Antimicrobial potential of Staphylococcus aureus bacteriophages in food biopreservation

Transcription

1 Antimicrobial potential of Staphylococcus aureus bacteriophages in food biopreservation Pilar García, Beatriz Martínez, Lorena Rodríguez and Ana Rodríguez Instituto de Productos Lácteos de Asturias (IPLA-CSIC), Villaviciosa, Asturias, Spain

2 - 9 Bacteriophages infecting Staphylococcus aureus Asturias (Northern Spain) Cow s milk production area ( x 1 liters/year) pain S. aureus is a common bovine mastitis agent Bovine mastitis caused by S. aureus 1-% total mastitis Percentage (%) 1 1 FB MA AP MY JUN JUL AUG SEP OCT NOV Time

3 Target: Staphylococcus aureus Mastitis Food poisoning: enterotoxins Antibiotic resistance Mastitis Staphylococcus aureus Food handler s Skin carriage Dairy products Biofilm in the dairy industry

4 Bacteriophages as biocontrol agents Staphylococcus aureus Quality and safety of dairy products

5 Bacteriophages as biocontrol agents Isolation of bacteriophages in dairy environment Efectiveness of phages for S. aureus biocontrol in dairy products φsaus-ipla lytic enzymes Endolysin (LysH) Peptidoglycan Hydrolase (HydH) Sinergistic effect endolysin + nisin

6 Bacteriophage isolation S. aureus from mastitic milk samples S. aureus Sa9 Shiphoviridae (order Caudovirales) dsdna kbp Phage isolation: * Enrichment cultures of dairy samples (milk and cheese) * Host range ΦH Isometric capsid ( ± 1 nm diameter) Non-contractil, flexible tail (1 ± nm) Baseplate with spikes ΦA Elongated head (9 ± nm long for ± 1 nm wide) Long non contractile tail (9 ± nm) Baseplate with spikes

7 Bacteriophage selection Challenge trials ºC 1 UHT whole milk 1 cfu/ml S. aureus Sa9 φh + φa (MOI 1) φipla + φipla (MOI 1) Control Temperate phages φh φa Lytic variants (sodium pyrophosphate) log CFU/ ml Lytic Temperate φsaus-ipla φsaus-ipla time (h) JM.Obeso, B. Martínez, A. Rodríguez, P. García (). Int. J. Food Microbiol. 1:1-1.

8 Bacteriophages as antimicrobials in milk Challenge trials (temperate phages) MOI = 1 ºC Pasteurized whole fat Semi- skimmed raw Whole fat raw log cfu/ ml log pfu/ ml 1 1 time (h) time (h) time (h) (,Staph. aureus Sa9), (,Staph. aureus + ΦA+ ΦH), (, phage titer) P. García, C. Madera, B. Martínez, A. Rodríguez, JE.Suárez (9). J. Dairy. Sci.9: 19-.

9 Bacteriophages as antimicrobials in curd Challenge trials (lytic phages) Pasteurised whole milk MOI = 1 log CFU ml -1 log PFU ml Acid curd S. aureus ph S.a. + L. lactis + (Φ+ Φ) 1 1 time (h) ºC S. a + L. lactis Phage titer ph log CFU ml -1 1 Phage titer Enzymatic curd S.a. + (Φ+ Φ) (curd, whey) 1 1 time (min) ºC ºC 1 S. a in curd S. a in whey log PFU ml -1

10 Bacteriophages as antimicrobials in cheese Challenge trials 9 Control Semi-hard cheese 9 MOI = 1 Log cfu/ml ph 1 Milk Curd Curd h Curd h Cheese d Cheese d Cheese 1 d Cheese d 1 Milk Curd Curd h Curd h Cheese d Cheese d Cheese 1 d Cheese d Total viable counts S. aureus ph Total viable counts S. aureus phage titer ph

11 Bacteriophages as antimicrobials in cheese Challenge trials Acid coagulated cheese Control MOI = 1 Phage treated Log cfu/ml 1 ph Log cfu/pfu/ml 1 ph Milk Milk h Milk h Curd h Cheese d Cheese 1d Milk Milk h Milk h Curd h Cheese d Cheese 1d total viable counts; S. aureus; ph; * phages

12 Bacteriophages genome sequence φsaus-ipla Lysogenic control Early genes Late genes Integration Lysis-lysogeny Replication Packaging Head Head-tail joining Tail Host lysis Integrase Excisionase ci* Cro Anti-repressor ssdna binding dutpase rinb rina Terminase Terminase Portal Scaffold Major head DNA packaging Major tail Tape measure Anti-receptor Hydrolase Holin Topoisomerase ori DNA replication Helicase Amidase DNA ds, bp pac Terminal redundant 1,9 bp

13 Bacteriophages genome sequence Lysogenic control Early genes φsaus-ipla Integration Lysis-lysogeny Replication Packaging Head Head-tail joining Tail Host lysis Integrase ci* Cro DNA polymerase dutpase Portal Major head Major tail Tail tape measure Holin Amidase Primase Methylase rinb Helicase Terminase Scaffold Nuclease rina HNH endonuclease Terminase DNA packaging ori cos DNA ds, bp cos -CGGCGGGGGC-

14 Phage-encoded antimicrobial proteins φsaus-ipla Integration Lysis-lysogeny Replication Packaging Head Head-tail joining Tail Host lysis Integrase Excisionase ci* Cro Anti-repressor ssdna binding dutpase rinb rina Terminase Terminase Portal Scaffold Major head DNA packaging Major tail Tape measure Anti-receptor Hydrolase Holin Topoisomerase ori DNA replication Helicase HydH LysH Amidase aa,. kda pi 9. 1 aa,. kda pi.

15 Heterologous production in Lactococcus lactis pnz-orf LysH ODnm S. aureus Sa9,,,,1 1 1 Time (s) pnz-orf1 Hyd Specific activity (U/mg) 1 1 ng/ml 1 ng/ml Specific activity (U/mg) ng/ml 1 ng/ml 1 Hours 1 Hours

16 Heterologous production in E. coli kda ,1 H -LysH H -HydH 19

17 Endolysin LysH characterization 1 1 Specific activity 1 Specific activity ph Temperature ( C) 1 1 % Activity 1, mm, mm, mm 1 mm % Activity 1 1 NaCl CaCl MgCl MnCl ZnCl 1 1 mm 1

18 Endolysin activity in milk pasteurised whole milk 1 + U/ml + 1 U/ml + U/ml S. aureus Sa9 Log cfu/ml 1 1 Time (h) Time (h) High Cell density Low

19 Endolysin-nisin sinergism, MIC lys U/ml 1, 1 MIC lys U/ml 1 1,,,,, Nis (μg/ml) Nis (μg/ml) MIC: lowest [Lys] Cells in buffer (1 9 /ml) Exponentially growing cells MIC: lowest [Lys] Cell lysis, 1 min, ºC Inhibition of S. aureus growth, 1 h,ºc

20 Endolysin-nisin sinergism in milk 1 1 Log cfu/ml Log cfu/ml 1 1 Time (h) Time (h) Control Lys.U Nis. Lys.+nis. Control Lys 1U Nis. Lys1+nis.

21 Spain INSTITUTO DE PRODUCTOS LÁCTEOS DE ASTURIAS (IPLA-CSIC). Asturias. Spain

22 ASTURIAS

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