INTRODUCTION

Infections due to gram-negative organisms are a significant cause of morbidity and mortality in critically ill patients.¹ Important aerobic or facultatively anaerobic gram-negative infections are often due to an opportunistic invasion by commensal intestinal flora, including Escherichia coli, Proteus spp, Pseudomonas aeruginosa, Serratia spp, and Klebsiella spp.^1,2 Less commonly, opportunistic infections are caused by environmental saprophytes that enter the body through wounds or the respiratory tract.³

GRAM-NEGATIVE CELL STRUCTURE AND PATHOGENICITY

In addition to having a cytoplasmic membrane and peptidoglycan layer similar to that found in gram-positive organisms, gram-negative bacteria possess unique factors that contribute to their ability to cause disease.^2-5 Among the bacterial products commonly implicated in the pathogenesis of gram-negative organisms is endotoxin, a unique lipopolysaccharide (LPS) that accounts for 75% of the outer surface of the gram-negative cell membrane.⁴ The role of LPS in triggering the cellular and physiologic host responses is well established.^2,3,5,6

Structurally, endotoxin consists of an outer polysaccharide chain that is bound to lipid A. Although it is buried deep in the bacterial cell wall, lipid A is known to be the toxic moiety of endotoxin.^3,4 Lipid A induces a wide range of proinflammatory responses (i.e., release of cytokines and activation of the compliment cascade) and endothelial dysfunction.^3,4,6 During minor or local infections with small numbers of bacteria, small amounts of LPS are released, leading to controlled cytokine production. The cytokines released promote body defenses by stimulating inflammation, fever, and appropriate protective immunologic responses.³ However, during severe systemic infections with large numbers of bacteria, increased amounts of LPS are released, resulting in excessive, and sometimes maladaptive, cytokine production by monocytes and macrophages.^2-5 Harmful effects of endotoxin include vasodilation, enhanced vascular permeability, tissue destruction, and activation of coagulation pathways.^2,3,6

Failure to contain or eradicate the microbe often results in further damage due to the inexorable progression of inflammation and infection.^2,4,5 Thus, of the many therapeutic interventions, early initiation of appropriate antibiotic therapy is of utmost importance to ensure a favorable outcome.^7,8

Gram-negative organisms also possess cellular structures that are often recognized as virulence factors.^2,3,5,9 Flagella are protein filaments that extend from the cell membrane. Flagella allow for locomotion, undulating in a coordinated manner to move the bacteria toward or away from a chemical gradient, a process called chemotaxis. Pili (also called fimbriae) are straight filaments arising from the bacterial cell wall. Pili most often serve as adherence factors, in which case they are referred to adhesins.^2,5 For many bacteria, adhesins are vital to their ability to cause disease. Capsules are protective walls, generally composed of simple sugar residues that surround the cell membranes.^2,3 Encapsulation enhances virulence by preventing bacterial phagocytosis by host neutrophils and macrophages.⁵

IDENTIFICATION OF GRAM-NEGATIVE BACTERIA OF MEDICAL IMPORTANCE

The classification of gram-negative bacteria is based on several criteria, including their appearance on selective media, utilization of carbohydrates (e.g., lactose), production of certain end products (e.g., acids and alcohols), and the presence or absence of specialized enzymes (e.g., oxidase).^3,5 Although the clinical relevance of these categories is a point for contention among clinicians, taxonomic schemes permit the microbiology laboratory to distinguish rapidly among commonly encountered bacteria.^3,5,10 For example, facultatively anaerobic oxidase-negative, gram-negative rods that grow on MacConkey agar are presumed to be members of the Enterobacteriaceae.^3,5,9 As more information accumulates, reclassification of bacteria among genera and species and the creation of new designations must be accepted as part of scientific progress.²

ENTEROBACTERIACEAE

Members of the family Enterobacteriaceae are the most frequently encountered gram-negative isolates recovered from clinical specimens.^1,3,7 These commensal organisms are found in soil and water, on plants and, as the family name implies, within the intestinal tract of animals and humans.^2,3

Before the advent of antibiotics, chemotherapy, and immunosuppressive measures, the infectious diseases caused by the Enterobacteriaceae were relatively well defined and typically characterized by diarrhea and other gastrointestinal syndromes.^2,5 However, members of the Enterobacteriaceae are now incriminated in virtually any type of infectious disease and may be recovered from any tissue or fluid specimen submitted to the laboratory.^3,5,9,11 By definition, commensal organisms colonize an individual without causing disease.¹² However, in a vulnerable host these “pathogenic commensals” have the capacity to produce disease.^1,7 Generally, enhanced bacterial virulence factors or damage to the mucosal barrier or immune system of the host is required for infection to occur.^2,5,9 Critically ill and immunocompromised patients are susceptible to hospital-acquired infections, following colonization with environmental strains or invasive procedures such as catheterization, endoscopy, and surgery.^2,5,7,12

Escherichia coli is the most commonly encountered bacteria ium clinical microbiology laboratories and is thought to be the most important of the facultative aerobic gram-negative species that comprise the normal flora of the alimentary tract in most dogs and cats.^5,9,13 Most strains of E. coli are of low virulence, but they may cause opportunistic infections in extraintestinal sites.^5,7,11,13

E. coli organisms were previously susceptible to select drugs. However, multidrug-resistant E. coli have emerged as a cause of opportunistic infections in companion animals.^14,15 Presently, the proportion of E. coli resistant to aminopenicillins, fluoroquinolones, and cephalosporins is increasing and causing great concern in both human and veterinary medicine.¹⁶ Indiscriminate use of antibiotics, inadequate hygiene, and extended hospital stays are among the proposed reasons for resistance to these commonly used agents (see Chapter 194, Antimicrobial Use in the Critical Care Patient).^7,12,14-18 Empirically, amoxicillin-clavulanic acid, ampicillin-sulbactam, or fluoroquinolones may be prescribed appropriately for first-time infections, pending susceptibility data. In critically ill hospitalized patients with a history of antibiotic therapy, the presence of multidrug-resistant (MDR) organisms should be presumed.^7,11 In such circumstances, the prescribing of a third-generation cephalosporin or aminoglycoside is considered appropriate while culture and susceptibility information is pending.

Infections with serovars of Salmonella enterica are uncommon in dogs and cats. Serovars of S. enterica can survive for relatively long periods in the environment, and transmission through food, water, or fomites contaminated by fecal material likely plays a role in disease pathogenesis. Importantly, the prevalence of Salmonella in canine fecal samples varies widely and does not correlate with clinical disease. Young dogs are more susceptible to infection and clinical signs.

Factors that increase susceptibility to salmonellosis include poor nutrition, anesthesia, overcrowding, concurrent disease, and prior or contemporaneous drug therapy. The severity of signs varies from none to subacute diarrhea and septic shock. Fever, lethargy, and anorexia may be followed by abdominal pain, vomiting, hemorrhagic diarrhea, and dehydration. Central nervous system (CNS) signs, polyarthritis, and pneumonia may be seen. Aggressive supportive care is the cornerstone of therapy, and appropriate antibiotic therapy might include chloramphenicol, amoxicillin, or the potentiated sulfonamides.

Among the 16 species included in the genus Enterobacter, E. aerogenes and E. cloacae are the species most commonly encountered in clinical infections. MDR E. cloacae has been recovered from the urinary tract, respiratory tract, and surgical wounds of veterinary patients.^12,18 E. cloacae strains are inherently resistant to amoxicillin, amoxicillin-clavulanate, narrow-spectrum cephalosporins, and cefoxitin. Additionally, E. cloacae may acquire resistance to broad-spectrum β-lactams, especially when they are subjected to antibiotic pressure.¹⁹ Pending final susceptibility data, the antimicrobial agents most indicated in the treatment of serious Enterobacter infections are carbapenems and fourth-generation cephalosporins. Aminoglycosides, fluoroquinolones, and the potentiated sulfonamides are frequently, although less predictably, effective. Third-generation cephalosporins frequently show good in vitro activity against these organisms but, as explained earlier, a significant risk of developing resistance during therapy exists.

Klebsiella spp are ubiquitous in nature and may be regarded as normal flora in the alimentary canal, biliary tract, and pharynx in dogs and cats.² K. pneumoniae is a primary pathogen; this property is thought to be related to its large antiphagocytic capsule. Patients with K. pneumoniae infection frequently have predisposing conditions, including immunosuppression, indwelling devices, chronic respiratory disease, or extended hospital stays.⁷ Although K. pneumoniae can cause severe pneumonia, it is more commonly the cause of hospital-acquired wound or urinary tract infections.^2,12 Extensive use of broad-spectrum antibiotics in hospitalized patients has led to both increased carriage of Klebsiella and, subsequently, the development of MDR strains that produce extended-spectrum β-lactamases (ESBLs).^7,20 The bowel is the most common site of colonization, with secondary infection of the urinary tract, respiratory tract, peritoneal cavity, biliary tract, wounds, and bloodstream.¹²

Proteus includes five species. The most common clinical isolates are P. vulgaris and P. mirabilis. Both are recovered from infected sites in immunocompromised hosts, particularly those receiving prolonged regimens of antibiotics. The recovery of an indole-negative Proteus spp can be presumptively identified as P. mirabilis. This is of clinical importance because most strains of P. mirabilis are sensitive to ampicillin and the cephalosporins, whereas P. vulgaris is predictably resistant to these drugs.

Serratia marcescens is recognized as an important opportunistic pathogen with invasive properties and a tendency to be resistant to many commonly used antibiotics. Serratia spp have been linked to nosocomial infections in both dogs and cats.²