Nanopore Genomic DNA MAP-003 kit (Oxford Nanopore, Oxford, United Kingdom), both following manufacturer instructions. For isolates TF81F4 and TF80G25, 2.5 ng of genomic DNA was used as input for Nextera XT sample preparation (Illumina, San Diego, CA), with all reactions prepared at half the specified volume. Barcoded Illumina pairedend sequencing was performed using a 2x80bp run on the MiSeq or 2x125bp run on the HiSeq 2500. Sequences were adapter- and quality-filtered (q30) and assemblies were performed using SPAdes v3.5 (St. Petersburg Academic University of the Russian Academy of Sciences, St. Petersburg, Russia), with annotation by Prokka v1.10 (Monash University, Melbourne, Australia), and visualization in Geneious v8.0 (Biomatters, Inc., Newark, NJ) (Bankevich et al., 2012; Seemann, 2014). Alignments were performed using MUSCLE or LASTZ with default parameters and phylogenies were generated using MrBayes v3.2.2 (Swedish Museum of Natural History, Stockholm, Sweden) with default parameters (Edgar, 2004; Ronquist et al., 2012). Genomic ring plots were prepared using BLAST Ring Image Generator (Alikhan et al., 2011). Single nucleotide polymorphisms (SNPs) were called by remapping the reads for each sample to the TF81F4 genome and required minimum coverage 5× with minimum variant frequency of 75%. Polymerase chain reaction (PCR) confirmation of low coverage and repeat sequences from TF81F4 was performed using primers in Table S1 and submitted for Sanger sequencing. 2.3. Accession numbers The complete genomes of P. apista TF80G25 (CP011279), TF81F4 (CP010518), and AU2161 (CP011501) have been deposited in Genbank. 3. Results 3.1. Case summary The patient was a 21-year-old male with advanced cystic fibrosis complicated by insulin-dependent pancreatic insufficiency resulting in iron-deficiency anemia and malnutrition with multiple hospitalizations annually for pulmonary exacerbations. At his initial University of California, San Francisco (UCSF) evaluation in March 1999, he was considered a potential candidate for lung transplant and added to the transplant waiting list. However, his subsequent medical history was complicated by a series of pulmonary exacerbations and pneumonias accompanied by the isolation of highly resistant organisms (Table S2). In July 1999, sputum cultures were positive for Staphylococcus aureus, a mucoid strain of Pseudomonas aeruginosa and P. apista (isolate TF80G25). The P. apista isolate was found to be susceptible to imipenem only among tested antibiotics. However, the patient responded to treatment with ceftazidime and tobramycin to which the P. aeruginosa and S. aureus were sensitive. In August 1999, he was readmitted with recurrent pneumonia and treated with imipenem and cilastatin for highly resistant Pseudomonas infection diagnosed at an outside hospital. He was again admitted in March 2000 and was treated with ceftazidime and tobramycin when cultures grew out pansensitive P. aeruginosa. During the subsequent hospitalization in April 2000, cultures grew 2 morphologies of P. aeruginosa sensitive only to ciprofloxacin or tobramycin, respectively, one of which was a mucoid strain, and P. apista (isolate TF81F4), again sensitive to imipenem only. He was treated with ciprofloxacin and tobramycin and discharged on home oxygen. He was readmitted twice in June 2000 and treated with cefepime, imipenem, and tobramycin for highly resistant Pseudomonas during the first admission and with ciprofloxaxcin, tobramycin, imipenem, colistin, and itraconazole for Pseudomonas and Aspergillus fumigatus during the second admission. P. apista was not isolated from cultures during the first admission, but was seen during the second admission in June (isolate AU2161). During his final admission in September 2000, he was treated
with multiple antibiotics and itraconazole with continued culture of resistant Pseudomonas and A. fumigatus. He developed progressive renal failure and septic physiology, and expired after withdrawal of medications to support his blood pressure. 3.2. Genome descriptions The de novo assembled chromosome of P. apista isolate TF81F4 from April 2000 using Illumina and Sanger sequencing is 5,582,097 base pairs (bp) with a GC content of 62.6% and average coverage of 86×. It contains 3 ribosomal RNA loci along with 68 tRNA loci, for a total of 5047 genes with 4972 coding DNA sequences. Comparison to P. apista strain DSM 16535 (CP013481), the only other complete genome assembly for this species, showed 94.0% pairwise identity, with 100% identity for 16S ribosomal RNA gene and 99.7% identity to gyrB. De novo assembly of P. apista isolate TF80G25 using only Illumina paired-end sequencing yielded 47 contigs of length greater than 200 bps for a total of 5,536,219 bp and N50 statistic of 276,426 bp. Gaps in the initial de novo assembly alignment were located in known integrase/transposase genes or low complexity, GC-rich regions and thus likely due to limitations of the paired-end-only sequence assembly. To bridge remaining gaps in the assembly, we also sequenced the TF80G25 isolate using third-generation nanopore sequencing from Oxford Nanopore. High-quality 2D “passing” reads (n = 2481) and “failing” reads (n = 596) were aligned to the TF80G25 draft genome using the LASTZ aligner. The additional Nanopore reads resulted in bridging of all gaps except for a single duplicated 99-bp region of an allantoicase gene. Error correction of spanning Nanopore reads and filling in this last remaining gap were performed by iterative remapping Illumina reads to the draft complete genome, yielding a single chromosomal scaffold for the TF80G25 genome with length of 5,609,637 bp with 5062 genes and 4987 coding sequences and average coverage of 277×. Isolate AU2161 (June 2000) was sequenced using Illumina pairedend sequencing and Nanopore sequencing. De novo assembly of 12,019,354 Nextera XT mate-pair reads with 1239 2D “passing” and 36,081 “failing” Nanopore reads yielded a single genome contig. The total genome size of AU2161 measured 5,574,863 bp with 5031 genes and 4953 coding sequences and average coverage of 118×. 3.3. The P. apista genome reveals novel loci Comparison of the isolate TF81F4 genome of P. apista with other P. species revealed between 75 and 80% nucleotide identity in areas with N10 kb of synteny. Over 90% of the P. apista genome was alignable with other Pandoraea species. Interestingly, genomic regions specific to P. apista that did not align to other Pandoraea species had significantly lowered GC content than the rest of the genome (55% versus 63%) (Fig. 1). These loci contained a number of genes that were variably alignable (30–70%) to bacterial proteins from other Betaproteobacteria genera by Blastx, including a locus from Nitrosomonas sp. containing a N-6 DNA methylase and several phage-related hypothetical proteins, several loci from B. pseudomallei containing a patatin-like phospholipase and additional hypothetical proteins, and a Ralstonia locus containing a lytic transglycosylase. 3.4. Comparison of the case patient isolates with other P. apista strains and Pandoraea species To understand how our isolates compared versus other P. apista strains, we constructed a phylogeny of 16S rRNA and gyrB sequences, the only 2 sequenced genes available from multiple P. apista strains (Fig. 2). Our 3 isolates showed 100% sequence identity with the other P. apista strains by 16S rRNA phylogenetic analysis, confirming that they belonged to this species. Sequence comparisons with the gyrB gene revealed 100% nucleotide identity among the 3 isolates. Other gyrB sequences available for P. apista showed 99.0–99.7% pairwise
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Fig. 1. BLAST ring plot of Pandoraea genomes demonstrates conserved regions within the genera and unique GC-poor loci of P. apista. The 5.58-Mb genome of P. apista isolate TF81F4 was compared with available Pandoraea spp. complete genomes using BRIG (Snell et al., 1993). Pandoraea species are organized by pairwise alignment identity to the P. apista genome from inner ring (most identical) to outer ring (least identical) with GC content plotted in the outermost ring (range, GC 30–80%). The vast majority of the genes in P. apista align to other Pandoraea species with N60% nucleotide identity. Genetic loci that are less likely to be represented in the other Pandoraea species are enriched for lower GC content.
identity for partial sequences (HI2804, HI2801, and HI2752) and full length sequence (DSM 16535).
3.5. The 3 sequenced P. apista genomes from the case patient are closely related The 3 P. apista genomes sequenced here were N99.999% identical by nucleotide. Although quite similar, a number of small differences were noted between the TF80G25 and TF81F4 genomes. A 27.5-kb region that was repeated 3 times in the TF81F4 was present in a copy number consistent with 4 copies in the TF80G25 genome. This region included genes for ornithine cyclodeaminase, fumarate hydratase, pyruvate ferredoxin oxidoreductase, 3 heme ABC transporters, hemin importer ATPbinding subunit, 2 LysR, and 1 AsnC family transcriptional regulators, along with 3 hypothetical proteins. This region was immediately downstream of a penicillin-binding protein 1C and α-2-macroglobulin genes. In addition to the 27.5-kb deletion, an additional 13 small nucleotide differences (single nucleotide polymorphisms or small insertions/deletions) were present between the TF80G25 and TF81F4 isolates (Fig. 3 and Table S3). Point mutations present in or near annotated genes in the TF80G25 genome relative to the TF81F4 genome included P358Q in a periplasmic sensor histidine kinase, A20 V in enoyl-CoA hydratase,
a G2040 A nucleotide change in 2 of the 3 23S rRNA sequences (corresponding to G2067 A in E. coli 23S rRNA), a single nucleotide deletion in a T homopolymer (octamer) immediate upstream of an aminoglycoside acetyltransferase, a 2-bp deletion disrupting the open reading frame for CorC magnesium and cobalt efflux protein, a single nucleotide deletion in a G homopolymer (decamer) disrupting the ORF of a hypothetical protein, a single nucleotide insertion restoring the ORF of a hypothetical YdcF-like protein, and a single nucleotide insertion in a T homopolymer (octamer) restoring the ORF for an acyltransferase family 3 protein. The June 2000 (AU2161) isolate genome demonstrated several additional changes relative to the TF81F4 and TF80G25 isolates. In particular, the AU2161 isolate contained a ~34.5-kb locus not present in either P. apista TF81F4 and TF80G25. This locus included an additional set of rRNA genes as well as genes encoding for fecR, fecI, and pupB ferric citrate transport, filamentous hemagglutinin family outer membrane protein, OPT oligopeptide transporter, methyl viologen resistance protein, penicillin-binding protein G protein, HTH-type transcriptional regulator GabR, a γ-glutamyl transpeptidase, M24 family metallopeptidase, and methionine importer-related genes metP, metQ, and metN (Fig. 3). Where the TF81F4 has 3 repeats each of a 12-kb and 15-kb locus, forming the 27.5-kb locus mentioned above, the AU2161 genome has 2 copies of the 12-kb locus containing pyruvate ferredoxin oxidoreductase, AsnC transcriptional regulator, putative membrane protein, 4
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Fig. 2. Phylogeny of Pandoraea genus. Phylogenies were constructed using available full length sequences from Pandoraea species for 16S rRNA sequence (A) and DNA gyrase B (B). To date, these are the only sequences available for multiple isolates of P. apista. 16S rRNA sequences demonstrated 100% identity to other P. apista strains. For the housekeeping gene gyrB, the 3 isolates from our case study aligned with 100% identity to each other but were 99.0–99.7% identical by nucleotide to other P. apista strains. Scale bar shows number of nucleotide substitutions per site.
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Fig. 3. Comparison of 3 sequentially isolated P. apista genomes isolated over a 12-month period. Compared to the initial isolate from July 1999 (TF80G25), the isolate from April 2000 (TF81F4) contained 13 small mutations (SNP or small insertion/deletion) and one 27.5-kb deletion. The isolate from July 2000 (AU2161) maintained 8 of the 13 small mutations, expanded the deleted region to 42 kb, has an insertion of 34.5 kb, and has an additional 38 small mutations. See Table S3 for detailed list of involved genes.
hemin transporter/binding protein-related genes, and 1 copy of the 15kb locus containing fumarate hydratase, choline dehydrogenase, major facilitator transporter, peptidase M38, tryptophanyl-tRNA synthetase, 2 LysR transcriptional regulators, and 2 hypothetical genes, leading to an overall deletion of 42 kb relative to TF80G25. Relative to TF81F4, the AU2161 isolate contained 50 total nucleotide changes, including P358Q, D359E change in a sensor histidine kinase, an additional G to A transition in the promoter of the aminoglycoside acetyltransferase, a G to C transversion in the promoter of a MarR family transcriptional regulator, a 60-bp deletion in the 3′ end of the tatA preprotein translocase coding sequence, a T to C transition 12-bp upstream of the frataxin gene, a G70R change in 50S ribosomal protein L4, a D1240G in a DNA-dependent RNA-polymerase β, a D423G in a putative membrane protein, a A287 T change in a glycerol-3-phosphate ABC transporter, a P13Q change in the emrB gene, a 588-bp deletion in a bacterial transcriptional activator domain containing protein, a T to G transversion leading to a truncation of the hslU ATP-dependent protease at amino acid 257, a G insertion leading to frame shift and early truncation at amino acid 363 of a 562 aa pentachorophenol monooxygenease, a Y472H change in isocitrate dehydrogenase, a E892K change in flagellar P-ring protein FlgI, a single nucleotide insertion restoring the ORF of a hypothetical YdcFlike protein, a A20 V mutation in enoyl-CoA hydratase, and a L836P change in cyanophycin synthase. The AU2616 isolate had an additional 5 small nucleotide mutations relative to TF81F4 that reverted back to original TF80G25 sequence. These were located within a NhaX stress response promoter region, oatA acyltransferase, phoR alkaline phosphatase synthesis sensor protein, and 2 hypothetical coding sequences with no known homology. The overall changes in the genomes of the 3 isolates were classified as synonymous SNP (single nucleotide polymorphism within a coding region without change in predicted amino acid sequence), nonsynonymous SNP (causing a change in predicted amino acid sequence), intragenic SNP (polymorphism between predicted coding sequences), insertion, and deletion (Table 1). 4. Discussion Here, we report the first bacterial genomes of P. apista associated with multiple episodes of pulmonary exacerbation in a hospitalized patient with advanced cystic fibrosis. Overall, the finding of 3 genetically similar isolates separated by a 12-month interval (July 1999 to July 2000) is consistent with a model in which a specific strain of P. apista chronically colonizes the upper airways of a cystic fibrosis patient.
Notably, Pseduomonas aeruginosa and B. multivorans have been shown to be powerful inhibitors of the growth of P. apista in culture (Costello et al., 2014). Thus, it is possible that the failure to culture P. apista during the 2 interim hospitalizations between July 1999 and April 2000 may be due to suppression by coinfecting P. aeruginosa. The microbiome of the cystic fibrosis lung niche is complex and influenced by a number of factors, including environmental exposures, patient clinical status, and antibiotic treatment, with the relative amounts of each organism changing over periods of time (Green and Jones, 2015). An important observation of this study is that the isolates accumulate and maintain point mutations over time, suggesting clonal bacterial evolution. While we have not fully excluded the possibility of recolonization of the lung from an external environmental source, this sequential accumulation of point mutations in subsequent isolates suggests in vivo strain evolution within the cystic fibrosis lung. Our data suggesting persistent colonization with P. apista are also consistent with a prior study based on repetitive element PCR of 2 cystic fibrosis patients that found that each patient was chronically colonized with their own unique P. apista strain over a period of several years (Atkinson et al., 2006). The closely related genomes of each of these P. apista isolates suggest that the genome is relatively stable in vivo, possibly with increased selective pressure during antibiotic treatment or cocolonization with other organisms. Our data suggest accumulation of approximately 20–250 mutations annually in the patient environment for a mutation rate of 4.5 × 10 −5 to 3.6 × 10 −6 mutations/bp/year, which is similar to the approximately 100 snp/year for P. aeruginosa seen in cystic fibrosis patients (Feliziana et al., 2014). The overall rate of synonymous SNP mutations appears to be lower than that of nonsynonymous changes, indicating that these colonizing organisms are continually functionally adapting to their environment over time. Interestingly, while 9 of the Table 1 Isolate evolution of P. apista isolates over time, shown by number of mutations at individual loci between each genome. Mutation type Synonymous SNP Nonsynonymous SNP Intragenic SNP Insertion Deletion Total
TF80G25 → TF81F4
TF81F4 → AU2161
AU2616 reversions to TF80G25
1 4
3 16
0 1
3 3 3 14
5 16 10 50
1 2 1 5
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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14 of mutations acquired in the TF81F4 stain were maintained over the course of several months in AU2161 isolate, 5 mutations in the TF81F4 isolate were seen later to revert to the original sequence, possibly indicating selective pressure at these loci. It is also possible that there are multiple strain lineages present in vivo, only some of which are isolated via culture, with occasional amplification or extinction of strain lineages that may share a variable number of mutations between them. Regional diversification in spatially isolated bacterial populations occurs for P. aeruginosa in the cystic fibrosis lung (Jorth et al., 2015), although our analysis of sputum culture isolates cannot address the original location within the lung of the P. apista isolates. It will require sequencing of substantial numbers of patient isolates and individual culture plate colonies in future studies to fully elucidate the factors affecting the mutation rate and strain relatedness over time. The main limitation of this study is the restriction to a single, longitudinal case report. While the first 3 genomes of P. apista will be helpful for future comparative genomic studies, without more outgroup data we cannot make conclusions about the genomic diversity within P. apista. Our study also cannot resolve questions about the potential pathogenicity of P. apista in cystic fibrosis patients. A Danish case series of 6 cystic fibrosis patients infected with P. apista at the same time documented a decline in their lung function after they were colonized with P. apista (Jørgensen et al., 2003). However, all patients were also chronically infected with P. aeruginosa at the same time they were infected with P. apista. Similarly, in our patient, sputum cultures were consistently positive for P. aeruginosa, and occasionally positive for A. fumigatus during each of his 4 hospitalizations from July 1999 to July 2000, and these may have been the major contributors to clinical symptoms. Coinfection with P. aeruginosa is commonly seen in patients colonized with P. apista (Atkinson et al., 2006; Jørgensen et al., 2003). The taxonomic relatedness of P. apista to members of the genus Burkholderia and the capacity of P. apista, like Burkholderia species, for disseminated bloodstream infection raises concerning questions regarding its potential pathogenicity. Cystic fibrosis patients can rapidly succumb to a fatal bacteremic illness caused by B. cepacia (“cepacia syndrome”) (Govan et al., 2007; Isles et al., 1984), whereas P. aeruginosa, rarely, if ever, causes bacteremia. Furthermore, cystic fibrosis patients infected or coinfected with B. cepacia have a substantially worse prognosis than those infected with P. aeruginosa alone (Muhdi et al., 1996; Whiteford et al., 1995). The practical importance of B. cepacia colonization in cystic fibrosis patients lies in the fact that many centers will refuse to perform lung transplantation on patients known to harbor B. cepacia because of high mortality rates associated with subsequent pneumonia and sepsis posttransplant (Aris et al., 2001; Snell et al., 1993). It remains to be seen whether P. apista chronic colonization and propensity for invasive bloodstream infection would also be a marker of poor outcome after transplantation, especially if the organism is highly resistant to antibiotics, as was the case here.
5. Conclusions The complete genome sequences of 3 P. apista isolates cultured from a cystic fibrosis patient over a 12-month period demonstrate N99.999% nucleotide identity, consistent with a model of chronic colonization with clonal evolution over time. A combination of first-, second- and third-generation sequencing methods enabled this first description of multiple assembled P. apista genomes, which have b80% identity to
other Pandoreae species and many undescribed loci. These isolates accumulated approximately 20–250 mutations annually, with the involved gene candidates for response to selective pressures of antibiotic treatment and competing species within the cystic fibrosis lung niche. Supplementary data to this article can be found online at doi:10. 1016/j.diagmicrobio.2016.10.013. Acknowledgments We would like to thank the members of the UCSF clinical microbiology laboratory for initial clinical isolate characterization and isolate preservation. Funding: this work was supported by the UCSF Department of Laboratory Medicine. References Alikhan N-F, Petty NK, Zakour NLB, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 2011;12:402. Aris RM, Routh JC, LiPuma JJ, Heath DG, Gilligan PH. Lung transplantation for cystic fibrosis patients with Burkholderia cepacia complex. Survival linked to genomovar type. Am J Respir Crit Care Med 2001;164:2102-2106. Atkinson RM, Lipuma JJ, Rosenbluth DB, Dunne WM. Chronic colonization with Pandoraea apista in cystic fibrosis patients determined by repetitive-element-sequence PCR. J Clin Microbiol 2006;44:833–836. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012;19:455–477. Caraher E, Collins J, Herbert G, Murphy PG, Gallagher CG, Crowe MJ, et al. Evaluation of in vitro virulence characteristics of the genus Pandoraea in lung epithelial cells. J Med Microbiol 2008;57:15–20. Chan K-G, Yin W-F, Goh S-Y. Complete genome sequence of Pandoraea pnomenusa 3kgm, a quorum-sensing strain isolated from a former landfill site. Genome Announc 2014; 2:e00427-14. Coenye T, Falsen E, Hoste B, Ohlén M, Goris J, Govan JR, et al. Description of Pandoraea gen. nov. with Pandoraea apista sp. nov., Pandoraea pulmonicola sp. nov., Pandoraea pnomenusa sp. nov., Pandoraea sputorum sp. nov. and Pandoraea norimbergensis comb. nov. Int J Syst Evol Microbiol 2000;50(Pt 2):887–899. Costello A, Reen FJ, O'Gara F, Callaghan M, McClean S. Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans. Microbiology 2014;160:1474-1487. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004;32:1792-1797. Feliziana S, Marvig RL, Lujan AM, Moyano AJ, Di Rienzo JA, Johansen HK, et al. Coexistence and within-host evolution of diversified lineages of hypermutable Pseudomonas aeruginosa in long-term cystic fibrosis infections. PLoS Genet 2014;10:e1004651. Govan JRW, Brown AR, Jones AM. Evolving epidemiology of Pseudomonas aeruginosa and the Burkholderia cepacia complex in cystic fibrosis lung infection. Future Microbiol 2007;2:153–164. Green H, Jones AM. The microbiome and emerging pathogens in cystic fibrosis and noncystic fibrosis bronchiectasis. Semin Respir Crit Care Med 2015;36:225–235. Isles A, Maclusky I, Corey M, Gold R, Prober C, Fleming P, et al. Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J Pediatr 1984;104:206–210. Jørgensen IM, Johansen HK, Frederiksen B, Pressler T, Hansen A, Vandamme P, et al. Epidemic spread of Pandoraea apista, a new pathogen causing severe lung disease in cystic fibrosis patients. Pediatr Pulmonol 2003;36:439–446. Jorth P, Staudinger BJ, X W, Hisert KB, Hayden H, Garudathri J, et al. Regional isolation drives bacterial diversification within cystic fibrosis lungs. Cell Host Microbe 2015. http://dx.doi.org/10.1016/j.chom.2015.07.006. [in press]. Muhdi K, Edenborough FP, Gumery L, O'Hickey S, Smith EG, Smith DL, et al. Outcome for patients colonised with Burkholderia cepacia in a Birmingham adult cystic fibrosis clinic and the end of an epidemic. Thorax 1996;51:374–377. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 2012;61:539–542. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics (Oxf Engl) 2014;30:2068-2069. Snell GI, de Hoyos A, Krajden M, Winton T, Maurer JR. Pseudomonas cepacia in lung transplant recipients with cystic fibrosis. Chest 1993;103:466–471. Whiteford ML, Wilkinson JD, McColl JH, Conlon FM, Michie JR, Evans TJ, et al. Outcome of Burkholderia (Pseudomonas) cepacia colonisation in children with cystic fibrosis following a hospital outbreak. Thorax 1995;50:1194-1198.
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic colonization with in vivo strain evolution over time Alexander L. Greninger a,b,c, Jessica Streithorst a, Jeffrey A. Golden d, Charles Y. Chiu a,b,e, Steve Miller a,b,⁎ a
Department of Laboratory Medicine, University of California San Francisco, San Francisco, CA, USA UCSF-Abbott Viral Diagnostics and Discovery Center, San Francisco, CA, USA c Department of Laboratory Medicine, University of Washington, Seattle, WA, USA d Department of Medicine, Division of Pulmonology, University of California San Francisco, San Francisco, CA, USA e Department of Medicine, Division of Infectious Diseases, University of California San Francisco, San Francisco, CA, USA b
a r t i c l e
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Article history: Received 31 August 2016 Received in revised form 7 October 2016 Accepted 7 October 2016 Available online xxxx Keywords: Pandoraea apista Genome sequence Strain evolution
a b s t r a c t Pandoraea apista in the family Burkholderiaceae is an emerging opportunistic pathogen in cystic fibrosis patients. Here, we describe a case from which 3 separate isolates of P. apista were recovered over a 1-year period. Using a combination of first-, second-, and third-generation sequencing technologies, we sequenced and de novo assembled the complete genomes of these 3 P. apista isolates. The genome of P. apista TF81F4 sequenced in this study was 5.58 Mb with a GC% of 62.3%, differed in sequence from other Pandoraea species by N20%, and included a number of previously undescribed loci. Three P. apista isolates cultured over a 12-month period were N99.999% identical by nucleotide, consistent with a model of chronic colonization by a single strain. Over time, the isolates accumulated point mutations, deletions, and insertions in a stepwise fashion, indicating in vivo strain evolution within the cystic fibrosis lung niche. © 2016 Elsevier Inc. All rights reserved.
1. Introduction Pandoraea is a genus of gram-negative nonfermenting bacteria in the family Burkholderiaceae and contains the emerging pathogens P. apista, P. pulmonicola, P. sputorum, and P. pnomenusa. All of these Pandoraea species have been associated with lung infections in cystic fibrosis patients. The genus was originally described in 2000 on the basis of polyphasic taxonomy integrating unique housekeeping gene sequence alignments and biochemical profiles of putative Burkholderia and Ralstonia spp. isolated from the sputa of cystic fibrosis patients across a broad geographic range (Coenye et al., 2000). Notably, Pandoraea species have the potential for invasion, with bloodstream infections being one of the most common sources of isolation, and have also been shown to translocate across cultures of lung epithelial cells and to induce a strong proinflammatory response (Caraher et al., 2008). The name Pandoraea was chosen to refer to Pandora's box given the surprising genetic and biochemical diversity of the members within the genus (Coenye et al., 2000). The diversity of the genus merits a genomic approach to better understand the full genetic basis of this diversity as well as evolution of the genus and contributions to pathogenicity. To date, complete genomes have been assembled for strains of P. pnomenusa, P. sputorum, P. pulmonicola, and P. apista isolated from ⁎ Corresponding author. Tel.: +1-415-353-9630. E-mail address: [email protected] (S. Miller).
human infections (Chan et al., 2014). Here, we describe a clinical case from which 3 isolates of P. apista were recovered over a 12-month interval. We leveraged a combination of Sanger (first generation), Illumina (second generation), and Oxford Nanopore (third generation) technologies to sequence the complete genome of all 3 isolates, revealing that they are nearly completely identical to each other but dissimilar from other P. apista strains within locally sequenced regions or other Pandoraea species. 2. Materials and methods 2.1. Bacterial isolates Bacterial isolates were derived from clinical sputum cultures and maintained in glycerol frozen stock suspensions. Initial identification was performed at the Burkholderia cepacia Research Laboratory and Repository, Michigan and University of Gent, Belgium (Jørgensen et al., 2003). This study was approved by the committee for human research at University of California, San Francisco. Consent for publication of this study was obtained from patient's next of kin. 2.2. Genomic sequencing and analysis More than 10 μg of DNA was extracted from pure bacterial colonies grown on blood agar plates of each of the 3 isolates using the DNA EZ1
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Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Tissue kit (Qiagen, Valencia, CA). For each isolate, 1 μg of genomic DNA was used for sample preparation using the Nextera mate-pair kit or Oxford Nanopore Genomic DNA MAP-003 kit (Oxford Nanopore, Oxford, United Kingdom), both following manufacturer instructions. For isolates TF81F4 and TF80G25, 2.5 ng of genomic DNA was used as input for Nextera XT sample preparation (Illumina, San Diego, CA), with all reactions prepared at half the specified volume. Barcoded Illumina pairedend sequencing was performed using a 2x80bp run on the MiSeq or 2x125bp run on the HiSeq 2500. Sequences were adapter- and quality-filtered (q30) and assemblies were performed using SPAdes v3.5 (St. Petersburg Academic University of the Russian Academy of Sciences, St. Petersburg, Russia), with annotation by Prokka v1.10 (Monash University, Melbourne, Australia), and visualization in Geneious v8.0 (Biomatters, Inc., Newark, NJ) (Bankevich et al., 2012; Seemann, 2014). Alignments were performed using MUSCLE or LASTZ with default parameters and phylogenies were generated using MrBayes v3.2.2 (Swedish Museum of Natural History, Stockholm, Sweden) with default parameters (Edgar, 2004; Ronquist et al., 2012). Genomic ring plots were prepared using BLAST Ring Image Generator (Alikhan et al., 2011). Single nucleotide polymorphisms (SNPs) were called by remapping the reads for each sample to the TF81F4 genome and required minimum coverage 5× with minimum variant frequency of 75%. Polymerase chain reaction (PCR) confirmation of low coverage and repeat sequences from TF81F4 was performed using primers in Table S1 and submitted for Sanger sequencing. 2.3. Accession numbers The complete genomes of P. apista TF80G25 (CP011279), TF81F4 (CP010518), and AU2161 (CP011501) have been deposited in Genbank. 3. Results 3.1. Case summary The patient was a 21-year-old male with advanced cystic fibrosis complicated by insulin-dependent pancreatic insufficiency resulting in iron-deficiency anemia and malnutrition with multiple hospitalizations annually for pulmonary exacerbations. At his initial University of California, San Francisco (UCSF) evaluation in March 1999, he was considered a potential candidate for lung transplant and added to the transplant waiting list. However, his subsequent medical history was complicated by a series of pulmonary exacerbations and pneumonias accompanied by the isolation of highly resistant organisms (Table S2). In July 1999, sputum cultures were positive for Staphylococcus aureus, a mucoid strain of Pseudomonas aeruginosa and P. apista (isolate TF80G25). The P. apista isolate was found to be susceptible to imipenem only among tested antibiotics. However, the patient responded to treatment with ceftazidime and tobramycin to which the P. aeruginosa and S. aureus were sensitive. In August 1999, he was readmitted with recurrent pneumonia and treated with imipenem and cilastatin for highly resistant Pseudomonas infection diagnosed at an outside hospital. He was again admitted in March 2000 and was treated with ceftazidime and tobramycin when cultures grew out pansensitive P. aeruginosa. During the subsequent hospitalization in April 2000, cultures grew 2 morphologies of P. aeruginosa sensitive only to ciprofloxacin or tobramycin, respectively, one of which was a mucoid strain, and P. apista (isolate TF81F4), again sensitive to imipenem only. He was treated with ciprofloxacin and tobramycin and discharged on home oxygen. He was readmitted twice in June 2000 and treated with cefepime, imipenem, and tobramycin for highly resistant Pseudomonas during the first admission and with ciprofloxaxcin, tobramycin, imipenem, colistin, and itraconazole for Pseudomonas and Aspergillus fumigatus during the second admission. P. apista was not isolated from cultures during the first admission, but was seen during the second admission in June (isolate AU2161). During his final admission in September 2000, he was treated
with multiple antibiotics and itraconazole with continued culture of resistant Pseudomonas and A. fumigatus. He developed progressive renal failure and septic physiology, and expired after withdrawal of medications to support his blood pressure. 3.2. Genome descriptions The de novo assembled chromosome of P. apista isolate TF81F4 from April 2000 using Illumina and Sanger sequencing is 5,582,097 base pairs (bp) with a GC content of 62.6% and average coverage of 86×. It contains 3 ribosomal RNA loci along with 68 tRNA loci, for a total of 5047 genes with 4972 coding DNA sequences. Comparison to P. apista strain DSM 16535 (CP013481), the only other complete genome assembly for this species, showed 94.0% pairwise identity, with 100% identity for 16S ribosomal RNA gene and 99.7% identity to gyrB. De novo assembly of P. apista isolate TF80G25 using only Illumina paired-end sequencing yielded 47 contigs of length greater than 200 bps for a total of 5,536,219 bp and N50 statistic of 276,426 bp. Gaps in the initial de novo assembly alignment were located in known integrase/transposase genes or low complexity, GC-rich regions and thus likely due to limitations of the paired-end-only sequence assembly. To bridge remaining gaps in the assembly, we also sequenced the TF80G25 isolate using third-generation nanopore sequencing from Oxford Nanopore. High-quality 2D “passing” reads (n = 2481) and “failing” reads (n = 596) were aligned to the TF80G25 draft genome using the LASTZ aligner. The additional Nanopore reads resulted in bridging of all gaps except for a single duplicated 99-bp region of an allantoicase gene. Error correction of spanning Nanopore reads and filling in this last remaining gap were performed by iterative remapping Illumina reads to the draft complete genome, yielding a single chromosomal scaffold for the TF80G25 genome with length of 5,609,637 bp with 5062 genes and 4987 coding sequences and average coverage of 277×. Isolate AU2161 (June 2000) was sequenced using Illumina pairedend sequencing and Nanopore sequencing. De novo assembly of 12,019,354 Nextera XT mate-pair reads with 1239 2D “passing” and 36,081 “failing” Nanopore reads yielded a single genome contig. The total genome size of AU2161 measured 5,574,863 bp with 5031 genes and 4953 coding sequences and average coverage of 118×. 3.3. The P. apista genome reveals novel loci Comparison of the isolate TF81F4 genome of P. apista with other P. species revealed between 75 and 80% nucleotide identity in areas with N10 kb of synteny. Over 90% of the P. apista genome was alignable with other Pandoraea species. Interestingly, genomic regions specific to P. apista that did not align to other Pandoraea species had significantly lowered GC content than the rest of the genome (55% versus 63%) (Fig. 1). These loci contained a number of genes that were variably alignable (30–70%) to bacterial proteins from other Betaproteobacteria genera by Blastx, including a locus from Nitrosomonas sp. containing a N-6 DNA methylase and several phage-related hypothetical proteins, several loci from B. pseudomallei containing a patatin-like phospholipase and additional hypothetical proteins, and a Ralstonia locus containing a lytic transglycosylase. 3.4. Comparison of the case patient isolates with other P. apista strains and Pandoraea species To understand how our isolates compared versus other P. apista strains, we constructed a phylogeny of 16S rRNA and gyrB sequences, the only 2 sequenced genes available from multiple P. apista strains (Fig. 2). Our 3 isolates showed 100% sequence identity with the other P. apista strains by 16S rRNA phylogenetic analysis, confirming that they belonged to this species. Sequence comparisons with the gyrB gene revealed 100% nucleotide identity among the 3 isolates. Other gyrB sequences available for P. apista showed 99.0–99.7% pairwise
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Fig. 1. BLAST ring plot of Pandoraea genomes demonstrates conserved regions within the genera and unique GC-poor loci of P. apista. The 5.58-Mb genome of P. apista isolate TF81F4 was compared with available Pandoraea spp. complete genomes using BRIG (Snell et al., 1993). Pandoraea species are organized by pairwise alignment identity to the P. apista genome from inner ring (most identical) to outer ring (least identical) with GC content plotted in the outermost ring (range, GC 30–80%). The vast majority of the genes in P. apista align to other Pandoraea species with N60% nucleotide identity. Genetic loci that are less likely to be represented in the other Pandoraea species are enriched for lower GC content.
identity for partial sequences (HI2804, HI2801, and HI2752) and full length sequence (DSM 16535).
3.5. The 3 sequenced P. apista genomes from the case patient are closely related The 3 P. apista genomes sequenced here were N99.999% identical by nucleotide. Although quite similar, a number of small differences were noted between the TF80G25 and TF81F4 genomes. A 27.5-kb region that was repeated 3 times in the TF81F4 was present in a copy number consistent with 4 copies in the TF80G25 genome. This region included genes for ornithine cyclodeaminase, fumarate hydratase, pyruvate ferredoxin oxidoreductase, 3 heme ABC transporters, hemin importer ATPbinding subunit, 2 LysR, and 1 AsnC family transcriptional regulators, along with 3 hypothetical proteins. This region was immediately downstream of a penicillin-binding protein 1C and α-2-macroglobulin genes. In addition to the 27.5-kb deletion, an additional 13 small nucleotide differences (single nucleotide polymorphisms or small insertions/deletions) were present between the TF80G25 and TF81F4 isolates (Fig. 3 and Table S3). Point mutations present in or near annotated genes in the TF80G25 genome relative to the TF81F4 genome included P358Q in a periplasmic sensor histidine kinase, A20 V in enoyl-CoA hydratase,
a G2040 A nucleotide change in 2 of the 3 23S rRNA sequences (corresponding to G2067 A in E. coli 23S rRNA), a single nucleotide deletion in a T homopolymer (octamer) immediate upstream of an aminoglycoside acetyltransferase, a 2-bp deletion disrupting the open reading frame for CorC magnesium and cobalt efflux protein, a single nucleotide deletion in a G homopolymer (decamer) disrupting the ORF of a hypothetical protein, a single nucleotide insertion restoring the ORF of a hypothetical YdcF-like protein, and a single nucleotide insertion in a T homopolymer (octamer) restoring the ORF for an acyltransferase family 3 protein. The June 2000 (AU2161) isolate genome demonstrated several additional changes relative to the TF81F4 and TF80G25 isolates. In particular, the AU2161 isolate contained a ~34.5-kb locus not present in either P. apista TF81F4 and TF80G25. This locus included an additional set of rRNA genes as well as genes encoding for fecR, fecI, and pupB ferric citrate transport, filamentous hemagglutinin family outer membrane protein, OPT oligopeptide transporter, methyl viologen resistance protein, penicillin-binding protein G protein, HTH-type transcriptional regulator GabR, a γ-glutamyl transpeptidase, M24 family metallopeptidase, and methionine importer-related genes metP, metQ, and metN (Fig. 3). Where the TF81F4 has 3 repeats each of a 12-kb and 15-kb locus, forming the 27.5-kb locus mentioned above, the AU2161 genome has 2 copies of the 12-kb locus containing pyruvate ferredoxin oxidoreductase, AsnC transcriptional regulator, putative membrane protein, 4
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Fig. 2. Phylogeny of Pandoraea genus. Phylogenies were constructed using available full length sequences from Pandoraea species for 16S rRNA sequence (A) and DNA gyrase B (B). To date, these are the only sequences available for multiple isolates of P. apista. 16S rRNA sequences demonstrated 100% identity to other P. apista strains. For the housekeeping gene gyrB, the 3 isolates from our case study aligned with 100% identity to each other but were 99.0–99.7% identical by nucleotide to other P. apista strains. Scale bar shows number of nucleotide substitutions per site.
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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Fig. 3. Comparison of 3 sequentially isolated P. apista genomes isolated over a 12-month period. Compared to the initial isolate from July 1999 (TF80G25), the isolate from April 2000 (TF81F4) contained 13 small mutations (SNP or small insertion/deletion) and one 27.5-kb deletion. The isolate from July 2000 (AU2161) maintained 8 of the 13 small mutations, expanded the deleted region to 42 kb, has an insertion of 34.5 kb, and has an additional 38 small mutations. See Table S3 for detailed list of involved genes.
hemin transporter/binding protein-related genes, and 1 copy of the 15kb locus containing fumarate hydratase, choline dehydrogenase, major facilitator transporter, peptidase M38, tryptophanyl-tRNA synthetase, 2 LysR transcriptional regulators, and 2 hypothetical genes, leading to an overall deletion of 42 kb relative to TF80G25. Relative to TF81F4, the AU2161 isolate contained 50 total nucleotide changes, including P358Q, D359E change in a sensor histidine kinase, an additional G to A transition in the promoter of the aminoglycoside acetyltransferase, a G to C transversion in the promoter of a MarR family transcriptional regulator, a 60-bp deletion in the 3′ end of the tatA preprotein translocase coding sequence, a T to C transition 12-bp upstream of the frataxin gene, a G70R change in 50S ribosomal protein L4, a D1240G in a DNA-dependent RNA-polymerase β, a D423G in a putative membrane protein, a A287 T change in a glycerol-3-phosphate ABC transporter, a P13Q change in the emrB gene, a 588-bp deletion in a bacterial transcriptional activator domain containing protein, a T to G transversion leading to a truncation of the hslU ATP-dependent protease at amino acid 257, a G insertion leading to frame shift and early truncation at amino acid 363 of a 562 aa pentachorophenol monooxygenease, a Y472H change in isocitrate dehydrogenase, a E892K change in flagellar P-ring protein FlgI, a single nucleotide insertion restoring the ORF of a hypothetical YdcFlike protein, a A20 V mutation in enoyl-CoA hydratase, and a L836P change in cyanophycin synthase. The AU2616 isolate had an additional 5 small nucleotide mutations relative to TF81F4 that reverted back to original TF80G25 sequence. These were located within a NhaX stress response promoter region, oatA acyltransferase, phoR alkaline phosphatase synthesis sensor protein, and 2 hypothetical coding sequences with no known homology. The overall changes in the genomes of the 3 isolates were classified as synonymous SNP (single nucleotide polymorphism within a coding region without change in predicted amino acid sequence), nonsynonymous SNP (causing a change in predicted amino acid sequence), intragenic SNP (polymorphism between predicted coding sequences), insertion, and deletion (Table 1). 4. Discussion Here, we report the first bacterial genomes of P. apista associated with multiple episodes of pulmonary exacerbation in a hospitalized patient with advanced cystic fibrosis. Overall, the finding of 3 genetically similar isolates separated by a 12-month interval (July 1999 to July 2000) is consistent with a model in which a specific strain of P. apista chronically colonizes the upper airways of a cystic fibrosis patient.
Notably, Pseduomonas aeruginosa and B. multivorans have been shown to be powerful inhibitors of the growth of P. apista in culture (Costello et al., 2014). Thus, it is possible that the failure to culture P. apista during the 2 interim hospitalizations between July 1999 and April 2000 may be due to suppression by coinfecting P. aeruginosa. The microbiome of the cystic fibrosis lung niche is complex and influenced by a number of factors, including environmental exposures, patient clinical status, and antibiotic treatment, with the relative amounts of each organism changing over periods of time (Green and Jones, 2015). An important observation of this study is that the isolates accumulate and maintain point mutations over time, suggesting clonal bacterial evolution. While we have not fully excluded the possibility of recolonization of the lung from an external environmental source, this sequential accumulation of point mutations in subsequent isolates suggests in vivo strain evolution within the cystic fibrosis lung. Our data suggesting persistent colonization with P. apista are also consistent with a prior study based on repetitive element PCR of 2 cystic fibrosis patients that found that each patient was chronically colonized with their own unique P. apista strain over a period of several years (Atkinson et al., 2006). The closely related genomes of each of these P. apista isolates suggest that the genome is relatively stable in vivo, possibly with increased selective pressure during antibiotic treatment or cocolonization with other organisms. Our data suggest accumulation of approximately 20–250 mutations annually in the patient environment for a mutation rate of 4.5 × 10 −5 to 3.6 × 10 −6 mutations/bp/year, which is similar to the approximately 100 snp/year for P. aeruginosa seen in cystic fibrosis patients (Feliziana et al., 2014). The overall rate of synonymous SNP mutations appears to be lower than that of nonsynonymous changes, indicating that these colonizing organisms are continually functionally adapting to their environment over time. Interestingly, while 9 of the Table 1 Isolate evolution of P. apista isolates over time, shown by number of mutations at individual loci between each genome. Mutation type Synonymous SNP Nonsynonymous SNP Intragenic SNP Insertion Deletion Total
TF80G25 → TF81F4
TF81F4 → AU2161
AU2616 reversions to TF80G25
1 4
3 16
0 1
3 3 3 14
5 16 10 50
1 2 1 5
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013
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14 of mutations acquired in the TF81F4 stain were maintained over the course of several months in AU2161 isolate, 5 mutations in the TF81F4 isolate were seen later to revert to the original sequence, possibly indicating selective pressure at these loci. It is also possible that there are multiple strain lineages present in vivo, only some of which are isolated via culture, with occasional amplification or extinction of strain lineages that may share a variable number of mutations between them. Regional diversification in spatially isolated bacterial populations occurs for P. aeruginosa in the cystic fibrosis lung (Jorth et al., 2015), although our analysis of sputum culture isolates cannot address the original location within the lung of the P. apista isolates. It will require sequencing of substantial numbers of patient isolates and individual culture plate colonies in future studies to fully elucidate the factors affecting the mutation rate and strain relatedness over time. The main limitation of this study is the restriction to a single, longitudinal case report. While the first 3 genomes of P. apista will be helpful for future comparative genomic studies, without more outgroup data we cannot make conclusions about the genomic diversity within P. apista. Our study also cannot resolve questions about the potential pathogenicity of P. apista in cystic fibrosis patients. A Danish case series of 6 cystic fibrosis patients infected with P. apista at the same time documented a decline in their lung function after they were colonized with P. apista (Jørgensen et al., 2003). However, all patients were also chronically infected with P. aeruginosa at the same time they were infected with P. apista. Similarly, in our patient, sputum cultures were consistently positive for P. aeruginosa, and occasionally positive for A. fumigatus during each of his 4 hospitalizations from July 1999 to July 2000, and these may have been the major contributors to clinical symptoms. Coinfection with P. aeruginosa is commonly seen in patients colonized with P. apista (Atkinson et al., 2006; Jørgensen et al., 2003). The taxonomic relatedness of P. apista to members of the genus Burkholderia and the capacity of P. apista, like Burkholderia species, for disseminated bloodstream infection raises concerning questions regarding its potential pathogenicity. Cystic fibrosis patients can rapidly succumb to a fatal bacteremic illness caused by B. cepacia (“cepacia syndrome”) (Govan et al., 2007; Isles et al., 1984), whereas P. aeruginosa, rarely, if ever, causes bacteremia. Furthermore, cystic fibrosis patients infected or coinfected with B. cepacia have a substantially worse prognosis than those infected with P. aeruginosa alone (Muhdi et al., 1996; Whiteford et al., 1995). The practical importance of B. cepacia colonization in cystic fibrosis patients lies in the fact that many centers will refuse to perform lung transplantation on patients known to harbor B. cepacia because of high mortality rates associated with subsequent pneumonia and sepsis posttransplant (Aris et al., 2001; Snell et al., 1993). It remains to be seen whether P. apista chronic colonization and propensity for invasive bloodstream infection would also be a marker of poor outcome after transplantation, especially if the organism is highly resistant to antibiotics, as was the case here.
5. Conclusions The complete genome sequences of 3 P. apista isolates cultured from a cystic fibrosis patient over a 12-month period demonstrate N99.999% nucleotide identity, consistent with a model of chronic colonization with clonal evolution over time. A combination of first-, second- and third-generation sequencing methods enabled this first description of multiple assembled P. apista genomes, which have b80% identity to
other Pandoreae species and many undescribed loci. These isolates accumulated approximately 20–250 mutations annually, with the involved gene candidates for response to selective pressures of antibiotic treatment and competing species within the cystic fibrosis lung niche. Supplementary data to this article can be found online at doi:10. 1016/j.diagmicrobio.2016.10.013. Acknowledgments We would like to thank the members of the UCSF clinical microbiology laboratory for initial clinical isolate characterization and isolate preservation. Funding: this work was supported by the UCSF Department of Laboratory Medicine. References Alikhan N-F, Petty NK, Zakour NLB, Beatson SA. BLAST Ring Image Generator (BRIG): simple prokaryote genome comparisons. BMC Genomics 2011;12:402. Aris RM, Routh JC, LiPuma JJ, Heath DG, Gilligan PH. Lung transplantation for cystic fibrosis patients with Burkholderia cepacia complex. Survival linked to genomovar type. Am J Respir Crit Care Med 2001;164:2102-2106. Atkinson RM, Lipuma JJ, Rosenbluth DB, Dunne WM. Chronic colonization with Pandoraea apista in cystic fibrosis patients determined by repetitive-element-sequence PCR. J Clin Microbiol 2006;44:833–836. Bankevich A, Nurk S, Antipov D, Gurevich AA, Dvorkin M, Kulikov AS, et al. SPAdes: a new genome assembly algorithm and its applications to single-cell sequencing. J Comput Biol 2012;19:455–477. Caraher E, Collins J, Herbert G, Murphy PG, Gallagher CG, Crowe MJ, et al. Evaluation of in vitro virulence characteristics of the genus Pandoraea in lung epithelial cells. J Med Microbiol 2008;57:15–20. Chan K-G, Yin W-F, Goh S-Y. Complete genome sequence of Pandoraea pnomenusa 3kgm, a quorum-sensing strain isolated from a former landfill site. Genome Announc 2014; 2:e00427-14. Coenye T, Falsen E, Hoste B, Ohlén M, Goris J, Govan JR, et al. Description of Pandoraea gen. nov. with Pandoraea apista sp. nov., Pandoraea pulmonicola sp. nov., Pandoraea pnomenusa sp. nov., Pandoraea sputorum sp. nov. and Pandoraea norimbergensis comb. nov. Int J Syst Evol Microbiol 2000;50(Pt 2):887–899. Costello A, Reen FJ, O'Gara F, Callaghan M, McClean S. Inhibition of co-colonizing cystic fibrosis-associated pathogens by Pseudomonas aeruginosa and Burkholderia multivorans. Microbiology 2014;160:1474-1487. Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004;32:1792-1797. Feliziana S, Marvig RL, Lujan AM, Moyano AJ, Di Rienzo JA, Johansen HK, et al. Coexistence and within-host evolution of diversified lineages of hypermutable Pseudomonas aeruginosa in long-term cystic fibrosis infections. PLoS Genet 2014;10:e1004651. Govan JRW, Brown AR, Jones AM. Evolving epidemiology of Pseudomonas aeruginosa and the Burkholderia cepacia complex in cystic fibrosis lung infection. Future Microbiol 2007;2:153–164. Green H, Jones AM. The microbiome and emerging pathogens in cystic fibrosis and noncystic fibrosis bronchiectasis. Semin Respir Crit Care Med 2015;36:225–235. Isles A, Maclusky I, Corey M, Gold R, Prober C, Fleming P, et al. Pseudomonas cepacia infection in cystic fibrosis: an emerging problem. J Pediatr 1984;104:206–210. Jørgensen IM, Johansen HK, Frederiksen B, Pressler T, Hansen A, Vandamme P, et al. Epidemic spread of Pandoraea apista, a new pathogen causing severe lung disease in cystic fibrosis patients. Pediatr Pulmonol 2003;36:439–446. Jorth P, Staudinger BJ, X W, Hisert KB, Hayden H, Garudathri J, et al. Regional isolation drives bacterial diversification within cystic fibrosis lungs. Cell Host Microbe 2015. http://dx.doi.org/10.1016/j.chom.2015.07.006. [in press]. Muhdi K, Edenborough FP, Gumery L, O'Hickey S, Smith EG, Smith DL, et al. Outcome for patients colonised with Burkholderia cepacia in a Birmingham adult cystic fibrosis clinic and the end of an epidemic. Thorax 1996;51:374–377. Ronquist F, Teslenko M, van der Mark P, Ayres DL, Darling A, Höhna S, et al. MrBayes 3.2: efficient Bayesian phylogenetic inference and model choice across a large model space. Syst Biol 2012;61:539–542. Seemann T. Prokka: rapid prokaryotic genome annotation. Bioinformatics (Oxf Engl) 2014;30:2068-2069. Snell GI, de Hoyos A, Krajden M, Winton T, Maurer JR. Pseudomonas cepacia in lung transplant recipients with cystic fibrosis. Chest 1993;103:466–471. Whiteford ML, Wilkinson JD, McColl JH, Conlon FM, Michie JR, Evans TJ, et al. Outcome of Burkholderia (Pseudomonas) cepacia colonisation in children with cystic fibrosis following a hospital outbreak. Thorax 1995;50:1194-1198.
Please cite this article as: Greninger AL, et al, Complete genome sequence of sequential Pandoraea apista isolates from the same cystic fibrosis patient supports a model of chronic..., Diagn Microbiol Infect Dis (2016), http://dx.doi.org/10.1016/j.diagmicrobio.2016.10.013