These gramegative rods are not classified with the Enterobacteriaceae, although they may be found normally in the GI tract. The most important is Pseudomonas aeruginosa. Pseudomonas is less common in community-acquired infection than with most of the major Enterobacteriaceae but becomes more frequent in hospital-acquired infections (about 10% of nosocomial infections). The conditions for which it is best known are urinary tract infection, septicemia, pneumonia (especially in patients with cystic fibrosis), extensive burn infection, malignant otitis externa, and infection associated with ocular contact lenses or physical therapy whirlpool tubs. P. aeruginosa is also known for its resistance to many antibiotics and for its high mortality rate.

In the environment, Pseudomonas organisms live in water or on moist surfaces. In human disease, Pseudomonas has many attributes similar to “opportunistic fungi” in that infection is rare in healthy persons; and even in hospitalized persons, colonization is more common than infection. Infection is most often superimposed on serious underlying diseases such as hematologic or nonhematologic malignancy (especially during chemotherapy), severe burns, wounds, and foreign bodies (tracheostomy, catheter), or in immunocompromised patients. P. aeruginosa is resistant to many of the standard antibiotics and therefore may become a secondary invader after antibiotic therapy for the original infection. This is most frequent in urinary tract infections. Pseudomonas may be found normally on the skin (as well as in the GI tract) and thus is a very important and frequent problem in severe burns. Pseudomonas septicemia is increasing as a complication or a terminal event in patients with malignancy or immunocompromised state. In some tertiary medical centers, Pseudomonas is reported involved in as many as 20% (range, 6%-25%) of bacteremic episodes.

Calymmatobacterium granulomatis (Donovania granulomatis)

Granuloma inguinale is a venereal disease caused by a gramegative rod bacterium that has some antigenic similarity to the Klebsiella group. Infection is transmitted by sexual contact. After incubation, an elevated irregular flattened granulomatous lesion develops, usually in or around the medial aspect of the inguinal area or on the labia. The organism is difficult to culture and requires special media, so culture is not usually done. Diagnosis is accomplished by demonstration of the organisms in the form of characteristic Donovan bodies, found in the cytoplasm of histiocytes. The best technique is to take a punch biopsy of the lesion, crush the fresh tissue between two glass slides, and make several smears with the crushed tissue. These smears are air-dried and stained with Wright’s stain. (It is possible to process a biopsy specimen in the routine manner and do special stains on tissue histologic sections, but this is not nearly as effective.) Granuloma inguinale is sometimes confused with lymphogranuloma venereum, a totally different disease, because of the similarity in names and because both are venereal diseases.

Haemophilus (hemophilus)

This genus is one of three in the family Pasteurellaceae (the other two are Pasteurella and Actinobacillus). The genus Haemophilus contains several species, most of which are normal nasopharyngeal inhabitants, that on Gram stain are very small gramegative rods (“coccobacilli”). The most important species is Haemophilus influenzae, which is the most common etiology of meningitis between the ages of 2 months and 5 years (about 70% of cases). H. influenzae occasionally produces a serious type of laryngitis (croup) in children known as acute epiglottitis and occasionally is associated with childhood otitis media, sinusitis, meningitis, bacteremia, and pneumonia. H. influenzae infection in adults is far less common than in early childhood, but the same types of diseases may occur. H. influenzae exists in both capsulated and noncapsulated forms. In early childhood, 90%-95% of cases of meningitis or bacteremia are caused by capsulated type B. Non-B encapsulated serotypes and nonencapsulated forms increase in frequency in localized pediatric infection and in adult infection. Currently, most typing of Haemophilus organisms is done with antibodies to the capsular antigens of H. influenzae. “Nontypable” H. influenzae means a noncapsulated strain and cannot be ignored, on this basis alone, as a cause for serious disease. Organisms in the genus Haemophilus require one or both substances called X and V factors. Therefore, the traditional H. influenzae culture plate contains blood agar (supplying the X factor) on which is a small area previously inoculated with S. aureus (“staph streak”), which supplies V factor (“satellite test”). However, other satisfactory culture and identification systems are now available. In addition to culture, latex agglutination tests are available to detect H. influenzae antigen in spinal fluid. These will be discussed in more detail in the chapter on CSF tests. In very limited studies involving patients with infections other than CSF, latex agglutination test results on urine were positive in 92%-100% of cases. Haemophilus influenzae is considered normal flora of the nasopharynx (3%-5% of children over age 6 months) and also sputum (which acquires these organisms as it passes through the oropharynx). Heavy growth or predominance of a single “normal flora” organism increases the suspicion that it may be pathogenic.

Other members of the Haemophilus genus that deserve special mention are H. aegyptius (Koch-Weeks bacillus), which produces purulent conjunctivitis; and H. ducreyi, which is the etiologic agent of the venereal disease chancroid. Haemophilus aphrophilus is being reported more often in recent years as a pathogen.


The most important organism in this genus is Pasteurella multocida. This is a small gramegative bacillus found normally in the mouth of many (but not all) cats and dogs. Dog bites and either cat bites or cat scratches inoculate the organism, leading to cellulitis and occasionally to osteomyelitis or infection of prosthetic devices. The organism grows on ordinary laboratory media. Isolation and identification is made much easier if the laboratory knows that the specimen is from a dog- or cat-inflicted wound.


For a long time these organisms were included in the genus Haemophilus. The most important of this group is Bordatella pertussis, the etiologic agent of pertussis (“whooping cough”). The disease is spread by aerosol droplets and is highly contagious, with attack rates in family members of patients ranging from 50% to over 90%. In one study about two thirds of those infected were asymptomatic. The incubation period averages 7 days (range, 6-20 days). In children there are three clinical stages. The catarrhal stage lasts 1-2 weeks (range, 1-3 weeks), associated with symptoms similar to a mild or moderate viral upper respiratory infection. The paroxysmal stage lasts 2-4 weeks (sometimes more), with paroxysms of coughing alternating with relatively asymptomatic periods being the main symptoms. The convalescent stage usually lasts 1-2 weeks but may persist up to 6 months. The most frequent complication is pneumonia (15% of cases in one study); this is responsible for over 90% of deaths. In adults, clinical symptoms most often consist of chronic cough. In one study, 20%-25% of adults with chronic cough had serological evidence of pertussis.

Laboratory findings consist mostly of leukocytosis (64% of cases in one study) that averages 21,000/mm3 (21 Ч 109/L) but can reach 70,000/mm3(70 x 109/L). There typically is an absolute lymphocytosis; since young children normally have more lymphocytes than neutrophils on peripheral smear, the percentage of lymphocytes is most often within the age-adjusted reference range for young children. There typically is a significant percentage of small lymphocytes with a clefted or distorted nucleus (12%-56% of the lymphocytes). Most of these are T-lymphocytes. In one study lymphocytosis persisted for more than 1 week in 70% of cases and for more than 2 weeks in 14% of cases. In young children, one study found an abnormal chest x-ray in 42% of patients.

Options for diagnosis include posterior nasopharyngeal culture, direct tests for antigen on nasopharyngeal swabs, and serologic tests. Material for culture or antigen tests should be obtained from the pharynx at the posterior end of the nose, using a calcium alginate or Dacron-tipped flexible wire swab. Cotton-tipped swabs decrease chances of a positive culture. A positive culture is most likely (80%-90% of cases) during the first and second weeks of the catarrhal stage; during the later part of the third week, and the beginning of the paroxysmal (fourth week) stage, culture positivity falls off rapidly. After the fifth week only 15%-20% of cases are culture-positive. The original (and still useful) culture medium was Bordet-Gengou; however, the newer Regan-Lowe media can increase culture yield by 20% or more. Reported rates of culture diagnosis vary considerably (30%-60%; range, 20%-90%), depending on the stage of disease culture was obtained and the details of culture technique. Culture takes 4-7 days. As noted previously, recovery results over 50% are most likely to be obtained in the first 3 weeks except during epidemics. Direct fluorescent antibody (DFA) tests on posterior nasopharyngeal specimens are less sensitive than culture in the early nontreated stage but may be more sensitive later in clinical illness or after antibiotic therapy. In general, DFA is 50%-90% as sensitive as culture (range, 11%-100% depending on circumstances). False positive rates of 7%-40% have been reported (although some of these may have been true but nonconfirmed positives). Serologic tests are available but difficult to obtain. The most useful clinically are IgG antibodies against pertussis toxin or filamentous hemagglutinins (FHA). Acute and convalescent specimens give best results. Sensitivity of FHA-IgG tests is reported to be 75%-80% (range, 54%-100%).


There are several species within this genus, of which the most important is Campylobacter fetus. This is a gramegative curved or spiral organism that originally was classified as a Vibrio. There are three subspecies (ssp.) of which two may infect humans. One of these is Campylobacter fetus ssp. fetus (formerly, ssp. intestinalis), which causes abortion in sheep and cattle and which can rarely infect humans. It produces disseminated disease without diarrhea, presenting as newborn or infant meningitis and childhood or adult septicemia. It also can produce thrombophlebitis. Patients usually are immunocompromised or have some debilitating disease such as alcoholism, cancer, or renal failure. Diagnosis is made by blood culture in septicemia and by spinal fluid culture in meningitis. Ordinary culture media can be used. The other Campylobacter organism, C. fetus ssp. jejuni, is much more common and infects cattle, dogs, and birds, in addition to humans. Poultry, especially turkeys, have been responsible for some epidemics. C. fetus ssp. jejuni produces enteric infection with diarrhea, which in some areas is responsible for as many cases as Salmonella and Shigella combined. Overall, both children and adults are affected in about equal proportion, although there is variance in different reports. Published studies indicate that C. fetus ssp. jejuni can be isolated from approximately 4%-8% of patients with diarrhea (literature range, 3%-32%).

Typical symptoms include a prodromal period of 12-24 hours with fever, headache, abdominal pain, and malaise, followed by diarrhea with crampy abdominal pain. Fever often disappears after onset of diarrhea. The diarrhea is sometimes grossly bloody, typically lasts 2-3 days, and usually is self-limited. In some patients it is more prolonged. The severity of illness varies considerably.

Laboratory tests. Helpful laboratory studies include tests for fecal blood and Gram stain of the feces for WBCs. About two thirds of patients demonstrate gross or occult stool blood (literature range, 60%-90%), and about the same number have segmented neutrophils in the stool. About two thirds of patients have peripheral blood leukocytosis. Fecal culture is the mainstay of diagnosis (blood cultures are usually negative in ssp. jejuni infection). However, routine stool culture will be negative, because the organism is microaerophilic and must be incubated 48-72 hours on special media in special gas mixtures. It grows best at 42°C but will grow at 37°C. Routine laboratories can perform the cultures, but it takes special effort and special techniques. The organism is extremely sensitive to drying. If specimens are sent to an outside laboratory, they should be inoculated into special transport media (not ordinary media) and sent with ice if more than 1 day’s travel is necessary. Gram stain of the feces can demonstrate Campylobacter in about 50% of cases (range, 43%-65%). The organism is gram negative and has different shapes; most typically curved, S-shaped, and “gull winged.” A few reports indicate that 1% aqueous basic fuchsin stain is more sensitive than Gram stain.


The Aeromonas organism, a gramegative rod, is frequently found in water (usually nonchlorinated although chlorinated water is not always safe) and in soil. It infects fish and amphibians as well as humans. Sources of human infection are water; uncooked or poorly cooked meat, poultry, shellfish, and fish; and raw milk. Clinical infection in humans is not common but has been reported in those with traveler’s diarrhea, immunosuppressed patients, and sporadic cases. Asymptomatic infection has been reported in 0.1%-27% of persons examined. Diarrhea and abdominal cramps are the most common symptoms. Persons of all ages can become infected; acute self-limited diarrhea is more common in infants and young children and chronic infection in adults. Occasional extraintestinal disease (osteomyelitis, urinary tract infection, septicemia, and others) has been reported. Diagnosis is most often made by stool culture; enrichment media followed by special selective media is said to give best overall results, although the organisms can grow on standard culture media.


Helicobacter pylori (formerly Campylobacter pylori or pyloridis) is an S-shaped (curved, or “gull-winged” like Campylobacter), small gramegative bacillus found in the mucus covering the gastric mucosa, with the organisms located next to the gastric lining cells of the surface and gastric pits. H. pylori is associated with acute and chronic gastritis in the gastric antrum and is present in about 90% (range, 70%-100%) of patients with duodenal ulcer, in 70%-75% (range, 40%-90%) of patients with gastric ulcer, about 50% (range, 30%-75%) of patients with nonulcer dyspepsia, and about 20%-25% of patients with gastric cancer. Significant H. pylori antibody levels can be detected in about 20%-25% of clinically normal U.S. and European adults (range, 5%-75% depending on age). This suggests that H. pylori infection is often subclinical. Indeed, in patients with clinical infection, biopsy of normal-appearing gastric mucosal areas often contains demonstrable H. pylori organisms. Chronic or recurrent duodenal ulcer is very highly associated with both excess acid production and H. pylori infection. However, one exception is duodenal ulcer due to the Zollinger-Ellison syndrome, in which incidence of H. pylori infection is zero.

Incidence of H. pylori is age-related; in one study 5% or less of adults age 25-45 had antibodies to H. pylori; 20% of adults age 45-55, 50% of adults 55-65, and 75% of adults over age 65 had the antibodies. African Americans are reported to have about twice the incidence seen in Europeans. Besides antral gastritis, H. pylori is associated to a lesser degree with patchy gastritis in the body of the stomach.

The traditional gold standard for diagnosis has been culture of gastric mucosal biopsy specimens. This should be placed in 2-3 ml of sterile isotonic saline, plated on enriched media such as chocolate agar or selective media such as Skirrow’s, and incubated 5-7 days at 37°C. However, based on comparison with results of other tests, culture only detects about 75% (range, 50%-95%) of cases. Giemsa stain of gastric mucosa biopsy tissue is reported to detect about 90% of cases (range, 88%-93%), although in my experience it has been less. Warthin-Starry silver stain has a little better sensitivity than Giemsa stain and Gram stain has somewhat less (about 75%; range, 69%-86%). H. pylori has considerable ability to metabolize urea by means of the enzyme urease that forms the basis for several other tests. The urea breath test using radioactive carbon-13 or carbon-14 incorporated into a urea test dose is administered orally and expired air is then tested for radioactivity 20-120 minutes later. This is probably the best test of all those available, with sensitivity in most reports over 95% (range, 90%-100%) and specificity over 95%. In fact, at present it is probably a better gold standard than culture. However, the test is available only in a relatively few large medical centers, is expensive, requires special equipment, and uses long-lived isotopes that are becoming serious disposal problems. Other tests are based on fresh gastric mucosal biopsy tissue placed in a urea-containing medium with some indicator system to signal metabolism of the urea. The best-studied of these is the CLO test; it is reported to have a sensitivity of about 90% (range, 86%-96%).

Some immunoassays for antibody to H. pylori have become commercially available, mostly in EIA format, which detects IgG, IgA, or IgM anti- body alone or total antibody. In experimental H. pylori infection, an IgM elevation first begins at about 3 weeks, becoming nondetectable about 8 weeks later. IgA and IgG levels rise at about 8-9 weeks after infection, with the IgG level remaining elevated a year or more. The height of elevation does not correlate well with severity of infection. IgM and IgA levels often fall in about 4 weeks after successful treatment, whereas IgG levels are not affected. Most of the antibody assays evaluated in the literature to date have been homemade (which often give better results than commercially available kits); these have reported sensitivity of 80%-100%. Several new commercial kits claim sensitivity of 95% or more. However, some of these sensitivity claims are less because the tests against which the commercial EIA was compared had sensitivity themselves less than 100%. Also, more evaluations must be done to get adequate information about each EIA kit. In general, most current interest seems to be toward IgG antibody. However, this antibody has the disadvantage that it often remains elevated for a year or more after adequate therapy.


Three members of the Brucella genus produce an uncommon febrile disease known as “brucellosis,” which sometimes must be considered in the differential diagnosis of fever of unknown origin. The Brucella organism is a gramegative coccobacillus with three main species. One species infects cattle, the second one infects goats, and the third is found in swine. Classic brucellosis was most often transmitted to humans by infected milk or milk products. However, persons most likely to contact the infection today in the United States are workers in the meat-processing industry, especially those working with swine. Veterinarians and dairy farmers are also at risk. Clinical symptoms include fever, chills, and myalgia. About 25% of patients develop single-joint arthralgia. Lymph nodes are usually not enlarged. Splenomegaly occurs in about 20% of cases. Some patients develop pneumonia. WBC counts are usually normal or decreased. Blood culture is positive in 30%-50% of cases, but the organisms need added carbon dioxide and grow very slowly on ordinary culture media, so that under ordinary circumstances the brucellae may either not grow at all or the culture bottle is discarded as negative before growth is evident. Special media are available that will assist growth. A slide agglutination test is the most frequent method for diagnosis. Test results greater than 1:80 are suspicious for brucellosis. After recovery, antibody titer elevation lasts 1-2 years.


Formerly included in the genus Pasteurella, the genus Francisella contains one medically important species, Francisella tularensis, which is the causative agent of tularemia. F. tularensis is a very small gramegative aerobic coccobacillus that requires special media for adequate growth. The major reservoir for human infection in the United States is wild animals, predominantly wild rabbits, and in some cases deerflies or ticks. Most persons who contract the disease are those who handle raw wild rabbit meat. Tularemia may exist in several clinical forms, but the most common symptoms are painful single-area lymphadenopathy (ulceroglandular form, comprising 75%-80% of cases) and prolonged low-grade fever. Much less common is a septicemic form (5%-15% of cases). Pneumonia may occur in 15% or more of either the ulceroglandular or the septicemic forms, especially the septicemic type. Rabbit-associated infection is usually associated with a small ulcerated skin lesion at the place of entry on the hand and also with axillary adenopathy, whereas in tick-associated infection the ulcer is located on the trunk or lower extremities and the lymph nodes affected are most commonly in the inguinal area. About 15% of affected persons lack either the ulcer or both the ulcer and the adenopathy. WBC counts are normal in at least 50% of patients and slightly or mildly elevated in the remainder. The differential cell count is usually normal. Inconsistent elevation of one or more liver function test results is present in about one half of the patients.

Tularemia must occasionally be considered in the differential diagnosis of fever of unknown origin. The organisms will not grow on standard laboratory culture media. Special media can be obtained and culture performed from lymph node aspiration, but this is rarely done because of an unusually high rate of laboratory technologist infection from the specimens. The standard diagnostic test is a slide agglutination procedure. Titers are said to be negative during the first week of illness and begin to rise at some time during the second week, become maximal in 4-8 weeks, and remain elevated for several years. A fourfold rising titer is necessary to prove acute infection. There is considerable disagreement regarding a single titer level that could be considered presumptive evidence of infection; 1:160 seems to be the least controversial value.


Vibrios are gramegative small curved rods. The most important species is Vibrio cholerae, which produces cholera by means of an enterotoxin. The V. cholerae species is subdivided into many serotypes, of which only one, the 01 strain, is associated with epidemic cholera in the United States. A different strain is found in Latin America than in the United States. About 75% of cholera organism infections are asymptomatic, about 20% produce mild to moderate diarrhea, and about 5% produce the severe watery diarrhea ordinarily associated with the name “cholera” that can lead to death from loss of fluid and electrolytes. Humans usually become infected by drinking contaminated water or eating shellfish from contaminated water. Diagnosis has traditionally been made by stool culture. The organism is very sensitive to drying, and if swabs are used, they must be placed in transport media as soon as the specimen is obtained. Also culture has been reported to miss 10%-20% of cases.

Recently, two stool tests have been developed by the same manufacturer using a monoclonal antibody called COLTA that is claimed to be specific for V. Cholerae 01 strain. CholeraScreen is a coagglutination slide test and CholeraSmart is a tube agglutination test. Both are said to be 95%-100% sensitive compared to culture. Only a few evaluations have been published to date.