The Enterobacteriaceae and Their Significance to the Food Industry

ILSI Europe Report Series

the Enterobacteriaceae and their Significance to the Food Industry

Report

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Prof. A. Boobis, Imperial College of London (UK) Mr. C. Davis, Kraft Foods (CH) Prof. P. Calder, University of Southampton (UK) Mr. R. Fletcher, Kellogg Europe (IE) Prof. G. Eisenbrand, University of Kaiserslautern (DE) Dr. M. Knowles, Coca-Cola Europe (BE) Prof. A. Grynberg, Université Paris Sud – INRA (FR) Dr. G. Kozianowski, Südzucker/BENEO Group (DE) Prof. em. G. Pascal, National Institute for Agricultural Research – Dr. G. Meijer, Unilever (NL) INRA (FR) Prof. J. O’Brien, Nestlé (CH) Prof. G. Rechkemmer, Max Rubner-Institut – Federal Research Prof. C. Shortt, McNeil Nutritionals (UK) Institute of Nutrition and Food (DE) Dr. J. Stowell, Danisco (UK) Dr. J. Schlundt, National Food Institute (DK) Dr. G. Thompson, Danone (FR) Prof. V. Tutelyan, National Nutrition Institute (RU) Dr. P. Weber, DSM (CH) Prof. G. Varela-Moreiras, University San Pablo-CEU of Madrid (ES)

ILSI Europe Emerging Microbiological Issues Task Force industry members

Barilla G. & R. Fratelli Campina Friesland H J Heinz Institut Mérieux Kraft Foods Mars Europe Nestlé Unilever

THE ENTEROBACTERIACEAE AND THEIR SIGNIFICANCE TO THE FOOD INDUSTRY

By Chris Baylis, Mieke Uyttendaele, Han Joosten and Andy Davies

Report Commissioned by the ILSI Europe Emerging Microbiological Issues Task Force

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ISBN 9789078637332 Contents T he

EXECUTIVE SUMMARY 4 en t

1. INTRODUCTION 5 erobac 2. CHANGES IN TAXONOMY 6

3. ENTEROBACTERIACEAE AND THEIR ROLE AS INDICATOR AND t eriaceae INDEX ORGANISMS IN FOODS 9 4. DETECTION AND ENUMERATION METHODS FOR ENTEROBACTERIACEAE IN FOODS 13

and 5. ENTEROBACTERIACEAE AS FOODBORNE PATHOGENS 17

5.1 Salmonella 19 t 5.2 Yersinia enterocolitica 21 heir 5.3 Yersinia pseudotuberculosis 21

5.4 Pathogenic Escherichia coli 22 significance 5.5 Cronobacter spp. 25 5.6 Shigella spp. 26 5.7 Opportunistic and emerging pathogenic Enterobacteriaceae 26 5.8 Extended-spectrum β-lactamase (ESβL)-producing Enterobacteriaceae 28

6. ENTEROBACTERIACEAE IN FOOD SPOILAGE 29 o

7. GROWTH, INACTIVATION AND SURVIVAL 31 he

7.1 Temperature 31 food

7.2 Water activity (aw) 32 7.3 pH 32

7.4 Gaseous atmosphere 33 indus 8. FURTHER READING 35 t ry 9. REFERENCES 36 GLOSSARY OF TERMS 42 ABBREVIATIONS 47

Authors: Chris Baylis, (UK), Mieke Uyttendaele, University of Ghent (BE), Han Joosten, Nestlé (CH) and Andy Davies, H J Heinz (UK) Scientific Reviewers: Tom Cheasty, Health Protection Agency (UK), Seamus Fanning, UCD Veterinary Sciences Centre (IE) and Stefano Morabito, Istituto Superiore di Sanità (IT) Report Series Editor: Kevin Yates (UK) Coordinator: Pratima Rao Jasti, ILSI Europe (BE)

3 ry t EXECUTIVE SUMMARY indus

he Enterobacteriaceae is a family of Gram-negative, non-spore-forming bacteria and is one of the most important groups of bacteria known to man. The food

T introduction of genetic methods, in particular the analysis of 16S rRNA gene he t

sequences, has revolutionised our understanding of these bacteria and the relationship that exists o t between the different genera and species. Consequently, there have been many changes in bacterial

taxonomy resulting in the introduction of new genera and species and the reclassification of some existing bacteria belonging to the Enterobacteriaceae. There are currently 48 genera and 219 species recognised within the Enterobacteriaceae and these numbers are likely to increase in the future. significance

This family includes a number of important foodborne pathogens such as Salmonella, Yersinia enterocolitica, pathogenic Escherichia coli (including E. coli O157:H7), Shigella spp. and Cronobacter heir t spp. Other members of the family are regarded as opportunistic pathogens, especially in clinical

settings (e.g., Klebsiella spp, Serratia spp. and Citrobacter spp.). It is not the intention of this report and

to provide detailed information on specific pathogens that belong to the Enterobacteriaceae. These have either been covered by separate ILSI reports or may instead be the topic of future publications.

In addition to their aetiology in foodborne illness, some members of the family are also associated eriaceae t with food spoilage and therefore contribute to significant economic losses for the agricultural and food industries. For example, Erwinia spp., and the more recently introduced Pectobacterium spp. and Brenneria spp., have long associations with plant and fruit diseases. Many other members of the erobac t Enterobacteriaceae are responsible for spoilage of a variety of foods including fruit and vegetables, en meats, poultry, eggs, milk and dairy products, as well as fish and other seafoods. he T Different numbers of Enterobacteriaceae can be cultured from a variety of raw materials, depending both on the origin of the raw material and on the control of hygiene. Raw materials entering the food chain are often subjected to further manipulations that impact on the microbial ecology. The impact of processing, product formulation and storage on Enterobacteriaceae in food will be discussed. It is important to note that the precise conditions required to support the growth and survival of a particular Enterobacteriaceae member can differ depending on several factors. These include prior exposure to intrinsic factors (i.e., acidity (pH), water activity and natural antimicrobial substances), extrinsic factors (i.e., temperature, relative humidity and gaseous atmosphere), and implicit conditions (i.e., interactions with other microbial populations, associated with a particular food product and the particular strain of bacterium). It is well-established that pathogenic strains of E. coli, Salmonella and Cronobacter demonstrate prolonged survival under adverse conditions. Thus Enterobacteriaceae demand particular attention both in perishable food and in processed foods with a long shelf-life.

The Enterobacteriaceae family includes genera with the ability to ferment lactose (termed coliform bacteria) and that have long been used as indicator organisms by the food and water industry. Nowadays, both Enterobactericeae and coliforms are isolated from foods to indicate evidence of poor hygiene or inadequate processing (especially heat-treatment), process failure and post-process contamination of foods. E. coli is commonly used to provide evidence of potential faecal contamination in certain foods and is used as an index organism for the presence of enteric pathogens such as Salmonella.

Methods for the detection and enumeration of Enterobacteriaceae have changed little since they were first introduced and many still rely on the growth of the bacterium in selective media along with the use of carbohydrate (e.g. glucose) as an energy source. In contrast, several rapid methods are now available for detection of specific pathogenic members of the Enterobacteriaceae found in foods including Salmonella and E. coli O157.

4 T 1. INTRODUCTION he

en t erobac he family Enterobacteriaceae comprises a large group of Gram-negative non-spore-forming bacteria typically 1-5 μm in length. They are facultative

T t

anaerobes and with the exception of Saccharobacter fermentans and some eriaceae strains of Yersinia and Erwinia, they share the ability to reduce nitrate to nitrite. These bacteria are generally motile by peritrichous flagella except for Shigella and Tatumella and some other non-motile members of this family. For example, Salmonella are typically motile, notable exceptions being the Salmonella serotypes Pullorum and Gallinarum. A common feature of the Enterobacteriaceae, and

which helps to differentiate them from other closely related bacteria, is the lack of cytochrome C t oxidase, although there are exceptions such as Plesiomonas spp. Enterobacteriaceae are catalase- heir positive with the exception of Shigella dysenteriae 1 and Xenorhabdus species. Enterobacteriaceae ferment a variety of carbohydrates, but their ability to produce acid and gas from the fermentation significance of D-glucose is one characteristic that remains an important diagnostic property and is commonly used as a basis for their detection and enumeration. Some members of the Enterobacteriaceae (e.g., Enterobacter spp., Escherichia coli, Citrobacter spp. and Klebsiella spp.) can be recognised using methods that exploit their ability to ferment lactose rapidly (usually within 24-48 h) producing

t acid and gas. These are collectively termed coliform bacteria and are often used as (faecal) o indicator organisms by the food and water industry (see below), because their normal habitat is the t he

gastrointestinal tract of mammals, birds etc. However, unlike the Enterobacteriaceae family, this is food not a well-defined taxonomic group.

Members of the Enterobacteriaceae are widely distributed. Although strains of some species are indus harmless commensals, such as some strains of E. coli, others are important human and animal pathogens, and some are pathogenic to plants and insects. Their ubiquitous distribution means t ry that it is inevitable that some members of the Enterobacteriaceae will enter the food chain. Members of the family are responsible for causing foodborne disease and some also cause food spoilage and therefore contribute to substantial economical losses and food wastage. The initial Enterobacteriaceae contamination level in the raw materials is predominantly governed by Good Agricultural Practices (GAP) during primary production and subsequently during slaughter of livestock at the abattoir. Further along the food supply chain, contamination by Enterobacteriaceae, including pathogens, must be prevented or controlled by the application of one or more of the acknowledged quality assurance systems including Hazard Analysis and Critical Control Point (HACCP) systems and Good Manufacturing Practices (GMP).

5 ry 2. CHANGES IN TAXONOMY t indus

he name Enterobacteriaceae was first proposed by Rahn (1937). The type genus is Escherichia. Enterobacteriaceae comprise a large group of genetically and food T biochemically related bacteria. Phylogenetic studies place them in the phylum he t

Proteobacteria, Class Gammaproteobacteria and Order Enterobacteriales (Brenner et al., 2005). Until o

t the early 1960s bacterial classification was largely based on phenotypic characteristics and culture-based

observations. The introduction of genetic methods, such as DNA-DNA hybridisation and guanine plus cytosine (G+C) determination revolutionised bacterial taxonomy and classification. More recently analysis of 16S rRNA gene sequences has been used to elucidate further the genetic relationships between members of the Enterobacteriaceae and their similarity to other closely related bacteria. Consequently,

significance there have been recent changes to the classification of members of this family. Indeed, the number of

genera and species of Enterobacteriaceae increased from 12 genera and 36 species in 1974 to at least heir

t 34 genera, 149 species and 21 subspecies in 2006 (Baylis, 2006). By the time of writing, these numbers

had grown to at least 48 genera, 219 species and 41 sub-species in the family Enterobacteriaceae and

(Table 1). This revised list excludes some additional genera that have been proposed but have yet to be validated and approved for inclusion. The situation is likely to continue to evolve as unpublished and as yet undescribed genera and species are added. eriaceae t Table 1. Genera, species and sub-species belonging to the Enterobacteriaceae Genus Species Sub species

erobac Alterococcus agarolyticus t Arsenophonus nasoniae en

Brenneria alni, nigrifluens, quercina, rubrifaciens, salicis he

T Buchnera aphidicola Budvicia aquatica Buttiauxella agrestis, brennerae, ferragutiae, gaviniae, izardii, noackiae, warmboldiae Candidatus fragariae Phlomobacter Cedecea davisae, lapagei, neteri Citrobacter amalonaticus, braakii, farmeri, freundii, gillenii, koseri (diversus), murliniae, rodentium, sedlakii, werkmanii, youngae Cosenzaea myxofaciens Cronobacter dublinensis,, malonaticus, muytjensii, sakazakii, dublinensis subsp. dublinensis, turicensis, Genomospecies I dublinensis subsp. lactardi, dublinensis subsp. lausannensis Dickeya chrysanthemi (Pectobacterium), dadantii, dianthicola, dieffenbachiae, paradisiaca, zeae Edwardsiella anguillimortifera, hoshinae, ictaluri, tarda Enterobacter aerogenes, amnigenus, arachidis, asburiae, cloacae subsp. cloacae, cancerogenus (Ent taylorae/Erwinia cancerogena), cloacae subsp. disslovens cloacae, (Erwinia disslovens/Ent disslovens), cowanii, gergoviae, helveticus, hormaechei, kobei, ludwigii, mori, nimipressuralis (Erwinia nimipressuralis), oryzae, pulveris, pyrinus, radicincitans, soli, turicensis Erwinia amylovora, aphidicola, billingiae, carnegieana, mallotivora, papayae, persicina, psidii, pyrifoliae, rhapontici, tasmaniensis, toletana, tracheiphila

6 T

Escherichia albertii, coli, coli inactive, fergusonii, hermannii, he

vulneris en

Ewingella americana t erobac Hafnia alvei, paralvei Klebsiella granulomatis, oxytoca, pneumonia, singaporensis, pneumoniae subsp. ozaenae, variicola pneumoniae subsp. pneumoniae, t

pneumoniae subsp. rhinoscleromatis, eriaceae Kluyvera ascorbata, cryocrescens, georgiana, intermedia Leclercia adecarboxylata (Escherichia)

Leminorella grimontii, richardii and Moellerella wisconsensis

Morganella morganii psychrotolerans morganii subsp. Morganii t heir morganii subsp. sibonii

Obesumbacterium proteus significance Pantoea agglomerans, allii, ananatis, anthophila, brenneri, stewartii subsp. indologenes, calida, conspicua, cypridedii, deleyi, dispersa, stewartii subsp. stewartii eucalypti, eucrina, gaviniae, stewartii, vagans Pectobacterium atrosepticum, betavasulorum, cacticida, carotovorum subsp. carotovorum, carotovorum, cypripedii, wasabiae carotovorum subsp. odoriferum

Photorhabdus asymbiotica, asymbiotica subsp. asymbiotica, o

(Xenorhabdus) luminescens temperata asymbiotica subsp. australis, t luminescens subsp. Akhurstii he

luminescens subsp. caribbeanensis food luminescens subsp. hainanensis luminescens subsp. Kayaii l

uminescens subsp. kleinii indus luminescens subsp. Laumondii luminescens subsp. Luminescens

luminescens subsp. thracensis t ry temperata subsp. Cinerea temperata subsp. Khanii temperata subsp. Stackebrandtii temperata subsp. Tasmaniensis temperata subsp. temperate temperata subsp. thracensis Plesiomonas shigelloides Pragia fontium Proteus hauseri, inconstans, mirabilis, penneri, vulgaris Providencia alcalifaciens, burhodogranariea, heimbachae, rettgeri, rustigianii, sneebia, stuartii, vermicola Rahnella aquatilis Raoultella ornithinolytica, planticola, terrigena Saccharobacter fermentatus Salmonella enterica, bongori enterica subsp. enterica, (Group I) enterica subsp. salamae, (Group II) enterica subsp. arizonae, (Group IIIa) enterica subsp. diarizonae, (Group IIIb) enterica subsp. houtenae, (Group IV) enterica subsp. indica, (Group V) Samsonia erythrinae Serratia entomophila*, ficaria, fonticola, glossinae, marcescens subsp. marcescens, grimesii*, liquefaciens, marcescens, marinorubra, marcescens subsp. sakuensis nematodiphila, odorifera, plymuthica, proteamaculans subsp. proteamaculans*, quinivorans, rubidaea, symbiotica, proteamaculans ureilytica * liquefaciens-like

7 ry Shigella boydii, dysenteriae, flexneri, sonnei t Shimwellia blattae, pseudoproteus Sodalis glossinidius indus

Tatumella citrea, morbirosei, ptyseous, punctata, terrea Thorsellia anophelis food

Trabulsiella guamensis, odontotermitis he t

Wigglesworthia glossinidia o t

Xenorhabdus beddingii, bovienii, budapestensis, cabanillasii, doucetiae, ehlersii, griffiniae, hominickii, indica, innexi, japonica, koppenhoeferi, kozodii, mauleonii, miraniensis, nematophila, poinarii, romanii, stockiae, szentirmaii, vietnamensis Yersinia aldovae, aleksiciae, bercovieri, enterocolitica, enterocolitica subsp. enterocolitica, significance

frederiksenii, intermedia, kristensenii, enterocolitica subsp. palearctica massiliensis, mollaretii, nurmii, pekkanenii, pestis,, heir pseudotuberculosis, rohdei, ruckeri, similis t

and Among the recent changes are the addition of new genera such as Consenzaea, Shimwellia, the insect

symbionts Arsenophonus, Buchnera and Wigglesworthia and the creation of the genus Cronobacter which includes the pathogen previously known as Enterobacter sakazakii (Iversen et al., 2007; 2008). In addition to Cronobacter sakazakii, this new genus includes five other species (malonaticus, turicensis, eriaceae t muytjensii, dublinensis and genomospecies I) along with 3 sub-species of Cronobacter dublinensis (subsp. dublinensis, subsp. lactaridi and subsp. lausannensis). Other new genera and species have also been created to accommodate changes in taxonomy. Erwinia spp. have always been important because erobac t of their association with plant diseases and this genus once contained a large number of species. More en

recently, some Erwinia spp. were re-assigned to either the genus Brenneria or Pectobacterium, which

he encompass necrotic phytopathogenic species and soft rotting phytopathogenic species, respectively. T

The genus Raoultella includes three former Klebsiella spp, including two (ornithinolytica, and planticola species), which are associated with histamine formation. Some strains of R. planticola were previously identified as histamine producing strains of K. pneumoniae or K. oxytoca (Kanki et al. 2002). Another former Klebsiella spp. (K. trevisanii) is now re-classified as a R. planticola. Following comparison of DNA relatedness data, the genus Photorhabdus was created to accommodate strains of Xenorhabdus and both genera now include several new species of Enterobacteriaceae.

Although genetic evidence reveals that Shigella spp. can be considered to represent metabolically inactive biogroups of E. coli, this genus has been retained because of the clinical importance associated with Shigellae and to avoid confusion that any reclassification would cause. Furthermore, comparison of 16S rRNA sequences has revealed a close phylogentic relationship between Salmonella, E. coli and Shigella (Christensen et al., 1998).

As a result of using genetic-based classification methods, the nomenclature of Salmonella has changed. The genus Salmonella comprises over 2,500 serotypes, which were once considered separate species based on a combination of somatic O and flagellar H antigens expressed by these bacteria. DNA-DNA hybridisation studies revealed that all Salmonella form a single DNA homology group comprising two species. The first species S. enterica comprises six groups or subspecies including S. enterica subsp. enterica (I), S. enterica subsp. salamae (II), S. enterica subsp. arizonae (IIIa), S. enterica subsp. diarizonae (IIIb), S. enterica subsp. houtenae (IV) and S. enterica subsp. indica (V). The seventh group, Salmonella bongori has become the second species.

8 T 3. ENTEROBACTERIACEAE AND THEIR ROLE AS he

INDICATOR AND INDEX ORGANISMS IN FOODS t erobac

ndicator organisms are bacteria that are used to provide evidence of poor hygiene, t I inadequate processing or post-process contamination of foods. They are often eriaceae chosen because they are relatively quick and simple to detect. Their absence in food provides a degree of assurance that the hygiene and food manufacturing process has been carried

out appropriately, whereas their presence usually indicates that a potential problem or failure in the and process has occurred. The Enterobacteriaceae and coliform bacteria within this family represent two of the most common groups of indicator organism used by the food industry. Historically, coliforms were t heir the most common indicator group tested for by the food industry, especially within the dairy sector. In

some countries, depending on regulatory requirements, the food industry has moved towards testing for significance Enterobacteriaceae. The ability of coliforms to ferment lactose rapidly is often employed by conventional culture methods for their detection and enumeration. Consequently coliforms are often defined by the method used. The genera normally regarded as coliforms include Enterobacter, Klebsiella, Citrobacter and Escherichia, particularly E. coli. However, others may include Hafnia alvei and strains belonging to

genera such as Buttiauxella, Leclercia, Pantoea, Serratia, Yersinia etc. (Figure 1). Bacteria outside the t o

Enterobacteriaceae, notably Aeromonas spp., can also ferment lactose and these can be falsely detected t as coliforms by some methods, if no additional confirmatory tests are performed. he

food Figure 1 Diagram showing the relationship between genera within the Enterobacteriaceae

and those in the coliform group. indus t ry

(a) Most species do not ferment lactose. Some exceptions exist. (b) Some species ferment lactose (variable or slowly). Not typical coliforms but some may be regarded as coliforms (depending on the method used). (c) High proportion ferment lactose (rapidly). Traditionally regarded as typical coliforms. Dotted circles show genera that include species or strains which commonly cross between two categories.

9 Moreover, species of other genera such as Erwinia and Serratia can also ferment lactose, albeit ry t slowly, whereas some strains of Citrobacter and Klebsiella, as well as some strains of Salmonella, notably Salmonella enterica subsp. arizonae (IIIa), and Hafnia alvei show delayed or variable lactose indus fermentation. food

Currently there is no consensus view as to how the coliform group should be defined or which

he genera or species should be included. The coliforms remain useful as indicators, even though there t is no taxonomic basis for this grouping; this lack of definition, however, can present problems for o t international trade. Microbiologists examining water attempted to improve the definition of coliforms by making the expression of β-galactosidase activity a requirement. Although this definition was intended as a practical working definition of coliform bacteria, it has no taxonomic value (Anon 1994). The criterion simply seeks to provide a working definition that is not dependent on methods that rely on rapid lactose fermentation. It now implies that slow lactose fermenting strains, which may not significance produce acid or gas by traditional methods, and those that demonstrate β-galactosidase activity when using chromogenic media can be regarded as coliforms. heir t

Testing foods and water for coliforms has remained popular, not least because specific guidelines and and regulations demand coliform testing. Whether testing foods for coliforms or Enterobacteriaceae, the significance of the results obtained must be put into context with the type of food matrix being analysed. This is especially important with foods of plant origin because of the natural associations that can exist eriaceae (Baylis and Petitt, 1997). The ability of some Enterobacteriaceae to multiply in certain foods, even t during chilled storage, can make interpretation of results more difficult because the numbers present may not always reflect the initial level of contamination. These psychrotrophic Enterobacteriaceae

erobac are widely distributed and can be found in a variety of foods including milk, meat and poultry and t other foods. Therefore, high levels of Enterobacteriaceae in some chilled foods may not necessarily en indicate temperature abuse or improper storage. For these reasons Enterobacteriaceae provide a he

T good indicator of overall GMP on the day of production but not throughout the shelf-life or at the end of shelf-life of some (refrigerated perishable) products.

Undoubtedly Enterobacteriaceae provide a valuable role as indicator organisms in processed foods, particularly those subjected to heat-treatment. Depending on the initial contamination level and treatment, they can provide a reliable indication of process failure, under-processing or post-process contamination Table 2 (page 11). In European legislation there are designated sampling plans and limits for the level of Enterobacteriaceae in certain foods for food business operators. These are laid down in Commission Regulation (EC) No. 2073/2005 on microbiological criteria for food-stuffs, as part of the process hygiene criteria. Examples of the sampling plans and the criteria for Enterobacteriaceae applied to specific food categories in this regulation are given in Table 3 (page 12).

With certain foods Enterobacteriaceae can also provide a measure of food quality and spoilage potential. However, because the Enterobacteriaceae is such a large and diverse group they may be useful indicators of overall GMP, but not necessarily faecal contamination, and their relevance in foods should be assessed and interpreted carefully. Some Enterobacteriaceae are commonly found in the gastrointestinal tract of animals including humans. These bacteria can be used as indicators of potential faecal contamination, although E. coli strains are perhaps the most common bacteria used for this purpose. Methods exist, which use elevated incubation temperatures (e.g. 44°C), to preferentially isolate these so-called faecal coliform bacteria, but the term is misleading and the term thermotolerant coliforms is therefore a more appropriate description for these bacteria.

As well as using indicator organisms for the above purposes, some groups or individual species of bacteria can be used to provide evidence of potential contamination of food or water by closely related pathogens. Such organisms have been given the term “index” or “marker” organisms (Mossel, 1978; 1982). This term should not be confused with “indicator organism”, which has a different

10 T

purpose. Bacteria such as E. coli can have a dual purpose in the same food – as an indicator of faecal he

contamination and as an index organism for enteric pathogens such as Salmonella. Regulation EC en 2073/2005 initially required testing of dried infant formulae for Salmonella and Cronobacter spp. t (E. sakazakii) if Enterobacteriaceae were detected. However, it was concluded by the Scientific Panel erobac on Biological Hazards of the European Food Safety Authority that it was not always possible to establish a correlation between Enterobacteriaceae and Salmonella and that no universal correlation t between Enterobacteriaceae and Cronobacter spp. exists. Therefore, EC Regulation 2073/2005 was eriaceae subsequently revised (Commission Regulation No 1441/2007).

Despite some limitations, testing foods for index organisms rather than pathogens is simple and and relatively cheap, with results often available in 24 h. By comparison, traditional culture methods for

pathogens such as Salmonella can take 3-7 days to obtain a result. Furthermore, pathogenic bacteria t heir in food are often heterogeneously distributed and present in low numbers making detection difficult.

Many food production sites also prefer not to isolate enteric pathogens in their on-site laboratory, significance but elect instead to have testing performed externally by an approved laboratory. In contrast, testing for indicator and index organisms is done routinely by most laboratories, including those on food manufacturing sites. Some Enterobacteriaceae can also cause spoilage of certain foods and this aspect is discussed further in Section 6.

t Table 2. Indicator functions associated with Enterobacteriaceae and E. coli o

t he

Production process or Indicator function Comments on choice of indicator food application group

Raw/unprocessed foods indus Slaughter hygiene & GMP/GHP Both Enterobacteriaceae and E. coli processing meat (raw) and fish Faecal contamination acceptable indicator groups t

Harvesting and processing of GAP Can have high/variable numbers of ry fruit and vegetables (raw) Environmental contamination Enterobacteriaceae compared to E. coli Harvesting and processing of GAP High levels of E. coli useful indicator fruit and vegetables (raw) Faecal contamination of potential faecal contamination Raw milk, fermented dairy GMP/GHP Limitations if products mixed or products, ice cream Hygiene seasoned with (dried) raw ingredients e.g. fruit. Some Enterobacteriaceae not associated with faeces (unlike E. coli) Pasteurised milk and dairy GMP/GHP Both Enterobacteriaceae and E. coli products Post-process contamination acceptable indicator groups Heat-treated foods Cooked meats, Pasteurised GMP/GHP Limitations if products mixed or milk and dairy products, milk Post-process contamination seasoned with (dried) raw ingredients powder, chocolate, Insufficient heat-treatment egg products, REPFEDs etc Canned foods Leakage or under-heating Both Enterobacteriaceae and E. coli Foods heated in package acceptable indicator groups Food production environments Hand hygiene GMP/GHP E. coli (preferred choice) Environmental swabs Faecal contamination

REPFED: Refrigerated Processed Foods of Extended Durability; GMP: Good Manufacturing Practice; GHP: Good Hygiene Practices; GAP: Good Agricultural Practices

11 ry Table 3. Examples of Enterobacteriaceae limits stated in the process hygiene criteria t of Commission Regulation 2073/2005 on microbiological criteria for foodstuffs and subsequent amendments (No. 1441/2007 and 365/2010) indus

Food category Sampling plan Limits Analytical Action in case of food

method unsatisfactory* results

he Meat and meat n c m M t prducts o t 2 2 Carcases of – – 1.5 log cfu/m 2.5 log cfu/m ISO 21528-2 Improvements in slaughter cattle, sheep, daily mean log daily mean log hygiene and review of goats and process controls horses Carcases of pigs – – 2.0 log cfu/m2 3.0 log cfu/m2 ISO 21528-2 Improvements in slaughter daily mean log daily mean log hygiene and review of significance

process controls Pasteurised 5 0 10 cfu/ml ISO 21528-2 Check on the efficiency heir t milk and other of heat-treatment

pasteurised and prevention of liquid dairy recontamination as well as and products the quality of raw materials Milk powder and 5 0 10 cfu/g ISO 21528-2 Check on the efficiency whey powder of heat treatment and prevention of eriaceae t recontamination Ice cream and 5 2 10 cfu/g 100 cfu/g ISO 21528-2 Improvements in production frozen dairy hygiene erobac desserts t

en Dried infant 10 0 Absent in 10g ISO 21528- 1 Improvements in production formulae and hygiene to minimise he dried dietary contamination. T foods for special medical purposes intended for infants below six months of age Dried follow-on 5 0 Absence in 10g ISO 21528- 1 Improvements in production formulae hygiene to minimise contamination. Egg products 5 2 10 cfu/g or ml 100 cfu/g or ml ISO 21528-2 Checks on the efficiency of the heat treatment and prevention of recontamination

The above criteria are applied to meat products at the carcass stage (after dressing but before chilling) and to dairy products at the end of the manufacturing process. n = number of units comprising the sample; c = number of sample units giving values between m= minimum and M=maximum. The analytical method to be used shall be the most recent edition of the standard. *Satisfactory, if all the values observed are < m. Acceptable, if a maximum of c/n values are between m and M, and the rest of the values observed are < m. Unsatisfactory, if one or more of the values observed are >M or more than c/n values are between m and M.

12 T 4. DETECTION AND ENUMERATION METHODS he

FOR ENTEROBACTERIACEAE IN FOODS t erobac

everal published standardised methods exist for the detection and enumeration t S of Enterobacteriaceae, coliforms and E. coli in foods including international eriaceae standard methods such as those published by the International Organization for Standardization (ISO) (Table 4). Most are quantitative because food manufacturers often impose

specifications or limits for these bacteria in their products. Detection methods are commonly used for and foods where the presence of low numbers needs to be confirmed, or for high-risk foods where zero tolerance is imposed. t heir

Table 4. significance a. Examples of Conventional Culture Methods used for the Detection/Enumeration of the Enterobacteriaceae in Foods

Test and medium Diagnostic Technique Incubation Appearance Examples or

property or positive standards t

reaction o

Enterobacteriaceae he

Enterobacteriaceae Glucose Detection or 30°C or 37°C Broth turns ISO 21528-1 food enrichment (EE) fermentation MPN: used in for 24 h turbid & (2004) broth conjunction yellowish green

with VRBGA (note: streak indus following pre- all broths enrichment in irrespective of BPW. colour) t ry Violet Red Bile Glucose Pour plate + 30°C or 37°C Typical colonies: ISO 21528-2 Glucose agar fermentation (pH overlay for 24 h red/purple with (2004) (VRBLA) indicator) halo Coliforms Lauryl sulphate Lactose Detection or 30°C or 37°C Gas collected ISO 4831 tryptose broth fermentation MPN for 24-48 h in Durham tube (2006) (LSTB) (gas production) (sub-cultured into BGBLB) Brilliant Green Lactose Detection or 30°C or 37°C Gas collected in ISO 4831 Bile Lactose broth fermentation MPN for 24-48 h Durham tube (2006) (BGBLB) (gas production) Violet red bile agar Lactose Pour plate + 30°C or 37°C Typical colonies ISO 4832 (VRBA) fermentation (pH overlay 24 h red/purple with (2006) indicator) halo Escherichia coli Tryptone Bile Agar Indole from Spread plate 4h at 37°C Pink cerise BS 5763-13 (TBA) tryptophan (with 85 mm + 18-20 h at colonies upon (1998) 0.45 µm 44°C addition of membrane) indole detecting agent Tryptone bile β-glucuronidase Spread plate 4h 37°C +1 Typical colonies: ISO 16649-1 X-glucuronide agar (BCIG) (with 85 mm 8-24 h at 44°C blue (2004) (TBX) 0.45 µm membrane) Tryptone bile β-glucuronidase Pour plate 44°C for Typical colonies: ISO 16649-2 X-glucuronide agar (BCIG) (optional 18-24 h blue (2004) (TBX) resuscitation 4 h at 37°C)

13 ry b. Examples of Alternative Methods with Tests available for Enterobacteriaceae, Coliforms t and E. coli indus Method Manufacturer Diagnostic property Tests available Direct plate/alternative plate count methods food Petrifilm™ 3M Health Care Carbohydrate fermentation (with/ Coliforms, E. coli/coliform, he without gas production) and Enterobacteriaceae t enzyme activity o t Compact Dry™ Nissui Pharmaceutical Carbohydrate fermentation and Coliforms, E. coli/coliform, Co. Ltd enzyme activity Enterobacteriaceae RIDA® Count r-BioPharm-Rhône Carbohydrate fermentation and Coliforms, E. coli/coliform, enzyme activity Enterobacteriaceae SimPlate® Coliforms/ BioControl Inc Binary detection technology (MPN Total Coliforms and E. coli

significance E. coli principle) using 100 well plate and fluorogenic/chromogenic substrates heir t Automated methods Soleris® Neogen Inc Optical assay measuring changes Enterobacteriaceae, and in pH and other metabolic activity Coliforms and E. coli TEMPO® bioMérieux Metabolic activity and enzyme Enterobacteriaceae, activity. Automated MPN Coliforms and E. coli procedure. eriaceae t Bactometer bioMérieux Impedance/conductance Enterobacteriaceae, Coliforms and E. coli Rapid automated Don Whitley Scientific Impedance Enterobacteriaceae, erobac

t bacterial impedance Coliforms and E. coli technique (RABIT) en

BacTrac 4300 SY-LAB Impedance Enterobacteriaceae, he

T Microbiological Coliforms and E. coli Analyser

Enzyme activity is commonly detected using chromogenic or fluorogenic substrates e.g. β-galactosidase activity (coliforms) and β-D-glucuronidase activity (E. coli). The above table excludes tests (including immunoassays, PCR test etc) for specific members of the Enterobacteriaceae e.g. E. coli O157, Cronobacter spp and Salmonella spp. Various chromogenic media specifically for the detection/enumeration of Enterobacteriaceae (including coliforms, E. coli, E. coli O157, Cronobacter spp (Enterobacter sakazakii) and Salmonella are available from culture media manufacturers.

Enumeration methods often involve direct plating on solid selective media prepared using a pour or spread plate technique. Liquid media can be used for detecting Enterobacteriaceae, coliforms and E. coli and for enumeration using the most probable number (MPN) technique (typically 3 or 5 tube MPN). The latter is particularly useful when low levels of these bacteria are present. Fermentation of glucose and lactose is the most common diagnostic feature exploited by traditional culture methods for Enterobacteriaceae and coliforms, respectively. Both carbon sources yield acid that is subsequently detected by a colour change in the medium. In liquid media, gas is collected using an inverted Durham tube placed in the tube containing the medium. Tubes are inspected after 24 h and if necessary after a further 48 h incubation. Bile salts are commonly used to inhibit Gram-positive and other non-bile tolerant bacteria, including some Gram-negative bacteria, especially those that do not belong to the Enterobacteriaceae. Alternative selective agents include detergents such as sodium dodecyl sulphate (lauryl sulphate).

Violet red bile glucose agar (VRBGA) and violet red bile lactose agar (VRBLA) are common media used to isolate Enterobacteriaceae and coliforms, respectively. Both are compositional variants of the MacConkey agar, developed for the detection of bile tolerant Gram-negative bacteria. Both VRBGA and VRBLA are used along with a pour plate technique with an overlay of the same medium to ensure fermentation of

14 T

the carbohydrates and to reduce the likelihood of oxidation. This approach improves the specificity of he

these media and reduces interference from motile strains or background flora. Plates are incubated en aerobically for 24 h at 37°C and inspected for purple-red colonies surrounded by a purplish halo. If t the purpose of the test is to include psychrotrophic coliforms or Enterobacteriaceae the incubation erobac temperature may be lowered to 30°C, but 37°C is the preferred temperature if the test is being used as a hygiene indicator. t eriaceae

Bacteria other than Enterobacteriaceae can also grow successfully on VRBLA and VRBGA, e.g., Aeromonas spp. and some Bacillus spp., although an oxidase test can be employed to distinguish these

from Enterobacteriaceae. These bacteria are often smaller and exhibit atypical colony morphologies. and If necessary further confirmatory tests can be performed including the production of gas at 37°C in

liquid media and growth in brilliant green bile lactose broth (BGBLB) (ICMSF, 1978). The ISO standard t heir methods 21528-2 (Anon, 2004a) and ISO 4832 (Anon, 2006a) are colony-counting methods designed for

Enterobacteriaceae using VRBGA and coliforms using VRBLA, respectively. ISO 21528-2:2004 stipulates significance biochemical identification of typical colonies whereas ISO 4832:2006 requires no confirmation of typical colonies, only confirmation of gas production by atypical colonies in BGBLB.

Several types of culture media and diagnostic tests have been developed specifically for E. coli detection

and enumeration, whilst others involve testing for coliforms followed by additional tests to confirm the t presence of E. coli. Many traditional tests use acid or gas-production at 44°C and production of indole o

t from tryptophan to indicate the presence of E. coli. Indole production is a feature of biotype I E. coli, he

which represent about 98% of E. coli strains (Farmer et al., 1985). food

There are standard methods for the detection and enumeration of presumptive E. coli, e.g., ISO 7251 indus (Anon 2005), and coliforms, e.g., ISO 4831 (Anon, 2006b), in foods using a MPN approach. For the detection and enumeration of Enterobacteriaceae, Mossel (1963) developed a method using pre- t enrichment in buffered peptone water followed by enrichment in buffered brilliant green-bile-glucose ry broth (EE broth) and subsequent streaking onto VRBGA. This method has become ISO standard 21528-1 (Anon 2004b) and is now a mandatory analytical method in European legislation that uses Enterobacteriaceae in process hygiene criteria for a range of food products. However, there is evidence that the combination of dyes and bile salts used in the EE broth can inhibit the growth of some Enterobacteriaceae, including certain strains of Cronobacter (Joosten et al., 2008). Consequently, this standard is likely to be revised.

Incorporating chromogenic and fluorogenic substrates into existing media compositions can greatly improve their specificity for identification of Enterobacteriaceae, especially coliforms and E. coli. These substrates detect enzyme activity unique to the target bacterium and are commonly used in many alternative and more rapid methods. Enzymes acting on chromogenic substrates generate chromophores, which are subsequently absorbed into the bacterial cell, producing colonies of a particular colour that can be distinguished easily from others. Fluorogenic substrates yield free fluorophores that diffuse into the surrounding medium and which fluoresce under ultra-violet light at a defined wavelength.

For coliforms, β-D-galactosidase, the enzyme that hydrolyses lactose to galactose and glucose, is a common target. The target enzyme for many E. coli methods is β-D-glucuronidase (GUD), which catalyses the hydrolysis of β-D-glucopyranosiduronic acids to their corresponding aglycones and D-glucuronic acid. This enzyme activity is reported to be present in about 97% of E. coli (Kilian and Bülow, 1976). However, GUD activity is not exclusive to E. coli; other members of the Enterobacteriaceae, notably some Salmonella and Shigella as well as Hafnia alvei and other genera, also possess this enzyme activity (Hartman, 1989; Baylis and Patrick, 1999). Furthermore, the majority of E. coli O157:H7 strains are GUD negative (Baylis et al., 2006). Incorporating chromogenic substrates for both of these enzymes enables simultaneous enumeration and differentiation of E. coli and total coliforms using the same culture medium.

15 Processed or dry foods are likely to contain sub-lethally injured cells; therefore, direct use of selective ry t media or elevated temperatures are inappropriate. Some methods include a resuscitation step typically involving pre-incubation at a lower temperature or in a medium with limited or no selective agents. indus Anderson and Baird-Parker (1975) developed a method to aid recovery of E. coli from frozen foods, which involved plating a homogenised sample onto a 0.45 µm membrane laid onto the surface of a non- food

selective or recovery medium (e.g., Minerals Modified Glutamate Agar) followed by incubation at 30°C

he or 37°C for 4 h. This step facilitates recovery of injured cells with the membrane then being transferred to t Tryptone Bile agar (TBA), containing bile salts, and incubated at 44°C. Tryptophan in this medium allows o t indole producing colonies (E. coli) to be counted. This protocol became standard method BS5763-13 in the UK (Anon, 1998). A chromogenic medium (Tryptone Bile X-glucuronide agar) can now be used instead of TBA, thus enabling E. coli (GUD-positive) to be identified and enumerated on the membrane or this medium. These approaches became international standard methods ISO 16649-1 (Anon 2001a) and ISO 16649-2 (Anon 2001b), respectively. significance

Various proprietary media and tests are now available for the detection and enumeration of heir t

Enterobacteriaceae Table 4 (page 14). The introduction of new chromogenic media and alternative methods have had a marked effect on testing for Enterobacteriaceae. Colony recognition is made and easier thus improving method specificity and reducing the amount of confirmation required. Traditional media such as VRBGA rely on the use of dyes and bile salts to make them selective for enteric bacteria. Consequently, these media can be inhibitory to sub-lethally injured cells and Enterobacteriaceae that eriaceae are sensitive to these compounds. In contrast, the composition of many new chromogenic media and t those used with the alternative methods has been optimised to improve the successful recovery of these bacteria. They are also less dependent on the selective agents to provide the desired specificity. The results

erobac from these newer media compositions can therefore yield higher and more accurate results compared t to some standard conventional methods that employ older traditional culture media designs. Greater en specificity is offered by molecular methods targeting the Enterobacteriaceae 16S rRNA gene. Methods he

T have been developed to detect total Enterobacteriaceae in foods using the polymerase chain reaction (PCR) technique (Nakano et al., 2003; Martinon et al., 2011) and to quantify coliforms (lactose-fermenting Enterobacteriaceae) using real time quantitative PCR (Martin et al., 2010) and total Enterobacteriaceae using specific 16S rRNA probes and the fluorescent in situ hybridisation (FISH) technique (Ootsubo et al., 2003). However, despite the advances, these methods are not yet widely used.

16 T 5. ENTEROBACTERIACEAE AS FOODBORNE he

PATHOGENS t erobac

he Enterobacteriaceae includes some of the most intensively studied t T microorganisms, including several important foodborne pathogens. Notable eriaceae examples are typhoid and non-typhoid Salmonellae, Shigella dysenteriae, Yersinia enterocolitica and a range of pathogenic E. coli, including E. coli associated with traveller’s

diarrhoea and E. coli O157:H7, which has become one of the most important foodborne pathogens. and Moreover, some Enterobacteriaceae have emerged or could potentially become pathogenic as a result of the acquisition of virulence associated genes (toxins, colonisation factors) carried on mobile t heir genetic elements such as transposons, plasmids, insertion sequences and bacteriophages.

Mechanisms of virulence are summarised in Table 5. significance

Table 5: Summary of virulence characteristics of pathogenic Enterobacteriaceae

Pathotype Pathogenicity associated Mechanism Clinical features

genes or factors t o

Enteroinvasive E. coli 140 MDa plasmid (pInv) Bacterial attachment and Ulceration of bowel, t

(EIEC) invasion of colonic enterocytes watery diarrhoea, he

Ipa encodes invasion via endocytosis, multiplication dysenteric stools plasmid antigens (IpA, causing host cell death and (bacillary dysentery) food IpB, IpC ,IpD and IpH) inflammation accompanied by

necrosis and ulceration of large Chromosomal genes bowel. indus Enterotoxigenic Plasmid encoded Colonisation of surface of small Acute watery diarrhoea,

E. coli (ETEC) Colonisation Factor bowel mucosa (CFA I-IV) and usually without blood t ry Antigens (CFAs) CFAI production of enterotoxins LTI, mucus or pus (rigid rod-like fimbriae), III LTII, STa, STb). (bundle forming group), II & IV (flexible fimbriae) and type IV related Longus pili Labile toxins: LTI, LTII ADP ribosylation of G proteins (plasmid encoded) → adenylate cyclase activation → increased cAMP secretion → reduced Na+ absorption/Cl- secretion → diarrhoea. Heat stable toxins: Guanylate cyclase (G C-C) STa, STb (plasmid and activation → increased cGMP transposon encoded) secretion →chloride secretion and/or inhibition of NaCl absorption → diarrhoea. Enteroaggregative Plasmid (60 MDa) Adherence and colonisation of Aggregative adherence E. coli (EAEC) encoded: intestinal mucosa facilitated by (AA) phenotype Aggregative adherence AAF/I &AAFII. fimbriae (AAF/I & AAFII), transcriptional regulator (AggR) E.coli heat stable-like Release of toxins and damage Persistent diarrhoea toxin-1 (EAST1) to host epithelial cells. Plasmid encoded toxin (Pet)

17 ry Diffusely Adherent Afa/Dr family adhesins DA phenotype facilitated by Diffusely adherent (DA) t E. coli (DAEC) (AIDA adhesins) surface fimbriae, e.g., F1845 phenotype encoded via daaC gene, or by EAST-1 other related adhesins, which indus are plasmid or chromosomally Set genes (enterotoxins) encoded. Watery diarrhoea, usually

food without blood Possible TTSS Events in pathogenesis remain he

t involvement unclear.

o Enteropathogenic E. coli (EPEC) t

Typical EPEC (tEPEC) 50-70 MDa plasmid Localised adherence (LA) via Acute diarrhoea (pEAF) encodes: Bundle BFP (especially in children <1 forming pilus (BFP) and year old) Per (plasmid encoded regulator). significance

Virulence factors mainly heir

t encoded by the pEAF and the LEE.

and Atypical EPC (aEPEC) Various virulence genes LA Like (LAL), diffuse Diarrhoea (often milder

(some found in EAEC, adherence, aggregative than caused by tEPEC) VTEC and others of adherence and no adherence unknown function). patterns Various adhesins. eriaceae t Both tEPEC and Chromosomal PAI: locus A/E histopathology; cytoskeletal A/E lesions aEPEC for enterocyte effacement rearrangement of host epithelial (LEE) cells involving TTSS. erobac t TTSS comprising intimin Intimate effacing adherence en

(eaeA), secreted proteins: mediated by intimin. Tir, EspA, B, D, F, G & he

T MAP Destruction of microvilli and interference with host cell EAST-1 signalling cascades.

Cytolethal distending toxin (CDT) in O86:H34 strains Enterohaemorrhagic Large (60 MDa) A/E histopathology and Bloody diarrhoea E. coli (EHEC) plasmid encodes: intimate adherence similar to (haemorrhagic colitis) enterohaemolysin (Ehx), EPEC. Alternative adherence LCT, EspP mechanisms (besides eae) known. TTSS aids pathogenesis, Verocytotoxin- Chromosomal PAI (LEE) toxins (Stx/VT) inhibit protein Haemolytic uraemic producing E. coli synthesis of host cells and syndrome (HUS) TTP (VTEC) Chromosomal (prophage) mediate different pathological encoded Shiga toxins effects. (Stx)/Verocytotoxin (VT) VT1, VT2 & VT2 variants Shigella spp. Virulence plasmid Invasion of the host’s epithelial Diarrhoea, fever, nausea, encodes 2 loci (ipa and cells via the host’s M cells vomiting, stomach mxi-spa). involves ipa and mxi-spa. IpaB cramps, flatulence. and IpaC produce pores in Ipa encodes invasion epithelial cells and IpaC and Dysentery: stool may plasmid antigens (IpA, IpaD facilitate uptake of the contain blood, mucus, IpB, IpC ,IpD and IpH) bacterium into a vacuole by the or pus epithelial cell. The TTSS delivers mxi-spa operon encodes Ipa proteins from the bacterium Seizures in children (rare) components of a TTSS to the host cell. Uptake of and reactive arthritis and mxi-spa the bacterium and replication induces the host inflammatory Shiga toxin response and the epithelial cells (S. dysenteriae1) ultimately lyse. Shiga toxin (see EHEC).

18 The enterobacteriaceae and their significance to the food industry Nausea, diarrhoea and Nausea, diarrhoea vomiting, fever and abdominal pain. diarrhoea, Fever, secondary bacteraemia of gut and reinvasion via the liver and gall Inflammation, bladder. ulceration and necrosis patches of the Peyer’s can lead to perforation or haemorrhage. Wide range of symptoms dependent on strain, dose and susceptibility and age of host. Gastrointestinal enteritis syndromes: enterocolitis, with fever, inflammation of the lymph glands, diarrhoea, abdominal disorders (pseudoappendicitis). reactive Although rare, arthritis can occur following infection. Neonatal meningitis (ventriculitis, brain abscess/cyst formation and development of hydrocephalus). Other clinical manifestations: neonatal enterocolitis. necrotising Bacteraemia. Translocation through M cells M cells through Translocation patches resulting of Peyer’s and in destruction of M cells adjacent epithelium enables the the Salmonella to cross A small intestine epithelium. into the TTSS exports proteins host cells to initiate cytoskeletal and bacterial rearrangement internalisation by macrophages. Multiplication in the intestine. the small from Transportation intestine to the mesenteric of the lymph nodes via M cells Dissemination is patch. Peyer’s via the lymphatic system and blood stream. Invasion of host cells is facilitated by pYV in conjunction with inv and ail gene. Bacteria colonise intestinal tract, transverse intestinal lumen, attach to and penetrate mucus layer of mucosal epithelial cells, to intestinal brush and adhere is membranes. YadA border involved in bacterial adherence. type III secretion Yesinia inhibit proteins system and Yop phagocytosis. of the bacterium Translocation plexus. the chordus through Cellular invasion aided by factors increases secretory permeability of the blood-brain barrier enabling access to the brain. 19 plasmid Salmonella virulence (spv) genes (invasive salmonellae) Salmonella pathogenicity island-1 (SPI-1) encodes a TTSS SPI-1 encoded proteins: SopE, SipA, SipC, StpP Vi antigen (Vi polysaccharide) pYV or Lcr plasmid (64- 75 kb) chromosomally encoded invasion genes ail) (inv, outer membrane Large and OMP (YadA) protein Plasmid encoded Yop virulon TTSS (Ysc), yersiniabactin, heat stable enterotoxins. factors: Secretory glycopeptides, elastases, collagenases and other proteases Endotoxins Salmonella cause two distinct forms these being non-typhoid salmonellosis and of disease, Salmonella (non-typhoid) Salmonella Paratyphi) (Typhi/ enterocolitica Yersinia spp. Cronobacter 5.1 Salmonella In many developed countries Salmonella is the second most common cause of bacterial foodborne with a diverse range of host widely distributed in nature Salmonella are illness after Campylobacter. animal recognised many are there Consequently, fish and reptiles. species including mammals, birds, been pathogen. Salmonella has recently zoonotic an important remains and Salmonella reservoirs basil, melons). Two lettuce, fresh tomatoes, (e.g., produce fresh of contamination with associated commonly associated S. enterica, which includes serotypes now recognised; Salmonella species are infections, and S. bongori, which is commonly associated with with the majority of food-related reptiles. typhoid and paratyphoid disease as explained below. pYV, plasmid associated with Yersinia virulence; Lcr, low calcium response; Yop, Yersinia outer membrane protein; membrane protein; outer Yersinia Yop, low calcium response; virulence; Lcr, plasmid associated with Yersinia pYV, invasin); ail, attachment invasion locus. invasion (encodes Yersinia inv, outer membrane protein; OMP, The enterobacteriaceae and their significance to the food industry with metastaticabscessesand cholecystitiscansometimesoccur. uncommon infection, Paratyphoid CisstillfoundintheFarEast.Clinicalfeatures includesepticaemia by typhoid-like illness, or asasevere gastroenteritis or illness with features ofboth.Althoughan spots are more abundant andlarger. in Africa,witharecent increase intheFarEastandIndia.Thediseaseissimilarto typhoidbutrose transmitted bycontaminatedwaterorfood.Infectionswith S.entericaserParatyphiAare common are closelyrelated andbothcausesimilardisease,includinggastroenteritis. Thesesalmonellaeare (previously Three Paratyphoid fever Typhoid iscausedbySalmonella Typhoid fever stream orthelymphaticsystemresulting insystemic infectionandmore severe illnessorevendeath. people recover withoutspecifictreatment, although occasionallythebacteriumcanenterblood generally appearwithin12to72handthedurationofillnessisusually4-7days.Mosthealthy numbers ofbacterialcellsingestedandtheagesusceptibilityhumanhost.Symptoms and diarrhoea.Theseverityofthesymptomswilldependonseveralfactorssuchasserotype, Humans infected by Salmonellatypically develop nausea, vomiting,fever, abdominalcramps response isinducedinthehost. resulting indiarrhoea.DuringinfectionbySalmonellaastrong innateimmune/inflammatory by thebacteriumthatprecipitates anetefflux of water andelectrolytes intotheintestinallumen invasion oftheintestinaltissue.Duringthisinvasiveprocess, aheat-labileenterotoxin issecreted characterised bycolonisationandattachmentofthebacteriatoepithelialcells,subsequent Salmonella generally cause illness by localised infection of the gastrointestinal tract. Infection is Non-Typhoid Salmonellosis Paratyphi B that do not ferment Paratyphi Bthatdonotferment into thefourthweek,feverbeginstosubsideandsurviving patientbeginstorecover. cholecystitis, endocarditis andosteitis.Inthefourthstage,towards theendofthird weekand Other complicationsincludesepticaemia,diffuse peritonitis,encephalitis,metastaticabscesses, fatal. However, intestinalperforationinthedistalileumisaseriouscomplication,whichoftenfatal. intestine causedbybleedingincongestedPeyer’s patcheswhich,althoughserious,isnotusually third weekoftyphoidfever, severalcomplicationscanoccur. although constipationisalsocommonandthespleenlivermaybecomeenlarged. Inthe rose spotsmayappearon thelowerchestandabdomen.Diarrhoeacanalsooccurduringthisstage the secondweek,patientliesprostrate withhighfever(upto40°C),deliriumiscommonand relative bradycardia (slowingoftheheartrate),malaise,headache,coughandabdominalpain.In of approximately one-week duration.Duringthefirstweek,there isaslowlyrisingtemperature with different organs throughout thebody. Typhoid fevercanbedividedintofourindividualstageseach the bacteriaspread viathe lymphaticsystemandbloodstream. Thisgivesthemaccesstothe destruction byneutrophils, complementandtheimmuneresponse. Whileinsidethemacrophages, through theintestinalwall andare phagocytosedbymacrophages, where theycanexistandavoid lymphoid tissueusuallyfoundinthelowestportionofileum).Thebacteriaperforateandenter lymph nodesisviamicrofoldMcells)ofthePeyer’s cells(alsotermed patches(aggregations of where multiplication occurs. Transportation of the bacteria from the small intestine to the mesenteric restricted tohumans.Once ingestedthebacteriapassthrough thestomachandenterintestine of infection.Unlikeothersalmonellae,there isnoknownanimalreservoir sothehostrangeis contaminated foodandwaterorcontactwithapatientcarrierofthediseaseare commonvehicles developing countries,althoughtheincidenceofdiseaseisfallingworldwide.Consumption Salmonella S. schotmulleri) andParatyphiC(previously S. enterica serotypes thatcauseparatyphoidfeverare ParatyphiA,B d -tartrate havebeendesignatedS.entericaser. enterica In Europe ParatyphoidBismore common.Thisischaracterised subsp. 20 enterica hirschfeldii). Strainsbelongingtoserotype serotype Typhi andiscommonin These includehaemorrhageofthe Java, althoughthey The enterobacteriaceae and their significance to the food industry can penetrate enterocolitica Y. 21 is the cause of chronic diarrhoea and mesenteric adenitis (inflammation is the cause of chronic pseudotuberculosis outer proteins (YOPs) that are involved in pathogenicity along with chromosomally along with chromosomally involved in pathogenicity (YOPs) that are outer proteins Yersinia 5.3 Yersinia pseudotuberculosis 5.3 Yersinia Yersinia to similar are symptoms humans, In animals. in abdomen) the in nodes lymph mesenteric the of (fever and right-sided abdominal pain), although diarrhoea enterocolitica those of infection with Y. mimic to diagnose and infections can is difficult the condition is often absent. Consequently, may cause the disease cases rare In adults. younger and children in especially appendicitis, into the and pain) or the bacteria can spread arthritis (joint stiffness skin complaints, reactive is now less common due pseudotuberculosis (bacteraemia). Infection of humans by Y. bloodstream has produce contamination of fresh However, in the hygiene of the water supply. to improvements in Finland in 2004 that for example, several cases of gastroenteritis been linked to this organism; possibly contaminated on the farm attributed to grated carrots, during storage. were ). The first stage of infection involves colonisation of the ail). The first stage of infection involves colonisation encoded invasion genes (e.g., inv and the intestinal lumen, attaches to and penetrates the intestinal gut and the bacterium transverses to the membranes. The bacterium localises brush border mucus barrier overlying the intestinal cell of Most (YadA). protein membrane outer large of a aid the with colon terminalproximal and ileum evading host cell defences and like Invasion involves occur in this region. their pathological effects bacterial species (e.g., Salmonella and Shigella), other enteroinvasive 5.2 Yersinia enterocolitica 5.2 Yersinia in soil, water and animals, and can be found in nature is widely distributed enterocolitica Yersinia biogroups enterocolitica of Y. number the bacterium is a commensal. A large notably pigs in which are serogroups these and humans to pathogenic are these of few a only but exist, serogroups and 1A are belonging to biogroup specific host (Bottone, 1997). Strains associated with a frequently 1B (O:8; O:4; within biogroup with human infections unlike serogroups not commonly associated 2, 3 and O:5, 27), 3 (O:1, 2 (O:9; O:5,27), biogroup and O:21), biogroup O:13a,13b; O:18; O:20 enterocolitica Yersinia 3; O:3 and O:1, 2, 3). In Europe, 5 (O:2, 4 (O:3) and biogroup biogroup associated with commonly O:9) are 2 (serogroup O:3) and biogroup 4 (serogroup biogroup commonly reported, less are sporadic infections 2007a). In the USA, where human disease (EFSA, is associated with carriage of a Pathogenicity 1B is commonly associated with outbreaks. biogroup which codes for a range of outer membrane proteins pYV, virulence (64-75 kb) plasmid designated termed and the phagocytic encoded gene products the M cells of the host with the aid of chromosomally a heat-stable enterotoxin strains also produce enterocolitica activity of the M cells. Pathogenic Y. controversial. in diarrhoeal disease remains of E. coli, although its role STa (Yst) that resembles symptoms that are can invoke a wide range of gastrointestinal enterocolitica Infection by Y. 5 years of under and age of the host. Children dependent on the strain, dose and susceptibility and inflammation of the gastroenteritis and adolescents susceptible and in children most age are enterocolitica the main symptoms. Pseudo-appendicitis is also associated with Y. lymph glands are the are disorders and abdominal especially in young adults. In adults, diarrhoea infection, in complications including skin disorders result can occasionally commonest symptoms. Infection and arthritis. pork . Raw or undercooked enterocolitica A variety of foods can become contaminated with Y. commonly associated with this pathogen, although unpasteurised milk and are and pork products sporadic, although in the water can also act as vehicles for infection. Most infections are untreated have been traced to contaminated pork and pork products. USA outbreaks The enterobacteriaceae and their significance to the food industry termed Verocytotoxin-producingE. colitermed E.coli (VTEC)orShigatoxin-producingE. coli(STEC). infections.TheEHECrepresentsassociated withfoodborne asub-group ofamuchlarger group of are knowntoalsohaveasource inanimals,especiallyruminants,andthispathotypehasoftenbeen pathotypes (such as enteropathogenic E. coli(IPEC)isresponsible for a rangeofdiarrhoealdiseasesandsyndromes inman.Mostofthe bacteraemia associatedwithE.colihaveincreased recently. Incomparison,theintestinalpathogenic meningitis inneonates.Additionally, E.colihasbeenimplicatedininfectionsoftheblood:reports of proportion ofurinarytract infections,andthemeningitisassociatedE.coli(MAEC),whichcause The extraintestinalE.coli,includeuropathogenic E. coli(UPEC),whichare responsible foralarge below stillprovide aconvenientwaytodescribepathogenicE.colistrains. the acquisitionofnewvirulence-associatedgenes.Nevertheless,different pathotypesdescribed simply beclassifiedasbelongingtoasinglepathotype,thepathotypecouldchangefollowing passed betweenbacteriaviamobilegeneticelementssuchasplasmids),E.colistrainscannot et al.,2006).Owingtothegeneticmobilityofsomevirulence-associatedgenes(i.e.,theycanbe that are responsible fordistinctiveclinicaldiseases orsyndromes (Nataro andKaper, 1998;Baylis This bacteriumisgeneticallydiverseandthere are severaltraditionalE.colipathotypesrecognised genes, theproduction ofdistincttoxinsandtheclinicalcharacteristicsassociatedwithinfection. via foodsorwater. reservoir andare predominantly diffused viathefaecal-oralroute andonlyoccasionallytransmitted E. coli(ETEC),enteroaggregative E.coli(EAEC)anddiffusely adherent E.coli(DAEC))haveahuman long been used as a convenient way to describe subsequent contaminationofwater, foodsandtheenvironment. Theconceptofpathotypeshas bacteria. Somecarrierscanharbourpathogenicstrainsandconsequentlyactasreservoirs for Most strainsarecommensalswhereby harmless thehostsremain asasymptomaticcarriersofthese animals. Thisbacteriumcanbeshed,ofteninhighnumbers,viathefaecesintoenvironment. Escherichia coliiscommonlyfoundinthegastrointestinal tractofhumansandawiderangeother 5.4 PathogenicEscherichiacoli carriage through milddiarrhoeatolife-threatening conditionssuchashaemorrhagic colitis(HC) Infection with VTECcanresult inarange of clinicalmanifestations. Theseincludeasymptomatic the hostcellsurface. an importantoutermembrane protein, intimin,which mediates direct bindingof thebacteriumto microvilli of the actin-rich pedestal. The and the formation the cytoskeletonofhostcellbeneathadherent organisms leadingtotheeffacement ofthe chaperones and effector proteins responsible for the characteristic re-arrangment of the actin in lesions inthelarge intestine. effacement (LEE)isanimportantclusterofgenesinthebacterialchromosome involvedinA/E EPEC. Transmitted byhorizontaltransfer, thelocusofenterocyte thepathogenicityisland(termed attaching andeffacing (A/E)lesionsonepithelialcellsusingsimilarmechanismsto thosefoundin that are involvedwithcolonisationandpathogenicity. SomeVTEC,notablyEHECstrains,cause or VT2aloneboth.BesidesVT, some VTECalsopossessadditionalvirulenceassociatedgenes generally elaborated by prophages of the lambda (λ) family and VTEC strains produce either VT1 VT2e, VT2fandVT2g)are recognised (Perssonetal.,2007).Thetoxin-encodinggenes(vtx/stx)are least 3subtypesofVT1(VT1,VT1candVT1d)sevenVT2(VT2,VT2c,VT2d,VT2d3, of VT(VT1and VT2) wereantigenically distinct forms initially identified (Scotland becauseoftheirstructuralsimilaritytoShigatoxinfromtoxins, sotermed Shigella The VTECare characterised bytheirabilitytoproduceVerocytotoxins potenttoxinstermed orShiga Verocytotoxin-producing E.coli(VTEC) It isonegroup inparticular, thesocalledEnterohaemorrhagic E.coli (EHEC),that The LEEencodesatypeIIIsecretion system(TTSS)andassociated E. coli (EPEC), enteroinvasive E. coli strains carrying specific virulence-associated 22 eaeA gene, locatedat the LEE, codes for E. coli (EIEC), enterotoxigenic dysenteriae I.Two et al., 1985). At The enterobacteriaceae and their significance to the food industry et al., et EAEC that produce EAEC that produce group. In turn, In group. coli E. In this outbreak, it was In this outbreak, , O104:H4, was unusual in many respects. respects. in many was unusual E. coli, O104:H4, 23 The strain was shown to share 93% of its genome sequence with The strain was shown to share Thrombotic thrombocytopenic purpura (TTP), which involves severe neurological neurological (TTP), which involves severe purpura thrombocytopenic Thrombotic In Germany, with more than 3,600 people reported ill, over 850 of them suffering from from over 850 of them suffering ill, than 3,600 people reported with more In Germany, ., 1998). EAEC and, according to research findings currently available, EAEC VTEC available, EAEC findings currently to research et al., 1998). EAEC and, according not just the observation of a rare serotype (O104:H4), but also and importantly the combination (O104:H4), serotype not just the observation of a rare strain that is to be noted. The outbreak strain, in the outbreak of virulence traits, that occurred E. coli factors of STEC/VTEC and enteroaggregative possessed an unusual combination of virulence described later. (EAEC), which are to as an strain was referred the outbreak isolated O104:H4 EAEC strain. For this reason a previously to E. coli (EAggEC VTEC). EAggEC strains can belong Verocytotoxin-producing Enteroaggregative . and EAEC VTEC have been found before serogroups a wide range of different be to reported been EHEC, have classical for markers genetic the harbour not do but VT2/Stx2, (Mossoro Republic African Central Bangui, in HIV with patients in HUS and HC in involved outbreak to be involved in a European also been reported 2002). Furthermore, EAEC VTEC have (Morabito animal reservoir. the “classical” VTEC strains have a predominantly whereas have a human reservoir, (defined as E. coli strains that carry one or multiple that VTEC strains It should be noted however, carry the VT genes, but do not possess the ability to VT genes) in sheep and cattle, which merely unlikely to be pathogenic to (as encoded by eae-gene in EHEC or aggR-gene in EAEC), are adhere disease severe cause to bacteria the for prerequisite a be to seems factor adhesion The humans. in humans. extended-spectrum produce to shown also was strain GermanThe O104:H4 outbreak VTEC EAEC to the E. coli bacteria resistant to as ESBL E. coli) that render β-lactamase enzymes (also referred This illustrates the potential for horizontal gene transfer within the E. antibiotics. many different of E. coli (including EAEC VTEC) with its In addition, the complexity of this VTEC group coli group. modes of pathogenicity, possessing a variety of virulence factors and different various serotypes, the characterising and defining in difficulties subsequent the in reflected is and in the detection and for food monitoring as targets in using the group this causes difficulties disease (diarrhoea, HUS, fatalities) in humans. identification of E. coli strains that can cause severe as a foodborneSince 1982, when it was first recognised pathogen, E. coli O157:H7 has become water and for sporadic disease and large one of the most important pathogens responsible the first foods to were burgers, foodborne Raw meats, especially beef and undercooked outbreaks. and contaminated fresh be associated with this pathogen. Unpasteurised milk and dairy products vehicles for E. coli O157:H7 also known seeds, lettuce and spinach) are (e.g., sprouted produce for VTEC, including serotype common reservoirs as well as other VTEC. Cattle and sheep are also commonly found in the environment. O157:H7, which are It carried one of the Verocytotoxin (Shiga toxin) genes, namely the Verocytotoxin 2 gene (vtx2a (Shiga toxin) genes, namely the Verocytotoxin It carried one of the Verocytotoxin gene. variant) but contained neither the eae gene nor the enterohaemolysin symptoms, may occur in a small proportion of the HUS cases, especially among elderly patients. of the HUS cases, especially in a small proportion symptoms, may occur dose can be as low as 10-100 cells. conditions can be fatal. The infectious Both HUS and TTP VT2 and eaeA and producing associated with strains carrying human disease is commonly Severe is often found in VTEC isolated associated with milder disease and of VT1 is often VT2c. Production associated with severe often strains, such as VTEC O157:H7 are animals and food. EHEC from plasmids. on large enterohaemolysin) of additional virulence factors (e.g., disease and the carriage been associated with human disease, have also O157, other VTEC serogroups serogroup Apart from O103 and O91 (EFSA being O26, O111, O145, VTEC serogroups the five dominant non-O157 infections; for example, in human be involved may also further VTEC serotypes 2007b). However, in Germany in cases in the EHEC infection outbreak from the O104:H4 strain of EHEC isolated . in an EHEC outbreak and never before in humans to be seen only rarely Spring 2011 was reported only one case identified as E. coli O104:H4 had been documented in Prior to the 2011 outbreak, . the literature strain of the implicated fatalities, including 40 HUS, and the severe complications of haemolytic uraemic syndrome (HUS) causing kidney damage and causing kidney (HUS) uraemic syndrome of haemolytic complications and the severe . failure renal The enterobacteriaceae and their significance to the food industry bowel canoccurinsevere cases. watery diarrhoeathatmayprecede dysentericstoolscontainingbloodandmucus.Ulceration ofthe IpaH containedonahigh(140MDa)molecularweightplasmid (pINV).Commonsymptomsinclude and destroy colonictissueisassociatedwithgenessuchastheinvasionplasmidantigens IpaAto been reported. Thesiteofinfectionispredominantly thecolonandabilityofEIECtoinvade is usually contaminated by infected (carrier) food handlers. Person-to-person transmission has also Infection occurs via thefaecal-oralroute with contaminated food andwatercommonvehicles.Food Outbreaks havebeenassociatedwithcontaminatedsoftcheese,potatosaladand guacamole. developed countries,EIEChaveoccasionallybeenimplicatedinfood-andwater-borne outbreaks. to betheprimarysource as there are noknownanimalreservoirs forEIEC.Althoughuncommonin ofdiarrhoeainhumansandinfectedappear EIEC. Bothcauseaninvasive,dysentericform lactose,albeitslowly.able toferment GeneticallyShigellaiscloselyrelated to E. coli,inparticular lactose. ferment usually non-motile,donotdecarboxylatelysineandapproximately 70%ofEIECstrainsdonot pathogenically similar. Enteroinvasive Enteroinvasive E.coli(EIEC) emerging pathogens. is knownaboutthesestrainsthantEPEC.IncommonwithVTECtheaEPECare recognised as meats butalsowithconsumptionofcontaminatedwater. contamination canbeassociatedwithaEPEC,outbreaks havecommonlybeenassociatedwithraw subsequent transmissiontohumansviacontaminatedfoods.Althoughanyfoodexposedfaecal related toVTECandshare similaritiessuchastheirassociationwitharangeofanimalhostsand In contrast, aEPEC, which are found in developed as well developing countries,are more closely inflammatory response. Salmonella andYersinia , tEPECstrainsare lessinvasive andunlikeETECorEAEC,theycausean rich pedestalswhichisalsoacommonfeature ofsomeEHECstrains.However, unlikeShigella, diarrhoea andduringinfections,EPECbacteriaadhereactin- tointestinalepithelialcellsandform and traditionally associated with infantile diarrhoea. The tEPEC cause either watery or bloody human astheyhavenotbeenfoundinotheranimals.Theyare transmittedbyinter-human contact The reservoirs fortEPEC,whichare almostexclusively associatedwithdevelopingcountries,are a diffuse adherenceaggregative pattern, ornodetectable adherence. LALike(LAL),whereascharacterised bylooseclustersofbacteria,termed otheraEPECcanexpress differentiates thesetwotypesofEPEC.Bycomparison, aEPECstrainsexhibitavariantLApattern the pEAFandassociatedplasmidencodedregulator (Per)intEPECanditsabsenceaEPEC is locatedona50-70MDaEPECadherence factor(EAF)-encodingplasmid(pEAF).Thepresence of pilus(BFP)encodedbythebfpAgene,which Adhesion oftEPECismediatedbythebundle-forming of tEPECistheiradherence tothesurfaceofHEp-2cellsinalocalisedadherence (LA)pattern. regulated bygenesoftheLEE,usingamechanismsimilartothatfoundinVTEC.Akeycharacteristic (aEPEC). BothtEPECandaEPEChavetheabilitytoproduce A/Elesions,afeature encodedand The EPECpathotypecanbedividedintotwogroups, typicalEPEC(tEPEC)andatypical Enteropathogenic Ecoli(EPEC) chromosomally encoded yet is similar to LTI both in mode of action and in structure. encoded andresembles thecholeratoxin produced cholerae. Another toxin(LTII) byVibrio is one ormore heat-stable(ST)orheat-labile(LT) enterotoxins. One ofthetoxins(LTI) isplasmid diarrhoea among infants and travellers. A characteristic feature of ETEC is their ability to express Common indevelopingcountries where sanitationispoor, ETECcontinues tobeamajorcauseof Enterotoxigenic E.coli(ETEC) E. coli(EIEC)closelyresemble Shigellaandare genetically, biochemicallyand These characteristics are similar to The EIECare atypicalcompared tothemajorityofE.colistrains.Theyare 24 Shigella, although some strains of The aEPECcausediarrhoea,althoughless Another toxin is S. sonnei are The enterobacteriaceae and their significance to the food industry Cronobacter Consumption spp, ) have been isolated from a variety a from isolated been have sakazakii) E. spp. and subsequent improper storage improper subsequent and spp. Cronobacter 25 These include plasmid-encoded aggregative adhesion fimbriae (AAF/I and adhesion fimbriae (AAF/I aggregative These include plasmid-encoded (EAEC) are a major cause of chronic infantile diarrhoea in some developing some diarrhoea in infantile chronic of cause a major are (EAEC) coli E. spp. are pathogens commonly associated with illness in neonates, especially low birth spp. are Cronobacter 1980 these pathogens infants. Before weight infants less than 2.5 kg and immuno-compromised cloacae because of the ability of strains to to as yellow pigmented Enterobacter referred were and blood agar yellow-pigmented colonies, especially at 25°C on trypticase soy agar, produce after the brain heart infusion agar after 48-72 h. In 1980, the bacterium was named E. sakazakii that E. sakazakii polyphasic analysis revealed recently, Japanese bacteriologist Riichi Sakazaki. More that these species be moved to a proposed species, and it was therefore consisted of six different ” (Iversen et al., 2007; 2008). Unlike most Enterobacter novel genus “Cronobacter 5.5 Cronobacter spp. 5.5 Cronobacter to elaborate an α-glucosidase activity and this has been used as an important diagnostic marker spp. and other Enterobacteriaceae. Enterobacter from distinguish Cronobacter name, previous their by (identified spp. Cronobacter of sources including the environment, clinical sources and a wide range of foods (Gurtler et al., and a wide clinical sources including the environment, of sources infant formula powdered (PIF) has 2005; Lehner and Stephan, 2004; Friedemann, 2007). However, and there been identified as one of the most epidemiologically important vehicles for transmission spp. infections linked of neonatal Cronobacter have been a number of documented outbreaks (Forsythe, 2005; Gurtler et al., 2005). PIF is not sterile and poor treatment to this food product by contamination environmental with together being in the product can result of the bacteria, leading to growth product, of the reconstituted distribution, the natural habitat of unsafe at the point of consumption. Despite its widespread unknown. spp. remains Cronobacter of contaminated foods, notably raw vegetables and soft cheeses, has been implicated in cases of has been implicated in cases of notably raw vegetables and soft cheeses, of contaminated foods, by ETEC. sporadic waterborne caused have been outbreaks infection and there Enteroaggregative Enteroaggregative pattern to the phenotype of EAEC is the ‘stacked-brick’ countries. A characteristic of adherence a of a 60 MDa plasmid. The EAEC is which is associated with the carriage surface of host cells, that are a range of putative virulence factors producing with strains group very heterogeneous . associated with disease the plasmid-encoded heat-stable enterotoxin (ST) of which there are two groups (STa/STI and STb/ (STa/STI two groups are there (ST) of which enterotoxin heat-stable the plasmid-encoded with little or no fever. is characterised by watery diarrhoea STII). Infection by ETEC E coli (DAEC) Adherent and Diffusely E. coli (EAggEC) Enteroaggregative in pathogenesis plays an important role AggR, which by transcriptional regulator AAF/II) regulated plasmid-encoded toxin (pet) and Shigella heat-stable toxin-1 (EAST-1), by EAEC. Enteroaggregative not all EAEC strains produce by strains of EAEC. However, also produced (ShET1) are enterotoxin-1 involved. whether other toxins or virulence factors are or Pet toxins and it is not known EAST-1 in results to the intestinal mucosa and colonisation. This Pathogenesis by EAEC involves adherence is usually and diarrhoea, which mucus production damage to the hosts’ epithelial cells, enhanced have been and often associated with abdominal pain. Some EAEC strains watery and protracted disease, including HUS. This was of VT2/Stx2 and the cause of severe associated with production strain in Germany in May/June 2011. highlighted by the EAEC O104:H4 outbreak to HEp-2 pattern characterised by a diffuse of adherence E. coli (DAEC) are adherent Diffusely virulence of absence the and (AIDA) adherence diffuse in involved adhesions of presence cells, set) and genes ( EAST-1 produce genes found in other E. coli pathotypes. Strains of DAEC may also E. coli pathotypes, little is known about the role encoding toxins found in Shigella spp. Unlike other include watery diarrhoea, usually without blood. of DAEC in disease. Clinical features The enterobacteriaceae and their significance to the food industry handlers. fresh produce (salads),fresh juicesandfoodsthatare susceptibletocontaminationbyinfectedfood shigellosisisuncommon.Foodsimplicatedinoutbreaksfoodborne intheUSAandS.Africainclude through person-to-person contactorviathefaecal-oralroute. humans andtheinfectivedoseisverylow(asfewas10cells).Shigellosiscommonlycontracted Shigella havebeenimplicatedasoneofthecausesreactive arthritis.Shigellaare adaptedto contain blood,mucus,orpus.Seizures canoccurinrare cases,especiallyinyoungchildren and destruction oftheepithelialcellsintestinalmucosaincaecumandrectum, thestoolmay stomach crampsandflatulence. such asProvidencia alcalifaciens, Besides thewell-knownpathogensthatbelongtoEnterobacteriaceae family, somemembers 5.7 Opportunisticandemergingpathogenic Enterobacteriaceae Common symptoms associated with vascular damageandHUSinsomepatients. VTEC (see above), but unlike VTEC this toxin gene is chromasomally encoded. Shiga toxin can cause also produce apotenttoxin (Shigatoxin),whichisstructurallyrelated toVT1produced bystrainsof the eventuallysisofepithelialcells.SomeShigellaproduce enterotoxin andS.dysenteriae1 epithelial cytoplasm.Bacterialcellreplication followsinducingthehostinflammatoryresponse and by IpaB-,andpossiblyIpaC-,inducedlysisofthevacuolerelease ofthebacteriainto the bacteriumtohostcell,triggeringbacterialinvasion.Uptakeofisfollowed operon, andthemxi-spa operon encodescomponents of aTTSSthatdeliversIpaproteins from by avirulenceplasmid.Invasionplasmidantigens(IpA,IpB,IpCandIpD)are encodedbytheipa of the host’s epithelialcells occurs via the M cellsand involves twoloci (ipa and mxi-spa) encoded infection isbacteraemiaandseizures are common,especiallywithS.dysenteriaeinfection.Invasion intestinal perforationcanoccasionallyoccur. limitedtothedistalileumandcolon.Althoughrare,flexneri, boydiiandsonnei).Infectionisnormally EIEC, with which they share a number of characteristics. There are 4 species of From ataxonomicalperspectiveShigellashouldberegarded asasub-group ofE.colialongwith 5.6 Shigellaspp. facilitate cellularinvasion. the chordus plexus,thesecretory factorsincrease oftheblood-brainbarrierand thepermeability of proteases, includingelastaseandcollagenase. variety of secretory factors that probably play a role in pathogenicity, e.g., endotoxins and a variety factors ofCronobacter spp.haveyettobefullyelucidated,althoughstrainsare knowntoproduce a by bacterialcolonisationoftheintestinaltract.Infectioncanalsoresult inbacteraemia.Thevirulence of anddevelopmentofhydrocephalus.formation Aclinicalmanifestationassociatedwithconsumption This canresult infurthercomplicationssuchasventriculitis,brainabscess,cerebral infarction, cyst Neonatal meningitisisthemostwidelyrecognised clinicalfeature ofCronobacter spp.infection. been implicated asacauseofdiarrhoea(Hawkey, 2006).However, there isnow goodevidence and animalfaeces.Thisbacterium wasonceassumed not tobeahumanpathogenalthough ithas Providencia alcalifaciensare widelydistributedinnature andcanbeisolatedfrom bothhuman Providencia alcalifaciens settings. meningitis, urinarytractinfectionsandwoundinfections, andare therefore importantinclinical implicated inhumandiseaseorcanbethecauseofopportunistic infectionsincludingbacteraemia, Cronobacter-contaminated PIF is the development of neonatal necrotising enterocolitis caused In casesofShigella-associateddysentery, whichresults inthe Klebsiella spp.,Serratiaspp.andCitrobacter spp.havebeen Shigella infection include diarrhoea, fever, nausea,vomiting, An importantcomplicationassociatedwithShigella 26 Once thebacteriumhastranslocatedthrough In manyEuropean countries, Shigella (dysenteriae, The enterobacteriaceae and their significance to the food industry . of C. freundii strains some as well as In a large outbreak of food poisoning in of food poisoning outbreak In a large C. rodentium rodentium C. 27 ., 2001). Although likely aetiological agent (Murata et al alcalifaciens was identified as the P. spp. are also widely distributed in the environment, being commonly found in water, soil and commonly found in water, being also widely distributed in the environment, Serratia spp. are nosocomial opportunistic and intestinal tract. They are plants as well as occasionally in the human . Contaminated soil and plants have been S. marcescens pathogens, with the most important being infections. linked to community-acquired Serratia spp. Citrobacter spp. Citrobacter commonly found in the intestinal and are the environment widely distributed in spp. are Citrobacter and C. koseri opportunistic pathogens spp. are other animals. Citrobacter tract of humans and spp. for meningitis. Citrobacter pathogen responsible would appear to be an important neonatal determinantshave been shown to carry various virulence pathogens. The eae gene found in other in found been has EHEC and EPEC in found that this species can cause diarrhoeal illness, especially in children, as well as being a cause of as being a cause as well in children, illness, especially can cause diarrhoeal that this species be a vehicle. that food could diarrhoea and traveller’s Japan, have not been fully alcalifaciens in diarrhoeal illness of P. of transmission and routes the sources as well as diarrhoea from bacterium in the faeces of patients suffering of this investigated, excretion contamination faecal and carriage hand Transient factors. risk potential are carriage asymptomatic of transmission. likely routes therefore of foods are An outbreak of HUS in Germanyin of HUS parsley containing butter of the consumption to linked was outbreak An et al., 1995). carrying the vtx2 gene (Tschäpe contaminated with C. freundii The enterobacteriaceae and their significance to the food industry Enterobacteriaceae 5.8 Extended-spectrumβ-lactamase(ESL)-producing extended-spectrum spp. andSalmonellaproduce plasmid-mediatedenzymes(classAβ-lactamases)termed antibiotic-resistant bacteriaamonghumans.SomeEnterobacteriaceae, particularlyE.coli, There is increasing that foods could act as potential vehicles for the dissemination of concern Person-to-person dissemination within the community is recognised as an important source of several antibiotics. for theoutbreakin2011wascarrying ESβL CTX-M-15andTEM-1makingitresistant inGermany to has alsobeenamore limited spread amongSalmonellaspp.TheVTECEAECO104:H4responsible E. coli andhavebecomeatleastasfrequent asTEMandSHVESβLsinhospitalKlebsiellaspp.There genetic escapesfrom the chromosomes ofKluyveraspp.TheCTX-MESβLshavespread among major sub-groups ofCTX-M ESβLs andover 90 minorvariants are known.Allappeartorepresent types, hasspread andare nowmore prevalent inE.colithantheearlierTEMandSHVvariants.Five contaminated equipment. However, of the century a new class of ESβLs, the CTX-M since the turn largely associatedwithnosocomial Klebsiellaspp.andtransmissionwaswithinhospitalsviastaff or were mutantsofthelong-established TEMandSHVplasmidmediatedpenicillinases.Thesewere oral antibioticinthecommunity. ESβLshavebeenknownsincethe1980sandmostearlytypes simpler infectionsresultingbetreated inhospitalisationofpatientswhowouldnormally withan producing Enterobacteriaceae andtheresistance theycausecomplicatesthetreatment ofmany severe infections,particularlybacteraemiasandnosocomialpneumonias.Thepresence ofESβL- ESβL-producing strainshavebeenassociatedwithincreased morbidityandmortalityinpatientswith other antibioticsincludingfluoroquinolones andaminoglycosides. resistant to commercially available penicillin-inhibitor combinations andare multiple resistant to are inhibitedbyβ-lactamaseinhibitors,e.g.,clavulanicacid,butmanyESβLproducing strainsare (e.g., aztreonam) andpenicillins(excepttemocillin),butnotcarbapenemsorcephamycins.ESβLs generation cephalosporins(e.g.,ceftazidime,cefotaxime,ceftriaxoneandcefepime),monobactam hydrolysis ofavarietyβ-lactamantimicrobial compounds,includingoxyimino-2nd,3rd and4th al., 2007;Rodriguezet2009;Kolar2010;Dhanji2010). ESβLs being isolated from foods, especially those of animal origin (Machado contribute tothewiderspread ofproducer strains,(Lavillaetal.,2008).There are severalreports of with CTX-MESβLsinthecommunity(Mesaetal.,2006;Lo2010).Foodcontaminationcould β lactamases(ESLs).Theseenzymescanconferresistance bycatalysingthe 28 et al., 2008; Jouini Klebsiella E. coli et The enterobacteriaceae and their significance to the food industry

and ) from ) from 3 , Proteus Klebsiella, Escherichia , are , are Hafnia and Yersinia . This results from from . This results carotovorum Erwinia) 29 can enter the food chain via faecal contamination chain via faecal contamination Salmonella can enter the food More important than their potential to cause deterioration in important than their potential to cause deterioration in More . and Enterobacteriaceae in fresh meats can utilise glucose and other simple meats can utilise glucose and in fresh Enterobacteriaceae S and to reduce trimethylamine oxide (TMAO) to trimethylamine (TMA), trimethylamine oxide (TMAO) to trimethylamine (TMA), S and to reduce S), which is a product of sulphur-containing amino acids, ammonia (NH of sulphur-containing S), which is a product 2 2 nterobacteriaceae may be present as the natural microflora of certain foods of certain foods the natural microflora as may be present nterobacteriaceae contamination. (post-)process of result as a be introduced can or Escherichia E are associated with defects including a condition termed in cottage cheese associated “slimy curd” are Erwinia spp., and especially Pectobacterium ( pectic enzymes breaking down pectins resulting in a characteristic mushy appearance, which is in a characteristic mushy appearance, which is down pectins resulting pectic enzymes breaking appearance. sometimes accompanied by a bad odour and water-soaked which has a strong “fishy” smell even at low concentrations.. Most species of Enterobacteriaceae, “fishy” smell even at low concentrations.. Most species of Enterobacteriaceae, which has a strong and TMAO to TMA (Barrett with the exception of Erwinia and some Shigella spp., can reduce Kwan, 1985) leading to spoilage the organoleptic properties of fish is the histidine decarboxylase activity of some members of the of fish is the histidine decarboxylase activity of some members of the properties the organoleptic of histamine in fish belonging to the Scombridae family leading to production Enterobacteriaceae poisoning (Baylis 2006). which in turn in scombroid (e.g., tuna, mackerel), can result type of spoilage of vegetables and fruits caused by Enterobacteriaceae, a common are Soft rots notably are commonly found in the environment (soil, water and plants). Erwinia, environment commonly found in the are Serratia and Panteoa so they can often be found in associated with plants, spp. are and Pectobacterium Brennaria Enterobacter, or plant origin. Other bacteria, such as foods of vegetable in foods. Enterobacteriaceae encountered commonly therefore and are also widely distributed meats, namely milk, dairy products, range of food products, can cause food spoilage in a broad and metabolic activity of fish, seafoods, fruit, vegetables and other foods. The growth poultry, odours, colour defects and other organoleptic in off-flavours, in food can result Enterobacteriaceae or lipids, production of proteins enzymatic breakdown deviations. These changes may arise from of volatile components and gas production. impart bitterness can by of Enterobacteriaceae some strains other dairy products, In milk and thermostabilityThe casein. down proteinases and lipases some of break that enzymes producing will not inactivate time (HTST) and ultra-heat (UHT) treatments suggests that high temperature-short notably Serratia spp., can produce some Enterobacteriaceae, these enzymes. Besides flavour defects, During 1998). (ICMSF, colour defects other bacteria may cause whilst other pigment, a reddish can also cause a spoilage Enterobacteriaceae cheese production the early ripening stage of fresh caused by or blown packs, in holes within the product, termed “early blowing” which can result , 1998). Proteus the fermentation of gas from of lactose (ICMSF, the production Enterobacter spp. and other members of the Enterobacteriaceae are associated are 2000). Serratia spp. and other members of the Enterobacteriaceae (Cousin, 1982; Jay, acid and gas following of the cream by clotting desserts, characterised cream fresh with spoilage of is favoured in meat and poultry of Enterobacteriaceae (Sutherland et al., 1986). Growth production by storage at 5°C and above. , Citrobacter and poultry. with particular foods such as meat and these may be associated acids and amino exhausted they can then make use of free carbohydrates and when these become compounds. When this occurs, noticeable odours and spoilage become simple nitrogenous related of amino acids to yield generally associated with the breakdown Foul odours in meats are apparent. sulphide (H hydrogen E D SPOILAG AE IN FOO TERIAC E ROBAC 6. ENTE many amino acids and indole from tryptophan (Jay, 2000). tryptophan (Jay, many amino acids and indole from of on ice is particularly susceptible to bacterial spoilage, although the role fish stored Fresh to other spoilage bacteria, notably Pseudomonas is less significant compared Enterobacteriaceae important characteristics of fish spoilage bacteria is their . Two spp. and Shewanella putrefaciens H ability to produce The enterobacteriaceae and their significance to the food industry have beenattributedtoEnterobacteriaceae withblackrots causedbyEscherichia Enterobacteriaceae have been associated with spoilage of maple syrup. Bacterial rots in shell eggs Obesumbacterium proteus isassociatedwithbeerspoilageandvariousmembersofthe red rots causedbyEnterobacter spp. (ICMSF, 1998). 30 and Proteus and The enterobacteriaceae and their significance to the food industry Hafnia, 70°C. Moreover, 70°C. Moreover, oxytoca, comprise Escherichia, An optimum growth . . Erwinia, Enterobacter, AND SURVIVAL TION varies with solute type (Mattick et al., 2001). w 31 is increased, particularly at temperatures ≥ particularly at temperatures is increased, w ) are often used in combination to ensure product stability and safety. stability and safety. product used in combination to ensure often ) are w rowth and survival characteristics of Enterobacteriaceae will determine if these of Enterobacteriaceae and survival characteristics rowth aof quality organoleptic the affect adversely or spoilage cause will bacteria susceptible to adverse more are Enterobacteriaceae particular food, or both. However, some cells can survive and may remain viable for long periods, so freezing some cells can survive and may remain However, . G . enterocolitica 1978) as well as some pathogenic Yersinia and Serratia species (ICMSF, Proteus However, the precise conditions that enable growth and survival or cause inactivation of a and survival or cause inactivation that enable growth conditions the precise However, . the extent of protection afforded by reduced a by reduced afforded the extent of protection The main objective of this so-called hurdle technology is to provide for an extended shelf-life, even technology is to provide The main objective of this so-called hurdle or survival of pathogens in the food. the growth and to prevent for so-called shelf-stable products, contribute may also contain intrinsically inhibitory substances that may also Some traditional products eugenol and carvacrol, as compounds such active their and oils essential include Examples stability. to although many Enterobacteriaceae towards activity known to exert an antimicrobial others, which are information of this section provides in foods. The remainder not always reproducible are their effects in foods. the Enterobacteriaceae and how these can affect on some key intrinsic and extrinsic factors, 7.1 Temperature mesophilic and thermotolerant bacteria. The majority include psychrotrophic, The Enterobacteriaceae temperatures including the foodborne mesophilic, pathogens Salmonella and EHEC, with growth are of 0-8°C forranging between 15 and 40°C. Rapid cooling to the normal temperatures refrigeration therefore inhibition (but not inactivation) of mesophilic Enterobacteriaceae, storage facilitates growth abuse when subjected to temperature they only multiply in perishable products strains (defined of faecal origin. Psychrotrophic of 37°C is typical for Enterobacteriaceae temperature growthoptimum an with and 0°C to down temperatures at growing of capable bacteria those as , of 22-30°C) include strains of Citrobacter temperature NACTIVA OWTH, I 7. GR conditions than Gram-positive bacteria. Their capacity to grow and cause food spoilage is mostly limited and cause bacteria. Their capacity to grow conditions than Gram-positive especially foods kept under refrigerated (i.e., those with a shelf-life < 10 days) to perishable foods storage particular strain of Enterobacteriaceae can differ significantly depending upon the genus and species, significantly depending upon can differ particular strain of Enterobacteriaceae (any factors), food processing characteristics of the food (intrinsic chemical and physical the inherent and and the storage conditions (extrinsic factors), product) action that substantially alters the initial other large populations within the food (implicit conditions). As with interactions with other microbial to make diversity; thus, it is difficult physiological show a large bacterial families, these organisms and survival. growth generalised statements regarding and and pH, modified atmosphere temperature reduced Intrinsic and extrinsic factors such as water activities (a appropriate Klebsiella, The thermotolerant which include strains of E. coli and Klebsiella Enterobacteriaceae, forminimum temperature and having a to 44°C up growth of mesophiles capable of a sub-group ceases at temperatures including Enterobacteriaceae, of micro-organisms, of > 7-8°C. Growth growth particularly susceptible are below -8°C. Along with most Gram-negative bacteria, Enterobacteriaceae which causes sub-lethal-injury along with a further gradual inactivation duringto damage by freezing, storage prolonged A 10-minute treatment at 55°C would normally destroy most psychrotrophic bacteria and heat- at 55°C would normally most psychrotrophic A 10-minute treatment destroy forming mesophiles, including non-spore heat-resistant the more sensitive mesophiles, whereas of micro-organisms at 65°C. Heat resistance and lactobacilli, would be destroyed some enterococci and the characteristics of the food, e.g., fat content and depends on the history of the strain however, Heat tolerance at lower a water activity. is not a reliable control measure or inactivation process during food manufacture or inactivation process measure control is not a reliable The enterobacteriaceae and their significance to the food industry some salmonellaecan divide atonlyonehalfof their maximalrateata foods withawateractivityproviding optimalconditionsforgrowth, especiallythosewitha pasteurisation practices(15sat72°C)anda5log ., 2002), which could be due to multiple sub-culturing and long-term storage storage strains (Bagamboula etal.,2002),which could beduetomultiplesub-culturing andlong-term collection strains,includingtype strainsaswasillustratedwithsomeShigella bacterium. Furthermore,food isolatesmayexhibitincreased resistance toacid compared toculture below theminimumpHforgrowth, becauseofthevariability ofthestrainsevenasinglespecies to defineeitherthepHtolerance ofaspecies,e.g.,Shigellaspp.,ortheirsurvivalunderconditions VTEC are sometimesreferred toasparticularlysuitedsurviveatlowpH.Itisnotpossibleprecisely onlyresponsible normally fordeteriorationoffoodswithneutral orslightlyacidicpH.Shigellaspp.and lactic acid)beingmore inhibitorythanmineralacids(e.g.hydrochloric acid).Enterobacteriaceae are 9.0. However, pHtoleranceisofteninfluencedbythenature oftheacidulant,withorganic acids(e.g. The minimumpHforgrowth ofEnterobacteriaceae is3.8withanupperlimitofapproximately pH 7.3 pH Most Enterobacteriaceae grow bestatabovea 7.2 Water activity(a the mostheat-resistant C.sakazakiistrainwillbereduced bymore than8log with anaverageD-valueat58°Cof9.9min(Edelson-MammelandBuchanan,2004).However, even reduction time(D-values)at58°Crangingfrom 0.39 to0.60min(Breeuwer etal.,2003)andsome Cronobacter spp.inreconstituted powderedisreported infantformula tobediversewithdecimal 63°C orequivalent,willdestroy allEnterobacteriaceae present. Theheatresistance displayedby Minimal time/temperature combinationforpasteurisationusedinthedairyindustry, e.g.,30minat enterica The lengthofsurvivalinthesefoodsmayvarydependingonthespecies.Forexample,Salmonella powdered infantformula. meatproductssemi-dry fermented suchassalamianddriedfoodsmilkpowder, flourand of sucrose, e.g.,chocolate, cake,biscuitsandconfectionery, andthosecontainingsalt,e.g.,dry Lowering thea associated withspoilageandfoodsafetyissuesinvolvingEnterobacteriaceae. 0.98 orabove,suchasfresh meatandfish,fresh fruitsandvegetablesmilkare mostcommonly at a destroy vegetativepathogenicbacteriaincludingEnterobacteriaceae. by the ACMSF Cooking burgers to70°Cfor2minorequivalenthasbeenupheldasarecommended heattreatment by theWorld HealthOrganization forreconstitution ofpowdered formula. 1999). InactivationofCronobacter spp.willoccurquicklyattemperatures above70°Casrecommend prolonged periods(several months)inlowa The pathogens competed bythenaturalmicroflora. greater disadvantagecompared tootherbacteria(particularlyGram-positive)andtheymaybeout- better survivalatrefrigeration temperatures thanatambienttemperature. granulated sugar(Samaraweeraetal.,2001).Survivalatlowa in saltedfoods(withequallowa of thematrixalsoplaysarole: infoodswithahighsugarcontentsurvivalisbettercompared tosurvival w 0.96 (ICMSF, 1980).Moreover, infoodswitha r. ser. Typhimurium havebeen shown to surviveforlongerperiodsof time than E. coli infine w Ad Hoc Group on Safe Cooking of Burgers (ACMSF, 2007). This treatment should will substantiallyreduce the growth rateofsomeEnterobacteriaceae Salmonella, Cronobacter spp. and pathogenic w w ) ). w w foods. Examples include foods containing high levels foods. Examplesincludefoodscontaininghighlevels 0.95, withaminimum 10 reduction by68°Cfor16s(Nazarowec-White etal., 32 w below 0.98Enterobacteriaceae are oftenata w also dependsonthetemperature, with E. coli, however, may survive for w w 0.97 and at about one-quarter 0.97 and ataboutone-quarter limit closeto0.94.Therefore,

The chemical composition The chemicalcomposition sonnei andS. flexneri 10 unitsbystandard . For example, For example, w values of values of

The enterobacteriaceae and their significance to the food industry . Packaging foodsPackaging In general, Gram- . . O157 strains,O157 coli E. However, upon temperature abuse, upon temperature However, . . In fresh meat and fish in MAP, lactic acid bacteria meat and fish in MAP, . In fresh 2 33 reduction in the USA) is required to ensure production of production ensure to required is USA) the in reduction 10 in the pack (and thus also its antimicrobial activity) will decrease, activity) will decrease, in the pack (and thus also its antimicrobial 2 O157:H7, which are reported to survive at pH levels below pH 3.8.pH below levels pH at survive to reported are which O157:H7, coli E. et al., 2001a). O157, acid resistance is an intrinsic characteristic; for others it canfor others characteristic; an intrinsic is resistance coli O157, acid Salmonella and E. are some of the most important spoilage organisms of proteinaceous foods, of proteinaceous some of the most important spoilage organisms Pseudomonas are Gaseous atmosphere will predominate and suppress growth of Enterobacteriaceae growth and suppress will predominate the concentration of dissolved CO 7.4 Although particularly meat and fish held at refrigeration temperature in air, psychrotrophic Enterobacteriaceae Enterobacteriaceae psychrotrophic in air, temperature particularly meat and fish held at refrigeration on can grow Enterobacteriaceae may also cause spoilage of these foods. Being facultative anaerobes, of strict aerobes, not usually inhibited by the growth the surface and in the interior of foods. They are Packing spoilage organisms. of aerobic inhibit the growth can of Enterobacteriaceae growth whereas storage, when used in combination with refrigerated meat and poultry under vacuum is effective Enterobacteriaceae by spoilage to prone are meats vacuum-packed although of these bacterial collections. Perhaps the most notable Enterobacteriaceae associated with acid associated with notable Enterobacteriaceae Perhaps the most collections. of these bacterial of strains are resistance risk for certain foods e.g. fermentedThese pose a food safety that meats. Survival under pH conditions enhancing with ambient temperature by temperature, is also influenced sub-optimal for growth are with faster inactivation at ambient temperature of et al., 2010). The same effect inactivation (Zhang of fermented sausage representative O157 kept under conditions was noted for E. coli temperature (Uyttendaele can exert a selective methods in the food chain and food preservation that changes It is recognised conditions, under challenging environmental and grow enabling them to adapt on microbes pressure acid-resistant of emergence The normallywould which growth. to inhibitory be is an example of strains adapting tothat can survive in contaminated fermented meat products, It is also the case inhibitory. otherwise considered that are survive under conditions (e.g. low pH) periods at low pH. This increased that some non-pathogenic E. coli strains can survive for prolonged combined with the low infectious dose has enabledpotential for survival under acidic conditions as yoghurt, juices and salami that such as a pathogen in acidic food products E. coli O157 to emerge For some strains of pathogenic bacteria, low pH. to be safe owing to their inherently assumed were including to history of the strain (i.e., prior exposure and the previous be classified as an adaptive response survival under harsh acidic conditions (Buchanan andsub-optimal pH) may enhance its subsequent et al., 2009). Edelson, 1996; Uyttendaele et al., 2001b; Tassou fermentationby either acid,lactic (e.g., acids weak of addition by or increased be can foods of Acidity of Enterobacteriaceae inactivation and inhibition fermentedIn acid). acetic acid, citric growth foods, used of acid by lactic acid bacteria, or other bacteria can be accomplished by the rapid production asadded or occurring naturally be can micro-organisms These fermentationsome in processes. In fermented fermentation initially induces rapid sub-lethal injurystarter cultures. meats, the process of fermented manufacture meat followed by inactivation. During commercial of Enterobacteriaceae log (5 a significant inactivation products, enabling growth of Enterobacteriaceae (Devlieghere et al., 2002). (Devlieghere of Enterobacteriaceae enabling growth a safe product. This is essential due to the potential risk of survival of low infectious dose pathogens of low of survival risk potential due to the is essential This a safe product. such as E. coli O157:H7. under modified atmospheric packaging (MAP) is an effective way of extending a product’s shelf-life product’s a extending of way effective an is (MAP) packaging atmospheric modified under used include 60-80% oxygen and 20-40% carbon dioxide Common gas mixtures susceptible to CO are negative microorganisms The enterobacteriaceae and their significance to the food industry living planttissue ripening activities.To prolong shelf-lifeitisimportanttomaintainphysiologicalactivityofthe Cutting fresh produce affects thephysiologicalprocesses oftheplanttissue,e.g.,respiration and dominate duringprolonged storageofpre-packed fresh produce The nutritionalcompositionofthefresh producethetypeofbacteriathat willalsodetermine an importantfactorthataffects growth. little ornoeffect ongrowth ofEnterobacteriaceae inpackagedfresh produce, whereas temperature is by different bacteria,includingEnterobacteriaceae retarding planttissuedie-off willretain someofthenaturaldefencesplanttissueagainstinfection concentrations (Jacxsensetal.,1999).Maintainingthephysiologicalactivityofplanttissuewhilst growth ofyeastsandmouldsinsomecaseslacticacidbacteria. suppress theEnterobacteriaceae vegetables suchascarrots andbellpeppersiscommonlyattributedtolacticacidbacteriathatwill Enterobacteriaceae willspoiltheless-acidicproduce e.g.leafyvegetables.Spoilageofsugar-rich . Fresh producepackedunderlowO isnormally . Intrinsic acidicproperties ofminimallyprocessed fruits favourthe . 34 Modifying thepackagingatmosphere alonehas . 2 (1–5%)andhighCO Gram-negative flora including Gram-negative floraincluding 2 (5–10%) (5–10%) The enterobacteriaceae and their significance to the food industry - sakazakii. Wash Enterobacter 35 G EADIN THER R ington D.C. ASM Press. John Wiley & Sons, Ltd. Edition) Chichester, foodborne pathogen. Foodborne Pathogens and Disease, 7: 339-350. - (Eds) Identification Methods in Applied and Environmen F.A. Jones, D. and Skinner, R.G., Board, Blackwell Scientific Publications. 127-149. , Oxford: tal Microbiology ASM Press. , 34: 103-113. of Food Microbiology . Garrity, G.M., Brenner, D.J., Krieg, N.R. and Staley, J.T., (Eds). New York, NY, NY, (Eds). New York, J.T., and Staley, D.J., Krieg, N.R. G.M., Brenner, atic Bacteriology. Garrity, pp 587-607. 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Two distinct toxins active on vero cells from cells from toxins active on vero distinct Two Rowe, B. (1985). S.M., Smith, H.R. and Scotland, The enterobacteriaceae and their significance to the food industry Commonly involvestheheart valves. Endocarditis: Inflammationorinfectionof the endocardium (innerlining oftheheartmuscle). Encephalitis: Inflammationofthebrain. certain bacteriaandisreleased upondestructionofthebacterialcell. anintegralpart ofthecellwall(lipopolysaccharidecomplex) Endotoxin: Atoxinthatforms Provides elasticityofstructures suchtheskin,bloodvessels,intestines,tendonsandligaments. Elastin: Aprotein thatcoilsandrecoils likeaspringwithintheelasticfibres ofconnectivetissue. Elastases: Anenzymethatcatalyzesthehydrolysis ofelastin. bacterium tothehostcellsurface. eaeA: Geneassociatedwiththeproduction oftheprotein intimin,whichmediatesbindingofthe number ofviablecellsorspores ofagivenmicroorganism to10%oftheinitial population. D value:Thetimerequired (usuallyexpressed inminutes)atagiventemperature toreduce the individual’s lifetime.Itcanberecruited andbrought intoactionbytheadaptiveimmunesystem. system (innateimmunesystem)thatisnotadaptableanddoeschangeoverthecourseofan antibodies todestroy pathogenic bacteriaandotherforeign cells.Itispartoftheimmune Complement: Groupbloodplasmathatcombinewith ofproteins foundinnormal normally another organism without adverselyaffecting orhelpingit. Commensal: Livinginarelationship in whichoneorganism derivesfoodorotherbenefitsfrom characterised bythepresence ofhydroxyproline andhydroxylysine. Collagen: Aprotein consistingoflarge proportions oftheaminoacidsglycineandproline and Collagenase: Proteolytic enzymethat catalyzes thedegradationofcollagen. Cholecystitis: Inflammationofthegallbladder’s wall. thatinfectsabacterium. Bacteriophage: Virus infection whichisclinicallymore significant). Bacteraemia: Presence ofbacteriaintheblood(incontrast:septicemiarefers tobloodstream areformation encodedbytheLEEpathogenicityisland. in thepathogenicityofEPECandEHECinfections.ThegenesinvolvedA/Elesion cytoskeletal proteinsbeneaththeadherent (pedestalformation) bacterium.Theyare essential wall, resulting inlocaliseddestructionofbrush-border microvilli andrearrangement ofhostcell certain bacteria(e.g.,EPEC,E.coliO157andotherEHEC)totheepithelialcellsofgutcell Attaching andeffacing (A/E)lesions:A/Elesionsare characterisedbytheintimateadherence of glossary ofterms 42 The enterobacteriaceae and their significance to the food industry 43 , required by the bacterium as by the bacterium E. coli, required strains of pathogenic by various secreted EspA: A protein to host cells surfaces. of attachment part of the process Extrinsic factors: Externalto a food e.g. temperature, (i.e. storage conditions) applied factors gaseous atmosphere. humidity, equally well but can grow aerobically, that normally grows e: A microorganism Facultative anaerob conditions. under anaerobic including the surface of certain bacteria extending from filaments Fimbriae: Thin proteinaceous bacterial associated with fimbriae are but with a similar structure, E. coli. Smaller than flagella adhesion. only one of motility found in motile bacteria. Certain bacteria express Flagella: Organelles cell surface. entire flagella over the numerous flagellum while others express Gene: The basic unit of heredity. cramps and bloody Haemorrhagic colitis (HC): An acute disease characterised by abdominal Attributed to a self-limited infection by VTEC. diarrhoea, usually without fever. VTEC from (HUS): A clinical condition, which sometimes arises Haemolytic Uraemic Syndrome and kidney failure. infection and is characterised by anaemia origin useful in the study of bacterial HEp-2 Cells: A human epithelial cell line of intestinal attachment and invasion. mixed of mutual interactions between the result Implicit conditions: Conditions that are i.e. in competition between microorganisms, These result populations of microorganisms. in the populations, that may result microbial different for nutrients and associations between populations. of certain species or microbial stimulation and inhibition of growth to the host cell by the bacterium to mediate intimate attachment required Intimin: A protein lesions by itself. attaching and effacing surface. Intimin cannot produce within a food, e.g., pH, water inherent Intrinsic factors: Factors (chemical and physical) that are salt and sugar content, preservatives. activity, of DNA chain length. 1 kb is for kilobase pairs or a measure Kilobase(s) (kb): The abbreviation 1000 base pairs. for 1000 Daltons. A Dalton is the unit of atomic or molecular KiloDaltons (kDa): The abbreviation mass. A molecule of 1 kDa has a molecular mass of 1000. of (LEE): Pathogenicity island found in the chromosome Effacement The Locus of Enterocyte , EPEC, and EHEC strains (including E. coli O157) as well as other VTEC. rodentium Citrobacter associated and system secretion III Type a encoding genes of cluster a by characterised is LEE The (A/E) lesions in the large for attaching and effacing responsible proteins and effector chaperones intestine. The enterobacteriaceae and their significance to the food industry Pathogenic: Pertainingtobehaviourasa pathogen. (infection of)thehost. Pathogen: Anymicroorganism thatcausesdiseaseinhumansoranimalsbydirect interactionwith process intheEuropean Unionisatleast71.7ºCfor15s. organisms inmilkandotherfoods,e.g.formilk,thelegalrequirement forthepasteurisation ofheattreatmentPasteurisation: Aform thatkillsvegetativepathogens and spoilagemicro- Osteitis: Inflammationofboneorbonytissue. was usedandbecameO-antigen. bacterial flagellaantigens.Where“Ohnehauch”(withouthauch) nohauchwasdetectedtheterm H-antigenfor hencetheterm resembling “hauch”inGerman, condensation onglass,termed Reaction ofananti-flagellaantibodywithastrainEscherichiacolicausedagglutination lipopolysaccharideorsomaticantigenandhasanunusualorigin. O-Antigen: Thisisalsotermed considered nosocomial. first appearing48hoursormore afterhospitaladmissionorwithin30daysdischarge are Nosocomial infection:Aninfectionacquired ina hospitalorotherhealthcare unit.Infections They playanimportantroleofacuteinflammation. intheearlystagesofmanyforms Neutrophil: Examples includetransposons,plasmids,insertionsequencesandbacteriophageelements. Mobile geneticelement:Aunitthatcaninsertintoachromosome, exit,andrelocate. bloodstream. site ofinfection,resulting from transportationofinfectiousparticlestootherlocationsviathe Metastatic abscesses:Secondaryabscessthatdevelopsatapointdistantfrom anoriginal Mesophilic: Amicroorganism withanoptimumgrowth temperature between20°Cand45°C. state. Malaise: Ageneralfeelingoflackwell-beingdiscomfort,illness,oftenassociatedwithadisease play arole inantigenpresentation toTandBlymphocytes. microorganisms. Theyalsorelease substancesthatstimulateothercellsoftheimmunesystemand Macrophages playanimportantrole intheimmune response toforeign invaderssuchasinfectious inflammation. Theyrequire thepresence ofactivatedTcells,ortheircomponentsforactivation. (in aprocess knownasphagocytosis).Macrophages becomeactivewhenstimulatedby Macrophage(s): Type ofwhitebloodcell(denotedasaleucocyte)thatingestsforeign material MDa: MegaDaltonsor1,000,000Daltons. barrier. Theyplayanimportantrole instimulatingmucosalimmunity. transporting organisms andparticlesfrom thegutlumen toimmunecellsacross theepithelial follicles thatactivelytakeupparticulatematterfrom theintestinalcontents.Responsiblefor M cells(microfold cells):SpecialepithelialcellsassociatedwithPeyer’s patchesandlymphoid An abundanttypeofgranularwhitebloodcellthatishighlydestructivemicroorganisms. 44 The enterobacteriaceae and their significance to the food industry 45 : The ability of a bacterium to cause disease. A measure of pathogenicity is termed of cause disease. A measure of a bacterium to : The ability Pathogenicity “virulence”. of the abdominal membrane which lines part peritoneum (serous Peritonitis : Inflammation of the cavity and viscera). of usually found in the lowest portion that are of lymphoid tissue Aggregations patches: Peyer’s that contains specialised by a special epithelium in humans). Covered the small intestine (ileum the lumen. Peyer’s from cells), which sample antigen directly cells (M cells called microfold in the immune response. role patches play an important of organisms among various groups Phylogenetic studies: The study of evolutionary relatedness molecular and morphological data matrices. through of engulfing and ingesting particles by the cell or a phagocyte (e.g., Phagocytosis: The process to form food vacuole). These fuse with lysosomes to become macrophage) a phagosome (or is eventually degraded or digested and either released phagolysosomes. The engulfed material further processing. intracellularly to undergo extracellularly via exocytosis, or released phage including biotype, serotype, Phenotype: The observable characteristics of an organism, type and bacteriocin-type. of the host cell chromosome. independently DNA that replicates Plasmid: Extrachromosomal they carry) may be transferable between cells. Plasmids (and the genetic characteristics copies of a target Polymerase Chain Reaction (PCR): A widely used technique to amplify multiple DNA sequence. as a linear part of the bacterial: A bacteriophage genome covalently integrated Prophage chromosome. into peptides or of proteins breakdown of enzymes that catalyze the hydrolytic : Group Proteases amino acids. e.g. 0-5°C, but at low temperatures that can survive and grow : A microorganism Psychrotrophic optimally between 15°C and 20°C. grows Pseudoappendicitis: A condition with symptoms like those of appendicitis but which do not result inflammation of the appendix. from a rRNA: Ribosomal RNA (rRNA) comprises the major part of RNA in the cell. It provides with transfer mechanism for decoding messenger RNA (mRNA) into amino acids and interacts peptidyl transferase activity. RNAs (tRNAs) during translation by providing : A method of distinguishing among bacteria on the basis of their antigenic properties Serotyping of a strain and in combination to known antisera). The O-antigen defines the serogroup (reaction of the strain. with the H-antigen defines the serotype of similarity degree that have a high of plants, animals or micro-organisms Species: A group so that they fertile offspring, only amongst themselves to produce and generally can interbreed other such groups. maintain their ‘separateness’ from The enterobacteriaceae and their significance to the food industry Water activity(a pathogens, whichisutilisedtodelivervirulenceeffector proteins directly intohostcells. Type IIIsecretion system:Aspecialisedsecretion systemfoundinmanyGram-negativebacterial Thrombocytopaenia: Lownumbersofplateletscirculating inthebloodstream. genus andspeciesbyeitherphenotypicand/orgenotypiccharacteristics. Strain: Anisolateorgroup ofisolatesthatcanbedistinguishedfrom otherisolatesofthesame RH of95%corresponds toana defined as1/100ththerelative humidity(RH%)oftheairinequilibriumwithsubstrate.E.g.,an w ): Amountof‘free’ or‘available’waterinagivensubstrate(e.g.food).Canbe w of0.95. 46 The enterobacteriaceae and their significance to the food industry 47 Escherichia coli Escherichia coli Escherichia coli Escherichia coli tions aggregative adherence fimbriae adherence aggregative attaching and effacing Safety of Food on the Microbiological Advisory Committee Escherichia coli attaching effacing bundle-forming pili base pair(s) Peptone Water Buffered Bile Lactose Broth Brilliant Green colony forming units colonisation factors colonisation factor antigens Escherichia coli adherent diffusely deoxyribonucleic acid gene Escherichia coli attaching and effacing factor EPEC adherence heat-stable toxin enteroaggregative Escherichia coli Enterohaemorrhagic protein E. coli secreted Points Critical Control Analysis Hazard haemorrhagic colitis haemolytic uraemic syndrome Specifications for Foods International Commission on Microbiological effacement locus of enterocyte heat labile enterotoxin Broth Lauryl Sulphate Tryptose number most probable Polymerase Chain Reaction Foods of Extended Durability Refrigerated Processed ribonucleic acid ribosomal RNA heat-stable enterotoxin E. coli Shiga-like toxin-producing Shiga toxin Shiga toxin-encoding gene agar bile X-glucuronide Tryptone Esp A/E ACMSF AEEC BFP substrate) (chromogenic β-glucuronide BCIG 5-bromo-4-chloro-3-indolyl- bp BPW BGBLB cfu CFs CFAs Da Dalton(s) DAEC DNA eae EAF EAST EHEC EPEC enteropathogenic ETEC enterotoxigenic EAggEC enteroaggregative HACCP HC HUS ICMSF kb kilobase(s) kDa kilodalton(s) LPS lipopolysaccharide LEE LT LSTB MPN PCR REPFED RNA rRNA ST STEC Stx stx TBX TMA trimethylamine evia abbr AAF EIEC enteroinvasive The enterobacteriaceae and their significance to the food industry vtx VT Verocytotoxin VRBLA VRBGA TTSS TMAO Verocytotoxin-encoding gene Violet RedBileLactoseAgar Violet RedBileGlucoseAgar type IIIprotein secretion system trimethylamine oxide 48 Other ILSI Europe Publications • Frontiers in Food Allergen Risk Assessment • Functional Foods in Europe – International Developments in Concise Monographs Science and Health Claims • Functional Foods – Scientific and Global Perspectives • Alcohol – Health Issues Related to Alcohol Consumption • Guidance for the Safety Assessment of Botanicals and Botanical • A Simple Guide to Understanding and Applying the Hazard Preparations for Use in Food and Food Supplements Analysis Critical Control Point Concept • Impact of Microbial Distributions on Food Safety • Calcium in Nutrition • Markers of Oxidative Damage and Antioxidant Protection: • Carbohydrates: Nutritional and Health Aspects Current status and relevance to disease • Caries Preventive Strategies • 3-MCPD Esters in Food Products • Concepts of Functional Foods • Method Development in Relation to Regulatory Requirements • Dietary Fibre for the Detection of GMOs in the Food Chain • Food Allergy • Micronutrient Landscape of Europe: Comparison of Intakes and • Food Biotechnology – An Introduction Methodologies with Particular Regard to Higher Consumption • Food, Glycaemic Response and Health • Mycobacterium avium subsp. paratuberculosis (MAP) and the • Functional Foods – From Science to Health and Claims Food Chain • Genetic Modification Technology and Food – Consumer Health • Nutrition in Children and Adolescents in Europe: What is the and Safety Scientific Basis? • Healthy Lifestyles – Nutrition and Physical Activity • Overview of the Health Issues Related to Alcohol Consumption • Microwave Ovens • Overweight and Obesity in European Children and Adolescents: • Nutrition and Genetics – Mapping Individual Health Causes and consequences – prevention and treatment • Nutrition and Immunity in Man • Packaging Materials: 1. 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