23       Enterobacteriaceae

 

Bacteria belonging to the family Enterobacteriaceae are Gram-negative rods which ferment glucose and other sugars and are oxidase-negative. They are catalase-positive, non-spore-forming, facultative anaerobes which grow well on MacConkey agar. The family contains more than 40 genera and over 180 species. Less than one-third of the genera are of veterinary importance.

Enterobacteria inhabit the intestinal tract of animals and humans and contaminate vegetation, soil and water. They can be arbitrarily grouped in three categories: major pathogens, opportunistic pathogens and non-pathogens. Opportunistic pathogens such as Proteus species, Klebsiella pneumoniae and Enterobacter aerogenes occasionally cause clinical disease in locations other than the alimentary tract. The major animal pathogens E. coli, Salmonella species and Yersinia species can cause both enteric and systemic disease. Selected criteria for differentiating pathogenic members are presented in Table 23.1. Apart from some strains of E. coli, few enterobacteria produce haemolysis on blood agar. Lactose fermentation in MacConkey agar is an important identifying feature of E. coli, Enterobacter aerogenes and Klebsiella pneumoniae. Colonies of these organisms and the surrounding medium are pink, due to acid production from lactose. Proteus species produce characteristic swarming on non-inhibitory media such as blood agar. Mucoid colonies are typical of Klebsiella and Enterobacter species. A variety of biochemical tests can be used to differentiate members of the Enterobacteriaceae and miniaturized strips comprising many different tests are frequently employed. However, results generated in these systems are unreliable for detection of Salmonella serotypes and reactions in triple sugar iron (TSI) agar and lysine decarboxylase production are used for confirming the identity of suspect Salmonella isolates. Serotyping, using slide agglutination tests with antisera, are used to detect the somatic (O) and flagellar (H) antigens of E. coli, Salmonella and Yersinia species. This procedure allows identification of the organisms involved in disease outbreaks. Many molecular techniques for identification and typing are available. PCR-based techniques, including multiplex and real-time PCR methods, are frequently employed for detection and identification of organisms. Typing methods include digestion with restriction enzymes followed by pulsed-field gel electrophoresis, multi-locus sequence typing and, in the case of E. coli, phylogenetic typing (see Chapter 4 for a description of these techniques).

Table 23.1 The clinical relevance and phenotypic characteristics of important members of the Enterobacteriaceae.

	Escherichia coli	Salmonella serotypes	Yersinia species	Proteus species	Enterobacter aerogenes	Klebsiella pneumoniae
Clinical importance	Major pathogen	Major pathogens	Major pathogens	Opportunistic pathogens	Opportunistic pathogen	Opportunistic pathogen
Cultural characteristics	Some strains haemolytic	Characteristic appearance on Salmonella-selective media	–	Swarming growtha	Mucoid	Mucoid
Motility at 30°C	Motile	Motile	Motileb	Motile	Motile	Non-motile
Lactose fermentation	+	–	–	–	+	+

^aWhen cultured on non-inhibitory medium.

^bExcept Y. pestis.

Particular members of the Enterobacteriaceae are sometimes involved in localized opportunistic infections in diverse anatomical locations. Faecal contamination of the environment contributes to the occurrence of opportunistic infection. The clinical conditions arising from infection with these opportunistic members are presented in Table 23.2. Klebsiella pneumoniae and Enterobacter aerogenes are two opportunistic pathogens commonly encountered in coliform mastitis in dairy cattle. These organisms usually gain entry to the mammary gland from contaminated environmental sources. Klebsiella pneumoniae is also reported to be one of the commonest causes of metritis in mares. Klebsiella species and Proteus species cause infections of the lower urinary tract in dogs. Proteus species are often implicated in otitis externa in dogs and sometimes in cats.

Table 23.2 Opportunistic pathogens in the Enterobacteriaceae and their associated clinical conditions.

Bacterial species	Clinical conditions
Enterobacter aerogenes	Coliform mastitis in cows and sows
Klebsiella pneumoniae	Coliform mastitis in cows; endometritis in mares; pneumonia in calves and foals; urinary tract infections in dogs
Proteus mirabilis and P. vulgaris	Urinary tract infections in dogs and horses; associated with otitis externa in dogs

Escherichia coli

Colonization of the mammalian intestinal tract by E. coli from environmental sources occurs shortly after birth and these organisms persist as important members of the normal flora of the intestine throughout life. Most strains of E. coli do not produce disease but may produce opportunistic infections in sites such as the mammary gland or in wounds. Predisposing factors which permit colonization include age, immune status, nature of diet and heavy exposure to pathogenic strains. As illustrated, pathogenic E. coli can be divided into strains causing intestinal and extraintestinal disease. Certain virulence factors are associated with particular pathotypes but the differentiation of E. coli strains is complex as many strains considered commensals also possess virulence determinants. Several pathotypes of intestinal E. coli are of importance in humans but a smaller number of types are responsible for disease in animals. In recent years, Shigatoxigenic E. coli have emerged as major food-borne zoonotic pathogens in humans, responsible for the haemorrhagic colitis and haemolytic uraemic syndrome.

Clinical infections in young animals may be limited to the intestines (enteric colibacillosis, neonatal diarrhoea), or may manifest as septicaemia or toxaemia. Extraintestinal localized infections in adult animals, many due to opportunistic invasion, can involve the urinary tract, mammary glands and uterus. The virulence factors of pathogenic strains of E. coli include capsules, endotoxin, structures responsible for colonization, enterotoxins and other secreted products. Capsular polysaccharides, which are produced by some strains of E. coli, interfere with the phagocytic uptake of these organisms. Endotoxin, a lipopolysaccharide (LPS) component of the cell wall of Gram-negative organisms, is released on death of the bacteria. The role of LPS in disease production includes pyrogenic activity, endothelial damage leading to disseminated intravascular coagulation and endotoxic shock. Fimbrial adhesins, which are present on many enterotoxigenic strains of E. coli, allow attachment to mucosal surfaces in the small intestine. The most significant adhesins in strains of E. coli producing disease in domestic animals are F4 (K88), F5 (K99), F6 (987P) and F41. The most common adhesin present in strains of E. coli infecting pigs is F4. The F5 and F41 adhesins occur in strains affecting calves and F5 in strains affecting lambs. The pathological effects of infection with pathogenic E. coli, other than those attributed to endotoxin, derive mainly from the production of enterotoxins, Shigatoxins or cytotoxic necrotizing factors. Unlike enterotoxins, which affect only the functional activity of enterocytes, Shigatoxins and cytotoxic necrotizing factors can produce demonstrable cell damage at their sites of action. To prevent enteric colibacillosis, neonatal diarrhoea and colisepticaemia, newborn animals should receive ample amounts of colostrum shortly after birth. Colostral antibodies can prevent colonization of the intestine by pathogenic E. coli. A clean, warm environment should be provided for newborn animals. Vaccination is of value for a limited number of diseases caused by E. coli.

Salmonella serotypes

The genus Salmonella contains more than 2,500 serotypes. Serotyping is based on the identification of somatic (O) and flagellar (H) antigens using specific antisera. These organisms occur worldwide and infect many mammals, birds and reptiles and are mainly excreted in faeces. Ingestion is the main route of infection. Organisms may be present in water, soil, animal feeds, raw meat and offal, and in vegetable material. The source of environmental contamination is invariably faeces. In poultry, some species such as Salmonella Enteritidis infect the ovaries and vertical transmission occurs.

Salmonellosis is of common occurrence in domestic animals and the consequences of infection range from subclinical carrier status to acute fatal septicaemia. Some Salmonella serotypes, such as Salmonella Pullorum in poultry, Salmonella Choleraesuis in pigs and Salmonella Dublin in cattle, are relatively host-specific. In contrast, Salmonella Typhimurium has a comparatively wide host range. The Salmonella serotypes of importance in domestic animals and the consequences of infection are indicated in Table 23.3. Salmonella Dublin causes a variety of clinical effects in cattle of different ages including acute or chronic enteric disease, septicaemia and abortion. In calves, joint ill, osteomyelitis and terminal dry gangrene may follow septicaemia or enteric disease. The virulence of salmonellae relates to their ability to invade host cells, replicate in them and resist both digestion by phagocytes and destruction by the complement components of plasma. Many of the virulence factors are encoded by clusters of virulence genes termed Salmonella Pathogenicity Islands (SPI). Although there are many of these islands, SPI-1 and SPI-2 are well characterized as SPI-1 is responsible for local invasion of intestinal cells and SPI-2 is responsible for systemic invasion. Salmonellae often localize in the mucosae of the ileum, caecum and colon and in the mesenteric lymph nodes of infected animals. Latent infections, in which the organisms colonize organs such as the mesenteric lymph nodes but are not excreted, also occur. Clinical disease may develop from subclinical and latent infections if affected animals are stressed by overcrowding, transportation or adverse environmental conditions.

Table 23.3 Selected Salmonella serotypes of clinical importance and the consequences of infection.

Salmonella serotype	Hosts	Consequences of infection
Salmonella Typhimurium	Many animal species Humans	Enterocolitis and septicaemia Food poisoning
Salmonella Dublin	Cattle Sheep, horses, dogs	Many disease conditions Enterocolitis and septicaemia
Salmonella Choleraesuis	Pigs	Enterocolitis and septicaemia
Salmonella Pullorum	Chicks	Pullorum disease (bacillary white diarrhoea)
Salmonella Gallinarum	Adult birds	Fowl typhoid
Salmonella Arizonae	Turkeys	Arizona or paracolon infection
Salmonella Enteritidis	Poultry Many other species Humans	Often subclinical in poultry Clinical disease in mammals Food poisoning
Salmonella Brandenburg	Sheep	Abortion

Enterocolitis caused by salmonella organisms can affect most species of farm animals, irrespective of age. Acute disease is characterized by fever, depression, anorexia and profuse foul-smelling diarrhoea often containing blood, mucus and epithelial casts. Dehydration and weight loss follow and pregnant animals may abort. Severely affected young animals become recumbent and may die within a few days of acquiring infection. Septicaemic salmonellosis can occur in all age groups but is most common in calves, in neonatal foals and in young pigs. Onset of clinical disease is sudden with high fever, depression and recumbency. If treatment is delayed, many young animals with septicaemia die within 48 hours.

Laboratory confirmation is required for salmonellosis. Specimens for submission should include faeces and blood from live animals. Intestinal contents and samples from tissue lesions should be submitted from dead animals and abomasal contents from aborted foetuses. Specimens should be cultured directly onto brilliant green and XLD agars and also added to selenite F or tetrathionate broth for enrichment and subculture. Suspicious colonies can be subjected to further biochemical tests and confirmed as salmonellae using commercially available specific antisera. Many PCR-based procedures are available for rapid detection of the organism in clinical specimens and in food.

Control of salmonellosis is based on reducing the risk of exposure to infection by implementing a closed-herd policy, purchasing animals from reliable sources and preventing contamination of foodstuffs and water. Vaccination procedures for enhancing resistance and reducing the likelihood of clinical disease are used in cattle, sheep, pigs and poultry.

Yersinia species

Although there are 18 Yersinia species currently described, only Y. pestis, Y. enterocolitica and Y. pseudotuberculosis are pathogenic for animals and humans. All three species can survive within macrophages and produce Yersinia outer proteins (Yops) which are important for inhibition of both phagocytosis and proinflammatory cytokine production. Yersinia pestis, which causes bubonic and pneumonic plague (‘black death’), is more invasive than Y. pseudotuberculosis and Y. enterocolitica and possesses additional virulence factors including an antiphagocytic protein capsule and a plasminogen activator which aids systemic spread. In endemic areas, wild rodents are important reservoirs of Y. pestis. Fleas, especially Xenopsylla cheopis, the oriental rat flea, transmit infection to humans and other animals. Feline plague, caused by Y. pestis, usually occurs when cats ingest infected rodents. Three clinical forms of the disease are recognized: bubonic, pneumonic and septicaemic. The most common form of the disease is characterized by enlarged lymph nodes (buboes) associated with lymphatic drainage from the site of infection. Septicaemia may occur without lymphadenopathy and is potentially fatal. In endemic areas, cats and dogs should be routinely treated for fleas and rodent control measures should be implemented. Yersinia pseudotuberculosis causes enteric infections in a wide variety of wild and domestic animals. A septicaemic form of the disease occurs in laboratory rodents and in caged birds.