Biocontrol of Foodborne Pathogens: the Pros and Cons

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Biocontrol of foodborne pathogens: the pros and cons

Jacques Mahillon

Laboratory of Food and Environmental Microbiology

Earth and Life Institute, UCL

IAFP 13th European Symposium on Food Safety

Brussels, March 29, 2017 Foodborne illnesses

Number of reported cases .

(EFSA, 2015) Foodborne safety

Is this enough ? Food safety: new challenges

Globalization of the food market • Large-scale processing and distribution • New microbial risks Increase of the more susceptible populations (YOPI) Consumer organizations Need to respond to the new consumer habits

ChangingAntibiogram consumer requirements

• Higher quality: preparation, shelf-life • Fresher: in flavour and texture • More “natural” food, with fewer additives • Ready-to-eat food

YOPI: Young, Old, Pregnant and Immuno-compromised people Food bio-control and bio-preservation

• Fermentation processes

• Probiotics and prebiotics Taming the good • Competitive exclusion microbes to (better) control the bad ones • Phages*

• Bacteriocins

• Quorum sensing inhibitors (quorum quenching)

• Enzymes degrading signals, signal analogues or antagonists

* Rees, Hagens & Schmelcher, Friday 31, morning session What is a bacteriophage ?

• 1031 - 1032 bacteriophages on Earth [ = Bacterial Virus ] • Present in every living ecosystems • Specific to bacteria, cannot infect eukaryotic cells

Myoviridae Lytic cycle Lysogenic cycle

Virulent phage Temperate phage Main milestones

Decline of interest in phage therapy with the advent of antibiotics (except in Soviet Union and some Eastern European countries) F. Twort

Use of phages as tools F. d’Herelle for molecular biology Renewed Host genome evolution

interest R. Bruynoghe & J. Maisin: Antibiotic resistance first publication on phage therapy

1915 1917 1919 1921 1940 1950 2000 2006 2014

Use of phages to treat bacterial Approval of the first diseases among soldiers during the phage-based product Second World War (ListShieldTM) in food

Phagoburn: beginning of clinical trials Eli Lilly Company Production of E.R. Squibb & Sons phage preparations Swan- Myers/Abbott Laboratories to clinical applications Bacteriophage applications

Human therapeutics Biopesticide Food safety Human therapeutics

Phage therapy

• Alternative or synergy to face the emergence of antibiotic-resistant bacteria

PhagoBioDerm®: biodegradable polyester amides impregnated with bacteriophages for wound healing

(Markoishvili et al. (2012) Int. J. Dermatol. 41: 453-458) Phages and food safety

Primary Processed food Sanitation Detection production Phages in the primary production

Livestock decontamination (2 to 5 log reduction)

Campylobacter jejuni Connerton et al. 2004, Carillo et al. 2005, Wagenaar et al. 2005, El- Shibiny et al. 2009

Salmonella spp. Berchieri et al. 1991, Higgins et al. 2005, Borie et al. 2008, Sillankorva et al. 2010, Wall et al. 2010, Callaway et al. 2011, Bardina et al. 2012, Lim et al. 2012

Escherichia coli O157:H7 Bach et al. 2009, Rozema et al. 2009, Rivas et al. 2010, Standford et al. 2010, Raya et al. 2011 Phages in the primary production

• Anti-E. coli & Anti-Salmonella (OmniLytics Inc., Sandy, Utah, USA)

Decontaminates animals prior to slaughter Approved by the FDA in 2007

• Finalyse (OmniLytics Inc. & Elanco Food Solutions)

Targets E. coli in meats Sprayed on cattle prior to slaughter

• Armanent (OmniLytics Inc. & Elanco Food Solutions)

Targets Salmonella on poultry

• BioTector (CJ CheilJedang, Seoul, Korea)

Replaces antibiotics in animal feed Controls Salmonella responsible for fowl typhoid and pullorum disease Phages in processed food

Decontamination of (postharvest) food

C. jejuni Atterbury et al. 2003, Hungaro et al. 2013

Salmonella spp. Leverentz et al. 2001, Modi et al. 2001, Genther et al. 2009, Hooten et al. 2011

E. coli O157:H7 O’Flynn et al. 2004, Viazis et al. 2010, Boyacioglu et al. 2013

Listeria monocytogenes Leverentz et al. 2003, Guenther et al. 2009, Bigot et al. 2011

Shigella spp., Cronobacter sakazakii Kim et al. 2007, Zuber et al. 2008, Endersen et al. 2012, Zhang et al. 2013 Phages in processed food

• ListShieldTM (Intralytics, Baltimore, MD, USA) 6 bacteriophages isolated from environment Reduces contamination by L. monocytogenes Sprayed onto Ready-to-Eat food Approved in 2006 by the FDA as food additive

• EcoShieldTM & SalmoFreshTM (Intralytics) Approved as GRAS by the FDA in 2013

• ListexTM P100 (Micreos Food Safety, Wageningen, The Netherlands) Approved by the FDA and USDA as processing aid Used in Europe as processing aid

• SalmonelexTM (Micreos Food Safety) Approved by the FDA and USDA as processing aid Phage biocontrol

Advantages and potential limitations

• Safe for humans – GRAS

• Ubiquitous Commensal of humans Naturally present in food

• Specificity No action against the microbiota

• Replicate themselves

• Potentially useful against biofilms

• No alteration of the general composition, taste, odour or colour of foods

• Phage-mediated gene transfer Selection of virulent phages

• Resistance to phage Formulation of a phage cocktail Phage future developments

Phage-derived enzymes

• Rapid bactericidal effect • Endolysin resistance never reported • Specific like bacteriophages • Mainly against Gram-positive bacteria • Higher production costs • Limited shelf-life

Immobilization of phages (enzymes) onto a cellulose membrane Bacteriophages: a credible alternative ?

• Decrease in antibiotic efficacy • Decline in the number of new drugs • Consumer demands for pathogens and synthetic chemicals free foods + antimicrobial phages properties

Numerous companies have already invest in the production of phage-based products Food bio-control and bio-preservation

• Fermentation processes

• Probiotics and prebiotics

• Competitive exclusion

• Phages

• Bacteriocins

• Quorum sensing inhibitors (quorum quenching)

• Enzymes degrading signal, signal analogues or antagonists Antimicrobial compounds

PKS: Polyketide synthesis, RP: Ribosomal peptides, NRS: non ribosomal peptides, VC, Volatile compounds Bacteriocins

• Discovered in 1925 by the Belgian microbiologist André Garcia (Colicin) • Ribosomally synthesized antimicrobial peptides • Synthesized by 30-99 % of bacteria and archaea • Four classes

Small-sized peptides Large-sized peptides < 10kDa > 10kDa

Class I Class II Class III Class IV Modified amino High heat Heat-labile Non-proteinaceous acids stability moieties Lanthibiotics Bacillus bacteriocins: diversity and applications 205

(a)

Bacillus bacteriocins: diversity and applications 205

(a)

(b) (c)

Fig. 2. (a) Comparison of subtilin and ericin structures with that of the lactococcal lantibiotic nisin A. Conserved residues at identical positions to all four bacteriocins are highlighted in green, while those conserved only in subtilin and ericins are depicted in yellow; other conserved residues are in light red. (b) Structures of the single-peptide lantibiotics sublancin, mersacidin and subtilosin A. The structure of the A1 subunit of haloduracin is also included for comparison with mersacidin. The conserved residues are highlighted in orange color. Cysteines involved in disulﬁde bridge formation are highlighted in blue. For subtilosin A, residues involved in sulfur to a-carbon linkages are shown in green, while residues involved in head-to-tail amide bond formation are in light blue. (c) Comparison of the two-peptide lantibiotics haloduracin and lichenicidin from Bacillus, and lacticin 3147 from Lactococcus lactis. Conserved residues of the A1 peptides are highlighted in orange and light yellow. A conserved Pro residue between lichenicidin A1 subunit and mersacidin is shown in light blue. Conserved residues of the A2 peptides are highlighted in green, deep yellow, light orange and violet. The C-terminal parts of the A1 subunits also share two conserved loops of 11 and eight amino acid residues. The C-terminal parts of A2 peptides share the same pattern of lanthionine (Ala–S–Ala) and methyllanthionine (Abu–S–Ala) bridges. Non-identical residues are in white.

for the secretion of the modiﬁed precursor (spaT, which an ABC transporter that exports the lantibiotic from the encodes the transporter SpaT) and genes for immunity cytoplasmic membrane, while SpaI is a lipoprotein that is against the cognate bacteriocin (spaIFEG). SpaFEG forms thought to interfere with the binding of bacteriocin

c FEMS Microbiol Rev 35 (2011) 201–232 2010 Federation of European Microbiological Societies Published by Blackwell Publishing Ltd. All rights reserved Nisin • From Lactococcus lactis • 34 amino acids residues • E234 • Limited action on Gram-positive bacteria Bacteriocins

• Bacteriostatic or bactericidal effect • Fast acting and active at low concentrations (nM) • Stable over a wide range of pH and temperature • Specific activity at nanomolar scales • Degradable, no secondary metabolite • No effect on human beings (protease sensibility) • No flavor or textural changes

• Low yield and high cost of production • Purification difficulties • Matrix interactions: pH, phospholipids, or enzymes • Regulation and approval: GRAS status needed “Good versus bad microbes” That’s the question APPLIED AND ENVIRONMENTAL MICROBIOLOGY, Jan. 2004, p. 631–634 Vol. 70, No. 1 0099-2240/04/$08.00ϩ0 DOI: 10.1128/AEM.70.1.631–634.2004 Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Identiﬁcation of Bacilysin, Chlorotetaine, and Iturin A Produced by Bacillus sp. Strain CS93 Isolated from Pozol, a Mexican Fermented Maize Dough Is the future in the past ? Trevor G. Phister,† Daniel J. O’Sullivan, and Larry L. McKay* Department of Food Science and Nutrition, University of Minnesota, St. Paul, Minnesota 55108

Received 21 April 2003/Accepted 10 October 2003

Three antimicrobial compounds produced by Bacillus sp. strain CS93 isolated from pozol were identiﬁed by Pozolusing high-performance (fermented liquidcorn, chromatography Maya) and massNatto spectrometry. (fermented The three soybean, compounds wereJapan) iturin, bacilysin, and chlorotetaine. Production of these compounds by CS93 could account for the medicinal properties attributed to pozol.

Pozol is a fermented maize dough that is consumed by the against all test organisms was inactivated by pronase E, sug- indigenous Mayan peoples of southeastern Mexico (2, 13). gesting that the compound(s) is proteinaceous (10). The ob- Present-day ethnic groups with Mayan ancestry use pozol as a jective of this study was to identify the compound(s) responsi- source of nutrients and, as did the early Mayans, in ceremonies ble for the antimicrobial activity of Bacillus sp. strain CS93 in promoting the growth and harvest of maize (2, 12, 13). The pozol that could account for the inhibitory effects of pozol and early Mayans consumed pozol since at least 1560 AD (3, 13). the medicinal significance of this food to the ancient Mayans. They also used pozol as a medicine to control diarrhea, to Inhibitory activity was produced in 250 ml of Fred Waksman reduce fever, and to cure intestinal infections. Pozol was used Basic 77 (K2HPO4 ⅐ 3H2O, 0.5 g; MgSO4 ⅐ 7H2O, 0.2 g; NaCl, on wounds as a poultice to prevent infection (16). Pozol was 0.2 g; MnSO4 ⅐ H2O, 0.2 g; FeCl3, 0.002 g; D-mannitol, 10 g; later found to inhibit a number of different bacteria, yeasts, distilled H2O, 1 liter) plus 1% NaNO3 and 1% proline in a and molds (4). 2-liter flask. Cultures were grown for 18 to 22 h at 37°C with The maize fermentation that produces pozol is uncontrolled shaking at 225 rpm. The cells were removed by centrifugation (Phister et al., 2004) and involves yeasts, molds, and bacteria (12, 14). The primary at 9,000 ϫ g for 10 min, and the supernatant was filtered fermentation organisms are lactic acid bacteria (6). Ampe et al. through a 0.45-␮m-pore-size filter and lyophilized for 18 h with (1) found that Lactococcus and Leuconostoc spp. were domi- a Hetovac VR-1 vacuum concentrator (Heto Lab Equipment nant at the start of the fermentation, but Lactobacillus and A/S Birkerod, Denmark). When 100 mg of lyophilized, active, Streptococcus spp. dominated by the end of the fermentation. cell-free CS93 culture supernatant was injected onto a pre-

Aerobic mesophilic bacteria and Enterobacteriaceae were also parative Econosil C18 reverse-phase high-performance liq- present during the fermentation (17). uid chromatography (HPLC) column (250 by 22 mm; Alltech A number of other bacteria have consistently been found to Associates Inc., Deerfield, Ill.), eight peaks were observed be part of pozol fermentation, including an organism initially (Fig. 1). The seventh peak (Fig. 1, second peak from the right) identified as Agrobacterium azotophilium (13). This organism exhibited activity in a disk assay against bacteria, yeasts, and was found to inhibit the growth of a number of gram-positive molds, while the eighth peak (Fig. 1, rightmost peak) exhibited bacteria, gram-negative bacteria, yeasts, and molds (15). activity against yeasts and molds (Fig. 2). After this purification The inhibitory activity of this organism could account for the step, 0.2 mg of the lyophilized peak produced a zone of inhi- early Mayan culture’s use of pozol as a medicine and for the bition that was three times as large as that produced by 1 mg inhibitory effect of pozol, as seen in early experiments by Her- of cell-free supernatant against E. coli and six times as large as rera and Ulloa (4). A. azotophilium was, however, misidentified that produced by 1 mg of cell-free supernatant versus mold. in these early experiments and has since been reisolated, re- This suggested that more than one antimicrobial compound is classified as Bacillus sp. strain CS93 by 16S rRNA sequencing, produced by CS93. The eighth peak was collected and sepa- and shown to produce broad-spectrum antimicrobial activity rated on an analytical C18 HPLC column. The sample con- (10). The strain was deposited with the Northern Regional tained peaks that corresponded to the peaks exhibited by a Research Laboratory, Peoria, Ill., as NRRL B-21974. commercial sample of iturin A subjected to the same chro- The inhibitory activity of strain CS93 was exhibited over a matographic conditions (data not shown). The presence of broad range of pH (3 to 11) and was heat stable (10). Activity iturin A was confirmed by using matrix-assisted laser desorp- tion–ionization time-of-flight mass spectrometry (MS) and comparing the strain CS93 iturin sample spectrum to that of * Corresponding author. Mailing address: Department of Food Sci- the commercial iturin A sample generated on the same mass ence and Nutrition, University of Minnesota, 1334 Eckles Ave., St. spectrometer. Both samples exhibited peaks having mass num- Paul, MN 55108. Phone: (612) 624-3090. Fax: (612) 625-5272. E-mail: m z [email protected]. bers of / 1,043.2, 1,065.2, and 1,079.3; however, the eighth † Present address: Department of Viticulture and Enology, Univer- peak was not as concentrated and the peak at m/z 1,057.2 was sity of California, Davis, Davis, CA 95616-8749. not present. The latter peak was present following concentra-

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