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PLEOMORPHIC GRAM NEGATIVE RODS HAEMOPHILUS BORDETELLA BRUCELLA PASTEURELLA LEGIONELLA HAEMOPHILUS The Haemophilus genus represents a large group of Gram-negative rods that like to grow on blood agar. The blood provides two factors which many Haemophilus species require for growth: X factor and V factor. Sometimes Haemophilus is cultured using something called the "Staph streak" technique. In this procedure, both Staphylococcus and Haemophilus organisms are grown together on blood agar. Haemophilus colonies will usually form small colonies called "satellites" around Staphylococccus colonies because Staph can provide the necessary factors required for optimium Haemophilus growth. Morphologically, Haemophilus bacteria usually appear as tiny coccobacilli under the microscope, but they are included with the Pleomorphic bacteria because of the many shapes they assume. A methylene blue stain of a smear can also help with identification. Haemophilus species are classified by their capsular properties into six different serological groups, (a-f). Species which possess a type b capsule are clinically significant because of their virulent properties. H. influenzae Infection by H. influenzae is common in children and its name may lead you to conclude that it is the causative agent of the flu. Actually, this bacterium causes a secondary respiratory infection that usually inflicts those who already have the flu. This species may exist with or without a pathogenic polysaccharide capsule. Although both strains occur as normal flora of the nose and pharynx, they can confer severe illness in patients that are immunosuppressed or that have pre-existing respiratory ailments. Strains that lack the capsule usually cause mild localized infections (otitis media, sinusitis), as opposed to the type b encapsulated strain of H. influenzae that can cause several serious infections. Most of these infections occur in unvaccinated children less than five years of age because they have not yet formed antibodies against the bacterium. H. influenzae infection can lead to a variety of diseases: Meningitis- H. influenzae is the most common cause of bacterial meningitis in children between the ages of five months and five years. The initial respiratory infection can spread to the blood stream and eventually the central nervous system. A stiff neck, lethargy, and the absence of the sucking reflex are common symptoms in infected babies. A vaccine is available, but is not always effective in very young children. Adult meningitis is much less common and usually only occurs in those predisposed to illness. Epiglottitis- H. influenzae is the number one cause of this potentially fatal disease, which may cause airway obstruction in children between the ages of 2 and 4. Haemophilus infection has also been associated with chronic bronchitis, pneumonia, bacteremia, conjuctivitis, and a host of other illnesses. For serious infections, third generation cephalosporins are the drug of choice. Resistance may develop when ampicillin is used in treatment. OTHER SPECIES H. aegyptius This bacterium is biochemically identical to H. influenzae. It is known to cause pinkeye (conjuctivitis) and is spread very easily, especially among children. H. ducreyi Chancroid, a sexually transmitted disease characterized by painful genital ulcers, is associated with this bacterium. H. ducreyi does not require the V factor for growth nor does it ferment glucose. BORDETELLA Bordetella organisms are small, Gram-negative coccobacilli which are strict aerobes. The three species of this genus vary in motility and certain biochemical characteristics. The most important species in this genus is B. pertussis, the organism which causes whooping cough. This highly contagious bacterium makes its way into the respiratory tract via inhalation and subsequently binds to and destroys the ciliated epithelial cells of the trachea and bronchi. It does this through the use of several toxins: Pertussis toxin- an exotoxin which enters target cells and activates their production of cAMP, a molecule that acts as a second messenger in cell protein synthesis regulation Tracheal cytotoxin- causes ciliated epithelial cell destruction Hemoagglutinin- a cell surface protein which helps the bacterium bind to the host cell surface Under the microscope, Bordetella are often bipolar stained and appear singly or in pairs. The best method for isolation of Bordetella is on Bordet-Gengou agar or Regan-Lowe medium. There are about 5000 cases of whooping cough per year in the United States, usually afflicting children less than a year old. A vaccine has reduced the incidence of this disease one hundred fold since its introduction! Despite this extraordinary rate of eradication, controversy exists regarding the safety of the vaccine. Antimicrobial therapy for whooping cough usually consists of erythromycin. Two other species of Bordetella are also of clinical importance. B. parapertussis is a respiratory pathogen that can cause mild pharyngitis. This bacterium is similar to B. pertussis but lacks some of the toxins which make its sibling so nasty. Biochemical testing can easily differentiate the two species. Bordetella bronchiseptica is usually a cause of pneumonia, otitis media, and other respiratory infections in animals. It is seldom known to be a human pathogen. Its motility makes it easy to distinguish from the other two organisms in this genus. DIFFERENTIATION OF BORDETELLA SPECIES Growth on Blood Free Peptone Urease Nitrate Reduction Motility Citrate B. pertussis - - - - - B. parapertussis + + - - + B. bronchiseptica + + + + + BRUCELLA Brucella is another strictly aerobic, Gram-negative coccobacillus which causes Brucellosis. This organism is sometimes carried by animals and only causes incidental infections in humans. The four species of this genus that can infect humans are named for the animal which they are most commonly found: B. abortus (cattle), B. suis (swine), B. melitensis (goats), B. canis (dogs). The cattle and dairy industries seem to be the primary source of infection in the United States. Brucella can enter the body via the skin, respiratory tract, or digestive tract. Once there, this intracellular organism can enter the blood and the lymphatics where it multiplies inside phagocytes and eventually cause bacteremia (bacterial blood infiltration). Symptoms vary from patient to patient but can include high fever, chills, and sweating. Afflicted individuals are usually treated with streptomycin or erythromycin. PASTEURELLA Organisms of the genus Pasteurella are Gram-negative, non-motile, facultatively anaerobic coccobacilli. Unlike most of the pleomorphic organisms we have spoken of, Pasteurella is not an intracellular parasite. P. multocida is the species which most commonly infects humans. Although most members infect animals, humans can acquire the organism from dog or cat bites. Patients tend to exhibit swelling, cellulitis, and some bloody drainage at the wound site. Infection may also move to nearby joints where it can cause swelling and arthritis (not to mention a lot of pain). Fortunately, P. multocida is susceptble to penicillin, tetracycline, and chloramphenicol. For identification, this organism can be cultured on chocolate agar and can produce a really foul odor. LABORATORY INDICATIONS: Oxidase + Non-motile Catalase + Non-hemolytic (some are beta-hemolytic) Indole + Ornithine decarboxylase + LEGIONELLA The first discovery of bacteria from genus Legionella came in 1976 when an outbreak of pneumonia at an American Legion convention led to 29 deaths. The causative agent, what would come to be known as Legionella pneumophila, was isolated and given its own genus. The organisms classified in this genus are Gram-negative bacteria that are considered intracellular parasites. They grow well on buffered charcoal yeast extract agar, but it takes about five days to get sufficient growth. When grown on this medium, Legionella colonies appear off-white in color and circular in shape. Laboratory identification can also include microscopic examination in conjunction with a direct flourescent antibody (DFA) test. Since the initial discovery many species have been added to the Legionella genus, but only two are within the scope of our discussion: L. pneumophila and L. micdadei. LABORATORY INDICATIONS: Motile Urease - Catalase + Nitrate - Gelatinase + L. pneumophila L. pneumophila is the bacterium associated with Legionnaires' disease and Pontiac fever. Respiratory transmission of this organism can lead to infection, which is usually characterized by a gradual onset of flu-like symptoms. Patients may experience fever, chills, and a dry cough as part of the early symptoms. Patients can develop severe pneumonia which is not responsive to penicillins or aminoglycosides. Legionnaires' disease also has the potential to spread into other organ-systems of the body such as the gastrointestinal tract and the central nervous system. Accordingly, patients with advanced infections may experience diarrhea, nausea, disorientation, and confusion. The 1200 or so cases of Legionnaires' disease per year in the United States usually involve middle-aged or immunosuppressed individuals. Pontiac fever is also caused by L. pneumophila but does not produce the severity of the symptoms found in Legionnaires' disease. The flu-like symptoms are still seen in Pontiac fever patients but pneumonia does not develop and infection does not spread beyond the lungs. Most L. pneomophila infections are easily treated with erythromycin. LABORATORY INDICATIONS: Beta-lactamase + Hippurate hydrolysis + L. micdadei L. micdadei is the second most commonly isolated member of Legionella. This bacterium can cause the same flu-like symptoms and pneomonia which characterize an L. pneumophila infection. Unlike its relative, L. micdadei is sensitive to the penicillins because it does not produce beta-lactamase. LABORATORY INDICATIONS: Beta-lactamase - Hippurate hydrolysis - Acid fast