Classification

E. coli, Proteus spp., and Klebsiella spp. are members of the family Enterobacteriaceae. They are non–spore-forming, non–acid-fast, facultative, anaerobic gram-negative rods that are natural inhabitants of the intestinal tracts of mammals. The Enterobacteriaceae family has 28 genera, with more than 100 well-defined species. Clinically important species include E. coli, Proteus mirabilis, Proteus vulgaris, and Klebsiella pneumoniae.

Pseudomonas spp. are ubiquitous, non-Enterobacteriaceae, gram-negative bacteria that are found in soil, water, decaying vegetation, and animals. The most important and ubiquitous pathogenic pseudomonad is Pseudomonas aeruginosa. In Asia, Eastern Europe, and Africa, Pseudomonas mallei is the cause of equine glanders; dogs and cats can become infected from the horse reservoir (see Chapter 44). Burkholderia (formerly Pseudomonas) pseudomallei is a soil organism found in Asia, central Africa, and Australia. In dogs and cats, it can cause syndromes ranging from a benign pulmonary infection to a lethal systemic disease (see Chapter 44).

Antigenic Structure

An internationally recognized serologic classification scheme has been developed for the Enterobacteriaceae based on antigenic differences in highly variable bacterial surface molecules. The serogroup is determined by the O antigens—sugars on the most external part of LPS (Fig. 35-2). The serotype is determined by flagellar H antigens. Additionally, K antigens can be identified in strains with a capsule. The antigenic formula of the strain indicates the presence of specific antigens (e.g., O55:K5:H21). However, for most strains, only O and H antigens are determined. At least 170 different serogroups have been identified for E. coli, and some correlation has been found between serogroup and virulence (e.g., O157:H7 and hemolytic uremic syndrome (HUS) in humans).

Virulence Factors

Some strains of E. coli are true pathogens; however, most E. coli, Klebsiella, Proteus, and Pseudomonas organisms are opportunistic pathogens. The pathogenicity of gram-negative bacteria depends on the presence of various virulence factors (e.g., adhesins, toxins, iron-acquisition systems, capsules) expressed by the organism.^25a It is the set of virulence genes carried and expressed by the organism that confers pathogenicity rather than the genus or species designation. Examples of virulence factors follow.

The establishment of infection first depends on bacteria adhering to an epithelium. Adhesins are located on bacterial pili (or fimbriae), the slender external structures that facilitate attachment to host cells. Colonization factor antigens I and II are specific pilus adhesins-hemagglutins that promote colonization by some enteropathogens. Another fimbrial antigen is the virantigen, which is associated with sepsis in some species. Bacterial capsules are polysaccharides, which protect the outer membrane from the membrane attack complex of complement and also inhibit the attachment of phagocytic cells. Colonization with Pseudomonas spp. occurs when the fibronectin coat covering host cells is disrupted by infection or mechanical trauma. The pili and exozyme S of Pseudomonas spp. promote attachment to epithelial cells, and the extracellular slime is antiphagocytic. Siderophores, such as aerobactin, aid the bacterium in competing with the host for iron in low-iron environments.

Production of various toxins is important in the pathogenesis of gram-negative infections. Secreted α-hemolysin damages host cell membranes. Endotoxin (LPS) stimulates various cytokine cascades, as well as the complement cascade. Excessive activation of these systems leads to sepsis and septic shock (see Chapter 36). Pseudomonas spp. variably produce collagenase, lecithinase, lipase, proteases, hemolysins, fibrinolysin, leukocidin (also called cytotoxin), and enterotoxin. Many strains also secrete exotoxin A, a cytotoxin that causes tissue necrosis, allowing the organism to colonize deep tissues in burn and trauma cases. As with the Enterobacteriaceae, Pseudomonas LPS can act as an endotoxin.

Virulence Factors Associated with Escherichia coli Intestinal Infections

The documentation of pathogenic E. coli infections is confusing because many of these organisms exist as normal enteric constituents and can be found in the microflora of clinically healthy animals. Analysis for specific toxin genes or molecular typing can be done to differentiate among the different isolates. Toxin analysis is important in addition to genetic studies because many of the genes identified may not be expressed by the organisms isolated. Pathogenic E. coli have been classified on the basis of their virulence properties into pathovars. Diarrheagenic pathovars include enteropathogenic E. coli (EPEC), enterohemorrhagic E. coli (EHEC), enterotoxigenic E. coli (ETEC), enteroinvasive E. coli (EIEC; including Shigella), enteroaggregative E. coli (EAEC) and diffusely adherent E. coli (DAEC), necrotoxigenic E. coli (NTEC), adherent invasive E. coli (AIEC), and cell-detaching E. coli (CDEC). Extraintestinal pathogenic E. coli (ExPEC) pathovars include uropathogenic E. coli (UPEC) and neonatal meningitis E. coli (NMEC) (Table 35-1, Web Table 35-2). Not all pathovars have been documented in dogs and cats (Fig. 35-3). Pathovars are not clear-cut distinctions; some enteropathogens isolated from dogs have predominantly “uropathogenic” traits, but they cause diarrhea. The pathovars are discussed in the following sections.

Uropathogenic Escherichia coli

E. coli is the most common isolate from dogs with first-time and recurrent UTIs and the second most common isolate from dogs with uroliths.83,102 Some uropathogenic strains of E. coli, designated UPEC, express virulence factors that facilitate the colonization of the urinary system. The major determinant of their pathogenicity is the ability to adhere to the uroepithelium via adhesins such as type 1 pili (mediated by fimbrial adhesion H at its tip) or P fimbriae, the gene of which (papG) has been identified in canine isolates.^69,71 After attachment, pili and endotoxin incite neutrophil migration and induce the release of cytokines, leading to an inflammatory response and characteristic signs of urgency, discomfort, and hematuria (see Chapter 90).

Exotoxins, especially hemolysins, may also be important in the development of clinical signs of lower UTIs. Iron acquisition is also an important virulence factor, as are bacterial capsules and antiphagocytic systems. Numerous virulence factors have been identified in canine and feline UPEC strains, including CNF1 and genes encoding adhesins, hemolysin, and aerobactin.37,163 The ability of some UPEC to actually invade uroepithelial cells has been reported in strains isolated from people. UPEC can replicate and form protective biofilm-like complexes within the cells. It is possible that this characteristic internalization of the organism may explain some relapsing UTIs in dogs and cats. UPEC are carried in the colons of many dogs; comparing phylogenetic background, O antigens, and extended virulence genotype, 54% of urinary isolates were also found in the gut. This is consistent with the idea that UPEC have a special type of pathogenicity, and UTIs are not the result of exposure to high numbers of “normal” E. coli.⁶⁸ Hemolytic E. coli have also been found in UTIs in cats.¹⁴³ Untreated UPEC cystitis can progress to pyelonephritis if the organism is motile and motility and the degree of type 1 pili expression have been shown to be inversely related. Within the kidney, pyelonephritis isolates often express P fimbriae and fewer motility organelles.^78,79,81,129

Studies based on the identification of genes encoding adhesins, hemolysins, CNF1, and aerobactin suggest that canine uropathic strains may be similar if not identical to some human uropathic strains.⁷¹ Similarly, P. mirabilis strains isolated from the urine of dogs have been characterized and found to be similar to human uropathogenic P. mirabilis strains. The significance of these findings for people and dogs is not known.⁴⁴ ExPEC can also infect almost any anatomic site. Pneumonia, sepsis, and meningitis are some of the infections that have been observed. Multidrug-resistant strains have been isolated from animals with recurrent infections that have been treated with several types of drugs.¹⁴²

Klebsiella Infections

Klebsiella organisms, which are found in the nasopharyngeal and intestinal flora, can be associated with clinically significant infections of the GI and genitourinary tracts and systemic bacteremia. Some strains of Klebsiella produce an enterotoxin similar to the ST of enterotoxigenic E. coli. Klebsiella pneumoniae–induced enteritis, pneumonia, and sepsis were reported in a Bordeaux mastiff breeding kennel. Seven of 15 adult dogs were affected and 4 died.¹¹⁸ Klebsiella pneumonia and septicemia with subsequent multiple organ dysfunction syndrome has also been reported in a dog.²⁰ Klebsiella organisms have been implicated in nosocomial infections, including intravenous catheter infections, in veterinary hospitals (see Chapter 93).⁴⁹

Pseudomonas Infections

P. aeruginosa is an environmentally stable organism that causes infection in animals whose immune systems or host defenses are impaired. Although it can be isolated from the upper respiratory, genital, and GI tracts and from intertriginous areas of the skin, numbers of organisms are usually controlled by competition from normal bacterial flora. Inflammation or injury to these mucosal areas or chronic antibacterial therapy can lead to a proliferation of P. aeruginosa. P. aeruginosa preferentially binds and enters the injured cells via the basolateral surface. The bacterium has been shown to facilitate its entry into cells by constitutively turning apical membranes into basolateral membranes by recruiting phosphatidylinositol-3-kinase and actin to the basolateral surface.⁷⁵ In addition, open wounds or surgical incisions can become contaminated and subsequently infected with antimicrobial-resistant strains from the hospital environment. Defective flexible endoscopes that became contaminated were incriminated as a source of an outbreak in people.¹³⁴ Pseudomonas bacteria also have the ability to invade a healthy cornea in its warm, moist state. Through small scratches, the organism can enter the cell cytoplasm, where it rapidly replicates. However, some strains produce an exoenzyme toxin that kills epithelial cells and macrophages. In dogs with otitis externa, P. aeruginosa and Proteus organisms could be cultured from the ears but were not found in the ears of clinically healthy dogs.¹⁶³ P. aeruginosa has been implicated as a cause of deep pyoderma in dogs, sometimes manifesting as acute onset dorsal truncal pain.⁶² Osteomyelitis has also been reported.^107a Pseudomonas bacteria are also isolated from mixed infections in dogs.¹³⁶ P. aeruginosa, which has few nutritional requirements, thrives in moist environments outside the host and creates microcolonies that allow it to grow on moist surfaces such as those in bath tubs and plumbing and drainage systems. Animals that are immunosuppressed or have implanted catheters or open wounds are particularly susceptible to contamination from these moist environments or surfaces. In hospital environments, many of the P. aeruginosa strains have acquired multidrug antimicrobial resistance. Exposure to quinolones in vitro or in vivo causes P. aeruginosa from dogs to develop resistance more rapidly than other bacteria.^19,141

Pseudomonas fluorescens is able to grow in packed erythrocyte blood units.^73a The growth occurs slowly at 4° C and more rapidly at 20° C. To prevent its occurrence, stored blood units must be monitored for discoloration and detection by microscopy and PCR confirmation. For further information on blood product infections, see Chapter 93.