Bacteriophages– ecology genomics & therapeutic potential

1. Bacteriophages–ecologygenomics & therapeutic potential Vijay Aswani, MD, PhD, FACP, FAAP October 27, 2017

2. Get this talk and papers! Scan this QR code or go to http://buffalo.edu/~vaswani to get this talk.

3. Outline • Are phages found in the anterior nares? (Aswani & Shukla, 2011) • Staphylococcal phage families and properties (Aswani et al, 2011) • Phage Genomics (Aswani et al, 2014, and in prep.) • History of Phage therapy (Aswani et al poster, 2014) • 2013-2015: West Africa Ebola Outbreak • Future directions (to pursue here in Buffalo) • Phages in the sputum of CF patients – lower respiratory tract • Phages as members of the microbiome – the virome • Ebola genomics and biology?

4. Research and Medicine Medical School Lamba G, Aswani V.. 2011. A severe laryngeal angioedema reaction from cefadroxil in a patient with no known allergies to penicillins. West Indian Med J. 2011 Jun;60(3):346-8. Lamba, Gurprit and Vijay Aswani. 2012. A Case Study of Mental Illness and Psychiatric Services on the Caribbean Island of Nevis. International Journal of Mental Health 41(4): 24-29. Winter 2012-13. Between medical school and residency Chandler RJ, Aswani V, Tsai MS, Falk M, Wehrli N, Stabler S, Allen R, Sedensky M, Kazazian HH, Venditti CP. Propionyl-CoA and adenosylcobalamin metabolism in Caenorhabditis elegans: Evidence for a role of methylmalonyl-CoA epimerase in intermediary metabolism. Mol Genet Metab. 2006 Sep-Oct; 89(1-2): 64-73, 2006. Residency Haas, Ron & Aswani, Vijay Adventures in CO-Oximetry: Apparent Methemoglobinemia. LabMedicine 38(11):1-4, 2007. Shukla SK, Aswani V, Stockwell PJ, Reed KD. Contribution of polymorphisms in ankA, gltA, and groESL in defining genetic variants of Anaplasma phagocytophilum. J Clin Microbiol. 2007 Jul; 45(7):2312-5, 2007. Practice Aswani V, Shukla SK. Two unusual pediatric cases of fungal infections in farming families. J Agromedicine. 2011 Apr;16(2):153-7. Chalasani S, Bettadahalli SS, Bhupathi SV, Aswani VH. 2013. A novel case of diabetic muscle necrosis in a patient with cystic fibrosis-related diabetes. Clin Med Res. 2013 Sep;11(3):113-6.

5. First Phage Observations? • First reported in 1896 by Hankin (Pasteur, Paris) • Observed that waters of the Ganges and Jumna rivers had potent anti-bacterial properties • Skin sores and other infections rapidly cleared when pilgrims bathed in the Ganges

6. Phage History • 1915-17 phage discovery: • Frederick Twort & micrococci; • Felix d'Herelle isolated phage from Shigella dysentery cases • 1919 d'Herelle uses phage in avian typhosis outbreak caused by Bacillus gallinarum, in France. • 75% of untreated chickens (60/80) died within 30 days. • 0% of phage treated chickens (20/20) died. Human patients soon followed: first human trial: 4 children with bacterial dysentery – all of whom survived.

7. What are bacteriophages? Bacteriophages are viruses that infect bacteria The ‘phages’ were discovered independently in 1915 and 1917 by Frederick W. Twort and Felix d’Herrelle, respectively D’Herrelle coined the term bacteriophage A bacteriophage attacking an E. coli cell (2)

8. Are phages found in the anterior nares? (Aswani & Shukla, 2011)

9. Staphylococci Gram positive cocci in clusters Rosenbach described the two clinically important species – aureus and albus (now epidermidis) in 1884 Catalase test distinguishes them from Streptococci (Staph is catalase positive)

10. The anterior nares - ecology nares are always heavily colonized, mostly with Staphylococcus epidermidis and corynebacteria, S. aureus is present in at least 30% of individuals in the normal population Lina et al (2003) analyzed the composition of the aerobic nasal flora of 216 healthy volunteers found that the S. aureus colonization rate in subjects colonized by Corynebacterium spp. and/or non-aureus staphylococci, especially S. epidermidis, was significantly lower than in subjects not colonized by these species, This suggests that both Corynebacterium spp. and S. epidermidis antagonize nasal colonization by S. aureus.

11. Background Kuehnert et al (2006) reported a national incidence of 32% for S aureus and 0.8% for MRSA. S. aureus is an important cause of community- and hospital-acquired infections (Lowry, 1998). Elimination of carriage in the anterior nares, the principal reservoirs of S. aureus, reduces the incidence of S. aureus infections (Holton et al, 1991). Leszczyński et al, 2006 used phage to treat MRSA-carrier status An anti-microbial product being developed for topical use in the UK based on a phage specific for S. aureus, including MRSA (O'Flaherty et al, 2005)

12. Background II There is no published data documenting the presence of bacteriophages in the anterior nares. What is the nasal carriage status of medical residents? Is it different from that of other healthcare workers and/or the general population? Do bacterteriophages play an ecological role in the anterior nares? Do they affect the nasal carriage status of S. aureus and/or MRSA?

13. Objectives and Hypotheses To detect and document the extent of staphylococcal bacteriophages in the anterior nares To determine the nasal carriage rates of S. aureus and MRSA in the house staff population at the Marshfield Clinic To determine if staphylococcal phages affect the nasal carriage status of S. aureus and MRSA in the anterior nares

14. Study Design A sample size of 200 individuals --Marshfield Clinic ambulatory patients and house staff was selected for studying the prevalence of phages and S. aureus. The proposal was approved by IRB before proceeding House staff (residents and fellows) and ambulatory patients of General Internal Medicine, Med/Peds and Family Practice departments were approached to participate in the research study. Swabs were coded by a study number and signed informed consent forms securely stored. Samples were processed and data analyzed ‘blindly’

15. Power of the study Sample size of n=200 selected based on: Prevalence of S. aureus in the anterior nares is estimated at 30% based on Kuehnert et al, 2006 Based on prevalence of bacteriophages in the oral cavity and gut (ranging from 3% to 33%) (Chibani-Chennoufi et al, 2004; Tylenda et al, 1985; Bachrach et al, 2003), I estimated a prevalence of 5% for lytic phages. Consequently, the estimated prevalence of S. aureus phages in the anterior nares is estimated at 0.5 x 0.3 = 0.015 or 1.5%. A sample size of n=200 would be estimated to yield at least 3 positive samples for bacteriophages against S. aureus.

16. Study flow-chart

17. Enrollment Rate

18. Population study Demographics

19. 11 positives 181 colonies picked: 98 α and 83 β 7/8 positive 56 positive 49 positive Study flow-chart 2 positive: type II and type IV-C

20. Healthcare vs Non-Healthcare

21. Conclusions: Prevalence • The prevalence of S. aureus nasal carriage in the population studied is 24.3% and 0.99% for MRSA • No significant difference was observed in the prevalence of S. aureus between subgroups studied. • No significant difference in the prevalence of S. aureus in the medical resident population or our healthcare-associated employees from the ambulatory pt population studied

22. Phage Results 72.7% (39 – 94%) of the positives came from medical residents

23. Conclusions: Phages • No lytic bacteriophages were isolated with activity against S. aureus • 11 bacteriophages with lytic activity against S. epidermidis belonging to at least 2 different families were isolated • Electron microscopy was able to demonstrate these phages attaching to S. epidermidis

24. Drawbacks of the study • The population size was insufficient for the subgroup analysis, so the lack of significant difference between subgroups must be interpreted with caution • We used 0.2 u filter to filter our samples for phages. The electron micrographs showed that some of the phages were even 0.5 u. • Concentrating the phage samples yielded more positives for phages; it is likely that we are underestimating the phage prevalence

25. Significance of this study • We demonstrate for the first time that lytic staphylococcal phages are found in the anterior nares of humans • Our study suggests an inverse relationship between prevalence of S. epidermidis phage and S. aureus nasal carriage • A preliminary survey of the literature suggests that this may be the first prevalence study of S. aureus and MRSA in house staff in the United States

26. Staphylococcal phage families and properties (Aswani et al, 2011)

27. Phage plates

30. Sample 5e Sample 6ec Big Siphoviridae These 2 phages might be different because of the tip of the tail that looks different. But it can be only that the phage tail at the left is bent. The picture at the right is very good. I put the picture at the left because we can see the icosahedric capsid (like a dice with 20 faces) Magnification: 150,000X

31. Phages adsorbed on the bacterial cells (sample 4e).

32. Phages adsorbed on the bacterial cells, closer look (sample 4e).

33. Phage Genomics (Aswani et al, 2014, and in prep.)

34. Aswani et al (in prep)

35. Aswani et al (in prep)

36. Aswani et al (in prep)

37. 15 17 19 21 23 25 27 29 14 16 18 20 22 24 26 28 30 2 4 6 8 10 12 1 3 5 7 9 11 13 0 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 Kilobases of Phage 6e v4 31 33 36 38 40 42 44 46 32 35 37 39 41 43 45 34 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 Kilobases of Phage 6e v4 48 50 52 54 47 49 51 53 55 57 59 61 63 65 67 69 71 73 75 77 79 56 58 60 62 64 66 68 70 72 74 76 78 80 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 Kilobases of Phage 6e v4 81 83 85 87 89 91 93 95 97 99 101 103 105 107 82 84 86 88 90 92 94 96 98 100 102 104 106 57 58 59 60 61 62 63 64 65 66 67 68 69 70 71 72 73 74 75 Kilobases of Phage 6e v4 131 133 135 137 139 141 143 130 132 134 136 138 140 142 109 111 113 115 117 119 121 123 125 127 129 108 110 112 114 116 118 120 122 124 126 128 76 77 78 79 80 81 82 Replication Packaging Head Head-tail joining tRNA-Pro Nuclease HP HP Phage Portal Protein HP HP HP Phage Prohead Protease Large SU Phage terminase Large SU Phage terminase Endodeoxy-ribonuclease Phage Major Capsid Protein Phage terminase Phage Head-tail joining protein Phage DNA Packaging Protein Phage Head-tail adaptor Tail HP StaphHP StaphHP conserved phage protein HP HP Phage preneck appendage protein Prophage endopeptidase tail Tail protein or tail measure length protein Phage Major tail protein Lipase acylhydrolase domain protein Phage Tail tape measure protein Tail tape measure chapterone protein Host lysis Integrase Replication Phage membrane protein phage MbpT protein guanylate kinase YpoX phage protein DNA polymerase phage HP Phage HP Phage HP StaphHP HP HP phage amidase StaphHP phage amidase 81 autolysin putative holin recombinase / integrase Homing HNH endonuclease Replication PhoH-related protein Single-stranded DNA specific exonuclease DNA polymerase (contd) Phage HP Phage HP Ribose NDP reductase subunit beta Phage HP Phage HP HP DNA helicase Phage HP HP Adenine-specific methyltransferase HP ribonuclease H Phage HP Replication Peak of high reads Phage protein Nrdl S epi phage protein Phage HP S epi HP Phage HP Phage HP Phage glutaredoxin protein S epi HP ATP/GTP binding protein HNH endonuclease Ribose NDP reductase subunit alpha HP HP Phage HP Phage HP Phage HP Phage HP Phage HP S epi Phage HP S epi Phage HP 83 84 85 86 87 88 89 90 91 92 93 Kilobases of Phage 6e v4

38. History of Phage therapy (Aswani et al, ASM poster, 2014)

41. 2013-2015: West Africa Ebola Outbreak (see presentations here) • Future directions (to pursue here in Buffalo) • Phages in the sputum of CF patients – lower respiratory tract • Ebola genomics and biology?

42. Thank You! Questions?