Biocontrol of foodborne Bacteria: Past, Present and future strategies

Scientific Review ArticleSCIENTIFIC SUPPLEMENT AUGUST/SEPTEMBER 2007Biocontrol of FoodborneBacteria: Past, Presentand Future StrategiesMcIntyre, L. 1Hudson, J.A. 1 ,Billington, C. 1Withers, H. 21 Institute of Environmental Science and Research Ltd., POBox 29 181, Christchurch 85402 AgResearch MIRINZ, Ruakura Research Centre,Private Bag 3123, HamiltonIntroductionConsumers dislike the use of chemical preservatives in theirfood, and with some there is an associated public health risk.This has increased the pressure for these chemicals to be removedfrom food and for the adoption of more ‘natural’ meansof preservation. While there is a variety of approaches to usingnatural preservatives, the most often adopted approach to datehas been to use biocontrol.The Parliamentary Commissioner for the Environment definesbiocontrol as ‘Using biological means (such as parasites,viruses or predators) to control a pest’ (http://www.pce.govt.nz/reports/pce_reports_glossary.shtml). For the purposes ofthis review the pest is primarily the pathogen of concern in aparticular food, but biocontrol can also be applied to spoilageorganisms and some examples of this will be given.Potential candidates for biocontrol in foods include bacteriophages,bacteriocins (peptides secreted by one species thatinhibit another), siderophores (molecules that sequester iron),quorum sensing (where the presence of an autoinducer of onespecies may influence the growth of another), competitive organisms,and various microbe- and plant-derived antimicrobials.Of these, siderophores might be considered as a candidatebiocontrol mechanism for lower iron foods such as seafood(Gram and Melchiorsen, 1996) and produce (Henry et al., 1991)but they are not discussed further here.What follows is a review of current and potential food-relatedapplications of selected biocontrol approaches.Viral predators – bacteriophagesBacteriophages (phages) are viruses that exclusively infectand kill bacteria. Their structure is simple, comprising nucleicacid contained within a protein capsule that may include a lipidcomponent. When a phage and a host meet (step 1, Figure 1)there needs to be a match between structures on both, and thisconfers specificity on the interaction, i.e. any given phage willonly infect a specific group of hosts. During infection nucleicacid is passed from the phage into the host through its cell wall(step 2). In the case of lytic phages, which kill their host soonafter infection, their nucleic acid is expressed to produce proteinswhich hijack the cell, forcing it to replicate phage nucleicAugust/September 200725

acid (step 3) and produce new phages (step 4). When, typically,around 100 new particles have been made, they are releasedfrom the host cell by fatal disruption of the cell wall (lysis, step5), and each progeny virus can then infect another cell.5Figure 1: The lytic cycle of bacteriophage infection (notto scale)The potential for using phages to kill bacteria has been recognisedfor almost 100 years, with early pioneering work beingcarried out at around the time of the First World War. However,phage therapy for human disease was not adopted in Westerncountries and the discovery of antibiotics saw an end tothe early era of phage therapeutics, apart from in the SovietUnion and Eastern Europe. More recently, there has been arenaissance of interest in phage therapy for human and animalclinical applications stimulated by the emergence of antibioticresistantbacteria.Phage biocontrol of foodborne pathogens is a rather morerecent idea, which seems to be gaining momentum. Several studieshave now been published describing such uses, includingthe application of phages to control Salmonella (a Salmonellaphage is shown in Figure 2) in bean sprouts, broiler carcasses,frankfurters, chicken skin, cheese and melon; Listeria monocytogeneson melon and cheese; Campylobacter on chicken skin;and Escherichia coli O157 on beef. These have recently beenreviewed (Hudson et al., 2005). Commercial production of thefirst phage for use in foods has begun in the Netherlands (www.ebifoodsafety.com), with their Listex P100 product (Carlton etal., 2005) launched to control Listeria in cheese and meat. Thispreparation has now received FDA approval (www.foodqualitynews.com/news/;story accessed 26 October 2006).Figure 2: Salmonella phages ready and waiting for theirnext victim1423In many food studies inactivation is only achieved by usinga high phage to host ratio (the multiplicity of infection, orMOI). In addition, inactivation has been reported to occur attemperatures beneath the growth minimum of the host. Thesefacts suggest that the inactivation reported may be due to lysisfrom without, whereby large numbers of attached phages killthe cell via the ‘death of a thousand cuts’. An example whichcould be explained by this is the inactivation of Salmonella onmelon which was reported to occur at 5°C and at an MOI of200 (Leverentz et al., 2001). However, other explanations arepossible. We have recently obtained some evidence suggestingthat lysis from without and infection can occur simultaneouslyand contribute to cell death.The host specificity of the phage may present a problemfor biocontrol if it is too narrow - within each pathogen speciesthere are numerous sub-types which all need to be controlled.An effective phage should therefore have a ‘Goldilocks’host range, not too narrow and not too broad. An example ofa phage with an almost perfect host range is Felix O1 whichlyses 96-99.5% of Salmonella serovars (Lindberg, 1967). Workwith spoilage organisms has shown that if the host range istoo narrow biocontrol does not work (Greer and Dilts, 1990).One possibility that would overcome this limitation is to usephage cocktails, and this has proved successful for controllingEscherichia coli O157:H7 on beef (O’Flynn et al., 2004).Public perception may also be an obstacle. Would the averageconsumer respond favourably to knowing that viruses hadbeen added to their food? Maybe they would. A recent Americanarticle indicates that some people see phages as ‘green’ andenvironmentally friendly (Fox, 2005). Phages may also be seenas a natural alternative to chemical preservatives. Early phagetherapy pioneers demonstrated safety by ingesting preparationsthemselves, and a recent publication reported the safe intake ofphage T4 by volunteers (Bruttin and Brüssow, 2005). Whateverthe perceptions concerning safety it is a fact that phages are anormal component of foods (Tsuei et al., 2007) and so are beingingested by everyone every day.The direct application of phages to foods is only one biocontrolapproach that might be adopted. For many pathogens,contamination of meat occurs either directly or indirectly fromthe animal’s faeces during processing. The use of phages todestroy pathogens within the intestinal tracts of food animalscould therefore be a way of reducing the number of pathogenson meat. While the use of phages to kill Salmonella in host gutshas historically met with mixed results, the inactivation of bothSalmonella and Campylobacter in chickens has been demonstrated(Loc Carrillo et al., 2005; Atterbury, 2007).Another approach is the application of purified phage-derivedantimicrobials. These are enzymes (endolysins) producedby lytic phages which degrade the cell wall from the inside out,allowing phages to burst out of the cell. These enzymes aregenerally very host specific (Fischetti, 2005) and can also workfrom the outside in. The use of endolysins is therefore best suitedfor Gram positive pathogens, because Gram negative hostshave an outer membrane which hinders access by the enzymeto the cell wall. However, this barrier is not insurmountable,with peptidoglycan degradation by a Pseudomonas endolysinrecently demonstrated (Paradis-Bleau et al., 2007).Phage and phage-derived technologies therefore present atoolbox of techniques using anything from intact viable phagesto enzymes derived from them, any of which could be appliedalong the farm-to-fork continuum. The trick will be to identifywhich tool is best suited to which segment of the value chain.Bacterial predators – BdellovibrioA number of predatory bacteria are also capable of attackingand killing bacteria in a manner similar to that described26Food New Zealand

previously for phages. The best characterised of these is theGram-negative Bdellovibrio bacteriovorus, so called because ofits parasitic leech-like behaviour (latin ‘bdello’) and curved ‘vibrio’shape. First isolated from soil by Stolp and Starr in 1962,this ubiquitous organism has since been found in a variety ofenvironments including fresh, salt and brackish waters, sewageand mammalian intestines.Current knowledge of the predatory cycle of B. bacteriovorushas recently been reviewed by Lambert et al. (2006) whodescribe a recent renaissance in research activity. This is attributed,in part, to the first description of the entire genomesequence of B. bacteriovorus strain HD100 by researchers at theMax-Plank-Institute for Developmental Biology (Rendulic et al.,2004). The replication cycle, is shown as a clock-wise series ofevents in Figure 3.In the ‘attack phase’, Bdellovibrio cells are small, monoflagellatedand extremely fast moving. Following a collision with aGram-negative prey cell (shown in blue; Figure 3), the attackingcell attaches to and invades prey using ‘twitching-type’ pili topull it through holes in the cell wall generated using a blend ofhydrolytic enzymes. Internalisation of Bdellovibrio in the preycell’s periplasm results in loss of the flagellum and a morphologychange to produce a non-motile elongated ‘growth phase’form in a joint predator:prey structure known as a bdelloplast.Replication then occurs in the bdelloplast, followed by preylysis and escape back into the environment.Figure 3: The predatory life cycle of Bdellovibrio bacteriovorus(Snježana Rendulic, Jürgen Berger, Stephan C.Schuster, PhD/Max-Plank-Institute)http://jama.ama-assn.org/cgi/reprint/291/10/1188Given the reported absence of horizontal gene transferduring predation and an inability to invade mammalian cells,Bdellovibrio has been suggested as a ‘living antibiotic’, or atthe very least a useful source of novel antimicrobial substances(Rendulic et al., 2004; Lambert et al., 2006). With its varied environmentalniches and a relatively broad host range, it hasparticular appeal in the battle against Gram-negative pathogenssuch as Salmonella and E. coli O157 in both food and water.Unsurprisingly, given the previous lack of interest in thisorganism, there are relatively few applied research publicationsto date. Fratamico and Cooke (1996) reported the abilityof Bdellovibrio isolates to control E. coli O157:H7 and Salmonellaspp. on stainless steel surfaces, suggesting the potentialfor application to food processing equipment and food. Theability of B. bacteriovorus strain 109J to attack E. coli biofilmswas reported some time later by Kadouri and O’Toole (2005).A 7 log 10reduction in planktonic E. coli numbers was foundafter 24 hours of exposure, while biofilm-associated pathogennumbers declined by 4 logs. Although cells in this biofilm wereclearly able to survive predation to a far greater extent, thiswork does suggest that Bdellovibrio could go some way towardsreducing the issues associated with biofilms in the foodindustry and in other situations.Some preliminary findings investigating food and water applicationsof a salt water Bdellovibrio isolate have recently beenreported by a New Zealand-based research group (Ahmed etal., 2006). In a king salmon shelf life trial, an extension of thelag phase during growth of the total microflora was evident inthe presence of Bdellovibrio at a storage temperature of 20°C,but no extension was observed at 10°C. The ability of Bdellovibrioto reduce coliform levels in polluted recreational waterswas also reported, but practical scale-up could be difficult.Further research of this type is clearly necessary to determinehow whole Bdellovibrio cells or derived antimicrobialscan be successfully applied to specific situations. It is howeveranticipated that further research findings will be forthcoming inthis area as researchers continue to look for naturally occurringways to control pathogens.Competitive exclusion, protective pultures and antimicrobialmetabolitesTo date, the biocontrol approach with the greatest consumeracceptance is the use of naturally occurring antagonistic bacteriaand/or their antimicrobial metabolites. These organismsuse their ability to outcompete pathogens (by outnumberingthem and creating an unfavourable environment) rather thanconducting physical attack and destroy missions. The use ofcompetitive exclusion (CE) cultures in animals, and protectivecultures and metabolites in foods are not new concepts (Andersonet al., 2006), and great efforts have been devoted to thecharacterisation and commercialisation of bacteria and metabolitesfor such purposes.The lactic acid bacteria (LAB) have been most extensivelyinvestigated as CE and protective culture candidates as they aremajor components of the microflora of both healthy mature animalsand foods. They also produce antimicrobial compoundsincluding acids, hydrogen peroxide, diacetyl and bacteriocinswhich gives them an additional edge against related organismsthat might otherwise predominate in a particular environmentalniche. However, other non-pathogenic microorganisms can alsobe suitable.In all cases, regardless of the application being considered,defining these microorganisms is important to ensure that CE/protective cultures do not contain potentially pathogenic subpopulationsor virulence and antimicrobial resistance genes thatcould be transferred to either the animal or human host microflora(Wagner, 2006).Bacterial antagonists offer a number of options in terms ofa farm-to-fork approach to food safety. CE cultures can be appliedpreharvest (Anderson et al., 2006) to prevent the colonisationof food animals with human pathogens such as Salmonellaand Campylobacter (Chaveerach et al., 2004). Postharvest useof protective cultures and/or antimicrobials during formulationand processing adds an additional hurdle by either manipulatingthe composition of the microflora to the detriment of pathogensand/or introducing inhibitory substances active againstthem. These approaches will be addressed separately below.CE culturesThe application of CE cultures to enhance animal productivityhas been promoted in part by the increased use of antibioticsand the consequent development of resistance amongstAugust/September 200727

microbes (Anderson et al., 2006; Diez-Gonzalez & Schamberger2006). CE cultures may act to control or inhibit other microbesvia mechanisms including (i) nutrient competition; (ii) productionof inhibitory compounds; (iii) immunostimulation; and/or(iv) competition for binding sites. Ideally, microbes used forCE applications should adhere well to epithelial cells to allowcolonisation to occur.Applications have included the inhibition of E. coli O157:H7 in cattle (Brashears et al., 2003; Stephens et al., 2007) andSalmonella, E. coli O157:H7 and Campylobacter in poultry usinga variety of microorganisms (Diez-Gonzalez & Schamberger,2006). Non-LAB organisms include ‘generic’ E. coli which caninhibit E. coli O157 through the production of colicins (Schamberger& Diez-Gonzalez 2002; 2004). Colicins have also beendescribed that inhibit other STEC serotypes such as O26 andO111 (Murinda et al., 1996).While successes have been reported, the choice of strain isan important factor (Stephens et al., 2007) along with its survivalcharacteristics, particularly under acidic conditions in thegut, and the method of administration (Anderson et al., 2006).Only one defined product known as PREEMPT has been approvedby the USFDA for poultry applications (Anderson et al.,2006) but production was halted in 2003 due to reduced carriageof Salmonella. Canadian biotech company CanBiocin currentlymarket an LAB-based product called Procin ® , developedto prevent E. coli scours in pigs.Protective cultures and antimicrobial metabolitesThe application of LAB as protective cultures to enhancefood safety has been extensively reviewed (Goktepe, 2006).Key considerations for this type of approach include the doserequired for effective antagonism in the food in question andthe potential effects on sensory properties. Several species ofbifidobacteria have been applied to fish, seafood, poultry anddairy foods to extend shelf life and protect against pathogens.Reported shelf life extensions range from 3 to 14 days dependingon the food and the mode of application, and can be enhancedby the combined use of acid salts such as sodium acetateand potassium sorbate (Goktepe, 2006). Similarly, lactobacillihave been exploited in a variety of food applications includingmeats, fish, vegetables and dairy products (Bredholt et al., 1999;2001; Scolaria & Vescovo, 2004; Millette et al., 2006). Their antimicrobialactivity has been attributed to the production of lacticacid and a variety of antimicrobial compounds.The elucidation of antimicrobial compounds produced bybacteria has been an area of intense research, particularly theproduction of bacteriocins, antimicrobial peptides which producespores in the cytoplasmic membrane, ultimately inhibitingenergy production and biosynthesis activities. These offer anothermeans of control, either through the direct use of culturesknown to produce certain compounds, or via the addition offermentates containing antimicrobials.The best known bacteriocin, nisin, is produced by Lactococcuslactis. It has an unusually broad host spectrum againstboth Gram-positive spoilage and pathogenic bacteria includingListeria monocytogenes, Bacillus and Clostridium, making it aparticularly attractive food additive for processed cheeses andcanned vegetable products. Although nisin has been approvedfor use in an estimated 50 countries, it does demonstrate somelimitations in relation to food type and pH (Deegan et al., 2006).Hence, research activities have continued unabated to isolateand characterise bacteriocins produced by other bacteria.Despite these efforts, the availability of commercialised bacterialcultures and metabolite-based antimicrobial products isnot as extensive as might be anticipated. Danisco produce andmarket a range of protective cultures and defined and undefinedfermentates containing antimicrobial metabolites for foodapplications. Cultures and undefined fermentates are seen asmore consumer-friendly approaches not requiring extensive labelling(Deegan et al., 2006). Defined (purified) fermentates onthe other hand need to be labelled as additives.The Danisco HOLDBAC range of protective cultures canbe used in dairy and meat products (including fermented versions)to control the growth of fungal and bacterial undesirablesincluding L. monocytogenes (Delves-Broughton et al., 2007).CanBiocin also market Micocin ® , a US Department of Agriculture-approvedLAB product with activity against Listeria for usein processed meat products. Examples of defined antimicrobialsinclude powdered nisin, marketed as Nisaplin ® , and Natamax® , a broad spectrum antifungal compound produced bythe fermentation of Streptomyces natalensis. Another definedbacteriocin, ALTA 2431, produced by Pediococcus acidilactici,is commercially manufactured by Kerry Bioscience (Deegan etal., 2006).Undefined fermentates are marketed as Microgard ® fermentedmilk and sugar powders which can be labelled as ‘culturedmilk’ or ‘cultured dextrose’ (Delves-Broughton et al., 2007).These products, typically produced by fermentations using Propionibacteriumfreudenreichii or Lactococcus lactis, are multifactorialin their activity, and can be used in a range of foodsincluding dairy, salad dressings, ready-to-eat meals and bakedgoods.While the use of antagonistic bacteria and/or bacteriocinsis not a total food safety solution, they can be combined withother preservation systems, e.g. modified atmospheres (Lu etal., 2004), bioactive packaging and high pressure processing(Deegan et al., 2006) and bacteriophages (Reynolds, 2007). Researchto date suggests that they are most useful as one hurdlein combination with other preservation options, and their futuresuccess relies on a greater understanding of the mechanisms ofaction occurring in food systems.Antimicrobials of plant originWhilst the potential for antimicrobial effects from medicinalplant extracts has been appreciated for centuries, it is only relativelyrecently that plant-derived food ingredients have beenexplored for these properties. The most frequently used formof plant extract tested for antimicrobial activity in foods is theessential oils (EO). These are aromatic oily liquids, composedof up to sixty individual components that are usually isolatedby steam distillation (Burt, 2004). EO have been reported to beactive in vitro against a wide range of foodborne pathogenicorganisms including E. coli O157:H7, Salmonella, Shigella,Campylobacter, L. monocytogenes, Staphylococcus aureus, Bacilluscereus and Vibrio species. They are generally more effectiveagainst Gram-positive than Gram-negative microorganisms,and can be active against fungi. The most frequently describedgroup of inhibitory compounds are phenolic based, with aromaticand isothiocyanate-based components also isolated. Synergismbetween individual components from the same EO ispossible, as single fractions are sometimes less efficacious thanwhole extracts. Some of the compounds are the same as thoseinvolved in plant host defence against microorganisms (Holleyand Patel, 2005).The mechanisms of action of the EO are not fully understoodat this time. It is known that phenolic components appearto integrate into the cytoplasmic membrane of the microorganismcausing increased proton and potassium permeability (Perez-Conesaet al., 2006). It has been suggested that a lowersensitivity of Gram-negative bacteria to EO may be due to theirrelatively impermeable outer membrane (Fisher and Phillips2006).The simplest and most reported method for delivery of EOantimicrobial activity is incorporation directly into the food, with28Food New Zealand

up to 8 log kills reported (Fisher et al., 2007). However, the fatcomposition of foods has been shown to influence the effectivenessof EO. For example in low fat cheese, bay, clove, cinnamonand thyme essential oils at 1% reduced L. monocytogenes to lessthan 1 log 10cfu ml -1 at 4°C, but only clove oil was effective infull fat cheese. In further experiments, S. Enteritidis incubated at10°C was less affected by fat content, with thyme oil being theonly ineffective EO in full fat cheese (Smith-Palmer et al., 2001).It is thought that the hydrophobic nature of EO means that theytend to partition in fat and oil phases and decrease the effectiveconcentration in the water phase. Micellar encapsulation ofEO may deliver a higher dose to the bacterial cell membranein these types of food environments (Gaysinsky et al., 2005),and this encapsulation strategy may also have potential for thedecontamination of surface adherent biofilms containing pathogens(Perez-Conesa et al., 2006).Direct addition of EO to food can result in reduced pathogenpopulations, but may also alter the food’s sensory characteristics.Incorporating EO into active packaging could be an alternativeway of delivering the antimicrobial action to the food withoutnegatively impacting on organoleptic properties. Whey proteinfilms containing 1-4% (w/v) of essential oils from oregano, rosemaryand garlic were effective in inhibiting E. coli O157:H7, S.aureus, S. Enteritidis, and L. monocytogenes on laboratory media(Seydim and Sarikus, 2006). A trial of alginate-based ediblefilms containing essential oils of oregano, cinnamon and savoryreduced S. Typhimurium and E. coli O157:H7 growth on beefmuscle slices by 1-2 log 10cfu cm 2 over 5 days at 4°C (Oussalah etal., 2006). The incorporation of EO and their active fractions wasalso found to significantly reduce tensile strength and elasticityof alginate-apple puree edible films, but did not affect vapouror oxygen permeability (Rojas-Grau et al., 2007). EO could alsobe used in combination with modified atmosphere packaging(Valero et al., 2006).Another potential intervention point for the use of EO is inthe feed of food animals. Several polysaccharide-emulsified essentialoils were evaluated for introduction into pig diets for thepre-slaughter control of Salmonella Typhimurium DT104 withinthe gut (Si et al., 2006a; Si et al., 2006b). EO were stable at lowpH and active at similar minimum bactericidal concentrations tothat of dietary antibiotics used in swine rearing in some countries(100-300 μg ml -1 ). Carvacrol and thymol were found to havesimilar inhibitory activity to cinnamaldehyde on laboratory media;however, after mixing with pig food, only cinnamaldehyderetained some activity against Salmonella.Other plant materials with potential antimicrobial use in foodsinclude extracts from berries, tea and wood smoke. Grape skins,juice and seed extracts have been found to be highly inhibitoryto L. monocytogenes, but not to other common food pathogens(Rhodes et al., 2006). Both Gram-negative and Gram-positivepathogens were inhibited by Nordic berry extracts (Puupponen-Pimia et al., 2005), with cloudberry and raspberry the best inhibitors.Whole tea infusions and flavanoid extracts were inhibitorywhen applied to Bacillus cereus cultures (Friedman et al., 2006).Whilst there are some concerns about the carcinogenic effects ofwood smoking on food, liquid wood smoke that has had suspectcompounds removed appears to retain antimicrobial activity andhas been tested successfully for inhibition of Listeria on salmonand trout, and moulds on cheese (Holley and Patel, 2005).Plant extracts are promising antimicrobial agents, with activitiesoften rivalling synthetic chemicals. However, a significantbarrier for the widespread adoption of essential oils or theircomponents is that at concentrations required for effective controlthe sensory quality of foods may be altered. The use of activepackaging, multi-hurdle strategies with lowered doses, orcombination with existing treatments offer promise to negatethese concerns.Biocontrol through quorum sensingBacteria live in complex environments that are constantlychanging. To survive, bacteria must be able to sense and interprettheir environment, making adaptive responses that promoteeither proliferation or survival. Those that cannot adaptor respond to change do not survive. Those that do are nowwell adapted to their new environment. This is especially truefor bacteria that appear in processed foods. Furthermore, individualbacteria do not live in isolation but within communitiesthat often contain more than one species of bacteria as well asother microorganisms such as fungi and yeasts.Cell signalling, or quorum sensing (QS) within these communitiesis a well-established phenomenon that orchestrates coordinatedbehaviour by bacteria through the detection of smallsignal molecules known as autoinducers (Kolenbrander et al.,2002). QS allows for cell-density-dependent regulation of manydifferent bacterial activities including surface attachment, biofilmformation, expression of virulence factors and secondarymetabolite production (Figure 4; Fuqua, 2001). Many of the QSregulated microbial activities are involved in food spoilage andsurvival of pathogens within the food matrix.Figure 4: Bacterial quorum sensing regulates target geneexpression, resulting in altered cell activitiesA range of cell signalling molecules have been identifiedthat allow bacteria to communicate with each other as well aswith other types of bacteria. In addition, small molecules producedby a host plant or animal can be detected by bacteriaand can influence the microbial composition of the residentmicroflora as well as affecting their day-to-day activities (Dudlerand Eberl, 2006). Many of the bacterial species found in foodare capable of producing and/or detecting signalling molecules(Liu et al., 2006; Medina-Martinez et al., 2006).Biocontrol strategies that exploit bacterial QS provide an opportunityto (a) down regulate microbial activity and increaseshelf life, and (b) alter microbial activity such that survival ofthe targeted microorganism is unlikely. The main advantagewith approach (a) is that it does not leave open an opportunityfor other undesirable microorganisms to colonise the niche butdown regulates the expression of enzymes such as proteases,thus limiting damage to the food. In the case of food-bornepathogens, approach (b) may be the most desirable outcome.Furthermore using the bacteria’s own QS system against itselfAugust/September 200729

decreases the possibility that the bacteria will adapt and becomeresistant to the QS inhibitor used. Interventions targetingbacterial QS in food are currently largely unexplored.A number of different classes of molecules that blockquorum sensing have been sought, including enzymes thatdegrade the signal (e.g. lactonases), signal analogues and signalantagonists as well as the use of naturally derived signalmolecules to stimulate an inappropriate response leadingto cell death (Dong et al., 2002; Castang et al., 2004; Müh etal., 2006; Ren et al., 2001; Qazi et al., 2006). Furthermore, anumber of these molecules have been derived from plantscommonly used in the food industry today. Extracts of garlichave been shown to block QS by Pseudomonas aeruginosa, ina cystic fibrosis mouse model, limiting the production of biofilmand thereby aiding clearance of the bacteria (Bjarnsholtet al., 2005). Similarly, vanilla extracts have been shown to interferewith quorum sensing of Chromobacterium violaceum,suggesting that the consumption of vanilla-containing foodsmay be beneficial (Choo et al., 2006). Another food additivethat exhibits QS inhibiting abilities is cinnamaldehyde (Niu etal., 2006). Low concentrations of cinnamaldehyde, similar tothose used in food, were shown to block detection of twodifferent bacterial cell signalling molecules by competing withthe signal molecule itself or signal precursors. Although, thesethree QS inhibitors are already used as food additives, theirantibacterial activity potential within different food substratesneeds to be explored.Medina-Martinez et al. (2007) showed that Yersinia enterocoliticaproduced QS signals when grown on meat and fishextracts but not on certain vegetable extracts, suggesting thatquorum sensing systems are active but are substrate- and mostprobably organism-dependent. Furthermore, not all QS inhibitorsare able to block QS systems and prevent food spoilage.Rasch et al. (2007) showed that some QS inhibitors were ableto decrease proteolytic activity of Pectobacterium, but theiractivity did not prevent spoilage of bean sprouts by these bacteria.Furthermore, garlic extract did not prevent spoilage ofbean sprouts by Pectobacterium but rather appeared to induceit. It is clear that spoilage is multi-factorial, with other regulatorypathways involved. It may be that QS inhibition (QSI)will be the ‘magic bullet’ in some situations while in others itmay not be successful. What is clear is that for each organismthe regulatory role played by QS needs to be understood andthe significance in specific food matrices evaluated; it is onlythen that QSI can be effective in the fight against bacteria inour food.ConclusionsConsumer expectations of pathogen-free food with anacceptable shelf life without the use of synthetic chemicalspresent major challenges for food manufacturers, given thecurrently available technologies. The biocontrol approachesreviewed here offer some particular advantages by being moretargeted than conventional treatments, both leaving the goodbugs alone and reducing wastage, and are perceived as more‘natural’ and ‘green’. While no one biocontrol approach currentlyoffers a complete solution, collectively they create atoolbox of options employing various mechanisms of attackwhich could be applied individually or in combination. Indeed,as our knowledge of the relationship between exposureand disease expands through quantitative risk assessment activities,it is becoming more apparent that ‘king hit’ reductionsare not necessarily the best approach to reducing human diseaseincidence. The use of biocontrol strategies in food thereforehas great potential to achieve more realistic outcomes,although much work is still to be done to bring these from thelab bench to market.ReferencesAhmed M, Fletcher G C, Maddox I, McIntyre L and Shu Q(2006) Can Bdellovibrionaceae be biological control agents? 51stMeeting of the New Zealand Microbiological Society; November21-24, 2006. Hamilton, New Zealand. (Oral presentation).Anderson R C, Genovese K J, Harvey R B, Callaway T Rand Nisbet D J (2006) Preharvest Food Safety Applications ofCompetitive Exclusion Cultures and Probiotics. In: Probiotics inFood Safety and Human Health. Eds. I Goktepe, V K Juneja &M Ahmedna. Taylor & Francis Group, LLC Boca Raton, FL, USA.pp 273-284.Atterbury R J, Van Bergen M A P, Oritz F, Lovell M A, Harris JA, De Boer A, Wagenaar J A, Allen V M and Barrow P A (2007)Bacteriophage therapy to reduce Salmonella colonization ofbroiler chickens. Appl Environ Microbiol 73, 4543-4549.Bjarnsholt T, Jensen P O, Rasmussen T B, Christophersen L,Calum H, Hentzer M, Hougen H P, Rygaard J, Moser C, Eberl L,Hoiby N, and Givskov M (2005) Garlic blocks quorum sensingand promotes rapid clearing of pulmonary Pseudomonas aeruginosainfections. Microbiol 151:3873-80.Brashears M M, Jaroni D and Trimble J (2003) Isolation, selection,and characterisation of lactic acid bacteria for a competitiveexclusion product to reduce shedding of Escherichiacoli O157:H7 in cattle. J Food Prot 66:355-363.Bredholt S, Nesbakken T and Holck A (1999) Protective culturesinhibit growth of Listeria monocytogenes and Escherichiacoli O157:H7 in cooked, sliced, vacuum- and gas- packagedmeat. Int J Food Microbiol 53:43-52.Bredholt S, Nesbakken T and Holck A (2001) Industrialapplication of an antilisterial strain of Lactobacillus sakei as aprotective culture and its effect on the sensory acceptability ofcooked, sliced, vacuum-packaged meats. Int J Food Microbiol66:191-196.Bruttin A and Brüssow H (2005) Human volunteers receivingEscherichia coli T4 orally: a safety test of phage therapy.Antimicrob Agents Chemother 49, 2874-2878.Burt S (2004) Essential oils: their antibacterial properties andpotential applications in foods – a review. Int. J. Food. Microbiol.94 (3), 223.Carlton R M, Noordman W H, Biswas B, de Meester E Dand Loessner M J (2005) Bacteriophage P100 for control of Listeriamonocytogenes in foods: Genome sequence, bioinformaticanalysis, oral toxicity study, and application. Regul ToxicolPharmacol 43, 301-312.Castang S, Chantegrel B, Deshayes C, Dolmazon R, GouetP, Haser R, Reverchon S, Nasser W, Hugouvieux-Cotte-PattatN, and Doutheau A. (2004) N-Sulfonyl homoserine lactones asantagonists of bacterial quorum sensing. Bioorg Med Chem Lett.14:5145-9.Choo J H, Rukayadi Y, and Hwang J K (2006) Inhibition ofbacterial quorum sensing by vanilla extract. Lett Appl Microbiol.42:637-41.30Food New Zealand

Deegan L H, Cotter P D, Hill C and Ross P (2006) Bacteriocins:Biological tools for bio-preservation and shelf-life extension.Int Dairy J 16:1058-1071.Delves-Broughton J, Weber G & Elsser D (2007) Naturalpreservation of foods using bacterial metabolites and live additionof bacteria. Food Science Central http://www.foodsciencecentral.com/fsc/ixid14740.Diez-Gonzalez F and Schamberger G P (2006) Use of Probioticsin Preharvest Food Safety Applications. In: Probiotics inFood Safety and Human Health. Eds. I Goktepe, V K Juneja &M Ahmedna. Taylor & Francis Group, LLC Boca Raton, FL, USA.pp 251-272.Dong Y H, Gusti A R, Zhang Q, Xu J L, and Zhang L H(2002) Identification of quorum-quenching N-acyl homoserinelactonases from Bacillus species. Appl Environ. Microbiol.68:1754-9.Dudler R and Eberl L (2006) Interactions between bacteriaand eukaryotes via small molecules. Curr Opin Biotechnol.17:268-73.Fisher K and Phillips C A (2006) The effect of lemon, orangeand bergamot essential oils and their components on thesurvival of Campylobacter jejuni, Escherichia coli O157, Listeriamonocytogenes, Bacillus cereus and Staphylococcus aureus invitro and in food systems. J Appl. Microbiol 101:1232-1240.Fisher K, Rowe C and Phillips C A (2007) The survival ofthree strains of Arcobacter butzleri in the presence of lemon,orange and bergamot essential oils and their components invitro and on food. Lett Appl Microbiol 44:495-9.Fischetti V A (2005) Bacteriophage lytic enzymes: novel antiinfectives.Trends Microbiol 13:491-496.Fox J L (2005) Therapy with phage: Mirage or potential barrageof products? ASM News 71:453-455.Fratamico P M and Cooke P H (1996) Isolation of Bdellovibriosthat prey on Escherichia coli O157:H7 and Salmonellaspecies and application for removal of prey from stainless steelsurfaces. J Food Safety 16:161-173.Friedman M, Henika P R, Levin C E, Mandrell R E and KozukueN (2006) Antimicrobial activities of tea catechins andtheaflavins and tea extracts against Bacillus cereus. J Food Prot69:354-61.Fuqua C, Parsek M R, and Greenberg E P (2001) Regulationof gene expression by cell-to-cell communication: acyl-homoserinelactone quorum sensing. Annu Rev Genet. 35:439-68.Gaysinsky S, Davidson P M, Bruce B D and Weiss J (2005)Growth inhibition of Escherichia coli O157:H7 and Listeriamonocytogenes by carvacrol and eugenol encapsulated in surfactantmicelles. J Food Prot 68:2559-66.Goktepe I (2006) Probiotics as Biopreservatives for EnhancingFood Safety. In: Probiotics in Food Safety and HumanHealth. Eds. I Goktepe, V K Juneja & M Ahmedna. Taylor &Francis Group, LLC Boca Raton, FL, USA. pp 285-307.Gram L and Melchiorsen J (1996) Interaction between fishspoilage bacteria Pseudomonas sp. and Shewanella putrefaciensin fish extracts and on fish tissue. J Appl Bacteriol 80:589-595.Greer G G and Dilts B D (1990) Inability of a bacteriophagepool to control beef spoilage. Int J Food Microbiol 10:331-342.Henry M B, Lynch J M and Fermor T R (1991) Role of siderophoresin the biocontrol of Pseudomonas tolaasii by fluorescentpseudomonad antagonists. J Appl Bacteriol 70:104-108.Holley R A and Patel D (2005) Improvement in shelf-lifeand safety of perishable foods by plant essential oils and smokeantimicrobials. Food Microbiol 22:273-292.Hudson J A, Billington C, Carey-Smith G and Greening G(2005) Bacteriophages as biocontrol agents in food. J Food Prot68:426-437.Kadouri D and O’Toole G A (2005) Susceptibility of Biofilmsto Bdellovibrio bacteriovorus Attack. Appl Environ Microbiol,71:4044-4051.Kolenbrander P E, Andersen R N, Blehert D S, Egland P G,Foster J S and Palmer R J Jr. (2002) Communication among oralbacteria. Microbiol Mol Biol Rev. 66:486-505.Lambert C, Morehouse K A, Chang C-Y and Sockett R E(2006) Bdellovibrio: growth and development during the predatorycycle. Curr Opinion Microbiol 9:639-644.Leverentz B, Conway W S, Alavidze Z, Janisiewicz W J, FuchsY, Camp M J, Chighladze E and Sulakvelidze A (2001) Examinationof bacteriophage as a biocontrol method for Salmonella onfresh-cut fruit: a model study. J Food Prot 64:1116-1121.Lindberg A A (1967) Studies of a receptor for Felix O-1phage in Salmonella minnesota. J Gen Microbiol 48:225-233.Liu M, Gray J M and Griffiths M W (2006) Occurrence ofproteolytic activity and N-acyl-homoserine lactone signals in thespoilage of aerobically chill-stored proteinaceous raw foods. JFood Prot 69:2729-37.Loc Carrillo C M, Atterbury R J, El-Shibiny A, Connerton PL, Dillon E, Scott A and Connerton I F (2005) Bacteriophagetherapy to reduce Campylobacter jejuni colonization of broilerchickens. Appl Environ Microbiol 71:6554-6563.Lu G, McIntyre L, Fletcher G C, Maddox I S and Shu Q (2004)Antagonistic activity of Lactobacillus reuteri DPC16 againstfoodborne pathogens. New Zealand Microbiological SocietyMeeting, Palmerston North, New Zealand, November 19th.Medina-Martinez M S, Uyttendaele M, Meireman S and DebevereJ (2006) Screening of N-acyl-L-homoserine lactone productionby bacteria isolated from fresh foods. Commun AgricAppl Biol Sci. 71:209-12.Medina-Martinez M S, Uyttendaele M, Meireman S, andDebevere J (2007) Relevance of N-acyl-L-homoserine lactoneproduction by Yersinia enterocolitica in fresh foods. J ApplMicrobiol. 102:1150-8.Millette M, Luquet F M and Lacroix M (2006) In vitro growthAugust/September 200731

control of selected pathogens by Lactobacillus acidophilus- andLactobacillus casei-fermented milk. Letts Appl Microbiol 44:314– 319.Müh U, Hare B J, Duerkop B A, Schuster M, Hanzelka BL, Heim R, Olson E R and Greenberg E P (2006) A structurallyunrelated mimic of a Pseudomonas aeruginosa acyl-homoserinelactone quorum-sensing signal. Proc Natl Acad Sci U S A.103:16948-52.Murinda S E, Roberts R F and Wilson R A (1996) Evaluationof colicins for inhibitory activity against diarrheagenicEscherichia coli strains including serotype O157. Appl EnvMicrobiol 62:3196-3202.Niu C, Afre S and Gilbert E S (2006) Subinhibitory concentrationsof cinnamaldehyde interfere with quorum sensing. LettAppl Microbiol. 43:489-494.O’Flynn G, Ross R P, Fitzgerald G F and Coffey A (2004)Evaluation of a cocktail of three bacteriophages for biocontrolof Escherichia coli O157:H7. Appl Environ Microbiol 70:3417-3424.Oussalah M, Caillet S, Salmieri S, Saucier L and Lacroix M(2006) Antimicrobial effects of alginate-based film containingessential oils for the preservation of whole beef muscle. J FoodProt 69:2364-9.Paradis-Bleau C, Cloutier I, Lemieux L, Sanschagrin F, LarocheJ, Auger M, Garnier A and Levesque R C (2007) Peptidoglycanlytic activity of the Pseudomonas aeruginosa phage fKZgp144 lytic transglycosylase. FEMS Microbiol. Lett. 266:201-209.Perez-Conesa D, McLandsborough L and Weiss J (2006)Inhibition and inactivation of Listeria monocytogenes and Escherichiacoli O157:H7 colony biofilms by micellar-encapsulatedeugenol and carvacrol. J Food Prot 69:2947-2954.Puupponen-Pimia R, Nohynek L, Hartmann-Schmidlin S,Kahkonen M, Heinonen M, Maatta-Riihinen K and Oksman-Caldentey K M (2005) Berry phenolics selectively inhibit thegrowth of intestinal pathogens. J Appl Microbiol 98:991-1000.Qazi S, Middleton B, Muharram S H, Cockayne A, Hill P,O’Shea P, Chhabra S R, Camara M and Williams P (2006) N-acyl homoserine lactones antagonize virulence gene expressionand quorum sensing in Staphylococcus aureus. Infect Immun74:910-919.Rasch M, Rasmussen T B, Andersen J B, Persson T, Nielsen J,Givskov M and Gram L (2007) Well-known quorum sensing inhibitorsdo not affect bacterial quorum sensing-regulated beansprout spoilage. J Appl Microbiol 102:826-37.Ren D, Sims J J and Wood T K (2001) Inhibition of biofilmformation and swarming of Escherichia coli by (5Z)-4-bromo-5-(bromomethylene)-3-butyl-2(5H)-furanone. Environ Microbiol3:731-6.Rendulic S, Jagtap P, Rosinus A et al.,. (2004) A PredatorUnmasked: Life Cycle of Bdellovibrio bacteriovorus from a GenomicPerspective. Science 303:689-692.Reynolds G (2007) Bacteria effective against fresh cut fruitcontamination. http://www.foodnavigator-usa.com/news/print-NewsBis.asp?id=75142.Rhodes P L, Mitchell J W, Wilson M W and Melton L D (2006)Antilisterial activity of grape juice and grape extracts derivedfrom Vitis vinifera variety Ribier. Int J Food Microbiol 107:281.Rojas-Grau M A, Avena-Bustillos R J, Olsen C, Friedman M,Henika P R, Martin-Belloso O, Pan Z and McHugh T H (2007)Effects of plant essential oils and oil compounds on mechanical,barrier and antimicrobial properties of alginate-apple pureeedible films. J Food Eng 81:634.Schamberger G P and Diez-Gonzalez F (2002) Selection ofrecently isolated colicinogenic Escherichia coli strains inhibitoryto Escherichia coli O157:H7. J Food Prot 65:1381-1387.Schamberger P and Diez-Gonzalez F (2004) Characterizationof colicinogenic Escherichia coli strains inhibitory to enterohemorrhagicEscherichia coli. J Food Prot 67:486-492.Scolari G and Vescovo M (2004). Microbial antagonism ofLactobacillus casei added to fresh vegetables. Italian J Food Sci16:465-475.Seydim A C and Sarikus G (2006) Antimicrobial activity ofwhey protein based edible films incorporated with oregano,rosemary and garlic essential oils. Food Research International39:639.Si W, Gong J, Chanas C, Cui S, Yu H, Caballero C and FriendshipR M (2006a) In vitro assessment of antimicrobial activity ofcarvacrol, thymol and cinnamaldehyde towards Salmonella serotypeTyphimurium DT104: effects of pig diets and emulsificationin hydrocolloids. J Appl Microbiol 101:1282-1291.Si W, Gong J, Tsao R, Zhou T, Yu H, Poppe C, Johnson R andDu Z (2006b) Antimicrobial activity of essential oils and structurallyrelated synthetic food additives towards selected pathogenicand beneficial gut bacteria. J Appl Microbiol 100:296-305.Smith-Palmer A, Stewart J and Fyfe L (2001) The potentialapplication of plant essential oils as natural food preservativesin soft cheese. Food Microbiol 18:463.Stephens T P, Loneragan G H, Chichester L M and BrashearsM M. (2007). Prevalence and enumeration of Escherichia coliO157 in steers receiving various strains of Lactobacillus-baseddirect-fed microbials. J Food Prot 70:1252-1255.Tsuei A-C, Carey-Smith G V, Hudson J A, Billington C andHeinemann J A (2007) Prevalence and numbers of coliphagesand Campylobacter jejuni bacteriophages in New Zealandfoods. Int J Food Microbiol 116:121-125.Valero D, Valverde J M, Martinez-Romero D, Guillen F, CastilloS and Serrano M (2006) The combination of modified atmospherepackaging with eugenol or thymol to maintain quality,safety and functional properties of table grapes. Postharvest Bioland Technol 41:317.Wagner R D (2006) Efficacy and food safety considerationsof poultry exclusion products. Mol Nutr Food Res, 50: 1061-1071.32Food New Zealand