Chapter 16: Pseudomonads and Acinetobacter

The pseudomonads and Acinetobacter species are widely distributed in soil and water. Pseudomonas aeruginosa sometimes colonizes humans and is the major human pathogen of the pseudomonads. P aeruginosa is invasive and toxigenic, produces infections in patients with abnormal host defenses, and is an important nosocomial pathogen. Of the Acinetobacter species, Acinetobacter baumannii is responsible for most human infections. It is a significant nosocomial pathogen, especially in critical or intensive care units, and is frequently resistant to multiple antibiotics.

The pseudomonads are Gram-negative, motile, aerobic rods, some of which produce water-soluble pigments. The pseudomonads occur widely in soil, water, plants, and animals. P aeruginosa is frequently present in small numbers in the normal intestinal flora and on the skin of humans, and is the major pathogen of the group. Other pseudomonads infrequently cause disease. The classification of pseudomonads is based on rRNA/DNA homology and common culture characteristics.

P aeruginosa is widely distributed in nature and is commonly present in moist environments in hospitals. It can colonize normal humans, in whom it is a saprophyte. It causes disease in humans with abnormal host defenses, especially in individuals with neutropenia.

Morphology and Identification

A. Typical Organisms

P aeruginosa is motile and rod shaped, measuring about 0.6 × 2 μm (Figure 16-1). It is Gram-negative and occurs as single bacteria, in pairs, and occasionally in short chains.

B. Culture

P aeruginosa is an obligate aerobe that grows readily on many types of culture media, sometimes producing a sweet or grape-like or corn taco–like odor. Some strains hemolyze blood. P aeruginosa forms smooth round colonies with a fluorescent greenish color. It often produces the nonfluorescent bluish pigment pyocyanin, which diffuses into the agar. Other Pseudomonas species do not produce pyocyanin. Many strains of P aeruginosa also produce the fluorescent pigment pyoverdin, which gives a greenish color to the agar (Figure 16-2). Some strains produce the dark red pigment pyorubin or the black pigment pyomelanin.

P aeruginosa in a culture can produce multiple colony types (Figure 16-3). P aeruginosa from different colony types may also have different biochemical and enzymatic activities and different antimicrobial susceptibility patterns. Sometimes, it is not clear if the colony types represent different strains of P aeruginosa or are variants of the same strain. Cultures from patients with cystic fibrosis (CF) often yield P aeruginosa organisms that form mucoid colonies as a result of overproduction of alginate, an exopolysaccharide. In CF patients, the exopolysaccharide appears to provide the matrix for the organisms to live in a biofilm (see Chapters 2 and 9).

C. Growth Characteristics

P aeruginosa grows well at 37–42°C; its growth at 42°C helps differentiate it from other Pseudomonas species that produce fluorescent pigments. It is oxidase positive. It does not ferment carbohydrates, but many strains oxidize glucose. Identification is usually based on colonial morphology, oxidase positivity, the presence of characteristic pigments, and growth at 42°C. Differentiation of P aeruginosa from other pseudomonads on the basis of biochemical activity requires testing with a large battery of substrates.

Antigenic Structure and Toxins

Pili (fimbriae) extend from the cell surface and promote attachment to host epithelial cells. An exopolysaccharide, alginate, is responsible for the mucoid colonies seen in cultures from patients with CF. Lipopolysaccharide, which exists in multiple immunotypes, is responsible for many of the endotoxic properties of the organism. P aeruginosa can be typed by lipopolysaccharide immunotype and by pyocin (bacteriocin) susceptibility. Most P aeruginosa isolates from clinical infections produce extracellular enzymes, including elastases, proteases, and two hemolysins (a heat-labile phospholipase C and a heat-stable glycolipid).

Many strains of P aeruginosa produce exotoxin A, which causes tissue necrosis and is lethal for animals when injected in purified form. The toxin blocks protein synthesis by a mechanism of action identical to that of diphtheria toxin, although the structures of the two toxins are not identical. Antitoxins to exotoxin A are found in some human sera, including those of patients who have recovered from serious P aeruginosa infections.

P aeruginosa produces four type III–secreted toxins that cause cell death or interfere with the host immune response to infection. Exoenzyme S and exoenzyme T are bifunctional enzymes with GTPase and ADP-ribosyl transferase activity; exoenzyme U is a phospholipase, and exoenzyme Y is an adenylyl cyclase.

Pathogenesis

P aeruginosa is pathogenic only when introduced into areas devoid of normal defenses, such as when mucous membranes and skin are disrupted by direct tissue damage as in the case of burn wounds; when intravenous or urinary catheters are used; or when neutropenia is present, as in cancer chemotherapy. The bacterium attaches to and colonizes the mucous membranes or skin, invades locally, and produces systemic disease. These processes are promoted by the pili, enzymes, and toxins described earlier. Lipopolysaccharide plays a direct role in causing fever, shock, oliguria, leukocytosis and leukopenia, disseminated intravascular coagulation, and adult respiratory distress syndrome. The propensity to form biofilms by P aeruginosa in the lumen of catheters and in the lungs of CF patients greatly contributes to the virulence of this organism.

P aeruginosa and other pseudomonads are resistant to many antimicrobial agents and therefore become dominant and important when more susceptible bacteria of the normal microbiota are suppressed.

Clinical Findings

P aeruginosa produces infection of wounds and burns, giving rise to blue-green pus; meningitis when introduced by lumbar puncture or during a neurosurgical procedure; and urinary tract infection when introduced by catheters and instruments or in irrigating solutions. Involvement of the respiratory tract, especially from contaminated respirators, results in necrotizing pneumonia. In CF patients, P aeruginosa causes a chronic pneumonia, a significant cause of morbidity and mortality in this population. The bacterium is often found in mild otitis externa in swimmers. It may cause invasive (malignant) otitis externa in patients with diabetes. Infection of the eye, which may lead to rapid destruction of the eye, occurs most commonly after injury or surgical procedures. In infants or debilitated persons, P aeruginosa may invade the bloodstream and result in fatal sepsis; this occurs commonly in patients with leukemia or lymphoma who have received antineoplastic drugs or radiation therapy and in patients with severe burns. In most P aeruginosa infections, the symptoms and signs are nonspecific and are related to the organ involved. Occasionally, verdoglobin (a breakdown product of hemoglobin) or fluorescent pigment can be detected in wounds, burns, or urine by ultraviolet fluorescence. Hemorrhagic necrosis of skin occurs often in sepsis caused by P aeruginosa; the lesions, called ecthyma gangrenosum, are surrounded by erythema and often do not contain pus. P aeruginosa can be seen on Gram-stained specimens from ecthyma lesions, and culture results are positive. Ecthyma gangrenosum is uncommon in bacteremia caused by organisms other than P aeruginosa. A form of folliculitis associated with poorly chlorinated hot tubs and swimming pools can be seen in otherwise healthy persons.

Diagnostic Laboratory Tests

A. Specimens

Specimens from skin lesions, pus, urine, blood, spinal fluid, sputum, and other material should be obtained as indicated by the type of infection.

B. Smears

Gram-negative rods are often seen in smears. No specific morphologic characteristics differentiate pseudomonads in specimens from enteric or other Gram-negative rods.

C. Culture

Specimens are plated on blood agar and the differential media commonly used to grow the enteric Gram-negative rods. Pseudomonads grow readily on most of these media, but they may grow more slowly than the enterics. P aeruginosa does not ferment lactose and is easily differentiated from the lactose-fermenting bacteria. Culture is the specific test for diagnosis of P aeruginosa infection.

Treatment

Traditionally, significant infections with P aeruginosa have not been treated with single-drug therapy because the success rate is low with such therapy and the bacteria can rapidly develop resistance when single drugs are used. An extended-spectrum penicillin such as piperacillin active against P aeruginosa is used in combination with an aminoglycoside, usually tobramycin. Other drugs active against P aeruginosa include aztreonam; carbapenems such as imipenem or meropenem; and the fluoroquinolones, including ciprofloxacin. Of the cephalosporins, ceftazidime, cefoperazone, and cefepime are active against P aeruginosa; ceftazidime is often used with an aminoglycoside in primary therapy of P aeruginosa infections, especially in patients with neutropenia. The susceptibility patterns of P aeruginosa vary geographically, and susceptibility tests should be done as an adjunct to selection of antimicrobial therapy. Multidrug resistance has become a major issue in the management of hospital-acquired infections with P aeruginosa because of acquisition of chromosomal β-lactamases, extended-spectrum β-lactamases, porin channel mutations, and efflux pumps.

Epidemiology and Control

P aeruginosa is primarily a nosocomial pathogen, and the methods for control of infection are similar to those for other nosocomial pathogens. Because Pseudomonas thrives in moist environments, special attention should be paid to sinks, water baths, showers, hot tubs, and other wet areas. For epidemiologic purposes, strains can be typed using molecular typing techniques.

Burkholderia pseudomallei is a small, motile, oxidase-positive, aerobic Gram-negative bacillus. It grows on standard bacteriologic media, forming colonies that vary from mucoid and smooth to rough and wrinkled and in color from cream to orange. It grows at 42°C and oxidizes glucose, lactose, and a variety of other carbohydrates. B pseudomallei causes melioidosis (or Whitmore’s disease), which occurs predominantly in Southeast Asia and northern Australia. The organism is a natural saprophyte that has been cultured from soil, fresh water, rice paddies, and vegetable produce. Human infection probably originates from these sources by contamination of skin abrasions and possibly by ingestion or inhalation. Epizootic B pseudomallei infection occurs in sheep, goats, swine, horses, and other animals, although animals do not appear to be a primary reservoir for the organism. B pseudomallei is considered a Category B agent of bioterrorism by the US Centers for Disease Control and Prevention because, if weaponized, the bacterium would be moderately easy to disseminate, resulting in moderate morbidity and low mortality.

Melioidosis may manifest itself as acute, subacute, or chronic infection. The incubation period can be as short as 2–3 days, but latent periods of months to years also occur. A localized suppurative infection can occur at the inoculation site where there is a break in the skin. This localized infection may lead to the acute septicemic form of infection with involvement of many organs. The signs and symptoms depend upon the major sites of involvement. The most common form of melioidosis is pulmonary infection, which may be a primary pneumonitis (B pseudomallei transmitted through the upper airway or nasopharynx) or subsequent to a localized suppurative infection and bacteremia. The patient may have fever and leukocytosis with consolidation of the upper lobes. Subsequently, the patient may become afebrile, while upper lobe cavities develop, yielding an appearance similar to that of tuberculosis on chest films. Some patients develop chronic suppurative infection with abscesses in skin, brain, lung, myocardium, liver, bone, and other sites. Patients with chronic suppurative infections may be afebrile and have indolent disease. Latent infection is sometimes reactivated as a result of immunosuppression.

The diagnosis of melioidosis should be considered for a patient from an endemic area who has fulminant upper lobe pulmonary or unexplained systemic disease. A Gram stain of an appropriate specimen will show small Gram-negative bacilli; bipolar staining (safety pin appearance) is seen with Wright’s stain or methylene blue stain. A positive culture result is diagnostic. A positive serologic test result is diagnostically helpful and constitutes evidence of past infection.

Melioidosis has a high mortality rate if untreated. Surgical drainage of localized infection may be necessary. B pseudomallei are generally susceptible to ceftazidime, imipenem, meropenem, and amoxicillin–clavulanic acid (also ceftriaxone and cefotaxime). B pseudomallei are commonly resistant to penicillin, ampicillin, first-generation and second-generation cephalosporins, and gentamicin and tobramycin. Depending on the clinical setting, the initial intensive therapy should be for a minimum of 10–14 days with ceftazidime, imipenem, or meropenem; sulfamethoxazole–trimethoprim can be considered in patients with severe allergy to β-lactam antimicrobials. Eradication therapy with sulfamethoxazole–trimethoprim or doxycycline should follow the intensive initial therapy and be continued for a minimum of 3 months. Recurrent disease because of failure of eradication can occur for several reasons, but the most important is noncompliance with the long-term eradication therapy.

Burkholderia cepacia and 17 other genomospecies comprise the B cepacia complex. The classification of these bacteria is complex; their specific identification is difficult. These are environmental organisms able to grow in water, soil, plants, animals, and decaying vegetable materials. In hospitals, members of the B cepacia complex have been isolated from a variety of water and environmental sources from which they can be transmitted to patients. People with CF and those patients with chronic granulomatous disease are particularly vulnerable to infection with bacteria in the B cepacia complex. It is likely that B cepacia can be transmitted from one CF patient to another by close contact. They may have asymptomatic carriage, progressive deterioration over a period of months, or rapidly progressive deterioration with necrotizing pneumonia and bacteremia. Although a relatively small percentage of CF patients become infected, the association with progressive disease makes B cepacia complex a major concern for these patients. A diagnosis of B cepacia infection in a CF patient may significantly alter the patient’s life because he or she may not be allowed association with other CF patients and may be removed from eligibility for lung transplantation.

B cepacia grows on most media used in culturing specimens for Gram-negative bacteria. Selective media containing colistin (eg, B cepacia selective agar) can be used and is recommended when culturing the sputum of patients with CF. B cepacia grows more slowly than enteric Gram-negative rods, and it may take 3 days before colonies are visible. B cepacia are oxidase positive and lysine decarboxylase positive and produce acid from glucose, but differentiating B cepacia from other pseudomonads, including Stenotrophomonas maltophilia, requires a battery of biochemical tests and can be difficult. Submission of isolates to reference laboratories is recommended because of the prognostic implications of colonization in CF patients. In the United States, the Cystic Fibrosis Foundation (http://www.cff.org) supports a reference laboratory that uses phenotypic and genotypic methods to confirm the identity of organisms within the B cepacia complex. Susceptibility tests should be done on B cepacia complex isolates, although slow growth may make routine testing difficult. B cepacia complex isolates recovered from CF patients often are multidrug resistant. Trimethoprim–sulfamethoxazole, meropenem, and ciprofloxacin, or alternatively minocycline, are effective treatments.

S maltophilia is a free-living Gram-negative rod that is widely distributed in the environment. On blood agar, colonies have a lavender-green or gray color. The organism is generally oxidase negative, and positive for lysine decarboxylase, DNase, and oxidation of glucose and maltose (hence the name “maltophilia”).

S maltophilia is an increasingly important cause of hospital-acquired infections in patients who are receiving antimicrobial therapy and in immunocompromised patients. It has been isolated from many anatomic sites, including respiratory tract secretions, urine, wounds, and blood. The isolates are often part of mixed flora present in the specimens. When blood culture results are positive, it is commonly in association with use of indwelling plastic intravenous catheters.

S maltophilia is usually susceptible to trimethoprim–sulfamethoxazole and ticarcillin–clavulanic acid and resistant to other commonly used antimicrobials, including cephalosporins, aminoglycosides, imipenem, and the quinolones. The widespread use of the drugs to which S maltophilia is resistant plays an important role in the increased frequency with which it causes disease.

Acinetobacter species are aerobic, Gram-negative bacteria that are widely distributed in soil and water and can occasionally be cultured from skin, mucous membranes, secretions, and the hospital environment.

A baumannii is the species most commonly isolated. Acinetobacter lwoffii and other species are isolated occasionally. Some isolates have not received species names but are referred to as genomospecies. Acinetobacters are usually coccobacillary or coccal in appearance; they resemble neisseriae on smears, because diplococcal forms predominate in body fluids and on solid media. Rod-shaped forms also occur, and occasionally the bacteria appear to be Gram-positive. Acinetobacter grows well on most types of media used to culture specimens from patients. Acinetobacter recovered from patients with meningitis and bacteremia has been mistaken for Neisseria meningitidis; similarly, Acinetobacter recovered from the female genital tract has been mistaken for Neisseria gonorrhoeae. However, the neisseriae produce oxidase, and Acinetobacter does not.

Acinetobacters often are commensals but occasionally cause nosocomial infection. A baumannii has been isolated from blood, sputum, skin, pleural fluid, and urine, usually in device-associated infections. Acinetobacters encountered in nosocomial pneumonias often originate in the water of room humidifiers or vaporizers. In patients with Acinetobacter bacteremias, intravenous catheters are almost always the source of infection. In patients with burns or with immune deficiencies, acinetobacters act as opportunistic pathogens and can produce sepsis. Acinetobacter strains are often multidrug resistant, and therapy of infection can be difficult. In many cases, the only active agent may be colistin. Such multidrug-resistant strains are a common cause of serious wound infections among wounded servicemen in Iraq. Susceptibility testing should be done to help select the best antimicrobial drugs for therapy. The more susceptible Acinetobacter strains respond most commonly to gentamicin, amikacin, or tobramycin and to extended-spectrum penicillins or cephalosporins.

• P aeruginosa is an oxidase-positive, frequently pigmented, Gram-negative, glucose-nonfermenting rod that elaborates enzymes, such as elastase, and other virulence factors that promote disease. This organism causes a broad range of infections from superficial skin disease, such as hot tub folliculitis, to gram-negative sepsis and ecthyma gangrenosum in neutropenic patients.

• B pseudomallei is found in the soil and water of Southeast Asia and northern Australia. Human infection with B pseudomallei can be acute, subacute, or chronic, and involves multiple organ systems. It is also considered a Category B agent of bioterrorism by the CDC.

• B cepacia complex are a group of closely related environmental organisms second only to P aeruginosa as a cause of morbidity and mortality in CF patients.

• Acinetobacter sp. and S maltophilia are two organisms frequently associated with hospital-acquired infections that are very drug resistant. In some cases, the only active agent for multidrug-resistant Acinetobacter is colistin.