Bacteriophage treatment of carbapenemase-producing Klebsiella pneumoniae in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities.
Journal: 2020/March - AIMS Microbiology
ISSN: 2471-1888
Abstract:
The p-traps of hospital handwashing sinks represent a potential reservoir for antimicrobial-resistant organisms of major public health concern, such as carbapenemase-producing KPC+ Klebsiella pneumoniae (CPKP). Bacteriophages have reemerged as potential biocontrol agents, particularly against biofilm-associated, drug-resistant microorganisms. The primary objective of our study was to formulate a phage cocktail capable of targeting a CPKP strain (CAV1016) at different stages of colonization within polymicrobial drinking water biofilms using a CDC biofilm reactor (CBR) p-trap model. A cocktail of four CAV1016 phages, all exhibiting depolymerase activity, were isolated from untreated wastewater using standard methods. Biofilms containing Pseudomonas aeruginosa, Micrococcus luteus, Stenotrophomonas maltophilia, Elizabethkingia anophelis, Cupriavidus metallidurans, and Methylobacterium fujisawaense were established in the CBR p-trap model for a period of 28 d. Subsequently, CAV1016 was inoculated into the p-trap model and monitored over a period of 21 d. Biofilms were treated for 2 h at either 25 °C or 37 °C with the phage cocktail (109 PFU/ml) at 7, 14, and 21 d post-inoculation. The effect of phage treatment on the viability of biofilm-associated CAV1016 was determined by plate count on m-Endo LES agar. Biofilm heterotrophic plate counts (HPC) were determined using R2A agar. Phage titers were determined by plaque assay. Phage treatment reduced biofilm-associated CAV1016 viability by 1 log10 CFU/cm2 (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log10 and 1.6 log10 CFU/cm2 (p < 0.05) at 7 and 14 d, respectively (25 °C). No significant reduction was observed at 21 d post-inoculation. Phage treatment had no significant effect on the biofilm HPCs (p > 0.05) at any time point or temperature. Supplementation with a non-ionic surfactant appears to enhance phage association within biofilms. The results of this study suggest the potential of phages to control CPKP and other carbapenemase-producing organisms associated with microbial biofilms in the healthcare environment.
Relations:
Content
References
(55)
Conditions
(1)
Chemicals
(3)
Genes
(1)
Organisms
(7)
Anatomy
(1)
Similar articles
Articles by the same authors
Discussion board
AIMS Microbiol 6(1): 43-63

Bacteriophage treatment of carbapenemase-producing <em>Klebsiella pneumoniae</em> in a multispecies biofilm: a potential biocontrol strategy for healthcare facilities

Clinical and Environmental Microbiology Branch, Division of Healthcare Quality Promotion, Centers for Disease Control and Prevention, Atlanta, GA, USA
* Correspondence: Email: vog.cdc@8dlr; Fax: +4046393822.
Received 2019 Nov 25; Accepted 2020 Feb 13.
This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0)

Abstract

The p-traps of hospital handwashing sinks represent a potential reservoir for antimicrobial-resistant organisms of major public health concern, such as carbapenemase-producing KPC+ Klebsiella pneumoniae (CPKP). Bacteriophages have reemerged as potential biocontrol agents, particularly against biofilm-associated, drug-resistant microorganisms. The primary objective of our study was to formulate a phage cocktail capable of targeting a CPKP strain (CAV1016) at different stages of colonization within polymicrobial drinking water biofilms using a CDC biofilm reactor (CBR) p-trap model. A cocktail of four CAV1016 phages, all exhibiting depolymerase activity, were isolated from untreated wastewater using standard methods. Biofilms containing Pseudomonas aeruginosa, Micrococcus luteus, Stenotrophomonas maltophilia, Elizabethkingia anophelis, Cupriavidus metallidurans, and Methylobacterium fujisawaense were established in the CBR p-trap model for a period of 28 d. Subsequently, CAV1016 was inoculated into the p-trap model and monitored over a period of 21 d. Biofilms were treated for 2 h at either 25 °C or 37 °C with the phage cocktail (10 PFU/ml) at 7, 14, and 21 d post-inoculation. The effect of phage treatment on the viability of biofilm-associated CAV1016 was determined by plate count on m-Endo LES agar. Biofilm heterotrophic plate counts (HPC) were determined using R2A agar. Phage titers were determined by plaque assay. Phage treatment reduced biofilm-associated CAV1016 viability by 1 log10 CFU/cm (p < 0.05) at 7 and 14 d (37 °C) and 1.4 log10 and 1.6 log10 CFU/cm (p < 0.05) at 7 and 14 d, respectively (25 °C). No significant reduction was observed at 21 d post-inoculation. Phage treatment had no significant effect on the biofilm HPCs (p > 0.05) at any time point or temperature. Supplementation with a non-ionic surfactant appears to enhance phage association within biofilms. The results of this study suggest the potential of phages to control CPKP and other carbapenemase-producing organisms associated with microbial biofilms in the healthcare environment.

Keywords: biofilms, bacteriophage, carbapenemase-producing Klebsiella pneumoniae, healthcare-associated infections
Abstract

Acknowledgments

This work was supported in part by internal funds provided under the Centers for Disease Control and Prevention's (CDC) Innovation Fund. Authors would like to acknowledge staff from Snapfinger Creek Advanced Wastewater Plant (Dekalb County, GA) and RL Sutton Wastewater Reclamation Facility (Cobb County, GA) for collecting wastewater samples. The use of trade names and commercial sources is for identification only and does not imply endorsement by the Public Health Service or the U.S. Department of Health and Human Services. The findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the U.S. CDC.

Acknowledgments

Abbreviations

ANOVA
analysis of variance
AR
antibiotic resistant
CBR
CDC biofilm reactor
CDC
Centers for Disease Control and Prevention
CRE
carbapenem-resistant Enterobacteriaceae
CPKP
carbapenemase-producing Klebsiella pneumonia
CV
crystal violet
KPC
Klebsiella pneumoniae carbapenemase
EPS
extracellular polymeric substance
HAIs
healthcare-associated infections
HPCs
heterotrophic plate counts
MOI
multiplicity of infection
PBS
phosphate buffered saline
PSB
phage storage buffer
TSA
trypticase soy agar
TSB
tryptic soy broth
USD
U. S. dollars
SAO
soft agar overlay
DI
deionized
SEM
standard error of the mean
Abbreviations

Footnotes

Conflicts of Interest: All authors declare no conflicts of interest in this paper.

Footnotes

References

  • 1. Leitner E, Zarfel G, Luxner J, et al Contaminated handwashing sinks as the source of a clonal outbreak of KPC-2-producing Klebsiella oxytoca on a hematology ward. Antimicrob Agents Ch. 2015;59:714–716.[Google Scholar]
  • 2. Regev-Yochay G, Smollan G, Tal I, et al Sink traps as the source of transmission of OXA-48–producing Serratia marcescens in an intensive care unit. Infect Control Hosp Epidemiol. 2018;39:1307–1315.[PubMed][Google Scholar]
  • 3. Donlan RMBiofilms: microbial life on surfaces. Emerging infect dis. 2002;8:881–890.[Google Scholar]
  • 4. Tofteland S, Naseer U, Lislevand JH, et al A long-term low-frequency hospital outbreak of KPC-producing Klebsiella pneumoniae involving intergenus plasmid diffusion and a persisting environmental reservoir. PLoS One. 2013;8:e59015.[Google Scholar]
  • 5. Lowe C, Willey B, O'Shaughnessy A, et al Outbreak of extended-spectrum β-lactamase-producing Klebsiella oxytoca infections associated with contaminated handwashing sinks (1) Emerging Infect Dis. 2012;18:1242–1247.[Google Scholar]
  • 6. Carling PCWastewater drains: epidemiology and interventions in 23 carbapenem-resistant organism outbreaks. Infect Control Hosp Epidemiol. 2018;39:972–979.[PubMed][Google Scholar]
  • 7. Vergara-López S, Domínguez MC, Conejo MC, et al Wastewater drainage system as an occult reservoir in a protracted clonal outbreak due to metallo-β-lactamase-producing Klebsiella oxytoca. Clin Microbiol Infect. 2013;19:E490–E498.[PubMed][Google Scholar]
  • 8. Stone PWEconomic burden of healthcare-associated infections: an American perspective. Expert Rev Pharmacoeconomics Outcomes Res. 2009;9:417–422.[Google Scholar]
  • 9. Al-Tawfiq JA, Tambyah PAHealthcare associated infections (HAI) perspectives. J Infect Public Health. 2014;7:339–344.[PubMed][Google Scholar]
  • 10. Logan LK, Weinstein RAThe epidemiology of carbapenem-resistant enterobacteriaceae: the impact and evolution of a global menace. J Infect Dis. 2017;215:S28–S36.[Google Scholar]
  • 11. Gupta N, Limbago BM, Patel JB, et al Carbapenem-resistant enterobacteriaceae: epidemiology and prevention. Clin Infect Dis. 2011;53:60–67.[PubMed][Google Scholar]
  • 12. Jacob J, Klein E, Laxminarayan R, et al Vital signs: carbapenem-resistant enterobacteriaceae. MMWR Morbid Mortal We. 2013;62:165–170.[Google Scholar]
  • 13. Kizny Gordon AE, Mathers AJ, Cheong EYL, et al The hospital water environment as a reservoir for carbapenem-resistant organisms causing hospital-acquired infections—a systematic review of the literature. Clinl Infect Dis. 2017;64:1435–1444.[PubMed][Google Scholar]
  • 14. De Geyter D, Blommaert L, Verbraeken N, et al The sink as a potential source of transmission of carbapenemase-producing Enterobacteriaceae in the intensive care unit. Antimicrob resist infect control. 2017;6:24–24.[Google Scholar]
  • 15. Roux D, Aubier B, Cochard H, et al Contaminated sinks in intensive care units: an underestimated source of extended-spectrum beta-lactamase-producing Enterobacteriaceae in the patient environment. J Hosp Infect. 2013;85:106–111.[PubMed][Google Scholar]
  • 16. Abedon STActive bacteriophage biocontrol and therapy on sub-millimeter scales towards removal of unwanted bacteria from foods and microbiomes. AIMS Microbiol. 2017;3:649–688.[Google Scholar]
  • 17. Ramirez K, Cazarez-Montoya C, Lopez-Moreno HS, et al Bacteriophage cocktail for biocontrol of Escherichia coli O157:H7: Stability and potential allergenicity study. PLoS One. 2018;13:e0195023.[Google Scholar]
  • 18. Lehman SM, Donlan RMBacteriophage-mediated control of a two-species biofilm formed by microorganisms causing catheter-associated urinary tract infections in an in vitro urinary catheter model. Antimicrob Agents Ch. 2015;59:1127–1137.[Google Scholar]
  • 19. Hughes KA, Sutherland IW, Jones MVBiofilm susceptibility to bacteriophage attack: the role of phage-borne polysaccharide depolymerase. Microbiology. 1998;144:3039–3047.[PubMed][Google Scholar]
  • 20. Azeredo J, Sutherland IWThe use of phages for the removal of infectious biofilms. Curr Pharm Biotechnol. 2008;9:261–266.[PubMed][Google Scholar]
  • 21. Majkowska-Skrobek G, Latka A, Berisio R, et al Phage-borne depolymerases decrease Klebsiella pneumoniae resistance to innate defense mechanisms. Front Microbiol. 2018;9:2517.[Google Scholar]
  • 22. Fish R, Kutter E, Wheat G, et al Bacteriophage treatment of intransigent diabetic toe ulcers: a case series. J Wound Care. 2016;25:S27–S33.[PubMed][Google Scholar]
  • 23. Mendes JJ, Leandro C, Corte-Real S, et al Wound healing potential of topical bacteriophage therapy on diabetic cutaneous wounds. Wound Repair Regen. 2013;21:595–603.[PubMed][Google Scholar]
  • 24. Carson L, Gorman SP, Gilmore BFThe use of lytic bacteriophages in the prevention and eradication of biofilms of Proteus mirabilis and Escherichia coli. Pathog Dis. 2010;59:447–455.[PubMed][Google Scholar]
  • 25. Lungren MP, Donlan RM, Kankotia R, et al Bacteriophage K antimicrobial-lock technique for treatment of Staphylococcus aureus central venous catheter–related infection: a leporine model efficacy analysis. J Vasc Interv Radiol. 2014;25:1627–1632.[Google Scholar]
  • 26. Mathers AJ, Cox HL, Bonatti H, et al Fatal cross infection by carbapenem-resistant Klebsiella in two liver transplant recipients. Transpl Infect Dis. 2009;11:257–265.[Google Scholar]
  • 27. Pierce VM, Simner PJ, Lonsway DR, et al Modified carbapenem inactivation method for phenotypic detection of carbapenemase production among enterobacteriaceae. J Clin Microbiol. 2017;55:2321.[Google Scholar]
  • 28. Adams MH Bacteriophages. London, United Kingdom: Interscience Publishers; 1959. [PubMed][Google Scholar]
  • 29. O'Toole GAMicrotiter dish biofilm formation assay. J Vis Exp. 2011;pii: 2437[PubMed][Google Scholar]
  • 30. Donlan RM, Elliot DL, Kapp NJ, et al Surfanctants for reducing bacterial adhesion onto surfaces. U.S., Patent, 6039965. 2000 editors.[Google Scholar]
  • 31. Mazher M, Santiago A, Donlan RControl of Carbapenem-Resistant Klebsiella pneumoniae biofilms using a nonionic surfactant. ASM Microbe 2018 Conference; 2018 Jun 6-11; Atlanta, GA. 2018. [PubMed]
  • 32. Donlan RM, Piede JA, Heyes CD, et al Model system for growing and quantifying Streptococcus pneumoniae biofilms in situ and in real time. Appl Environ Microbiol. 2004;70:4980–4988.[Google Scholar]
  • 33. Goeres DM, Loetterle LR, Hamilton MA, et al Statistical assessment of a laboratory method for growing biofilms. Microbiology. 2005;151:757–762.[PubMed][Google Scholar]
  • 34. Armbruster CS, Forster TM, Donlan R, et al A biofilm model developed to investigate survival and disinfection of Mycobacterium mucogenicum in potable water. Biofouling. 2012;28:1129–1139.[PubMed][Google Scholar]
  • 35. Kotay S, Chai W, Guilford W, et al Spread from the sink to the patient: in situ study using green fluorescent protein (GFP)-expressing Escherichia coli to model bacterial dispersion from hand-washing sink-trap reservoirs. Appl Environ Microbiol. 2017;83:e03327–03316.[Google Scholar]
  • 36. Kotsanas D, Wijesooriya WRPLI, Korman TM, et al ‘Down the drain’: carbapenem-resistant bacteria in intensive care unit patients and handwashing sinks. Med J Aust. 2013;198:267–269.[PubMed][Google Scholar]
  • 37. Kotay SM, Donlan RM, Ganim C, et al Droplet-rather than aerosol-mediated dispersion is the primary mechanism of bacterial transmission from contaminated hand-washing sink traps. Appl Environ Microbiol. 2019;85:e01997–01918.[Google Scholar]
  • 38. Chan BK, Abedon STBacteriophages and their enzymes in biofilm control. Curr Pharm Design. 2015;21:85–99.[PubMed][Google Scholar]
  • 39. Cornelissen A, Ceyssens P-J, T'Syen J, et al The T7-related Pseudomonas putida phage φ15 displays virion-associated biofilm degradation properties. PLoS OneE. 2011;6:e18597.[Google Scholar]
  • 40. Bodratti AM, Alexandridis PFormulation of poloxamers for drug delivery. J Funct Biomater. 2018;9:11.[Google Scholar]
  • 41. Pitto-Barry A, Barry NPEPluronic® block-copolymers in medicine: from chemical and biological versatility to rationalisation and clinical advances. Polym Chem. 2014;5:3291–3297.[PubMed][Google Scholar]
  • 42. Percival SL, Mayer D, Malone M, et al Surfactants and their role in wound cleansing and biofilm management. J Wound Care. 2017;26:680–690.[PubMed][Google Scholar]
  • 43. Das Ghatak P, Mathew-Steiner SS, Pandey P, et al A surfactant polymer dressing potentiates antimicrobial efficacy in biofilm disruption. Sci Rep. 2018;8:873–873.[Google Scholar]
  • 44. Seo Y, Bishop PLInfluence of nonionic surfactant on attached biofilm formation and phenanthrene bioavailability during simulated surfactant enhanced bioremediation. Environ Sci Technol. 2007;41:7107–7113.[PubMed][Google Scholar]
  • 45. Simmons M, Drescher K, Nadell CD, et al Phage mobility is a core determinant of phage-bacteria coexistence in biofilms. ISME J. 2018;12:531–543.[Google Scholar]
  • 46. Tait K, Skillman LC, Sutherland IWThe efficacy of bacteriophage as a method of biofilm eradication. Biofouling. 2002;18:305–311.[PubMed][Google Scholar]
  • 47. Baghal Asghari F, Nikaeen M, Mirhendi HRapid monitoring of Pseudomonas aeruginosa in hospital water systems: a key priority in prevention of nosocomial infection. FEMS Microbiol Lett. 2013;343:77–81.[PubMed][Google Scholar]
  • 48. Cieplak T, Soffer N, Sulakvelidze A, et al A bacteriophage cocktail targeting Escherichia coli reduces E. coli in simulated gut conditions, while preserving a non-targeted representative commensal normal microbiota. Gut Microbes. 2018;9:391–399.[Google Scholar]
  • 49. Doolittle MM, Cooney JJ, Caldwell DETracing the interaction of bacteriophage with bacterial biofilms using fluorescent and chromogenic probes. J Ind Microbiol. 1996;16:331–341.[PubMed][Google Scholar]
  • 50. Alexandridis P, Alan Hatton TPoly (ethylene oxide) poly (propylene oxide) poly (ethylene oxide) block copolymer surfactants in aqueous solutions and at interfaces: thermodynamics, structure, dynamics, and modeling. Colloids Surf A. 1995;96:1–46.[PubMed][Google Scholar]
  • 51. Choi YC, Morgenroth EMonitoring biofilm detachment under dynamic changes in shear stress using laser-based particle size analysis and mass fractionation. Water Sci Technol. 2003;47:69–76.[PubMed][Google Scholar]
  • 52. Roy B, Ackermann HW, Pandian S, et al Biological inactivation of adhering Listeria monocytogenes by listeriaphages and a quaternary ammonium compound. Appl Environ Microbiol. 1993;59:2914–2917.[Google Scholar]
  • 53. Sillankorva S, Neubauer P, Azeredo JPhage control of dual species biofilms of Pseudomonas fluorescens and Staphylococcus lentus. Biofouling. 2010;26:567–575.[PubMed][Google Scholar]
  • 54. Chhibber S, Bansal S, Kaur SDisrupting the mixed-species biofilm of Klebsiella pneumoniae B5055 and Pseudomonas aeruginosa PAO using bacteriophages alone or in combination with xylitol. Microbiology. 2015;161:1369–1377.[PubMed][Google Scholar]
  • 55. Lenski RE. Dynamics of Interactions between Bacteria and Virulent Bacteriophage. In: Marshall KC, editor. Advances in microbial ecology. Boston, MA: Springer US; 1988. pp. 1–44. [PubMed]
  • 56. Donlan RMPreventing biofilms of clinically relevant organisms using bacteriophage. Trends Microbiol. 2009;17:66–72.[PubMed][Google Scholar]
  • 57. Meaden S, Koskella BExploring the risks of phage application in the environment. Front Microbiol. 2013;4:358–358.[Google Scholar]
  • 58. Colomer-Lluch M, Jofre J, Muniesa MAntibiotic resistance genes in the bacteriophage DNA fraction of environmental samples. PLoS One. 2011;6:e17549.[Google Scholar]
  • 59. Nilsson ASPharmacological limitations of phage therapy. Upsala J Med Sci. 2019;124:218–227.[Google Scholar]
  • 60. Wang H, Edwards MA, Falkinham JO, et al Probiotic approach to pathogen control in premise plumbing systems? a review. Environ Sci Technol. 2013;47:10117–10128.[PubMed][Google Scholar]
  • 61. Camper AK, LeChevallier MW, Broadaway SC, et al Growth and persistence of pathogens on granular activated carbon filters. Appl Environ Microbiol. 1985;50:1378.[Google Scholar]
  • 62. Hu YOO, Hugerth LW, Bengtsson C, et al Bacteriophages synergize with the gut microbial community to combat Salmonella. mSystems. 2018;3:e00119–00118.[Google Scholar]
  • 63. Zimlichman E, Henderson D, Tamir O, et al Health care–associated Infections: a meta-analysis of costs and financial impact on the US Health Care System. JAMA Int Med. 2013;173:2039–2046.[PubMed][Google Scholar]
  • 64. Kim BR, Anderson JE, Mueller SA, et al Literature review—efficacy of various disinfectants against Legionella in water systems. Water Res. 2002;36:4433–4444.[PubMed][Google Scholar]
Collaboration tool especially designed for Life Science professionals.Drag-and-drop any entity to your messages.