Resistente micro-organismen: Voorkomen en controle met hordentechnologie

Transcription

1 Resistente micro-organismen: Voorkomen en controle met hordentechnologie Prof. Dr. Ir. Chris Michiels Labo Levensmiddelenmicrobiologie Faculteit Bio-ingenieurswetenschappen KU Leuven , Antwerpen 1

2 Resistant foodborne microorganisms 1. Heat resistance 1. High pressure resistance 2. Acid tolerance Occurrence / relevance in foods Cellular mechanisms Control by hurdle technology 2

3 1. Heat resistant bacteria 1.1 Natural occurrence Salmonella Senftenberg 775W Isolated from egg powder in 1946! More heat resistant than any other Salmonella strain Can survive USDA minimum pasteurisation requirements: Whole egg: C Egg white: C Am. J. Pub. Hlth 36:451 (1946) 3

4 1. Heat resistant bacteria 1.1 Natural occurrence Cronobacter sakazakii Some strains are much more heat resistant than others May survive production of powdered infant formula May survive heating during reconstitution of PIF J. Food Safety 29:287 (2009) 4

5 1. Heat resistant bacteria 1.1 Natural occurrence E. coli J. Appl. Microbiol. 110:840 (2001) Isolates from environment where carcasses are steamtreated in 2011 More heat resistant than other E. coli strains Survive in beef patties heated to internal T = 71 C 5

6 1. Heat resistant bacteria 1.2 Resistance mechanism Cronobacter sakazakii Proteomics 5:4161 (2005) Specific protein present in heatresistant strains but not in heatsensitive strains (2005) Function unknown 6

7 1. Heat resistant bacteria 1.2 Resistance mechanism Heat-resistant strains of several Enterobacteriaceae share a locus of heat resistance Heat resistance requires several genes of this locus Locus also called: transmissible locus for protein quality control (TLPQC) Occurs in 2% of all sequenced E. coli genomes Nguyen et al., msystems 2:e (2017) 7

8 1. Heat resistant bacteria 1.2 Resistance mechanism E. coli strains without locus of heat resistance can also develop heat resistance Overexpression of rpoh and/or rpos Appl. Environ. Microbiol. 82:6656 (2016) 8

9 1. Heat resistant bacteria 1.1 Heat resistance variation in bacterial spores Bacillus subtilis group: B. subtilis, B. licheniformis, B. amyloliquefaciens Strains with heat resistant spores have spova genes on a Tn1546 transposon Food. Microbiol. 45:18 (2015) Front. Microbiol. doi: /fmicb

10 1. Heat resistant bacteria 1.3 Control by hurdle technology Heat + natural antimicrobials Essential oil compounds Synergistic effect at very low concentration Nanomaterials 7:65 (2017) Food Bioprocess. Technol. 7:471 (2014) Log 10 of times for 5 log reduction of E. coli O157:H7 at different temperatures in orange juice, with no Eos (filled circles), orange EO (open circles) or limonene (open squares) Survival curves of S. Senftenberg in TSB broth at 55 C (B) with 0 (closed squares), 0.1 (circles), 0.5 (open squares) 10 and 1 (triangles) mm D-limonene nanoemulsified

11 1. Heat resistant bacteria 1.3 Control by hurdle technology Food Microbiol. 48:35 (2015) B. cereus spore inactivation at 100 C (control, closed circles) and with 0.5 mm thymol in heating medium (closed squares) or in recovery medium (open squares) Int J Food Microbiol 141:242 (2010) Log D of C. sporogenes spores inactivation at 90C with different concentrations of fatty acids in recovery medium. Palmitic (closed circles), stearic (closed triangles), palmitoleic (open circles), and oleic (open triangles) = Exploitation of injury of spores 11

12 2. High pressure resistant bacteria > 250 million L HP juices sold in

13 2. High pressure resistant bacteria 2.1 Natural occurrence Very large strain variability (more than for heat) At low p, L. mono is on average more resistant than E. coli At high p, E. coli is on average more resistant than L. mono Some natural strains of E. coli are extremely HP resistant E. coli and some (but not all!) bacteria can acquire extreme HP resistance E. coli L. monocytogenes Compilation of literature data on inactivation of E. coli and L. monocytogenes in neutral ph buffer, C, 10 min treatment 13

14 2. High pressure resistant bacteria 2.1 Natural occurrence E. coli can acquire extreme HP resistance by natural selection (1996) Strains resist HP up to 2 GPa Appl Environ Microbiol 63:945 (1997) Inactivation of wild-type E. coli strain MG1655 and its HP resistant mutant LMM1010 by 15 min treatment at different pressure-temperature combinations 14

15 2. High pressure resistant bacteria 2.2 Resistance mechanism 300 MPa Different mechanisms exist Increased RpoS expression (cfr. heat resistance) Loss of cya/crp catabolite repression system 600 MPa Int J Food Microbiol 163:28 (2013) 56 C Inactivation of wild-type E. coli O157:H7 strain (left) and four selected HP resistant mutants by (A) pressure (300 MPa), (B) pressure (600 MPa) and (C) heat (56 C), 15

16 2. High pressure resistant bacteria 2.2 Control by hurdle technology Combined treatment HP + different EO compounds Limited number of compounds were synergistic carvacrol eugenol Innov Food Sci Em Technol 27:26 (2015) thymol allyl isothiocyanate t-cinnamaldehyde linalool oxide Front. Microbiol. doi: /fmicb geraniol citral linalool eucalyptol α- terpineol α-pinene 16

17 2. High pressure resistant bacteria 2.2 Control by hurdle technology Strong synergy of HP + cinnamaldehyde at MIC level (4 mm) Works on all tested bacteria, including HP resistant strains trans-cinnamaldehyde No treatment t-cin alone HP alone HP + t-cin 17

18 2. High pressure resistant bacteria 2.2 Control by hurdle technology Most synergistic compounds are electrophiles that react with thiol groups (proteins, AcCoA, glutathion ) HP treatment is known to disrupt bacterial thiol-disulfide homeostasis Some synergistic compounds have other mode of action (e.g. linalool oxide) BUT: Compounds from plant essential oils have strong flavour α, β unsaturated carbonyls Isothiocyanates t-cinnamaldehyde allyl isothiocyanate citral Other thiol-reactive electrophiles with less side-effects, or with beneficial properties? 18

19 2. High pressure resistant bacteria 2.2 Control by hurdle technology MG MPa The lactoperoxidase system (LPER) Int J Food Microbiol 81:211 (2003) Milk enzyme: SCN - + H 2 O 2 OSCN - + H 2 O OSCN - reacts with thiol groups LPER has a mostly bacteriostatic effect Applied in milk in remote areas of tropical countries Nontoxic in animals; no off-flavour LMM MPa LMM MPa Combination HP + LPER HP sensitizes bacteria for LPER system HP-sensitized bacteria undergo rapid inactivation after HP treatment Also HP resistant mutants are sensitized 6-D reduction of ALL vegetative cells can be achieved at p < 600 MPa and T < 40 C in food LMM MPa Survival during storage at 20 C of E. coli strains after HP treatment in milk ( ), milk with H 2 O 2 ( ), and milk with the LPER system ( ) 19

20 Untreated 1 mm SUL HP HP + SUL Lab Food Microbiology 2. High pressure resistant bacteria 2.2 Control by hurdle technology Sulphoraphane Isothiocyanate (reactive with thiols) Moderate flavour Health-promoting properties Reuterin Aldehyde that reacts with thiol groups Produced by Lactobacillus reuteri No taste or flavour L. reuteri is used as probiotic Can be converted to acrolein (cancerogen!) 20

21 2. High pressure resistant bacteria 2.2 Control by hurdle technology Innov Food Sci Em Technol 16:26 (2012) L. monocytogenes counts in smoked salmon after treatment with HP + LPER during storage at 5 C 21

22 3. Acid tolerant bacteria 3.1 Occurrence of acid resistance in E. coli Survival of extreme acid challenge has been well studied in E. coli and Salmonella Growth at (moderately) low ph has been less studied Acid resistance (AR) systems that support survival of E. coli at extremely low ph (ph ) 22

23 3. Acid tolerant bacteria 3.1 Occurrence of acid tolerance (growth) in E. coli Food Microbiol 45:222 (2015) Predicted growth no growth interfaces of 180 E. coli strains at low ph and different temperatures without or with lactic acid (25 mm) 23

24 3. Acid tolerant bacteria 3.2 Mechanisms of acid tolerance: role of butanediol fermentation Fermentation pathways in Enterobacteriaceae Mixed acid fermenters E. coli, Salmonella, MR+ VP- Butanediol fermenters Enterobacter, Klebsiella, MR- VP+ 24

25 3. Acid tolerant bacteria 3.2 Mechanisms of acid tolerance: role of butanediol fermentation Int J Food Microbiol 175:36 (2014) Growth of S. plymuthica control strain (black) and budab knockout strain (grey) in tomato juice at 20 C 25

26 3. Acid tolerant bacteria 3.2 Mechanisms of acid tolerance: role of butanediol fermentation Appl Environ Microbiol 80:6054 (2014) VRBG McConkey VRBG McConkey E. coli control E. coli + budab genes Challenge test of E. coli control strain (grey) and strain with budab genes (black) in tomato juice at 30 C Appearance of E. coli control strain (left) and strain with budab genes (right) on four differential plating media Do natural butanediol-fermenting stains of E. coli (Salmonella, Shigella ) strains exist? 26

27 Conclusions and outlook Bacteria can develop resistance against processing and preservation hurdles This is not a surprise The speed and extent of resistance development varies depending on the stress It will be important to continue monitoring resistance development Hurdle technology based on synergistic processing/preservation factors can control bacteria that developed resistance to one factor Hurdle technology may prevent resistance development because resistance development against multiple hurdles simultaneously is improbable or impossible 27