Therapeutic potential of bacteriophages for staphylococcal infections and selected methods for in vitro susceptibility testing of staphylococci

Česká verze

Authors: M. Dvořáčková ¹; F. Růžička ¹; M. Dvořáková Heroldová ¹; L. Vacek ¹; D. Bezděková ²; M. Benešík ²; P. Petráš ³; R. Pantůček ²
Authors place of work: Mikrobiologický ústav Lékařské fakulty Masarykovy univerzity a Fakultní nemocnice u sv. Anny v Brně ¹; Ústav experimentální biologie Přírodovědecké fakulty Masarykovy univerzity, Brno ²; Národní referenční laboratoř pro stafylokoky SZÚ, Praha ³
Published in the journal: Epidemiol. Mikrobiol. Imunol. 69, 2020, č. 1, s. 10-18
Category: Původní práce

Summary

Aim: Staphylococcus aureus strains are the cause of frightening hospital and community infections, especially when they are resistant to antimicrobials, have important pathogenicity factors, or have biofilm production ability. Looking for novel therapeutic options which would be effective against such strains is one of the highest priorities of medicine and medical research.

The study aim was to describe the occurrence of S. aureus strains and proportion of methicillin resistant strains (MRSA) detected in laboratories of the Microbiological Institute, Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno in 2011–2018. Selected strains of S. aureus were tested for biofilm production ability and susceptibility to antimicrobials and Stafal®, a phage therapeutic agent. A prerequisite was to develop a simple routine method suitable for phage susceptibility testing of bacteria.

Material and methods: Altogether 867 clinical isolates of S. aureus and 132 strains of other species of the genus Staphylococcus (isolated in 2011–2017) were tested for susceptibility to the phage therapy preparation Stafal® using the double-layer agar method. All strains of S. aureus were tested for biofilm production ability by the modified Christensen method with the use of titration microplates and for susceptibility to antistaphylococcal antibiotics by the disk diffusion test. For 95 S. aureus strains, the outcome of the double-layer agar method (DAM) was compared with that of our newly designed method (ODM) based on optical density decrease of the bacterial suspension.

Results: During the study period, the laboratories of the Faculty of Medicine, Masaryk University (FM MU) and St. Anne's University Hospital, Brno detected 2900 strains of S. aureus per year on average. The proportion of MRSA among S. aureus isolates from blood culture and venous catheters ranged between 8.8–15.2 %.

S. aureus strains recovered from venous catheters and blood culture were confirmed as stronger biofilm producers than those from other clinical specimens. MRSA strains showed higher biofilm production than methicillin susceptible strains (MSSA).

As many as 90.4 % of S. aureus strains tested susceptible to the Stafal® preparation. Even a higher proportion, i.e. 99.0 %, of MRSA strains were Stafal® susceptible. No relationship was found between Stafal® susceptibility and biofilm production ability. Although Stafal® targets primarily S. aureus, some susceptibility (26.5 %) was also found for other staphylococcal species.

A novel simple method designed for routine testing of susceptibility to phage therapy preparations based on optical density decrease was comparably sensitive and reliable as the commonly used double-layer agar method (DAM) and, in addition to being easy and rapid to perform, after prolonged suspension culture and at higher measurement frequency, it has an extra advantage of providing the possibility for monitoring also phage action dynamics.

Conclusions: The proportion of MRSA strains detected in this study is comparable to that reported for the whole Czech Republic, and the biofilm production data are consistent with scientific evidence.

The host range of the Stafal® preparation is relatively wide and covers most strains of S. aureus and some coagulase negative staphylococci. The highest efficiency of Stafal® (99.4 %) was observed against MRSA strains with multiple types of antibiotic resistance.

In vitro testing of 867 strains of S. aureus and 132 other staphylococcal species has shown the phage therapy preparation Stafal® to be a suitable candidate therapeutic option for the treatment of staphylococcal infections, especially in case of failure of conventional antibiotic therapy. Moreover, a simple method for routine phage susceptibility testing of clinical bacterial isolates has been designed, which is an essential tool to be used in phage therapy.

Keywords:

Staphylococcus – Phage therapy – biofilm – MRSA – Stafal®

Zdroje

1. Hormozi SF, Vasei N, Aminianfar M, et al. Antibiotic resistance in patients suffering from nosocomial infections in Besat Hospital. Eur J Transl Myol, 2018;28(3):7594.

2. Miller MA, Hyland M, Ofner-Agostini M, et al. Canadian Hospital Epidemiology Committee. Morbidity, Mortality, and Healthcare Burden of Nosocomial Clostridium Difficile-Associated Diarrhea in Canadian Hospitals. Infect Control Hosp Epidemiol, 2002;23(03):137–140.

3. Ziebuhr W, Hennig S, Eckart M, et al. Nosocomial infections by Staphylococcus epidermidis: how a commensal bacterium turns into a pathogen. J Antimicrob Agents, 2006;28:14–20.

4. Liu GY. Molecular Pathogenesis of Staphylococcus aureus Infection. Pediatric Research, 2009;65(5 Part 2):71R–77R.

5. Wertheim HF, Melles DC, Vos MC, et al. The role of nasal carriage in Staphylococcus aureus infections. Lancet Infect Dis, 2005;5(12):751–762.

6. Grundmann H, Aires-de-Sousa M, Boyce J, et al. Emergence and resurgence of meticillin-resistant Staphylococcus aureus as a public-health threat. Lancet, 2006;368(9538):874–885.

7. Lowy FD. Antimicrobial resistance: the example of Staphylococcus aureus. J Clin Invest, 2003;111(9):1265–1273.

8. Perichon B, Courvalin P. Heterologous Expression of the Enterococcal vanA Operon in Methicillin-Resistant Staphylococcus aureus. Antimicrob. Agents Chemother, 2004;48(11):4281–4285.

9. Mitka M. Seeking Medicare Audit. JAMA, 2010;303(6):499.

10. Otero LH, Rojas-Altuve A, Llarrull LI, et al. How allosteric control of Staphylococcus aureus penicillin binding protein 2a enables methicillin resistance and physiological function. Proc Natl Acad Sci U S A, 2013;110(42):16808–16813.

11. Alder JD. Daptomycin, a new drug class for the treatment of Gram-positive infections. Drugs Today, 2005;41(2):81.

12. Nyč O. Novinky a trendy v antibiotické léčbě. Interní Medicína pro Praxi, 2017;19(3):142–144.

13. Kurlenda J, Grinholc M. Alternative therapies in Staphylococcus aureus diseases. Acta Biochim Pol, 2012;59(2):171–184.

14. Chudobova D, Cihalova K, Dostalova S, et al. Comparison of the effects of silver phosphate and selenium nanoparticles on Staphylococcus aureus growth reveals potential for selenium particles to prevent infection. FEMS Microbiol Lett, 2014;351(2):195–201.

15. Grinholc M, Kawiak A, Kurlenda J, et al. Photodynamic effect of protoporphyrin diarginate (PPArg2) on methicillin-resistant Staphylococcus aureus and human dermal fibroblasts. Acta Biochim Pol, 2008;55(1):85–90.

16. Senna JPM, Roth DM, Oliveira JS, et al. Protective immune response against methicillin resistant Staphylococcus aureus in a murine model using a DNA vaccine approach. Vaccine, 2003;21(19–20):2661–2666.

17. Summers WC. The strange history of phage therapy. Bacteriophage, 2012;2(2):130–133.

18. Furfaro LL, Payne MS, Chang BJ. Bacteriophage Therapy: Clinical Trials and Regulatory Hurdles. Front Cell Infect Microbiol, 2018;8:376.

19. Hodyra-Stefaniak K, Miernikiewicz P, Drapała J, et al. Mammalian Host-Versus-Phage immune response determines phage fate in vivo. Scientific Reports, 2015;5:14802.

20. Lin DM, Koskella B, Lin HC. Phage therapy: An alternative to antibiotics in the age of multi-drug resistance. World J Gastrointest Pharmacol Ther, 2017;8(3):162–173.

21. Sulakvelidze A, Alavidze Z, Morris JG. Bacteriophage Therapy. Antimicrob. Agents Chemother, 2001;45(3):649–659.

22. Wittebole X, De Roock S, Opal SM. A historical overview of bacteriophage therapy as an alternative to antibiotics for the treatment of bacterial pathogens. Virulence, 2014;5(1):226–235.

23. Loc-Carrillo C, Abedon ST. Pros and cons of phage therapy. Bacteriophage, 2011;1(2):111–114.

24. Merril CR, Biswas B, Carlton R, et al. Long-circulating bacteriophage as antibacterial agents. Proc Natl Acad Sci, 1996;93(8):3188–3192.

25. Chhibber S, Kumari S. Application of Therapeutic Phages in Medicine. In: Kurtbke pek, ed. Bacteriophages, InTech; 2012:141–158.

26. Górski A, Jończyk-Matysiak E, Międzybrodzki R, et al. Phage Therapy: Beyond Antibacterial Action. Front Med, 2018;5:146.

27. Expert round table on acceptance and re-implementation of bacteriophage therapy. Silk route to the acceptance and re-implementa-tion of bacteriophage therapy. Biotechnol J, 2016;11(5):595–600.

28. Rohde C, Resch G, Pirnay JP, et al. Expert Opinion on Three Phage Therapy Related Topics: Bacterial Phage Resistance, Phage Training and Prophages in Bacterial Production Strains. Viruses, 2018;10(4):178.

29. Pillich J, Výmola F. Antistafylokokový fágový lyzát pro místní použití, PV-1156-79. Patent 203719, A61K39/12. Úřad pro vynálezy a objevy, Praha 1979.

30. Botka T, Pantůček R, Mašlaňová I, et al. Lytic and genomic properties of spontaneous host-range Kayvirus mutants prove their suitability for upgrading phage therapeutics against staphylococci. Sci Rep, 2019;9(1):5475.

31. ŠÚKL-Stafal, Detail lieku [online]. 2018-02-20 [cit. 2019-02-11]. Dostupné na www: <https://www.sukl.sk/hlavna-stranka/slovenska-verzia/pomocne-stranky/detail-lieku?page_id=386&lie_id=24546>

32. SÚKL-Stafal, sol 1×10, SPC [online]. 2014-01-01 [cit. 2019-02-11]. Dostupné na www: <http://www.sukl.cz/modules/medication/detail.php?code=0185891&tab=texts>

33. Zelenková H. Antistafylokokový fágový lyzát v liečbe chronických rán predkolenia na podklade chronickej venóznej insuficiencie a diabetes mellitus. Kazuistiky v diabetologii, 2014;12(2):15–19.

34. Jarčuška P. Ekonomické aspekty liečby stafylokokových infekcií kože a mäkkých tkanív protistafylokokovým fágovým lyzátom. Slovenská Chirurgia, 2015;12(1):15–18.

35. Dvořáčková M, Růžička F, Benešík M, et al. Antimicrobial effect of commercial phage preparation Stafal^® on biofilm and planktonic forms of methicillin-resistant Staphylococcus aureus. Folia Microbiol, 2019;64(1):121–126.

36. Drilling A, Morales S, Jardeleza C, et al. Bacteriophage Reduces Biofilm of Staphylococcus aureus Ex Vivo Isolates from Chronic Rhinosinusitis Patients. American Journal of Rhinology & Allergy, 2014;28(1):3–11.

37. Stepanović S, Vuković D, Hola V, et al. Quantification of biofilm in microtiter plates: overview of testing conditions and practical recom-mendations for assessment of biofilm production by staphylococci. APMIS, 2007;115(8):891–899.

38. Surveillance Atlas of Infectious Diseases – MRSA, European Centre for Disease Prevention and Control [online]. 2018-11-15 [cit. 2019-02-11]. Dostupné na www: <https://atlas.ecdc.europa.eu/public/index.aspx?Dataset=27&HealthTopic=4&Indicator=299444&GeoResolution=2&TimeResolution=Year&StartTime=2000&EndTime=2017>

39. Factsheet for experts, European Centre for Disease Prevention and Control [online]. 2018-11-15 [cit. 2019-02-11]. Dostupné na www: <https://antibiotic.ecdc.europa.eu/en/get-informed/factsheets/factsheet-experts>

40. Ackermann G. Drugs of the 21st century: telithromycin (HMR 3647) – The first ketolide. J Antimicrob Chemother, 2003;51(3):497–511.

41. Revdiwala S, Rajdev BM, Mulla S. Characterization of Bacterial Etiologic Agents of Biofilm Formation in Medical Devices in Critical Care Setup. Crit Care Res Pract, 2012;2012:1–6.

42. Patel F, Goswami P, Khara R. Detection of Biofilm formation in device associated clinical bacterial isolates in cancer patients. SLJID, 2016;6(1):43.

43. Hassan A, Usman J, Kaleem F, et al. Evaluation of different detection methods of biofilm formation in the clinical isolates. Braz J Infect Dis, 2011;15(4):305–311.

44. Manandhar S, Singh A, Varma A, et al. Biofilm Producing Clinical Staphylococcus aureus Isolates Augmented Prevalence of Antibiotic Resistant Cases in Tertiary Care Hospitals of Nepal. Front Med, 2018;9:2749.

45. Stewart PS, Costerton JW. Antibiotic resistance of bacteria in biofilms. Lancet, 2001;358(9276):135–138.

46. Ghasemian A, Najar Peerayeh S, Bakhshi B, et al. Comparison of Biofilm Formation between Methicillin-Resistant and Methicillin--Susceptible Isolates of Staphylococcus aureus. Iran Biomed J, 2016;20(3):175–181.

47. Sugimoto S, Sato F, Miyakawa R, et al. Broad impact of extracellular DNA on biofilm formation by clinically isolated Methicillin-resistant and -sensitive strains of Staphylococcus aureus. Scientific Reports, 2018;8(1):2254.

48. Donlan RM. Biofilm Formation: A Clinically Relevant Microbiological Process. Clin Infect Dis, 2001;33(8):1387–1392.

49. Rao RS, Karthika RU, Singh SP, et al. Correlation between biofilm production and multiple drug resistance in imipenem resistant clinical isolates of Acinetobacter baumannii. Indian J Med Microbiol, 2008;26(4):333–337.

50. Chaudhry WN, Concepción-Acevedo J, Park T, Andleeb S, Bull JJ, Levin BR. Synergy and Order Effects of Antibiotics and Phages in Killing Pseudomonas aeruginosa Biofilms. PLoS One, 2017;12(1):e0168615.

51. Pantůček R, Rosypalová A, Doškař J, et al. The Polyvalent Staphylococcal Phage 812:Its Host-Range Mutants and Related Phages. Virology, 1998;246(2):241–252.

52. Vandersteegen K, Kropinski AM, Nash JHE, et al. Two Phage Isolates Representing a Distinct Clade within the Twortlikevirus Genus, Display Suitable Properties for Phage Therapy Applications. J Virol, 2013;87(6):3237–3247.

53. O’Flaherty S, Coffey A, Meaney W, et al. The Recombinant Phage Lysin LysK Has a Broad Spectrum of Lytic Activity against Clinically Relevant Staphylococci, Including Methicillin-Resistant Staphylococcus aureus. J Bacteriol, 2005;187(20):7161–7164.

54. Alves DR, Gaudion A, Bean JE, et al. Combined Use of Bacteriophage K and a Novel Bacteriophage To Reduce Staphylococcus aureus Biofilm Formation. J Appl Environ Microbiol, 2014;80(21):6694–6703.

55. Hsieh SE, Lo HH, Chen ST, et al. Wide Host Range and Strong Lytic Activity of Staphylococcus aureus Lytic Phage Stau2. J Appl Environ Microbiol, 2011;77(3):756–761.

56. Freyberger H, He Y, Roth A, et al. A. Effects of Staphylococcus aureus Bacteriophage K on Expression of Cytokines and Activation Markers by Human Dendritic Cells in vitro. Viruses, 2018;10(11):617.

57. Mattila S, Ruotsalainen P, Jalasvuori M. On-Demand Isolation of Bacteriophages Against Drug-Resistant Bacteria for Personalized Phage Therapy. Front Med, 2015;6:1271.

58. Sahin F, Karasartova D, Ozsan TM, et al. Identification of a novel lytic bacteriophage obtained from clinical MRSA isolates and evaluation of its antibacterial activity. Mikrobiol Bul, 2013;47(1):27–34.

59. Lehman S, Mearns G, Rankin D, et al. Design and Preclinical Development of a Phage Product for the Treatment of Antibiotic-Resistant Staphylococcus aureus Infections. Viruses, 2019;11(1):88.

60. Maharjan R, Ferenci T. The fitness costs and benefits of antibiotic resistance in drug-free microenvironments encountered in the human body: Fitness costs of antibiotic resistance. Environ Microbiol Rep, 2017;9(5):635–641.

61. Pirnay J-P, Blasdel BG, Bretaudeau L, et al. Quality and Safety Requirements for Sustainable Phage Therapy Products. Pharm Res, 2015;32(7):2173–2179.

62. Kaur S, Harjai K, Chhibber S. Methicillin-Resistant Staphylococcus aureus Phage Plaque Size Enhancement Using Sublethal Concentrations of Antibiotics. J Appl Environ Microbiol, 2012;78(23):8227–8233.

63. Delbrück M. The Growth of Bacteriophage and Lysis of the Host. J Gen Physiol, 1940;23(5):643–660.

64. Rodríguez-Rubio L, Martínez B, Donovan DM, et al. Bacteriophage virion-associated peptidoglycan hydrolases: potential new enzybiotics. Crit Rev Microbiol, 2013;39(4):427–434.

65. Xie Y, Wahab L, Gill J. Development and Validation of a Microtiter Plate-Based Assay for Determination of Bacteriophage Host Range and Virulence. Viruses, 2018;10(4):189.