1887

Abstract

Several members of the Gram-negative environmental bacterial genus are associated with serious infections, with being the most common. Despite their pathogenic potential, little is understood about these intrinsically drug-resistant bacteria and their role in disease, leading to suboptimal diagnosis and management. Here, we performed comparative genomics for 158 spp. genomes to robustly identify species boundaries, reassign several incorrectly speciated taxa and identify genetic sequences specific for the genus and for . Next, we developed a Black Hole Quencher probe-based duplex real-time PCR assay, Ac-Ax, for the rapid and simultaneous detection of spp. and from both purified colonies and polymicrobial clinical specimens. Ac-Ax was tested on 119 isolates identified as spp. using phenotypic or genotypic methods. In comparison to these routine diagnostic methods, the duplex assay showed superior identification of spp. and , with five isolates failing to amplify with Ac-Ax confirmed to be different genera according to 16S rRNA gene sequencing. Ac-Ax quantified both spp. and down to ~110 genome equivalents and detected down to ~12 and ~1 genome equivalent(s), respectively. Extensive analysis, and laboratory testing of 34 non- isolates and 38 adult cystic fibrosis sputa, confirmed duplex assay specificity and sensitivity. We demonstrate that the Ac-Ax duplex assay provides a robust, sensitive and cost-effective method for the simultaneous detection of all spp. and and will facilitate the rapid and accurate diagnosis of this important group of pathogens.

Funding
This study was supported by the:
  • Erin P. Price , University of the Sunshine Coast
  • Timothy J. Kidd , National Health and Medical Research Council , (Award 1088448)
  • Derek S. Sarovich , Advance Queensland , (Award AQRF13016-17RD2)
  • Erin P. Price , Advance Queensland , (Award AQIRF0362018)
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2020-07-15
2020-10-20
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References

  1. Dumolin C, Peeters C, Ehsani E, Tahon G, De Canck E et al. Achromobacter veterisilvae sp. nov., from a mixed hydrogen-oxidizing bacteria enrichment reactor for microbial protein production. Int J Syst Evol Microbiol 2020; 70:530–536 [CrossRef][PubMed]
    [Google Scholar]
  2. Amoureux L, Bador J, Fardeheb S, Mabille C, Couchot C et al. Detection of Achromobacter xylosoxidans in hospital, domestic, and outdoor environmental samples and comparison with human clinical isolates. Appl Environ Microbiol 2013; 79:7142–7149 [CrossRef][PubMed]
    [Google Scholar]
  3. Nakamoto S, Sakamoto M, Sugimura K, Honmura Y, Yamamoto Y et al. Environmental distribution and drug susceptibility of Achromobacter xylosoxidans isolated from outdoor and indoor environments. Yonago Acta Med 2017; 60:67–70[PubMed]
    [Google Scholar]
  4. Amoureux L, Bador J, Verrier T, Mjahed H, DE Curraize C et al. Achromobacter xylosoxidans is the predominant Achromobacter species isolated from diverse non-respiratory samples. Epidemiol Infect 2016; 144:3527–3530 [CrossRef][PubMed]
    [Google Scholar]
  5. Swenson CE, Sadikot RT. Achromobacter respiratory infections. Ann Am Thorac Soc 2015; 12:252–258 [CrossRef][PubMed]
    [Google Scholar]
  6. Duggan JM, Goldstein SJ, Chenoweth CE, Kauffman CA, Bradley SF. Achromobacter xylosoxidans bacteremia: report of four cases and review of the literature. Clin Infect Dis 1996; 23:569–576 [CrossRef][PubMed]
    [Google Scholar]
  7. Al-Jasser AM, Al-Anazi KA. Complicated septic shock caused by Achromobacter xylosoxidans bacteremia in a patient with acute lymphoblastic leukaemia. Libyan J Med 2007; 2:218–219 [CrossRef][PubMed]
    [Google Scholar]
  8. Molina-Cabrillana J, Santana-Reyes C, González-García A, Bordes-Benítez A, Horcajada I. Outbreak of Achromobacter xylosoxidans pseudobacteremia in a neonatal care unit related to contaminated chlorhexidine solution. Eur J Clin Microbiol Infect Dis 2007; 26:435–437 [CrossRef][PubMed]
    [Google Scholar]
  9. Parkins MD, Floto RA. Emerging bacterial pathogens and changing concepts of bacterial pathogenesis in cystic fibrosis. J Cyst Fibros 2015; 14:293–304 [CrossRef][PubMed]
    [Google Scholar]
  10. Recio R, Brañas P, Martínez MT, Chaves F, Orellana MA. Effect of respiratory Achromobacter spp. infection on pulmonary function in patients with cystic fibrosis. J Med Microbiol 2018; 67:952–956 [CrossRef][PubMed]
    [Google Scholar]
  11. Llorca Otero L, Girón Moreno R, Buendía Moreno B, Valenzuela C, Guiu Martínez A et al. Achromobacter xylosoxidans infection in an adult cystic fibrosis unit in Madrid. Enferm Infecc Microbiol Clin 2016; 34:184–187 [CrossRef][PubMed]
    [Google Scholar]
  12. Edwards BD, Greysson-Wong J, Somayaji R, Waddell B, Whelan FJ et al. Prevalence and outcomes of Achromobacter species infections in adults with cystic fibrosis: a North American cohort study. J Clin Microbiol 2017; 55:2074–2085 [CrossRef][PubMed]
    [Google Scholar]
  13. Qvist T, Taylor-Robinson D, Waldmann E, Olesen HV, Hansen CR et al. Comparing the harmful effects of nontuberculous mycobacteria and Gram negative bacteria on lung function in patients with cystic fibrosis. J Cyst Fibros 2016; 15:380–385 [CrossRef][PubMed]
    [Google Scholar]
  14. Conway SP, Brownlee KG, Denton M, Peckham DG. Antibiotic treatment of multidrug-resistant organisms in cystic fibrosis. Am J Respir Med 2003; 2:321–332 [CrossRef][PubMed]
    [Google Scholar]
  15. Pereira RHV, Leão RS, Carvalho-Assef AP, Albano RM, Rodrigues ERA et al. Patterns of virulence factor expression and antimicrobial resistance in Achromobacter xylosoxidans and Achromobacter ruhlandii isolates from patients with cystic fibrosis. Epidemiol Infect 2017; 145:600–606 [CrossRef][PubMed]
    [Google Scholar]
  16. Emerson J, McNamara S, Buccat AM, Worrell K, Burns JL. Changes in cystic fibrosis sputum microbiology in the United States between 1995 and 2008. Pediatr Pulmonol 2010; 45:363–370 [CrossRef][PubMed]
    [Google Scholar]
  17. Papalia M, Steffanowski C, Traglia G, Almuzara M, Martina P et al. Diversity of Achromobacter species recovered from patients with cystic fibrosis, in Argentina. Rev Argent Microbiol 2020; 52:S0325–7541 [CrossRef][PubMed]
    [Google Scholar]
  18. Coward A, Kenna DTD, Perry C, Martin K, Doumith M et al. Use of nrdA gene sequence clustering to estimate the prevalence of different Achromobacter species among cystic fibrosis patients in the UK. J Cyst Fibros 2016; 15:479–485 [CrossRef][PubMed]
    [Google Scholar]
  19. Spilker T, Vandamme P, Lipuma JJ. Identification and distribution of Achromobacter species in cystic fibrosis. J Cyst Fibros 2013; 12:298–301 [CrossRef][PubMed]
    [Google Scholar]
  20. Rodrigues CG, Rays J, Kanegae MY. Native-valve endocarditis caused by Achromobacter xylosoxidans: a case report and review of literature. Autops Case Rep 2017; 7:50–55 [CrossRef][PubMed]
    [Google Scholar]
  21. Gomila M, Prince-Manzano C, Svensson-Stadler L, Busquets A, Erhard M et al. Genotypic and phenotypic applications for the differentiation and species-level identification of Achromobacter for clinical diagnoses. PLoS One 2014; 9:e114356 [CrossRef][PubMed]
    [Google Scholar]
  22. Marko DC, Saffert RT, Cunningham SA, Hyman J, Walsh J et al. Evaluation of the Bruker Biotyper and Vitek MS matrix-assisted laser desorption ionization-time of flight mass spectrometry systems for identification of nonfermenting gram-negative bacilli isolated from cultures from cystic fibrosis patients. J Clin Microbiol 2012; 50:2034–2039 [CrossRef][PubMed]
    [Google Scholar]
  23. Rocchetti TT, Silbert S, Gostnell A, Kubasek C, Jerris R et al. Rapid detection of four non-fermenting Gram-negative bacteria directly from cystic fibrosis patient's respiratory samples on the BD MAX system. Pract Lab Med 2018; 12:e00102 [CrossRef][PubMed]
    [Google Scholar]
  24. Rocca MF, Barrios R, Zintgraff J, Martínez C, Irazu L et al. Utility of platforms Viteks MS and Microflex LT for the identification of complex clinical isolates that require molecular methods for their taxonomic classification. PLoS One 2019; 14:e0218077 [CrossRef][PubMed]
    [Google Scholar]
  25. McElvania TeKippe E, Burnham C-AD. Evaluation of the Bruker Biotyper and VITEK MS MALDI-TOF MS systems for the identification of unusual and/or difficult-to-identify microorganisms isolated from clinical specimens. Eur J Clin Microbiol Infect Dis 2014; 33:2163–2171 [CrossRef][PubMed]
    [Google Scholar]
  26. Bador J, Amoureux L, Blanc E, Neuwirth C. Innate aminoglycoside resistance of Achromobacter xylosoxidans is due to AxyXY-OprZ, an RND-type multidrug efflux pump. Antimicrob Agents Chemother 2013; 57:603–605 [CrossRef][PubMed]
    [Google Scholar]
  27. Febbraro F, Rodio DM, Puggioni G, Antonelli G, Pietropaolo V et al. MALDI-TOF MS versus VITEK®2: comparison of systems for the identification of microorganisms responsible for bacteremia. Curr Microbiol 2016; 73:843–850 [CrossRef][PubMed]
    [Google Scholar]
  28. Wattal C, Oberoi JK, Goel N, Raveendran R, Khanna S. Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) for rapid identification of micro-organisms in the routine clinical microbiology laboratory. Eur J Clin Microbiol Infect Dis 2017; 36:807–812 [CrossRef][PubMed]
    [Google Scholar]
  29. Segonds C, Heulin T, Marty N, Chabanon G. Differentiation of Burkholderia species by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene and application to cystic fibrosis isolates. J Clin Microbiol 1999; 37:2201–2208 [CrossRef][PubMed]
    [Google Scholar]
  30. Spilker T, Vandamme P, Lipuma JJ. A multilocus sequence typing scheme implies population structure and reveals several putative novel Achromobacter species. J Clin Microbiol 2012; 50:3010–3015 [CrossRef][PubMed]
    [Google Scholar]
  31. Huang W, Li L, Myers JR, Marth GT. ART: a next-generation sequencing read simulator. Bioinformatics 2012; 28:593–594 [CrossRef][PubMed]
    [Google Scholar]
  32. Bolger AM, Lohse M, Usadel B. Trimmomatic: a flexible trimmer for illumina sequence data. Bioinformatics 2014; 30:2114–2120 [CrossRef][PubMed]
    [Google Scholar]
  33. Price EP, Viberg LT, Kidd TJ, Bell SC, Currie BJ et al. Transcriptomic analysis of longitudinal Burkholderia pseudomallei infecting the cystic fibrosis lung. Microb Genom 2018; 4: [CrossRef][PubMed]
    [Google Scholar]
  34. Price EP, Sarovich DS, Webb JR, Ginther JL, Mayo M et al. Accurate and rapid identification of the Burkholderia pseudomallei near-neighbour, Burkholderia ubonensis, using real-time PCR. PLoS One 2013; 8:e71647 [CrossRef][PubMed]
    [Google Scholar]
  35. Price EP, Harris TM, Spargo J, Nosworthy E, Beissbarth J et al. Simultaneous identification of Haemophilus influenzae and Haemophilus haemolyticus using real-time PCR. Future Microbiol 2017; 12:585–593 [CrossRef][PubMed]
    [Google Scholar]
  36. Sarovich DS, Price EP. SPANDx: a genomics pipeline for comparative analysis of large haploid whole genome re-sequencing datasets. BMC Res Notes 2014; 7:618 [CrossRef][PubMed]
    [Google Scholar]
  37. Swofford DL. PAUP*. Phylogenetic Analysis Using Parsimony (*and Other Methods) Sunderland, Massachusetts: Sinauer Associates; 2002
    [Google Scholar]
  38. Quinlan AR, Hall IM. BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 2010; 26:841–842 [CrossRef][PubMed]
    [Google Scholar]
  39. Fraser TA, Bell MG, Harris PNA, Bell SC, Bergh H et al. Quantitative real-time PCR assay for the rapid identification of the intrinsically multidrug-resistant bacterial pathogen Stenotrophomonas maltophilia . Microb Genom 2019; 5:TBA [CrossRef][PubMed]
    [Google Scholar]
  40. Yabuuchi E, Oyama A. Achromobacter xylosoxidans n. sp. from human ear discharge. Jpn J Microbiol 1971; 15:477–481 [CrossRef][PubMed]
    [Google Scholar]
  41. Vandamme P, Moore ERB, Cnockaert M, Peeters C, Svensson-Stadler L et al. Classification of Achromobacter genogroups 2, 5, 7 and 14 as Achromobacter insuavis sp. nov., Achromobacter aegrifaciens sp. nov., Achromobacter anxifer sp. nov. and Achromobacter dolens sp. nov., respectively. Syst Appl Microbiol 2013; 36:474–482 [CrossRef][PubMed]
    [Google Scholar]
  42. Packer L, Vishniac W. Hydrogen metabolism in a Hydrogenomonas sp. Bacteriol Proc 1954111–112
    [Google Scholar]
  43. Jeukens J, Freschi L, Vincent AT, Emond-Rheault J-G, Kukavica-Ibrulj I et al. A pan-genomic approach to understand the basis of host adaptation in Achromobacter . Genome Biol Evol 2017; 9:10301046 [CrossRef][PubMed]
    [Google Scholar]
  44. Wood ME, Stockwell RE, Johnson GR, Ramsay KA, Sherrard LJ et al. Cystic fibrosis pathogens survive for extended periods within cough-generated droplet nuclei. Thorax 2019; 74:87–90 [CrossRef][PubMed]
    [Google Scholar]
  45. Hall TA. BioEdit: a user-friendly biological sequence alignment editor and analysis program for windows 95/98/NT. Nucl Acids Symp Ser 1999; 41:95–98
    [Google Scholar]
  46. Nadkarni MA, Martin FE, Jacques NA, Hunter N. Determination of bacterial load by real-time PCR using a broad-range (universal) probe and primers set. Microbiology 2002; 148:257–266 [CrossRef][PubMed]
    [Google Scholar]
  47. Kidd TJ, Ramsay KA, Hu H, Bye PTP, Elkins MR et al. Low rates of Pseudomonas aeruginosa misidentification in isolates from cystic fibrosis patients. J Clin Microbiol 2009; 47:1503–1509 [CrossRef][PubMed]
    [Google Scholar]
  48. Wellinghausen N, Köthe J, Wirths B, Sigge A, Poppert S. Superiority of molecular techniques for identification of gram-negative, oxidase-positive rods, including morphologically nontypical Pseudomonas aeruginosa, from patients with cystic fibrosis. J Clin Microbiol 2005; 43:4070–4075 [CrossRef][PubMed]
    [Google Scholar]
  49. Bosshard PP, Zbinden R, Abels S, Böddinghaus B, Altwegg M et al. 16S rRNA gene sequencing versus the API 20 Ne system and the Vitek 2 ID-GNB card for identification of nonfermenting gram-negative bacteria in the clinical laboratory. J Clin Microbiol 2006; 44:1359–1366 [CrossRef][PubMed]
    [Google Scholar]
  50. Almuzara M, Barberis C, Traglia G, Sly G, Procopio A et al. Isolation of Bordetella species from unusual infection sites. JMM Case Rep 2015; 2:e000029 [CrossRef]
    [Google Scholar]
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