Enterobacteriaceae

Chapter 17


Enterobacteriaceae


Most members of the family Enterobacteriaceae share the following characteristics: Gram-negative, medium-sized rods (0.4–0.6 × 2–3 µm; Fig. 17.1); peritrichate arrangement of flagella, if motile; facultatively anaerobic and ferment, rather than oxidize, glucose; catalase-positive and oxidase-negative; reduce nitrate to nitrite and are able to grow on non-enriched media such as nutrient agar. There are a few exceptions to these general properties, for example, Shigella dysenteriae is catalase-negative; Tatumella ptyseos is motile by polar, subpolar or lateral flagella and Photorhabdus species do not regularly reduce nitrate.





Nomenclature


There are, at present, more than 40 genera and over 180 well-defined species in the Enterobacteriaceae. Traditionally the genera and species of the family have been distinguished biochemically and this is convenient for identification of clinical isolates. However, genetic means of defining species, based on DNA–DNA homology, has led to the recognition of numerous new species, some previously regarded as aberrant biotypes, and also to the recognition of genetically closely related members as single genomic species.


The terms ‘coliform’ or ‘coliform bacteria’ have no taxonomic significance but are used to refer to those members of the Enterobacteriaceae that usually ferment lactose, such as Escherichia coli, Klebsiella and Enterobacter species and sometimes to describe other members of the family.




Differentiation of the Enterobacteriaceae



Conventional microbiology


All enterobacteria will grow on blood and MacConkey agars and these are used routinely to isolate them in diagnostic laboratories. Although MacConkey agar is a selective medium, it is relatively permissive and allows the growth of some other Gram-negative bacteria as well as the enterobacteria. Brilliant green agar and xylose-lysine-deoxycholate (XLD) medium are more selective and used for the isolation of salmonellae, although some other enterobacteria are able to grow on them. A number of other selective agars are frequently used for the isolation of salmonellae and these are detailed under the relevant section. Table 17.1 gives the reactions of some members of the Enterobacteriaceae on MacConkey agar, brilliant green agar and XLD medium. The uninoculated media (Fig. 17.2) are illustrated together with the appearance of 11 enterobacteria and Pseudomonas aeruginosa on these selective/indicator media (Figs 17.3 to 17.14 inclusive). A summary of isolation methods for the detection and presumptive identification of important members of the Enterobacteriaceae is given in Figure 17.15.




















XLD medium



Fermentable sugars: lactose, sucrose and xylose.


pH indicator: phenol red (red at pH 8.2 and yellow at pH 6.4).


Other substrates: lysine and chemicals for detecting hydrogen sulphide (H2S) production.


Inhibitor: bile salts (sodium deoxycholate).


Reactions: salmonellae will first ferment the xylose creating a temporary acid reaction but this is reversed by the subsequent decarboxylation of lysine with alkaline metabolic products. Superimposed on the red (alkaline) colonies is the production of hydrogen sulphide, so most salmonellae have red colonies with a black centre (Fig. 17.3). Edwardsiella tarda also gives this reaction although the H2S production is less marked and the periphery of the colonies tends to be a yellowish-red colour. The large amount of acid produced by enterobacteria that can ferment either lactose or sucrose, or both, prevents the reversion to alkaline conditions even if the bacterium is able to decarboxylate the lysine.



Triple sugar iron (TSI) agar

This is an indicator medium only and does not contain an inhibitor. A brief description of the medium and technique for inoculation is given in Chapter 2. It is prepared in tubes with a slant.



Bacteria that are unable to ferment either lactose or sucrose, after the depletion of the limited amount of glucose, will use the peptone in the medium. This is a less efficient method of producing energy and occurs mainly at the surface of the slant in the presence of atmospheric oxygen. The metabolites of peptone are alkaline and this causes the slant to revert back to the original red colour. Some members of the Enterobacteriaceae, including most Salmonella spp., are able to produce hydrogen sulphide. This reaction is superimposed over the sugar fermentations and is seen as a blackening of the medium.


The general interpretation of the reactions is as follows:



The reactions are illustrated in Figure 17.16, while Figure 17.17 gives a summary of the differentiation of some of the enterobacteria by their reactions in TSI agar and lysine decarboxylase broth.





Pathogenicity


The Enterobacteriaceae can be divided into three groups based on their pathogenicity for animals:



• Major pathogens of animals such as Salmonella species, Escherichia coli and three of the Yersinia species.


• Opportunistic pathogens that are known to occasionally cause infections in animals. These include species within the genera Klebsiella, Enterobacter, Proteus, Serratia, Edwardsiella, Citrobacter, Morganella and Shigella. Shigella species cause disease in humans and other primates.


• Organisms of uncertain significance for animals. These include species from 17 genera of the Enterobacteriaceae and they are summarized in Table 17.2. As some of them may be isolated from clinical specimens a range of their biochemical reactions is given in Table 17.3.



Table 17.2


Genera of the Enterobacteriaceae whose species are of uncertain significance for animals






















































Genus (species) Site of isolation and possible pathogenicity
Budvicia aquatica Water and human faeces
Buttiauxella agrestis Water
Cedecea species Rare isolates from human clinical specimens (most commonly from the respiratory tract). Bacteraemia in humans has been reported
Ewingella americana Rare isolate from human clinical specimens
Erwinia herbicola This and other Erwinia species are associated with plants as pathogens or saprophytes
Kluyvera species Water, sewage, soil and milk. Occasionally isolated from human clinical specimens
Yokenella regensburgei (Koserella trabulsii) Human respiratory tract, wounds, urine and faeces but pathogenicity uncertain
Leclercia (Escherichia) adecarboxylata Environment, food and water. It has been isolated from human clinical specimens
Lemiorella species Isolated from human faeces and urine
Moellerella wisconsensis Human faeces
Obesumbacterium proteus Found only in contaminated beer. Biogroup 1 is thought to be a brewery-adapted biochemical variant of Hafnia alvei. Unlikely to be pathogenic for animals
Photorhabdus species Pathogenic for nematodes
Pragia fontium Isolated from water
Providencia species Urinary tract of compromised or catheterized human patients, patients suffering from burn infections and diarrhoea. Rarely isolated from faeces of healthy humans
Rahnella aquatilis Water and from human burn wounds
Tatumella ptyseos Occasionally isolated from human clinical specimens, mainly from the respiratory tract


All Gram-negative bacteria, including the members of the Enterobacteriaceae, have lipopolysaccharides in the outer membrane of the cell wall that are potent endotoxins, the main endotoxic principle being lipid A. The bacteria must die and lyse before the endotoxin is released. The effects of endotoxin in the animal body include fever, leukopaenia followed by leukocytosis and hyperglycaemia with a subsequent fall in blood sugar and lethal shock after a latent period. The more pathogenic members of the Enterobacteriaceae have other virulence factors such as adhesins to attach to host cells, capsules that are antiphagocytic, siderophores that aid the bacterium in its competition with the host for iron and exotoxins that include enterotoxins and cytotoxins. These will be reviewed in the relevant sections on each genus/organism.



Escherichia coli




Pathogenesis and Pathogenicity



Predisposing causes


Although E. coli strains possess a number of virulence factors which assist them in colonizing the intestine, invading the host and producing disease, predisposing causes are of paramount importance and largely determine whether or not clinical signs of illness will occur:



• Neonates obtaining insufficient passive immunity (antibodies) from colostrum. This might be due to either a quantitative or qualitative deficiency.


• Intensive husbandry practices lend themselves to rapid transmission of the pathogenic E. coli strains.


• Poor hygiene that allows a build-up of pathogenic strains in the environment of the young animal. A large dose of pathogenic E. coli may overcome colostral immunity.


• Young neonates, under one week of age, are particularly susceptible because:



• Recently weaned pigs are subject to stress factors such as altered surroundings, companions and diet. Heavy grain diets in particular can lead to a massive colonization of the anterior small intestine by enterotoxigenic strains of E. coli.


• Oedema disease occurs most commonly in young weanling pigs but the disease can occur in older pigs. The following factors are often present prior to the occurrence of oedema disease in pigs:




Types of pathogenic E. coli


Escherichia coli strains, normally regarded as non-pathogenic, can cause opportunistic infections in various sites of the body such as mammary glands (mastitis) and uterus (metritis). Pathogenic E. coli strains are classified according to the type of disease that they produce and according to the virulence determinants which they possess. However, as more is learnt about the virulence attributes of all strains of E. coli, it is becoming increasingly clear that the possession of particular virulence genes may not be the only feature that differentiates pathogenic and non-pathogenic strains; the level of expression of those genes is also likely to be of crucial importance.


Escherichia coli strains can be divided into those causing extraintestinal disease and those causing enteric infections. Extraintestinal diseases result from infection with strains causing invasive conditions such as septicaemia (SEPEC), and also include uropathogenic E. coli (UPEC) and avian pathogenic E. coli (APEC). These strains may be collectively referred to as extraintestinal pathogenic E. coli or ExPEC. There are also suggestions for two new animal pathogenic groups: those causing infections of the mammary gland, mammary pathogenic E. coli (MPEC) and those affecting the uterus, endometrial pathogenic E. coli (EnPEC) (Köhler & Dobrindt, 2011). The types of E. coli causing enteric disease include enterotoxigenic strains (ETEC) and attaching and effacing E. coli (AEEC). The latter group includes the enteropathogenic E. coli (EPEC) and Vero- or Shiga-toxin producing strains (VTEC or STEC). Enterohaemorrhagic E. coli and strains of E coli producing oedema disease are subgroups of STEC. Of the disease syndromes caused by these pathogenic types, enteric disease produced by ETEC, oedema disease of pigs and extraintestinal diseases, including septicaemia, are the best characterized types in animals. In contrast to ETEC strains, ExPEC and EPEC strains form part of the normal flora in animals and are considered opportunistic pathogens (Gyles & Fairbrother, 2010).



Enterotoxigenic E. coli

These strains cause the majority of cases of neonatal colibacillosis in calves, lambs and piglets. They do not appear to be an important cause of diarrhoea in other domestic animals, for reasons which are as yet unclear. Pathogenicity is correlated with the presence of adhesins and the production of enterotoxins. The toxins function by reducing absorption and increasing secretion without damaging the intestinal epithelium. The first step in the production of disease is the adherence of the ETEC to the intestinal epithelium. The structures by which the ETEC adhere are most commonly fimbriae and these are classified according to properties such as their amino acid composition and their ability to agglutinate red blood cells in the presence or absence of D-mannose. The fimbriae described in pigs and calves include F4 (K88), F5(K99), F6 (987P), F17, F18 and F41 (Nagy & Fekete 1999). ETEC strains are host-specific and usually cause disease in the first week of life only. Host specificity can be explained by the presence or absence of genes encoding for fimbrial receptors in the intestinal lining of the host. Age-related resistance may be due to the degree of expression of host receptors. It appears that some of the receptors are over-expressed as the age of the animal increases, thus leading to shedding of receptors into the intestinal lumen. These free receptors coat the ETEC strains in the intestinal contents, thus preventing their adherence to the intestinal lining (Dean et al. 1989). F18 receptors are not produced in newborn pigs but are increasingly expressed up to approximately four weeks of age, thus helping to explain why ETEC are a cause of diarrhoea in pigs up to this age.


Following adherence, ETEC produce protein enterotoxins. The toxins can be subdivided into two main groups: Large heat-labile toxins of approximately 88 kDa in size (LT) and smaller, heat-stable toxins (ST) containing 11–48 amino acids. Both toxin types have two subgroups. Porcine strains of ETEC produce mostly LT1 and Sta whereas strains of ETEC occurring in calves usually produce Sta. STb is associated with porcine strains of ETEC although it may be produced by some strains of ETEC isolated from calves, chickens and humans. LT has a similar mechanism of action to the heat-labile toxin produced by Vibrio cholerae. It consists of an A domain and five B subunits. The B subunits bind to the cell and part of the A domain then enters the endoplasmic reticulum of the cell and activates the adenylate cyclase system. The resulting increase in cAMP levels leads to increased fluid and electrolyte secretion and decreased absorption. STa acts by increasing guanylate cyclase activity leading to elevated levels of cGMP in the cell. Increased levels of cAMP or cGMP activate protein kinases which induce phosphorylation of the cystic fibrosis transmembrane regulator (CFTR). This in turn causes secretion of chloride and bicarbonate ions. Protein kinases also inhibit reabsorption of sodium ions (Dubreuil 2012). There is a resultant reduction in absorption of water and electrolytes at the villus tips and an elevated secretion of chloride and water in crypt cells. The mechanism of action of STb differs from that of LT and STa as it does not activate adelyate or guanylate cyclases but phosphorylates CFTR through a different mechanism. Interference with the enteric nervous system may also be important in the secretory diarrhoea induced by E. coli enterotoxins (Dubreuil 2012).



Enteropathogenic E.coli

These strains are included in the attaching and effacing E. coli (AEEC) because of the nature of the lesions they produce in the intestinal lining. Classical or typical EPEC strains were first described as a cause of diarrhoea in infants and these strains are strict human pathogens and possess a specific (EPEC) adherence factor plasmid (EAF) which is not found in animal isolates of EPEC. Atypical EPEC cause diarrhoea in piglets, lambs, calves and pups. The virulence factors of EPEC are encoded by a pathogenicity island known as the locus of enterocyte effacement (LEE). The genes on this island encode a number of components including the outer membrane protein intimin and its receptor, known as translocated intimin receptor (Tir). Tir is inserted into a host cell membrane where it functions as a receptor for intimin and thus allows close attachment of the E. coli cell to the host cell. A characteristic pedestal formation and effacement of microvilli then follow. In addition, other effectors produced by the bacterium cause increased levels of intracellular calcium, secretion of chloride ions, impairment of tight junctions and recruitment of neutrophils.



Shigatoxigenic E. coli

Oedema disease in pigs is often associated with E. coli O139 and O141 and these strains are usually haemolytic and produce Shiga toxin. These toxins are similar in activity to the Shiga toxin (cytotoxin) of Shigella species and inhibit protein synthesis in host cells following interaction with the 60S ribosomal subunit resulting in death of the cell. There are two types of Shiga toxins, Stx1 that is neutralized by antibody specific for Shiga toxin and Stx2 which contains a number of subtypes. Subtype Stx2e is associated with oedema disease in pigs whereas strains producing Stx1, Stx2, Stx2c and Stx2d are associated with haemorrhagic enteritis in humans. The toxin Stx2e is produced in the intestine but is absorbed and carried via the bloodstream to the target cells, usually endothelial cells of the small arteries. These oedema disease strains of E. coli are normally present in the large intestine of pigs, but they appear to multiply rapidly under conditions of stress, particularly a change of diet.




Septicaemic E. coli

Septicaemic (SEPEC) strains are responsible for septicaemia in their hosts. These strains may possess a wide range of virulence factors but few of these virulence factors are common to all strains (Mokady et al. 2005). It appears that each step of the disease process can be mediated by a number of alternative virulence factors and a particular invasive strain may have a unique combination of pathogenic attributes. Adherence to the intestinal lining is the first step in the invasion process. Adherence may be mediated by fimbrial adhesins, for example, F5 as in ETEC strains, by other fimbriae such as long polar fimbriae or by non-fimbrial adhesins. Septicaemic strains carry a plasmid encoding Colicin V (Col V plasmid). This plasmid encodes for type IV pili which have been shown to be important for adherence and invasion in Salmonella Typhi. In addition it encodes for serum resistance and the aerobactin iron uptake system, both important virulence factors for systemic survival of E. coli strains. Recently the presence of another iron uptake system has been demonstrated, similar to that found in Yersinia species, and dependent on the biosynthesis of the siderophore yersiniabactin. Different capsular types are present depending on strain and these are important for survival of the organisms following invasion. The immune response of the host leads to the death of some organisms and endotoxin is released, with resultant clinical signs of pyrexia, weakness, depression and tachycardia.


The diseases caused by E. coli in domestic animals are summarized in Table 17.4. Major virulence factors are given in Table 17.5.





Laboratory Diagnosis



Diagnosis of the opportunistic infections caused by E. coli


In this case it is sufficient to isolate E. coli in an almost pure growth from carefully taken samples such as cervical swabs, mastitic milk samples and midstream urine. The culture and presumptive identification methods are shown in Figure 17.15. Pathogenic strains of E. coli are often haemolytic (Fig. 17.18) and as they are strong lactose-fermenters the colonies on MacConkey agar are bright-pink (Fig. 17.19). Eosin methylene blue (EMB) agar is occasionally used in diagnostic laboratories and on this medium E. coli colonies have a unique and characteristic metallic sheen (Fig. 17.20). Other chromogenic agars for the easy identification of E. coli are available commercially. The ‘IMViC’ test (indole+/ MR+/ VP−/ citrate−) is a quick presumptive method of identifying E. coli (Fig. 17.21) as almost no other lactose-positive member of the Enterobacteriaceae gives this combination of results. Biochemical reactions of some clinically significant members of the Enterobacteriaceae, including E. coli, are given in Table 17.6. Molecular virulence typing can be used for isolates such as UPEC which possess defined virulence attributes.









Demonstration of the enterotoxigenic strains

The enterotoxigenic strains of E. coli are present in large numbers in the small intestine, and in this case it is insufficient to merely isolate and identify the E. coli. Demonstration of the significant fimbrial antigens (F4, F5, F6, F17, F18 and F41) or the enterotoxin itself is necessary. Alternatively, molecular detection of virulence genes can be employed.



• Fimbrial antigens. Fimbriae are expressed poorly on selective and some types of non-selective laboratory media. E medium (Francis et al. 1982) is advised for F4, F5 and F41. Minca medium (BBL) has been found satisfactory for F6, F5 and F41. Commercial test kits are available for the detection of fimbrial antigens such as a latex agglutination test (Fig. 17.22). Specific antiserum can be obtained for use in a slide agglutination test. Enzyme-linked immunosorbent assays (ELISA) are available for directly measuring the presence of fimbriae-expressing E. coli in faecal extracts. The fluorescent antibody technique, using conjugates prepared against each of the common colonizing-associated antigens, can be used on smears made from scrapings from the ileum of a fresh carcass.


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Jul 18, 2016 | Posted by in PHARMACOLOGY, TOXICOLOGY & THERAPEUTICS | Comments Off on Enterobacteriaceae

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