1887

Abstract

Overuse of antibiotics is contributing to an emerging antimicrobial resistance crisis. To better understand how bacteria adapt tolerance and resist antibiotic treatment, Pseudomonas aeruginosa isolates obtained from infection sites sampled from companion animals were collected and evaluated for phenotypic differences. Selected pairs of clonal isolates were obtained from individual infection samples and were assessed for antibiotic susceptibility, cyclic di-GMP levels, biofilm production, motility and genetic-relatedness. A total of 18 samples from equine, feline and canine origin were characterized. A sample from canine otitis media produced a phenotypically heterogeneous pair of P. aeruginosa isolates, 42121A and 42121B, which during growth on culture medium respectively exhibited hyper dye-binding small colony morphology and wild-type phenotypes. Antibiotic susceptibility to gentamicin and ciprofloxacin also differed between this pair of clonal isolates. Sequence analysis of gyrA, a gene known to be involved in ciprofloxacin resistance, indicated that 42121A and 42121B both contained mutations that confer ciprofloxacin resistance, but this did not explain the differences in ciprofloxacin resistance that were observed. Cyclic di-GMP levels also varied between this pair of isolates and were shown to contribute to the observed colony morphology variation and ability to form a biofilm. Our results demonstrate the role of cyclic di-GMP in generating the observed morphological phenotypes that are known to contribute to biofilm-mediated antibiotic tolerance. The generation of phenotypic diversity may go unnoticed during standard diagnostic evaluation, which potentially impacts the therapeutic strategy chosen to treat the corresponding infection and may contribute to the spread of antibiotic resistance.

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2017-10-16
2019-10-19
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References

  1. Marston HD, Dixon DM, Knisely JM, Palmore TN, Fauci AS et al. Antimicrobial resistance. JAMA 2016;316:1193–1204 [CrossRef][PubMed]
    [Google Scholar]
  2. Llor C, Bjerrum L. Antimicrobial resistance: risk associated with antibiotic overuse and initiatives to reduce the problem. Ther Adv Drug Saf 2014;5:229–241 [CrossRef][PubMed]
    [Google Scholar]
  3. Caniaux I, van Belkum A, Zambardi G, Poirel L, Gros MF. MCR: modern colistin resistance. Eur J Clin Microbiol Infect Dis 2017;36:415–420 [CrossRef][PubMed]
    [Google Scholar]
  4. Amin AN, Deruelle D. Healthcare-associated infections, infection control and the potential of new antibiotics in development in the USA. Future Microbiol 2015;10:1049–1062 [CrossRef][PubMed]
    [Google Scholar]
  5. Fernandes P. The global challenge of new classes of antibacterial agents: an industry perspective. Curr Opin Pharmacol 2015;24:7–11 [CrossRef][PubMed]
    [Google Scholar]
  6. Tommasi R, Brown DG, Walkup GK, Manchester JI, Miller AA. ESKAPEing the labyrinth of antibacterial discovery. Nat Rev Drug Discov 2015;14:529–542 [CrossRef][PubMed]
    [Google Scholar]
  7. Guardabassi L, Damborg P, Stamm I, Kopp PA, Broens EM et al. Diagnostic microbiology in veterinary dermatology: present and future. Vet Dermatol 2017;28:146–e30 [CrossRef][PubMed]
    [Google Scholar]
  8. Pinchbeck LR, Cole LK, Hillier A, Kowalski JJ, Rajala-Schultz PJ et al. Pulsed-field gel electrophoresis patterns and antimicrobial susceptibility phenotypes for coagulase-positive staphylococcal isolates from pustules and carriage sites in dogs with superficial bacterial folliculitis. Am J Vet Res 2007;68:535–542 [CrossRef][PubMed]
    [Google Scholar]
  9. Peters BM, Jabra-Rizk MA, O'May GA, Costerton JW, Shirtliff ME. Polymicrobial interactions: impact on pathogenesis and human disease. Clin Microbiol Rev 2012;25:193–213 [CrossRef][PubMed]
    [Google Scholar]
  10. Stacy A, Mcnally L, Darch SE, Brown SP, Whiteley M. The biogeography of polymicrobial infection. Nat Rev Microbiol 2016;14:93–105 [CrossRef][PubMed]
    [Google Scholar]
  11. Filkins LM, O'Toole GA. Cystic fibrosis lung infections: polymicrobial, complex, and hard to treat. PLoS Pathog 2015;11:e1005258 [CrossRef][PubMed]
    [Google Scholar]
  12. Flemming HC. EPS-then and now. Microorganisms 2016;4:41 [CrossRef][PubMed]
    [Google Scholar]
  13. Stewart PS, Franklin MJ. Physiological heterogeneity in biofilms. Nat Rev Microbiol 2008;6:199–210 [CrossRef][PubMed]
    [Google Scholar]
  14. Workentine ML, Harrison JJ, Weljie AM, Tran VA, Stenroos PU et al. Phenotypic and metabolic profiling of colony morphology variants evolved from Pseudomonas fluorescens biofilms. Environ Microbiol 2010;12:1565–1577 [CrossRef][PubMed]
    [Google Scholar]
  15. Ferris RA, Mccue PM, Borlee GI, Loncar KD, Hennet ML et al. In vitro efficacy of nonantibiotic treatments on biofilm disruption of Gram-negative pathogens and an in vivo model of infectious endometritis utilizing isolates from the equine uterus. J Clin Microbiol 2016;54:631–639 [CrossRef][PubMed]
    [Google Scholar]
  16. Holcombe LJ, Patel N, Colyer A, Deusch O, O'Flynn C et al. Early canine plaque biofilms: characterization of key bacterial interactions involved in initial colonization of enamel. PLoS One 2014;9:e113744 [CrossRef][PubMed]
    [Google Scholar]
  17. Maddox TW, Clegg PD, Williams NJ, Pinchbeck GL. Antimicrobial resistance in bacteria from horses: Epidemiology of antimicrobial resistance. Equine Vet J 2015;47:756–765 [CrossRef][PubMed]
    [Google Scholar]
  18. Pye CC, Yu AA, Weese JS. Evaluation of biofilm production by Pseudomonas aeruginosa from canine ears and the impact of biofilm on antimicrobial susceptibility in vitro. Vet Dermatol 2013;24:446e98–e9999 [CrossRef][PubMed]
    [Google Scholar]
  19. Westgate SJ, Percival SL, Knottenbelt DC, Clegg PD, Cochrane CA. Microbiology of equine wounds and evidence of bacterial biofilms. Vet Microbiol 2011;150:152–159 [CrossRef][PubMed]
    [Google Scholar]
  20. Hickman JW, Tifrea DF, Harwood CS. A chemosensory system that regulates biofilm formation through modulation of cyclic diguanylate levels. Proc Natl Acad Sci USA 2005;102:14422–14427 [CrossRef][PubMed]
    [Google Scholar]
  21. Paul R, Weiser S, Amiot NC, Chan C, Schirmer T et al. Cell cycle-dependent dynamic localization of a bacterial response regulator with a novel di-guanylate cyclase output domain. Genes Dev 2004;18:715–727 [CrossRef][PubMed]
    [Google Scholar]
  22. Borlee BR, Goldman AD, Murakami K, Samudrala R, Wozniak DJ et al. Pseudomonas aeruginosa uses a cyclic-di-GMP-regulated adhesin to reinforce the biofilm extracellular matrix. Mol Microbiol 2010;75:827–842 [CrossRef][PubMed]
    [Google Scholar]
  23. Starkey M, Hickman JH, Ma L, Zhang N, de Long S et al. Pseudomonas aeruginosa rugose small-colony variants have adaptations that likely promote persistence in the cystic fibrosis lung. J Bacteriol 2009;191:3492–3503 [CrossRef][PubMed]
    [Google Scholar]
  24. Cohen D, Mechold U, Nevenzal H, Yarmiyhu Y, Randall TE et al. Oligoribonuclease is a central feature of cyclic diguanylate signaling in Pseudomonas aeruginosa. Proc Natl Acad Sci USA 2015;112:11359–11364 [CrossRef][PubMed]
    [Google Scholar]
  25. Kulasakara H, Lee V, Brencic A, Liberati N, Urbach J et al. Analysis of Pseudomonas aeruginosa diguanylate cyclases and phosphodiesterases reveals a role for bis-(3'-5')-cyclic-GMP in virulence. Proc Natl Acad Sci USA 2006;103:2839–2844 [CrossRef][PubMed]
    [Google Scholar]
  26. Orr MW, Donaldson GP, Severin GB, Wang J, Sintim HO et al. Oligoribonuclease is the primary degradative enzyme for pGpG in Pseudomonas aeruginosa that is required for cyclic-di-GMP turnover. Proc Natl Acad Sci USA 2015;112:E5048E5057 [CrossRef][PubMed]
    [Google Scholar]
  27. Römling U, Galperin MY, Gomelsky M. Cyclic di-GMP: the first 25 years of a universal bacterial second messenger. Microbiol Mol Biol Rev 2013;77:1–52 [CrossRef][PubMed]
    [Google Scholar]
  28. Vogel HJ, Bonner DM. Acetylornithinase of Escherichia coli: partial purification and some properties. J Biol Chem 1956;218:97–106[PubMed]
    [Google Scholar]
  29. Rybtke MT, Borlee BR, Murakami K, Irie Y, Hentzer M et al. Fluorescence-based reporter for gauging cyclic di-GMP levels in Pseudomonas aeruginosa. Appl Environ Microbiol 2012;78:5060–5069 [CrossRef][PubMed]
    [Google Scholar]
  30. Plumley BA, Martin KH, Borlee GI, Marlenee NL, Burtnick MN et al. Thermoregulation of biofilm formation in Burkholderia pseudomallei is disrupted by mutation of a putative diguanylate cyclase. J Bacteriol 2017;199:e00780-16 [CrossRef][PubMed]
    [Google Scholar]
  31. Hickman JW, Harwood CS. Identification of FleQ from Pseudomonas aeruginosa as a c-di-GMP-responsive transcription factor. Mol Microbiol 2008;69:376–389 [CrossRef][PubMed]
    [Google Scholar]
  32. Nanvazadeh F, Khosravi AD, Zolfaghari MR, Parhizgari N. Genotyping of Pseudomonas aeruginosa strains isolated from burn patients by RAPD-PCR. Burns 2013;39:1409–1413 [CrossRef][PubMed]
    [Google Scholar]
  33. Institute CALS Performance standards for antimicrobial disk and dilution susceptibility tests for bacteria isolated from animals; Second Informational Supplement. , 33(VET01-S2). Clinical and Laboratory Standards Institute; 2013
  34. Kumar K, Awasthi D, Lee SY, Cummings JE, Knudson SE et al. Benzimidazole-based antibacterial agents against Francisella tularensis. Bioorg Med Chem 2013;21:3318–3326 [CrossRef][PubMed]
    [Google Scholar]
  35. O'Brien J, Wilson I, Orton T, Pognan F. Investigation of the Alamar Blue (resazurin) fluorescent dye for the assessment of mammalian cell cytotoxicity. Eur J Biochem 2000;267:5421–5426 [CrossRef][PubMed]
    [Google Scholar]
  36. Friedman L, Kolter R. Two genetic loci produce distinct carbohydrate-rich structural components of the Pseudomonas aeruginosa biofilm matrix. J Bacteriol 2004;186:4457–4465 [CrossRef][PubMed]
    [Google Scholar]
  37. D'Argenio DA, Calfee MW, Rainey PB, Pesci EC. Autolysis and autoaggregation in Pseudomonas aeruginosa colony morphology mutants. J Bacteriol 2002;184:6481–6489 [CrossRef][PubMed]
    [Google Scholar]
  38. Rotcheewaphan S, Belisle JT, Webb KJ, Kim HJ, Spencer JS et al. Diguanylate cyclase activity of the Mycobacterium leprae T cell antigen ML1419c. Microbiology 2016;162:1651–1661 [CrossRef][PubMed]
    [Google Scholar]
  39. Jørgensen KM, Wassermann T, Jensen , Hengzuang W, Molin S et al. Sublethal ciprofloxacin treatment leads to rapid development of high-level ciprofloxacin resistance during long-term experimental evolution of Pseudomonas aeruginosa. Antimicrob Agents Chemother 2013;57:4215–4221 [CrossRef][PubMed]
    [Google Scholar]
  40. Takenouchi T, Sakagawa E, Sugawara M. Detection of gyrA mutations among 335 Pseudomonas aeruginosa strains isolated in Japan and their susceptibilities to fluoroquinolones. Antimicrob Agents Chemother 1999;43:406–409[PubMed]
    [Google Scholar]
  41. Silva IN, Santos PM, Santos MR, Zlosnik JE, Speert DP et al. Long-term evolution of Burkholderia multivorans during a chronic cystic fibrosis infection reveals shifting forces of selection. mSystems 2016;1:e00029-16 [CrossRef][PubMed]
    [Google Scholar]
  42. Amiel E, Lovewell RR, O'Toole GA, Hogan DA, Berwin B. Pseudomonas aeruginosa evasion of phagocytosis is mediated by loss of swimming motility and is independent of flagellum expression. Infect Immun 2010;78:2937–2945 [CrossRef][PubMed]
    [Google Scholar]
  43. Lovewell RR, Collins RM, Acker JL, O'Toole GA, Wargo MJ et al. Step-wise loss of bacterial flagellar torsion confers progressive phagocytic evasion. PLoS Pathog 2011;7:e1002253 [CrossRef][PubMed]
    [Google Scholar]
  44. Boles BR, Thoendel M, Singh PK. Self-generated diversity produces "insurance effects" in biofilm communities. Proc Natl Acad Sci USA 2004;101:16630–16635 [CrossRef][PubMed]
    [Google Scholar]
  45. Parsek MR, Singh PK. Bacterial biofilms: an emerging link to disease pathogenesis. Annu Rev Microbiol 2003;57:677–701 [CrossRef][PubMed]
    [Google Scholar]
  46. Vorachit M, Lam K, Jayanetra P, Costerton JW. Resistance of Pseudomonas pseudomallei growing as a biofilm on silastic discs to ceftazidime and co-trimoxazole. Antimicrob Agents Chemother 1993;37:2000–2002 [CrossRef][PubMed]
    [Google Scholar]
  47. Kirisits MJ, Prost L, Starkey M, Parsek MR. Characterization of colony morphology variants isolated from Pseudomonas aeruginosa biofilms. Appl Environ Microbiol 2005;71:4809–4821 [CrossRef][PubMed]
    [Google Scholar]
  48. Holmes AH, Moore LS, Sundsfjord A, Steinbakk M, Regmi S et al. Understanding the mechanisms and drivers of antimicrobial resistance. Lancet 2016;387:176–187 [CrossRef][PubMed]
    [Google Scholar]
  49. Casagrande Proietti P, Stefanetti V, Hyatt DR, Marenzoni ML, Capomaccio S et al. Phenotypic and genotypic characterization of canine pyoderma isolates of Staphylococcus pseudintermedius for biofilm formation. J Vet Med Sci 2015;77:945–951 [CrossRef][PubMed]
    [Google Scholar]
  50. Prescott JF, Hanna WJ, Reid-Smith R, Drost K. Antimicrobial drug use and resistance in dogs. Can Vet J 2002;43:107–116[PubMed]
    [Google Scholar]
  51. Petersen AD, Walker RD, Bowman MM, Schott HC, Rosser EJ. Frequency of isolation and antimicrobial susceptibility patterns of Staphylococcus intermedius and Pseudomonas aeruginosa isolates from canine skin and ear samples over a 6-year period (1992–1997). J Am Anim Hosp Assoc 2002;38:407–413 [CrossRef][PubMed]
    [Google Scholar]
  52. Martinez M, Mcdermott P, Walker R. Pharmacology of the fluoroquinolones: a perspective for the use in domestic animals. Vet J 2006;172:10–28 [CrossRef][PubMed]
    [Google Scholar]
  53. Tyczkowska K, Hedeen KM, Aucoin DP, Aronson AL. High-performance liquid chromatographic method for the simultaneous determination of enrofloxacin and its primary metabolite ciprofloxacin in canine serum and prostatic tissue. J Chromatogr 1989;493:337–346 [CrossRef][PubMed]
    [Google Scholar]
  54. Rubin J, Walker RD, Blickenstaff K, Bodeis-Jones S, Zhao S. Antimicrobial resistance and genetic characterization of fluoroquinolone resistance of Pseudomonas aeruginosa isolated from canine infections. Vet Microbiol 2008;131:164–172 [CrossRef][PubMed]
    [Google Scholar]
  55. Haenni M, Hocquet D, Ponsin C, Cholley P, Guyeux C et al. Population structure and antimicrobial susceptibility of Pseudomonas aeruginosa from animal infections in France. BMC Vet Res 2015;11:9 [CrossRef][PubMed]
    [Google Scholar]
  56. Gasink LB, Fishman NO, Weiner MG, Nachamkin I, Bilker WB et al. Fluoroquinolone-resistant Pseudomonas aeruginosa: assessment of risk factors and clinical impact. Am J Med 2006;119:526.e19–526526 [CrossRef][PubMed]
    [Google Scholar]
  57. Damborg P, Olsen KE, Møller Nielsen E, Guardabassi L. Occurrence of Campylobacter jejuni in pets living with human patients infected with C. jejuni. J Clin Microbiol 2004;42:1363–1364 [CrossRef][PubMed]
    [Google Scholar]
  58. Guardabassi L, Loeber ME, Jacobson A. Transmission of multiple antimicrobial-resistant Staphylococcus intermedius between dogs affected by deep pyoderma and their owners. Vet Microbiol 2004;98:23–27 [CrossRef][PubMed]
    [Google Scholar]
  59. Rodrigues J, Thomazini CM, Lopes CA, Dantas LO. Concurrent infection in a dog and colonization in a child with a human enteropathogenic Escherichia coli clone. J Clin Microbiol 2004;42:1388–1389 [CrossRef][PubMed]
    [Google Scholar]
  60. Gundelly P, Suzuki Y, Ribes JA, Thornton A. Differences in Rhodococcus equi Infections based on immune status and antibiotic susceptibility of clinical isolates in a case series of 12 patients and cases in the literature. Biomed Res Int 2016;2016:1–9 [CrossRef][PubMed]
    [Google Scholar]
  61. Gurry GA, Campion V, Premawardena C, Woolley I, Shortt J et al. High rates of potentially infectious exposures between immunocompromised patients and their companion animals: an unmet need for education. Intern Med J 2017;47:333–335 [CrossRef][PubMed]
    [Google Scholar]
  62. Beier RC, Foley SL, Davidson MK, White DG, Mcdermott PF et al. Characterization of antibiotic and disinfectant susceptibility profiles among Pseudomonas aeruginosa veterinary isolates recovered during 1994–2003. J Appl Microbiol 2015;118:326–342 [CrossRef][PubMed]
    [Google Scholar]
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