Chapter 35: Pseudomonas and Other Opportunistic Gram-negative Bacilli

There is a large number of Pseudomonas species, the most important of which is P aeruginosa. The number of human infections produced by the other species together is far lower than that produced by P aeruginosa alone. Pseudomonas species are most frequently seen as colonizers and contaminants, but are able to cause opportunistic infections. The assignment of species names has little clinical importance beyond differentiation from P aeruginosa. Reports vary regarding the frequency of their isolation from cases of bacteremia, arthritis, abscesses, wounds, conjunctivitis, and urinary tract infections. In general, unless isolated in pure culture from a high-quality (direct) specimen, it is difficult to attach pathogenic significance to any of the miscellaneous Pseudomonas species.

BACTERIOLOGY

Pseudomonas aeruginosa is an aerobic, motile, gram-negative rod that is slimmer and more pale-staining than members of the Enterobacteriaceae. Its most striking bacteriologic feature is the production of colorful water-soluble pigments. P aeruginosa also demonstrates the most consistent resistance to antimicrobial agents of all the medically important bacteria.

P aeruginosa is sufficiently versatile in its growth and energy requirements to use simple molecules such as ammonia and carbon dioxide as sole nitrogen and carbon sources. Thus, it does not require enriched media for growth and can survive and multiply over a wide temperature range (20-42°C) in almost any environment, including one with a high salt content. The organism uses oxidative energy-producing mechanisms and has high levels of cytochrome oxidase (“oxidase-positive”). Although an aerobic atmosphere is necessary for optimal growth and metabolism, most strains multiply slowly in an anaerobic environment if nitrate is present as an electron acceptor.

Growth on all common isolation media is luxurious, and colonies have a delicate, fringed edge. Confluent growth often has a characteristic metallic sheen and emits an intense fruity odor. Hemolysis is usually produced on blood agar. The positive oxidase reaction of P aeruginosa differentiates it from the Enterobacteriaceae, and its production of blue, yellow, or rust-colored pigments differentiates it from most other gram-negative bacteria. The blue pigment, pyocyanin, is produced only by P aeruginosa. Fluorescein, a yellow pigment that fluoresces under ultraviolet light is produced by P aeruginosa and other free-living, less pathogenic Pseudomonas species. Pyocyanin and fluorescein combined produce a bright green color that diffuses throughout the medium.

Lipopolysaccharide (LPS) is present in the outer membrane, as are porin proteins, which differ from those of the Enterobacteriaceae family in offering much less permeability to a wide range of molecules, including antibiotics. Pili composed of repeating monomers of the pilin structural subunit extend from the cell surface. A single polar flagellum rapidly propels the organism and assists in binding to host tissues.

A mucoid exopolysaccharide slime layer is present outside the cell wall in some strains. This layer is created by secretion of alginate, a copolymer of mannuronic and glucuronic acids. It is created by the action of several enzymes that effectively channel carbohydrate intermediates into the alginate polymer. All P aeruginosa produce moderate amounts of alginate, but those with mutations in regulatory genes overproduce the polymer. Such mutants appear as striking mucoid colonies in cultures from the respiratory tract of patients with cystic fibrosis (CF).

Most strains of P aeruginosa produce multiple extracellular products, including exotoxin A (ExoA) and other enzymes with phospholipase, collagenase, adenylate cyclase, or elastase activity. ExoA is a secreted protein that inactivates eukaryotic elongation factor 2 (EF-2) by ADP-ribosylation. This arrests translation leading to shutdown of protein synthesis and cell death. Although this action is the same as diphtheria toxin, the two toxins are otherwise unrelated. The elastase acts on a variety of biologically important substrates, including elastin, human IgA and IgG, complement components, and some collagens. Exoenzyme S (ExoS) and a number of other proteins (ExoT, ExoY, ExoU) are transported directly into host cells by an injection (type III) secretion system. Inside the cell, ExoS acts on regulatory G proteins affecting the cytoskeleton, signaling pathways, and inducing apoptosis.

P AERUGINOSA DISEASE

EPIDEMIOLOGY

The primary habitat of P aeruginosa is the environment. It is found in water, soil, and various types of vegetation throughout the world. P aeruginosa has been isolated from the throat and stool of 2% to 10% of healthy persons. Colonization rates may be higher in hospitalized patients. Infection with P aeruginosa, rare in previously healthy persons, is one of the most important causes of invasive infection in hospitalized patients with serious underlying disease, such as leukemia, CF, and extensive burns (Figure 35–1).

The ability of P aeruginosa to survive and proliferate in water with minimal nutrients can lead to heavy contamination of any nonsterile fluid, such as that in the humidifiers of respirators. Inhalation of aerosols from such sources can bypass the normal respiratory defense mechanisms and initiate pulmonary infection. Infections have resulted from the growth of Pseudomonas in medications, contact lens solutions, and even some disinfectants. Sinks and faucet aerators may be heavily contaminated and serve as the environmental source for contamination of other items. The presence of P aeruginosa in drinking water or food is not a cause for alarm. The risk lies in the proximity between items susceptible to contamination and persons uniquely predisposed to infection.

P aeruginosa is now the most common bacterial pathogen to complicate the management of patients with CF, an inherited defect in chloride ion transport that leads to a buildup of thick mucus in ducts and the tracheobronchial tree. In a high percentage of patients, the respiratory tract eventually becomes colonized with P aeruginosa, which once established becomes almost impossible to eradicate. This infection is a leading cause of morbidity and eventual death of these patients.

PATHOGENESIS

Although P aeruginosa is an opportunistic pathogen, it is one of particular virulence. The organism usually requires a significant break in first-line defenses (such as a wound) or a route past them (such as a contaminated solution or endotracheal tube) to initiate infection. Attachment to epithelial cells is the first step in infection and is likely mediated by pili, flagella, and the extracellular polysaccharide slime. The receptors include sialic acid and N-acetylglucosamine borne by cell surface glycolipids. Attachment is favored by loss of surface fibronectin, which explains in part the propensity for debilitated persons.

Given the proper susceptible host, the virulence of P aeruginosa is not unexpected, given its myriad enzymes and other factors (Figure 35–2). The importance of ExoA is supported by studies in humans and animals, which correlate its presence with a fatal outcome and an antibody against it with survival. The effect of ExoA is not immediate, since it is one of a number of virulence factors activated through a gene-regulating system called quorum sensing. Under these conditions, lactones and/or quinolones secreted by P aeruginosa signal their presence to the other bacterial cells. The system is quantitative so when the Pseudomonas cell population reaches a certain threshold, the signals direct the cytotoxin gene to be transcribed, and the toxin is then produced by the entire population at once. No diphtheria-like systemic effect of ExoA has been demonstrated, but its action correlates with the primarily invasive and locally destructive lesions seen in P aeruginosa infections.

Elastase and phospholipase degrade proteins and lipids, respectively, allowing the organism to acquire nutrients from the host and disseminate from the local site. The many biologically important substrates of elastase argue for its importance, particularly its namesake, elastin. Elastin is found at some sites that P aeruginosa preferentially attacks, such as the lung and blood vessels. Hemorrhagic destruction, including the walls of blood vessels (Figure 35–3), is the histologic hallmark of Pseudomonas infection. The intracellular dysfunction caused by ExoS and other factors injected by the secretion system begin immediately upon contact with the host cell. ExoS is associated with dissemination from burn wounds and with actions destructive to cells, including its action on the cytoskeleton. The blue pigment pyocyanin has been detected in human lesions and shown to have a toxic effect on respiratory ciliary function.

Pseudomonas aeruginosa and Cystic Fibrosis

P aeruginosa is the most persistent of the infectious agents that complicate the course of CF. Initial colonization may be aided by the fact that cells from CF patients are less highly sialylated than normal epithelial cells, providing improved access to receptors suitable for P aeruginosa attachment; defects in the epithelia of CF patients may also impede bacterial clearance by desquamation. The most striking feature of this host–pathogen relationship is the appearance of strains with multiple mutations in regulatory genes, causing overproduction of the thick alginate polymer. The colonization of the bronchi then becomes a biofilm with microcolonies of bacteria and debris imbedded in the alginate (Figure 35–4). The high osmolarity of characteristically thick CF secretions facilitates expression of these alginate hyperproducing mutants. For P aeruginosa, biofilm confers highly advantageous protection from the immune system (complement, antibody, phagocytes) and antimicrobial agents. The global regulatory networks responsible for quorum sensing, and their effects on alginate production and other virulence-related behaviors, remain a central focus in the search for novel therapeutics.

IMMUNITY

Human immunity to Pseudomonas infection is not well understood. Inferences from animal studies and clinical observations suggest that both humoral and cell-mediated immunity are important. The strong propensity of P aeruginosa to infect those with defective cell-mediated immunity indicates that these responses are particularly important.

P AERUGINOSA DISEASE: CLINICAL ASPECTS

MANIFESTATIONS

P aeruginosa can produce any of the opportunistic extraintestinal infections caused by members of the Enterobacteriaceae. Burn, wound, urinary tract, skin, eye, ear, and respiratory infections all occur and may give rise to bacteremia. P aeruginosa is also one of the most common causes of infection in environmentally contaminated wounds (eg, osteomyelitis after compound fractures or nail puncture wounds of the foot).

Particularly in patients with neutropenia, P aeruginosa pneumonia is a rapid, destructive infection associated with alveolar necrosis, vascular invasion, infarcts, and bacteremia. Pulmonary infection in CF patients is different; it is a chronic infection that alternates between a state of colonization and more overt bronchitis or pneumonia (Figure 35–5). Although the more aggressive features of Pseudomonas infection in the immunocompromised are not common in CF, the infection is still serious enough to be a leading cause of death in CF patients.

P aeruginosa is also a common cause of otitis externa, including “swimmer’s ear” and a rare but life-threatening malignant otitis externa seen in patients with diabetes. Folliculitis of the skin may follow soaking in hot tubs that have become heavily contaminated with the organism. P aeruginosa can cause conjunctivitis, keratitis, or endophthalmitis when introduced into the eye by trauma or contaminated medication or contact lens solution. Keratitis can progress rapidly and destroy the cornea within 24 to 48 hours. In some cases of P aeruginosa bacteremia, cutaneous papules develop which progress to black, necrotic ulcers—a condition called ecthyma gangrenosum. The lesions are the result of direct invasion and destruction of blood vessel walls by the organism.

DIAGNOSIS

P aeruginosa is readily grown in culture. The combination of characteristic oxidase-positive colonies, pyocyanin production (Figure 35–6), and the ability to grow at 42°C is sufficient to distinguish P aeruginosa from other Pseudomonas species. Although biochemical test can identify other species, such tests are usually not routinely done unless the clinical evidence for infection is very strong.

TREATMENT

Of the pathogenic bacteria, P aeruginosa is the organism most consistently resistant to many antimicrobials. Inherent resistance is due to the porins that restrict entry of antibiotic compounds to the periplasmic space. P aeruginosa strains are uniformly resistant to penicillin, ampicillin, cephalothin, tetracycline, chloramphenicol, sulfonamides, and the earlier aminoglycosides (streptomycin, kanamycin). Much effort has been directed toward the development of antimicrobials with anti-Pseudomonas activity. All treatment must be guided by antimicrobial susceptibility testing as resistance patterns are highly variable. The aminoglycosides in current use—gentamicin, tobramycin, and amikacin—all are still active against most strains. Of the β-lactams, piperacillin/tazobactam, cefepime, ceftazidime, imipenem/cilastatin, meropenem, and doripenem have the best prospects for success; in contrast, aztreonam has poor rates of effectiveness. In general, urinary infections may be treated with a single drug, but more serious systemic P aeruginosa infections are initially treated with a combination of an anti-pseudomonal β-lactam and an aminoglycoside, particularly in neutropenic patients. Fluoroquinolones may also be used if susceptible.

The treatment for P aeruginosa infection in CF presents special problems because most of the effective antimicrobials are only given intravenously. To avoid hospitalization, oral agents are often used to manage mild exacerbations. Patients that have persistent or progressive symptoms will then be admitted for “cleanout” with multiple intravenous antibiotics. The chronic nature of P aeruginosa colonization leads to progressive development of resistance over the course of patients’ disease. Aerosolized tobramycin has been used in many CF patients, with evidence of clinical effectiveness in improving pulmonary function and decreasing risk of hospitalization.

PREVENTION

Vaccines incorporating somatic antigens from multiple P aeruginosa serotypes have been developed and proved immunogenic in humans. The primary candidates for such preparations are patients with burn injuries, CF, or immunosuppression. Although some protection has been demonstrated, these preparations have generally proven disappointing.

Burkholderia pseudomallei is a saprophyte in soil, ponds, rice paddies, and vegetables located in Southeast Asia, the Philippines, Indonesia, and other tropical areas. Infection is acquired by direct inoculation or by inhalation of aerosols or dust containing the bacteria. The resultant disease, melioidosis, is usually an acute pneumonia; however, it is sufficiently variable that subacute, chronic, and even relapsing infections may follow systemic spread. Some American soldiers relapsed years after their return from Vietnam. The clinical and radiologic features may resemble tuberculosis. In fulminant cases of melioidosis, rapid respiratory failure may ensue and metastatic abscesses develop in the skin or other sites. Though intrinsically resistant to a wide range of antibacterials, B pseudomallei may be susceptible to tetracycline, sulfonamides, and trimethoprim-sulfamethoxazole (as well as chloramphenicol, though its toxicity precludes its use in many developed countries). Burkholderia cepacia complex is a group of opportunistic species that has been found to contaminate reagents, disinfectants, and medical devices in much the same manner as P aeruginosa. They have also complicated the course of CF but do not produce the mucoid polymer seen with P aeruginosa.

The genus Acinetobacter comprises gram-negative coccobacilli that occasionally appear sufficiently round on Gram smears to be confused with Neisseria. On primary isolation, they closely resemble Enterobacteriaceae in growth pattern and colonial morphology, but are distinguished by their failure to ferment carbohydrates or reduce nitrates. As with most of the organisms discussed in this chapter, the isolation of Acinetobacter from specimens other than normally sterile sites (blood, bronchoalveolar lavage fluid) does not define infection because these bacteria appear frequently as skin and respiratory colonizers. They are most frequently found as contaminants of almost anything wet, including soaps and some disinfectant solutions. Pneumonia is the most common infection, followed by urinary tract and soft tissue infections. Nosocomial respiratory infections have been traced to contaminated inhalation therapy equipment, and bacteremia to infected intravenous catheters. While treatment is frequently complicated by resistance to penicillins, cephalosporins, and occasionally aminoglycosides, virtually pan-resistant Acinetobacter isolates have produced outbreaks in intensive care units and military hospitals abroad.

Moraxella is another genus of coccobacillary, gram-negative rods that are usually paired end-to-end. Some species require enriched media, such as blood or chocolate agar. Their morphology, fastidious growth, and positive oxidase reaction can result in confusion with Neisseria in the laboratory. Moraxella catarrhalis is found in the normal oropharyngeal flora, and it is an occasional cause of lower respiratory tract infection and otitis media. In otitis media cases, M catarrhalis has been detected in mixed culture with pathogens like Haemophilus influenzae and Streptococcus pneumoniae; because M catarrhalis frequently produces β-lactamase, it has been blamed for “protecting” the other pathogens when β-lactam treatment fails.

The genera Aeromonas and Plesiomonas have bacteriologic features similar to those of the Enterobacteriaceae, Vibrio, and Pseudomonas. They are aerobic and facultatively anaerobic, attack carbohydrates fermentatively, and demonstrate various other biochemical reactions. Aeromonas colonies are typically β-hemolytic. The resemblance of Aeromonas and Plesiomonas to Pseudomonas arises from their shared oxidase positivity and polar flagella. Their habitat is basically environmental (water and soil), but they can occasionally be found in the human intestinal tract.

Aeromonas is an uncommon but highly virulent cause of wound infections acquired in fresh or salt water. The onset can be as rapid as 8 hours after the injury, and the cellulitis can progress rapidly to fasciitis, myonecrosis, and bacteremia in less than a day. Aeromonas is also the leading cause of infections associated with the medical use of leeches, owing to its regular presence in the leech foregut. In addition to opportunistic infection, some evidence suggests an occasional role for Aeromonas in gastroenteritis through production of toxins with enterotoxic and cytotoxic properties. Plesiomonas is also associated with an enterotoxic diarrhea. These associations are not yet strong enough to warrant efforts to routinely isolate Aeromonas and Plesiomonas from diarrheal stools. Resistance to penicillins and cephalosporins is common. Most strains show susceptibility to tetracycline, with variable susceptibility to aminoglycosides, including gentamicin.

“HACEK” GROUP

The HACEK acronym denotes species from the genera Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella that have been implicated in up to 3% of all cases of infective endocarditis. These pathogens typically produce infection in individuals with prosthetic heart valves or other underlying heart disease. These species are part of the microbiota of the oral cavity and upper respiratory tract in humans, and tend to produce syndromes that are insidious in onset and challenging to diagnose. Treatment with third-generation cephalosporins produces a favorable outcome in 80% to 90% of cases.

OTHER GRAM-NEGATIVE RODS

There are many other gram-negative rods that rarely cause disease in humans. Some are members of the resident flora, and others come from the environment. Because many of these do not ferment carbohydrates or react in many of the tests routinely used to characterize bacteria, their identification is frequently delayed while additional tests are tried or the organism is sent to a reference laboratory. The clinical significance of all these organisms is essentially the same. The clinician usually receives report of a “nonfermenter” or another descriptive term and a susceptibility test result, and the significance of the isolate must then be determined on clinical grounds. The major characteristics of some of these organisms are shown in Table 35–1. The types of infection listed represent the most common among scattered case reports and should not be interpreted as typical for each organism.

Some gram-negative bacilli fail to conform to any of the species currently recognized. If clinically important, such strains are sent to reference centers, such as the Centers for Disease Control and Prevention (CDC) in Atlanta, Georgia. Eventually, some are given designations such as “CDC group IIF,” which may appear in clinical reports. Much later, a new genus and/or species name may be issued if agreement among taxonomists is sufficient.