526 Modern Food Microbiology Table 22–1 Some of the Phenotypic Responses Demonstrated to Occur in Some Gram-Negative Bacteria as a Consequence of Quorum Sensing (Taken from the Literature) Organisms Vibrio fischeri Escherichia coli strains Escherichia coli LuxS mutant Escherichia coli Escherichia coli EHEC and EPEC strains of E coli Serratia liquefaciens Serratia marcescens Pantoea stewartii Pectobacterium carotovorum Pectobacterium chrysanthemi Burkholderia cepacia Aeromonas hydrophila Pseudomonas aeruginosa et al Demonstrated Responses Bioluminescence Stx toxin production Decreased swimming speed Formation of att/eff SOS response Type III secretion system Swarming motility Prodigiosin production; carbapenum synthesis Increased polysaccharide synthesis Plant wall degrading enzymes produced Pectate lyases produced Proteases, siderophores produced Exoprotease produced Normal biofilm structure but the one on the right is bioluminescent as a result of acquiring a “quorum” of AI that binds to LuxR as noted above Autoinducer-2 (AI-2) is an alternate quorum signal compound in V harveyi where it regulates bioluminescence in conjunction with AI-1 The AI-2 system has been demonstrated in a number of Gram-negative pathogens The minimum number of cells needed to produce a “quorum” is rarely reported but in one study of psychrotrophic Enterobacteriaceae of food origin, at least 106 cfu/g were found to be necessary to elicit a positive response to the biosensors that were used.51 The best known and most widely studied AI substances for Gram-negative bacteria are Nacetylhomoserine lactones (AHLs) These compounds are composed of homoserine (with a lactone ring) + an acyl side chain that varies from to >10 carbons; and the structures of two are in Figure 22–5 Not all Gram-negative bacteria employ the LuxI-LuxR For example, V harveyi, E coli, and S Typhimurium employ a related but different autoinducer production system.94 In addition to the AHLs, some Gram-negative bacteria produce cyclic(cyclo) dipeptides that are involved in quorum sensing either alone or in combination with AHLs.34,54 Structures of two of the cyclo-dipeptides identified by Degrassi et al.34 are presented in Figure 22–5 Among foodborne bacteria, an increase in Stx toxins has been demonstrated, and the Stx genes were induced by quorum sensing.93 Although it is similar to the LuxI system (which produces AI-1), LuxS produces AI-2 and it has been demonstrated in E coli 0157:H7, S Typhimurium, and Campylobacter jejuni during their growth in milk and chicken broth.23 As noted above, a number of Gram-negative psychrotrophs have been shown to produce AHLs in naturally contaminated foods when cell numbers reached 105 –107 cfu/g.51 Quorum sensing is important in biofilm formation, and this is discussed further below For a review of quorum sensing in Gram-negative bacteria, see references 35 and 47 Although most studied in Gram-negative bacteria, quorum sensing occurs among Gram-positive bacteria.38 The AIs for Gram positives are peptides and peptide pheromones, and nisin is perhaps the best known As described by Kleerebezem et al.,62 cell density regulation in these systems appears to follow a common theme in which the signal molecule is a post-translationally processed peptide that Introduction to Foodborne Pathogens 527 Figure 22–5 Structures of four quorum sensing autoinducers A = N-butanoyl-l-homoserine lactone; B = Nhexanoyl-l-homoserine lactose; C = cyclo(l-Tyr-l-Pro); D = cyclo(l-Leu-l-Pro) Structures C and D are from reference 34 is secreted by a dedicated ATP-binding-cassette exporter The secreted peptide pheromone functions as the input signal for a specific sensor component of a two-component signal-transduction system Interestingly, some Gram-positive bacteria such as Bacillus spp produce lactonases that specifically degrade AHLs produced by Gram-negative bacteria.36 Among other phenotypic and physiologic activities demonstrated among Gram-positive bacteria is a virulence response in Staphylococcus aureus,59 and the production of antimicrobial peptides other than nisin Nisin has been shown to induce its own synthesis.62 Quorum sensing has been demonstrated in S aureus and S epidermidis, and when the two were co-cultured, S epidermidis appeared to be favored leading to the suggestion that this could be the reason why this species is more predominant on the skin where autoinducing pheromones are more likely to be effective than when inside the body.78 In S aureus, an octapeptide pheromone effects virulence by activating the expression of the agr locus.59 The extent to which quorum sensing occurs in vivo is problematic because of the general lack of opportunities for AI substances to reach a quorum (see Biofilms section below) BIOFILMS The importance of biofilms in food safety warrants a better understanding of their biology, structure, and function They are covered in this chapter relative to virulence properties of certain pathogens A biofilm consists of the growth of bacteria, fungi, and/or protozoa alone or in combination bound together by an extracellular matrix that is attached to a solid or firm surface Common examples include the slimy surfaces on rocks or logs in bodies of running water, dental plaques, and the slime layer on refrigerator-spoiled fresh meats, fish, and poultry They form on surfaces in large part because nutrients are found in higher concentrations than in the open liquid (planktonic) area In laboratory studies, surface adherence is best in rich media.11 Attachment is facilitated by the microbial excretion of an exopolysaccharide matrix sometimes referred to as a glycocalyx Microcolonies form within this microenvironment in a manner that leads to microbial communities that allow water channels to form between and around the microcolonies The latter has been likened to a primitive circulatory system 528 Modern Food Microbiology where nutrients are brought in and toxic by-products are carried out Microbial cells in liquids that are not in a biofilm are in a planktonic (free-floating) state From the standpoint of food safety and spoilage, biofilms are important because of their accumulation on foods, food utensils, and surfaces; and because of the difficulty of their removal While under natural conditions, biofilms tend to be composed of mixed cultures, pure culture systems are often used in laboratory studies Some of the solid surfaces employed to study foodborne bacteria include floor sealant, glass slides, nylon, polycarbonate, polypropylene, rubber, stainless steel, and Teflon Glass and stainless steel are widely used From some of the many studies that have been reported in food environments, the following summaries can be made: Although biofilm formation by single cultures in rich media (e.g., tryptic soy broth) may be evident after 24 hours when appropriate growth temperatures are used, 3–4 days or more are necessary for maximum development On glass slides in a culture medium for days at 24◦ C, L monocytogenes grew to about 6–7 log10 /cm21 Not all strains of the same species are equally capable of initiating biofilm formation,74 and surface attachment and biofilm development are different processes.63 Microorganisms in biofilms may exhibit different physiologic reactions than planktonic forms, and the biofilm may contain cells in the viable but nonculturable state.18,22 Microorganisms in biofilms are considerably more resistant to removal by commonly used cleaning and sanitizing agents, and cleaners and sanitizers used in combination appear to be more effective in removing biofilm growth.1,77 The attachment of a given pathogen to surfaces may be aided by the formation of a mixed-culture biofilm,17,68,88 and an example is presented below In a biofilm with L monocytogenes and a Flavobacterium sp growing together on stainless steel, the attachment of L monocytogenes was increased and persisted longer in mixed culture than when it grew alone.15 In addition, sublethally injured L monocytogenes cells increased significantly in mixed culture Shewanella putrefaciens readily formed biofilms on inert food processing surfaces, and when nutrients were supplied, multilayered structures were formed.7 Three strains of L monocytogenes, each from a human outbreak, produced unique “honeycomb” structured biofilms on stainless steel coupons, and strain Scott A was the most conspicuous.71 A laboratory strain did not form a biofilm In another study, the L monocytogenes strains that produced the most extra polymeric substance (EPS) produced a three-dimensional biofilm structure in contrast to control strains.14 Biofilm formation could not be correlated with serotypes Employing a strain of Pseudomonas aeruginosa, extracellular DNA was found to be essential for biofilm formation using a flow-chamber system.103 Although the source of DNA was not determined, the application of DNase I dissolved the biofilm, suggesting that DNA was an integral part of the biofilm structure As noted above, cells in the viable but nonculturable (VBNC) state can form biofilms, and the VBNC cells of Enterococcus faecalis adhered to Caco-2 and Girardi heart cells, but at a reduced capacity compared to controls.82 The inhibition of biofilm formation by Bacillus subtilis has been demonstrated using furanone ([5Z ]4-brome-5-[bromomethyhlene]-3-butyl-2[5H ]-furanone), and it inhibited both growth and swarming motility of B subtilis.83 It was originally isolated from a marine alga For more on biofilms, see references 26, 45, and 109 Introduction to Foodborne Pathogens 529 Apparent Role of Quorum Sensing The first published demonstration of the possible role of quorum sensing in biofilms was that of McLean et al.73 who recovered AHLs from aquatic biofilms growing on submerged stones in the San Marcos River in Texas The direct involvement of AHLs was demonstrated with Pseudomonas aeruginosa where a mutant that could not synthesize AHL produced atypical biofilms (contained no water channels) that were sensitive to sodium dodecyl sulfate in contrast to wild-type strains.30 Biofilm formation on indwelling medical devices and by organisms such as P aeruginosa and Burkholderia cepacia that wreak havoc on cystic fibrosis patients is well documented (as is biofilm formation by L monocytogenes in food processing environments) The relationship between biofilm formation, quorum sensing, virulence or pathogenicity of foodborne pathogens is unclear, but it is not inconceivable that relationships exist SIGMA (δ) FACTORS Sigma is one of the four subunits of RNA polymerase, and its role is in the recognition of the promoter (where RNA polymerase binds to DNA and transcription begins) Sigma is involved only in the initial RNA polymerase-DNA complex After a small portion of mRNA has formed, δ dissociates δ A (or δ 70 , the number refers to the molecular size in kilodaltons) recognizes the majority of genes that encode essential cell functions, and its closest homologue is δ S Among the known sigma factors are the following: δ 28 is involved in flagellar synthesis in Salmonella, and also in the type III secretion system δ 32 (RpoH) is involved in heat shock proteins (HSPs), some of which are molecular chaperones or proteases that eliminate those that cannot be repaired δ 54 (RpoN) regulates harp (hypersensitive response and pathogenicity) genes in at least some Pseudomonas syringae pathovars δ B endows L monocytogenes with resistance to lethal acidic conditions It is more abundant at 25◦ C than at 42◦ C in E coli It is involved in stress responses in Bacillus subtilis δ S is present in the γ -subclass of the Proteobacteria including the vibrios It aids V vulnificus in its resistance to adverse environmental conditions It is discussed further below under Alternative Sigma Factors A change in the cell’s environment that is stressful (starvation, low pH, increased osmotic pressure, etc.) leads to the induction of alternative sigma factors that aid the cell in coping with its unfavorable state (see reference 5) The general responses that bacteria make upon exposure to acidic conditions consist of the following:27 (1) proton pumps come into play where a proton motive force (PMF) can facilitate the extrusion of protons from the cytoplasm, which results in a drop in intracellular pH; (2) repair of macromolecules such as DNA, and proteins such as RecA; (3) changes in cell membrane components (e.g., fatty acids); (4) regulation of gene expression by alternative sigma factors; (5) cell density and biofilm formation (which protects cells from certain adverse environmental influences); and (6) alteration of metabolic pathways Biofilms are covered in the previous section of this chapter, and alternative sigma factors are discussed further below Alternative Sigma Factors The alternative sigma factor, δ 38 (sigma-38, δ S ) is encoded by the rpoS gene, and it regulates at least 30 proteins, and it along with δ B is discussed in this section Exposure to acid stress leads to the synthesis of proteins that protect the bacterium With log-phase cells at or below pH 4.5, at least 530 Modern Food Microbiology 43 proteins are induced When stationary-phase cells are shifted to or below pH 4.5, they synthesize 15 proteins that are distinct from log-phase cells Acid Tolerance Response With respect to the acid tolerance response (ATR) of L monocytogenes, the pH minimum for growth of two strains was 3.5 and 4.0 in a chemically defined medium using HCl.81 pH values below these were lethal unless the strains were previously exposed to pH of 4.8 and 3.5 With mutants of L monocytogenes that showed increased ATR, they demonstrated increased lethality for mice compared to wild type strains76 , suggesting that acid conditions could be selective for strains with increased virulence The mutants were recovered after exposure to pH 3.5 for up to hours at 37◦ C.76 Similarly, acid-adapted Yersinia enterocolitica cells grown at pH 7.5, then shifted to pH 5.0, were significantly more enteropathogenic than controls when tested using a suckling mouse model.106 δ B has been identified in L monocytogenes, B subtilis, and S aureus; and its function has been compared to those of RpoS/δ S in Gram-negative bacteria In B subtilis, δ B influences the regulon of 100 genes in response to environmental and energy stresses B subtilis mutants are more sensitive to heat, ethanol, acid, freezing, drying, etc.27 One δ B reduces virulence in L monocytogenes Interestingly, a hydrostatic pressure resistant strain of L monocytogenes (survived 400 MPa for 20 minutes) displayed increased resistance to heat, acid, and hydrogen peroxide.61 It has been found that the prior adaptation of L monocytogenes that leads to acid resistance may depend upon a number of other growth parameters.65 The δ B protein is necessary for full resistance of L monocytogenes to lethal acid exposure, and it along with δ S is associated with general stress responses in both Gram-positive and Gram-negative bacteria.44 After acid adaptation (pH 3.0, HCl), L monocytogenes strain Scott A, 11 proteins showed induced expression while 12 were repressed.31 On the other hand, acid adaptation did not protect this species on beef treated with 2% lactic or acetic acid.56 L monocytogenes on fresh meats may be made more acid sensitive by the Gram-negative bacteria of the fresh-meat biota.86 As noted above, δ B plays a role in acid resistance of stationary phase cells, oxidative and osmotic stress resistance, and in low-temperature growth of L monocytogenes Acid adaptation of L monocytogenes provides protection against HHP and freezing.102 Acid-adapted strains (pH 5.0–3.25, brain heart infusion, BHI, broth) of Shigella flexneri and S sonnei survived up to 14 days in tomato and apple juice stored at 7◦ C.6 The minimum pH for growth in acidified BHI was 4.75 and 4.5 for S flexneri and S sonnei, respectively In one study, acid-adapted (pH 2.5, tryptic soy broth), E coli 0157:H7 remained on beef carcasses after a 2% acetic acid wash more so than nonadapted cells.9 In yet another study of acid tolerance in E coli 0157:H7, acid tolerance decreased after exposure to non-acid washes.85 Acid resistance (percentage of cells that survive exposure to pH 2.5 for hours) is well studied in shigellae Gorden and Small50 found that among the cultures they examined, of 12 shigellae were acid resistant; 11 of 15 generic E coli (including strain K-12) showed the same level of acid resistance; of enteroinvasive (EIEC) strains were resistant but none of enteropathogenic (EPEC) strains or 12 salmonellae were acid resistant As to why so few shigellae cells are needed to cause disease, these investigators hypothesized that after these organisms leave the colon, they enter the stationary phase outside the host Upon ingestion by another host, they are already acid resistant, and low numbers can survive through the acidity of the stomach.50 In another study, Stx-producing strains of E coli that could not survive at pH 2.5 were made acid resistant by the introduction of the rpoS gene on a plasmid.100 When Stx-producing strains of E coli were grown in broth at pH 4.6–4.7, they became 1.1- to 2.0-fold more resistant to radiation than control strains.16 It has been suggested that this response may lower the number of cells needed to initiate infection.97 For instance, it has been ... monocytogenes was increased and persisted longer in mixed culture than when it grew alone.15 In addition, sublethally injured L monocytogenes cells increased significantly in mixed culture Shewanella... formed biofilms on inert food processing surfaces, and when nutrients were supplied, multilayered structures were formed.7 Three strains of L monocytogenes, each from a human outbreak, produced... conditions, biofilms tend to be composed of mixed cultures, pure culture systems are often used in laboratory studies Some of the solid surfaces employed to study foodborne bacteria include floor sealant,