GRAM NEGATIVE COCCI UNCOMMON GRAM NEGATIVE BACILLI Assoc

GRAM NEGATIVE COCCI & UNCOMMON GRAM NEGATIVE BACILLI Assoc. Prof. Dr. Yesim Gurol

Gram negative cocci and coccobacilli (2 hours): Learning Objectives 1. • • • Defines ‘’Gram negative cocci’’, ‘’Gram negative coccobacilli’’ 1. 1 Lists Gram negative cocci and Gram negative coccobacilli in normal flora. 1. 2. Lists pathogenic Gram negative cocci and Gram negative coccobacilli for human. 1. 3. Lists virulance factors, defines tissue damage mechanisms. 2. Lists the clinical tables related with Gram negative cocci and Gram negative coccobacilli and defines pathogenetic mechanisms. • • • 2. 1. Defines the clinical importance of Gram negative cocci and Gram negative coccobacilli (N. gonorrhoeae, N. meningitidis, Haemophilus spp, Aggregatibacter actinomycetemcomitans, Pasteurella multocida , Eikenella corrodens , Kingella kingae, Bordetella pertussis, Francisella tularensis, Brucella spp. ) 2. 2. Lists the syndromes related with Neisseria spp. Like Waterhouse Friederichson, Fitz-Hugh. Curtis syndrome 2. 3. Lists the diagnostic methods of Gram negative cocci and Gram negative coccobacilli 2. 4. Defines the prevention methods for infections related with Gram negative cocci and Gram negative coccobacilli

Neisseria meningitidis Biology, Virulence, and Disease • • Gram-negative diplococci with fastidious growth requirements Grows best at 35° C to 37° C in a humid atmosphere Oxidase and catalase positive; acid produced from glucose and maltose oxidatively Outer surface antigens include polysaccharide capsule, pili, and lipooligosaccharide (LOS) Capsule protects bacteria from antibody-mediated phagocytosis Specific receptors for meningococcal pili allow colonization of nasopharynx Bacteria can survive intracellular killing in the absence of humoral immunity Endotoxin mediates most clinical manifestations

Epidemiology • • • Humans are the only natural hosts Person-to-person spread occurs via aerosolization of respiratory tract secretions Highest incidence of disease is in children younger than 5 years, institutionalized people, and patients with late complement deficiencies Meningitis and meningococcemia most commonly caused by serogroups B, C, and Y; pneumonia most commonly caused by serogroups Y and W 135; serogroups A and W 135 associated with disease in underdeveloped countries Disease occurs worldwide, most commonly in the dry, cold months of the year

Diagnosis • • • Gram stain of cerebrospinal fluid is sensitive and specific but is of limited value for blood specimens (too few organisms are generally present, except in overwhelming sepsis) Culture is definitive, but organism is fastidious and dies rapidly when exposed to cold or dry conditions Tests to detect meningococcal antigens are insensitive and nonspecific Treatment, Prevention, and Control • • Breast-fed infants have passive immunity (first 6 months) Treatment is with penicillin (drug of choice) Chemoprophylaxis for contact with persons with the disease is with rifampin, ciprofloxacin, or ceftriaxone For immunoprophylaxis, vaccination is an adjunct to chemoprophylaxis; it is used only for serogroups A, C, Y, and W 135; no effective vaccine is available for serogroup B

• Neisseria species are aerobic, Gram-negative bacteria, typically coccoid shaped (0. 6 to 1. 0 μm in diameter) and arranged in pairs (diplococci) with adjacent sides flattened together (resembling coffee beans). • The bacteria are not motile and do not form endospores. • All species are oxidase positive, and most produce catalase-properties that combined with the Gram-stain morphology allow for a rapid, presumptive identification of a clinical isolate. Acid is produced by oxidation of carbohydrates (not by fermentation). • N. gonorrhoeae strains produce acid by oxidizing glucose, and N. meningitidis strains oxidize both glucose and maltose. Other carbohydrates are not oxidized. This pattern of carbohydrate utilization is useful for differentiating these pathogenic strains from other Neisseria species.

• Nonpathogenic species of Neisseria can grow on nutrient agar incubated at 35° C to 37° C. In contrast, N. meningitidis has variable growth on nutrient agar, and N. gonorrhoeae is a fastidious organism, requiring complex media for growth; it is adversely affected by exposure to dry conditions or fatty acids. • All strains of N. gonorrhoeae require cystine and an energy source (e. g. , glucose, pyruvate, and lactate) for growth, and many strains require supplementation of media with amino acids, purines, pyrimidines, and vitamins. Soluble starch is added to the media to neutralize the toxic effect of the fatty acids. • Thus N. gonorrhoeae does not grow on blood agar but does grow on chocolate agar and other enriched supplemented media. The optimum growth temperature is 35° C to 37° C, with poor survival of the organism at cooler temperatures. • A humid atmosphere supplemented with 5% carbon dioxide (CO 2) is either required or enhances growth of N. gonorrhoeae. Although the fastidious nature of this organism makes recovery from clinical specimens difficult, it is nevertheless easy for the organism to be transmitted sexually from person to person.

• The structure of N. gonorrhoeae and N. meningitidis is typical of gram-negative bacteria, with the thin peptidoglycan layer sandwiched between the inner cytoplasmic membrane and the outer membrane. • The major virulence factor for N. meningitidis is the polysaccharide capsule. • Although the outer surface of N. gonorrhoeae is not covered with a true carbohydrate capsule, the cell surface of N. gonorrhoeae has a capsule-like negative charge. • Antigenic differences in the polysaccharide capsule of N. meningitidis are the basis for serogrouping these bacteria. Thirteen serogroups are currently recognized (A, B, C, D, H, I, K, L, W-135, X, Y, Z, 29 E), with most infections caused by serogroups A, B, C, Y, and W 135.

• Pathogenic and nonpathogenic strains of Neisseria have pili that extend from the cytoplasmic membrane through the outer membrane. Pili mediate a number of functions, including attachment to host cells, transfer of genetic material, and motility, and the presence of pili in N. gonorrhoeae and N. meningitidis appears to be important for pathogenesis. • The pili are composed of repeating protein subunits (pilins), whose expression is controlled by the pil gene complex. Pili expression is associated with virulence, in part because the pili mediate attachment to nonciliated epithelial cells and provide resistance to killing by neutrophils. • Pilin proteins have a conserved region at the amino terminal end a highly variable region at the exposed carboxyl terminus. The carboxyl terminal portion of the pilin protein can be phosphorylated and glycosylated and is associated with a second protein, Pil. C, which contributes to its antigenic diversity. • The lack of immunity to reinfection with N. gonorrhoeae results partially from the antigenic variation among the pilin proteins and partially from the phase variation in pilin expression; these factors complicate attempts to develop effective vaccines for gonorrhea.

• Gonorrhea occurs naturally only in humans; it has no other known reservoir. • It is second only to chlamydia as the most commonly reported sexually transmitted disease in the United States. Infection rates are the same in males and females, are disproportionately higher in blacks than in Hispanic Americans and whites, and are highest in the southeastern United States. • The peak incidence of the disease is in the age group 15 to 24 years. The incidence of disease decreased from 1978 to 1997; however, between 1998 and 2006, the incidence of gonorrhea has remained relatively constant. • In 2006, almost 360, 000 new infections were reported in the United States. • However, even this large number is an underestimation of the true incidence of disease because the diagnosis and reporting of gonococcal infections are incomplete. • Public health officials believe that at least half of the new infections are not reported.

• N. gonorrhoeae is transmitted primarily by sexual contact. Women have a 50% risk of acquiring the infection as the result of a single exposure to an infected man, whereas men have a risk of approximately 20% as the result of a single exposure to an infected woman. • The risk of infection rises as the person has more sexual encounters with infected partners. • The major reservoir for gonococci is the asymptomatically infected person. Asymptomatic carriage is more common in women than in men.

• As many as half of all infected women have mild or asymptomatic infections, whereas most men are initially symptomatic. • The symptoms generally clear within a few weeks in people with untreated disease, and asymptomatic carriage may then become established. • The site of infection also determines whether carriage occurs, with rectal and pharyngeal infections more commonly asymptomatic than genital infections.

• Endemic meningococcal disease occurs worldwide, and epidemics are common in developing countries. Epidemic spread of disease results from the introduction of a new, virulent strain into an immunologically naïve population. Pandemics of disease have been uncommon in developed countries since World War II. • Of the 13 serogroups, almost all infections are caused by serogroups A, B, C, Y, and W 135. In Europe and the Americas, serogroups B, C, and Y predominate in meningitis or meningococcemia; serogroups A and W 135 predominate in developing countries. • Serogroups Y and W 135 are most commonly associated with meningococcal pneumonia. N. meningitidis is transmitted by respiratory droplets among people in prolonged close contact, such as family members living in the same household and soldiers living together in military barracks.

• Classmates in schools and hospital employees are not considered close contacts and are not at significantly higher risk of acquiring the disease, unless they are in direct contact with the respiratory secretions of an infected person. • Humans are the only natural carriers for N. meningitidis. Studies of the asymptomatic carriage of N. meningitidis have shown that there is a tremendous variation in its prevalence, from less than 1% to almost 40%. • The oral and nasopharyngeal carriage rates are highest for school-age children and young adults, are higher in lower socioeconomic populations (caused by person-to-person spread in crowded areas), and do not vary with the seasons, even though disease is most common during the dry, cold months of the year. • Carriage is typically transient, with clearance occurring after specific antibodies develop. Endemic disease is most common in children younger than 5 years, particularly infants, and teenagers and young adults. • People who are immunocompromised, the elderly, or those who live in closed populations (e. g. , military barracks, prisons) are prone to infection during epidemics.

Neisseria gonorrhoeae • • • Gonorrhea: characterized by purulent discharge for involved site (e. g. , urethra, cervix, epididymis, prostate, anus) after 2 - to 5 -day incubation period Disseminated infections: spread of infection from genitourinary tract through blood to skin or joints; characterized by pustular rash with erythematous base and suppurative arthritis in involved joints Ophthalmia neonatorum: purulent ocular infection acquired by neonate at birth Neisseria meningitidis • • • Meningitis: purulent inflammation of meninges associated with headache, meningeal signs, and fever; high mortality rate unless promptly treated with effective antibiotics Meningococcemia: disseminated infection characterized by thrombosis of small blood vessels and multiorgan involvement; small, petechial skin lesions coalesce into larger hemorrhagic lesions Pneumonia: milder form of meningococcal disease characterized by bronchopneumonia in patients with underlying pulmonary disease Eikenella corrodens • • Human bite wounds: infection associated with traumatic (e. g. , bite, fistfight injury) introduction of oral organisms into deep tissue Subacute endocarditis: infection of endocardium characterized by gradual onset of low grade fevers, night sweats, and chills Kingella kingae • Subacute endocarditis: as with E. corrodens

Meningococcemia • • • Septicemia (meningococcemia) with or without meningitis is a life-threatening disease. Thrombosis of small blood vessels and multiorgan involvement are the characteristic clinical features. Small, petechial skin lesions on the trunk and lower extremities are common and may coalesce to form larger hemorrhagic lesions. • Overwhelming disseminated intravascular coagulation with shock, together with the bilateral destruction of the adrenal glands (Waterhouse-Friderichsen syndrome), may ensue. A milder, chronic septicemia has also been observed. Bacteremia can persist for days or weeks, and the only signs of infection are a low-grade fever, arthritis, and petechial skin lesions. The response to antibiotic therapy in patients with this form of the disease is generally excellent. • • •

Additional infections caused by N. meningitidis • • • pneumonia, arthritis, urethritis. • Meningococcal pneumonia is usually preceded by a respiratory tract infection. Symptoms include cough, chest pain, rales, fever, and chills. Evidence of pharyngitis is observed in most affected patients. The prognosis in patients with meningococcal pneumonia is good. Other diseases associated with N. gonorrhoeae are perihepatitis (Fitz-Hugh-Curtis syndrome); purulent conjunctivitis , particularly in newborns infected during vaginal delivery (ophthalmia neonatorum); anorectal gonorrhea in homosexual men; and pharyngitis.

Microscopy • • • Gram stain is very sensitive (greater than 90%) and specific (98%) in detecting gonococcal infection in men with purulent urethritis. Its sensitivity in detecting infection in asymptomatic men is 60% or less. The test is also relatively insensitive in detecting gonococcal cervicitis in both symptomatic and asymptomatic women, although a positive result is considered reliable when an experienced microscopist sees gram-negative diplococci within polymorphonuclear leukocytes. • Gram stain can be reliably used to diagnose infections in men with purulent urethritis and women with cervicitis, but all negative results in women and asymptomatic men must be confirmed by culture. • Gram stain is also useful for the early diagnosis of purulent arthritis but is insensitive and nonspecific for the detection of N. gonorrhoeae in patients with skin lesions, anorectal infections, or pharyngitis. Commensal Neisseria species in the oropharynx and morphologically similar bacteria in the gastrointestinal tract can be confused with N. gonorrhoeae. N. meningitidis can be readily seen in the cerebrospinal fluid (CSF) of patients with meningitis unless the patient has received prior antimicrobial therapy. • • • Most patients with bacteremia caused by other organisms have so few organisms present in their blood that the Gram stain has no value. • In contrast, patients with overwhelming meningococcal disease commonly have large numbers of organisms in their blood, which can be seen when the peripheral blood leukocytes are Gram stained.

Antigen testing for the detection of N. Gonorrhoeae • is less sensitive than culture or nucleic acid amplification tests • not recommended unless confirmatory tests are performed on negative specimens. • Commercial tests to detect N. meningitidis capsular antigens in CSF, blood, and urine (where the antigens are excreted) were widely used in the past but have fallen into disfavor in recent years because the tests are less sensitive than Gram stains, and false-positive reactions, particularly with urine specimens, can occur. Nucleic acid amplification (NAA) assays specific for N. gonorrhoeae have been developed for the direct detection of bacteria in clinical specimens. Tests using these assays are sensitive, specific, and rapid (results are available in 4 hours). Combination NAA assays for both N. gonorrhoeae and Chlamydia organisms are available and have replaced culture in most labs. The primary problem with this approach is that it cannot be used to monitor antibiotic resistance of the identified pathogens.

Culture N. gonorrhoeae can be readily isolated from genital specimens if care is taken in collecting and processing the specimens. Because other commensal organisms normally colonize mucosal surfaces, all genital, rectal, and pharyngeal specimens must be inoculated onto both non-selective media (e. g. , chocolate blood agar) and selective media that suppress the growth of contaminating organisms (e. g. , modified Thayer-Martin medium). A nonselective medium should be used because some gonococcal strains are inhibited by the vancomycin present in most selective media. The gonococci die rapidly if specimens are allowed to dry. Therefore drying and cold temperatures should be avoided by directly inoculating the specimen onto prewarmed media at the time of collection.

The endocervix must be properly exposed to ensure that an adequate specimen is collected. Although the endocervix is the most common site of infection in women, the rectal specimen may be the only one positive for gonococci in women who have asymptomatic infections, as well as in homosexual and bisexual men. Blood culture results are generally positive for gonococci only during the first week of the infection in patients with disseminated disease. In addition, special handling of blood specimens is required to ensure the adequate recovery of gonococci, because supplements present in the blood culture media can be toxic to Neisseria. Culture results of specimens from infected joints are positive for the organism if the specimens are collected at the time the arthritis develops, but skin specimen cultures are generally unrewarding.

Vaccines • • • directed against the group-specific capsular polysaccharides have been developed for antibody-mediated immunoprophylaxis. A polyvalent polysaccharide-protein conjugate vaccine effective against serogroups A, C, Y, and W 135 was licensed in the United States in 2005. In 2007, the Advisory Committee on Immunization Practices (ACIP) recommended routine vaccination with one dose of this vaccine for all persons aged 11 to 18 years and other persons at increased risk for meningococcal disease. Unfortunately the group B polysaccharide is a weak immunogen and cannot induce a protective antibody response. Thus immunity to group B N. meningitidis must develop naturally after exposure to cross-reacting antigens. Vaccination with a suspension containing serogroup A can be used for control of an outbreak of disease, for travelers to hyperendemic areas, and for people at increased risk for disease (e. g. , patients with complement deficiency).

Eikenella corrodens • In the early 1960 s, a collection of small, fastidious, gram-negative rods were classified by workers at the CDC as members of the HB group (named after the patient infected with the original isolate). • The organisms were subsequently subdivided into subgroup HB-1 (now known as Eikenella corrodens), subgroup HB-2 (Aggregatibacter [Haemophilus] aphrophilus), and subgroups HB-3 and HB-4 (Aggregatibacter [Actinobacillus] actinomycetemcomitans). • In addition to being morphologically similar, these organisms colonize the human oropharynx; in the setting of preexisting heart disease, they can cause subacute bacterial endocarditis. In fact, the group of fastidious, gram-negative rods associated with subacute endocarditis is known by the now taxonomically incorrect acronym HACEK (H. aphrophilus, A. actinomycetemcomitans, Cardiobacterium hominis, E. corrodens, and K. kingae).

• E. corrodens is a moderate-sized (0. 2 × 2. 0 μm), nonmotile, non-spore-forming, facultatively anaerobic, gram-negative rod. The organism is named after Eiken, who characterized the bacterium and observed the ability of the organism to pit or "corrode" agar (from its ability to split polygalacturonic acid). E. corrodens is a normal inhabitant of the human upper respiratory tract, but because of its fastidious growth requirements, it is difficult to detect unless specific selective culture media are used .

• It is an opportunistic pathogen that causes infections in patients who are immunocompromised or have diseases or trauma of the oral cavity. • E. corrodens is most commonly isolated in the settings of a human bite wound or fistfight injury. Other infections • endocarditis, • sinusitis, • meningitis, • brain abscesses, • pneumonia, • lung abscesses.

• Because most infections originate from the oropharynx, polymicrobial mixtures of aerobic and anaerobic bacteria are often present in cultures. • A slow-growing, fastidious organism, E. corrodens requires 5% to 10% carbon dioxide to grow. Small (0. 5 to 1 mm) colonies are observed after 48 hours of incubation on blood or chocolate agar, but the organism grows poorly or not at all on selective media for gram-negative rods. • Pitting in agar is a useful differential characteristic, but fewer than half of all isolates exhibit pitting. The organism also produces a characteristic bleachlike odor. Thus if a slow-growing, gram-negative rod is found to pit blood agar and produce a bleachlike order, a preliminary identification of the organism can be made. • E. corrodens is resistant to many antibiotics that are selected empirically to treat bite-wound infections.

Kingella kingae • Kingella species are small, gram-negative coccobacilli that morphologically resemble Neisseria species and reside in the human oropharynx. The bacteria are facultatively anaerobic, ferment carbohydrates, and have fastidious growth requirements. • K. kingae, the most commonly isolated species, has been primarily responsible for septic arthritis in children and endocarditis in patients of all ages. Because the organism grows slowly, it may take 3 or more days of incubation for the organism to be detected in clinical specimens. • Most strains are susceptible to β-lactam antibiotics, including penicillin, tetracyclines, erythromycin, fluoroquinolones, and aminoglycosides.

Pasteurellaceae Haemophilus influenzae • Meningitis: primarily a disease of unimmunized children; characterized by fever, severe headache, and systemic signs • Epiglottitis: primarily a disease of unimmunized children; characterized by initial pharyngitis, fever, and difficulty breathing, and progressing to cellulitis and swelling of the supraglottic tissues, with obstruction of the airways possible • Pneumonia: inflammation and consolidation of the lungs observed primarily in the elderly with underlying chronic pulmonary disease; typically caused by nontypeable strains Haemophilus aegyptius • Conjunctivitis: an acute, purulent conjunctivitis ("pink eye") Haemophilus ducreyi • Chancroid: sexually transmitted disease characterized by a tender papule with an erythematous base, progressing to painful ulceration with associated lymphadenopathy Aggregatibacter actinomycetemcomitans • Endocarditis: responsible for subacute form of endocarditis in patients with underlying damage to the heart valve Aggregatibacter aphrophilus • Endocarditis: as with A. actinomycetemcomitans Pasteurella multocida • Bite wound: most common manifestation is infected cat- or dog-bite wound; particularly common with cat bites, because the wounds are deep and difficult to disinfect

• Haemophilae are small, sometimes pleomorphic, gram-negative rods present on the mucous membranes of humans. • Haemophilus influenzae is the species most commonly associated with disease, with infections most often reported in pediatric patients before the introduction of the H. influenzae type b (HIB) vaccine. • Haemophilus aegyptius is an important cause of acute, purulent conjunctivitis. • Haemophilus ducreyi is well recognized as the etiologic agent of the sexually transmitted disease soft chancre, or chancroid. • The other members of the genus are commonly isolated in clinical specimens (e. g. , Haemophilus parainfluenzae is the most common species in the mouth) but are rarely pathogenic, being responsible primarily for opportunistic infections.

The growth of most species of Haemophilus requires supplementation of media with one or both of the following growth-stimulating factors: • hemin (also called X factor for unknown factor), • nicotinamide adenine dinucleotide (NAD; also called V factor for "vitamin"). • Although both factors are present in blood-enriched media, sheep blood agar must be gently heated to destroy the inhibitors of V factor. For this reason, heated blood ("chocolate") agar is used for the in vitro isolation of Haemophilus.

• Lipopolysaccharide with endotoxin activity is present in the cell wall, and strain-specific and species-specific proteins are found in the outer membrane. • The surface of many but not all strains of H. influenzae is covered with a polysaccharide capsule, and six antigenic serotypes (a through f) have been identified. • Before the introduction of the HIB vaccine, H. influenzae serotype b was responsible for more than 95% of all invasive Haemophilus infections. After the introduction of the vaccine, most disease caused by this serotype disappeared, and more than half of all invasive disease is now caused by nonencapsulated (nontypeable) strains.

Haemophilus Biology, Virulence, and Disease • Small, pleomorphic, gram-negative rods or coccobacilli • Facultative anaerobes, fermentative • Most species require X and/or V factor for growth • • • H. influenzae subdivided serologically (types a to f) and biochemically (biotypes I to VIII) H. influenzae type b is clinically most virulent (with PRP, [polyribitol phosphate] in capsule) Haemophilus adhere to host cells via pili and nonpilus structures Epidemiology • Haemophilus species commonly colonized in humans although encapsulated Haemophilus species, particularly H. influenzae type b, are uncommon members of normal flora • Disease caused by H. influenzae type b was primarily a pediatric problem; eliminated in immunized populations • H. ducreyi disease is uncommon in the United States • With the exception of H. ducreyi, which is spread by sexual contact, most Haemophilus infections are caused by the patient's oropharyngeal flora (endogenous infections) • Patients at greatest risk for disease are those with inadequate levels of protective antibodies, those with depleted complement, and those who have undergone splenectomy

Diagnosis • Microscopy is a sensitive test for detecting H. influenzae in cerebrospinal fluid (CSF), synovial fluid, and lower respiratory specimens but not from other sites • Culture is performed using chocolate agar • Antigen tests are specific for H. influenzae type b; therefore, these tests are nonreactive for infections caused by other organisms Treatment, Prevention, and Control • Haemophilus infections are treated with broad-spectrum cephalosporins, azithromycin, or fluoroquinolones; many strains are resistant to ampicillin • Active immunization with conjugated PRP vaccines prevents most H. influenzae type b infections

Laboratory Diagnosis • • Specimen Collection and Transport Because most Haemophilus infections in vaccinated individuals originate from the oropharynx and are restricted to the upper and lower respiratory tract, contamination of the specimen with oral secretions should be avoided. • Direct needle aspiration should be used for the microbiologic diagnosis of sinusitis or otitis, and sputum produced from the lower airways is used for the diagnosis of pneumonia. • • Culture of blood for patients with pneumonia may be useful but would be predictably negative in patients with upper respiratory infections. Both blood and cerebrospinal fluid (CSF) should be collected from nonimmune children with the diagnosis of meningitis. Because there approximately 107 bacteria per m. L of CSF in patients with untreated meningitis, 1 to 2 m. L of fluid is generally adequate for microscopy, culture, and antigen-detection tests. • Microscopy and culture are less sensitive if the patient has been exposed to antibiotics before the CSF is collected.

• Blood cultures should also be collected for the diagnosis of epiglottitis, cellulitis, and arthritis. Specimens should not be collected from the posterior pharynx in patients with suspected epiglottitis because the procedure may stimulate coughing and obstruct the airway. • Specimens for the detection of H. ducreyi should be collected with a moistened swab from the base or margin of the ulcer. Culture of pus collected by aspiration from an enlarged lymph node can be performed but is generally less sensitive than culture of the ulcer. • The laboratory should be notified that H. ducreyi is suspected, because special culture techniques must be used for recovery of the organism.

• If microscopy is performed carefully, the detection of Haemophilus species in clinical specimens is both sensitive and specific. Gram-negative rods ranging in shape from coccobacilli to long, pleomorphic filaments can be detected in more than 80% of CSF specimens from patients with untreated Haemophilus meningitis. • The microscopic examination of Gram-stained specimens is also useful for the rapid diagnosis of the organism in arthritis and lower respiratory tract disease. • Chocolate agar or Levinthal agar is used in most laboratories. However, if chocolate agar is overheated during preparation, V factor is destroyed, and Haemophilus species requiring this growth factor (e. g. , H. influenzae, H. aegyptius, H. parainfluenzae) will not grow. The bacteria appear as 1 - to 2 -mm, smooth, opaque colonies after 24 hours of incubation. They can also be detected growing around colonies of Staphylococcus aureus on unheated blood agar (satellite phenomenon). • • The staphylococci provide the requisite growth factors by lysing the erythrocytes in the medium and releasing intracellular heme (X factor) and excreting NAD (V factor). The colonies of H. influenzae in these cultures are much smaller than they are on chocolate agar, because the V factor inhibitors present in blood are not inactivated.

• Actinobacillus species are small, facultatively anaerobic, gram-negative rods that grow slowly (generally requiring 2 to 3 days of incubation). Actinobacillus actinomycetemcomitans was the most important human pathogen in this genus; however, in 2006 this species and Haemophilus aphrophilus were transferred into a new genus, Aggregatibacter. The remaining members of the genus Actinobacillus colonize the oropharynx of humans and animals and are rare causes of periodontitis, endocarditis, bite wound infections, and opportunistic infections. Two members of this genus are important human pathogens: • A. actinomycetemcomitans and A. aphrophilus Both species colonize the human mouth and can spread from the mouth into the blood and then stick to a previously damaged heart valve or artificial valve, leading to the development of endocarditis. • Endocarditis caused by these bacteria is particularly difficult to diagnose, because clinical signs and symptoms develop slowly (subacute endocarditis), and the bacteria grow slowly in blood cultures. Both species form adherent colonies that can be observed on the glass surface of the blood culture bottles and on agar media. The treatment of choice for endocarditis caused by these organisms is a cephalosporin such as ceftriaxone .

• Pasteurella are small, facultatively anaerobic, fermentative coccobacilli commonly found as commensals in the oropharynx of healthy animals. • Most human infections result from animal contact (e. g. , animal bites, scratches, shared food). • Pasteurella multocida (the most common isolate) and Pasteurella canis are human pathogens; the other Pasteurella species are rarely associated with human infections. • • The following three general forms of disease are reported: (1) localized cellulitis and lymphadenitis that occur after an animal bite or scratch (P. multocida from contact with cats or dogs; P. canis from dogs); (2) an exacerbation of chronic respiratory disease in patients with underlying pulmonary dysfunction (presumably related to colonization of the patient's oropharynx followed by the aspiration of oral secretions); (3) a systemic infection in immunocompromised patients, particularly those with underlying hepatic disease. • •

• Bordetella is an extremely small (0. 2 to 0. 5 × 1 μm), strictly aerobic, gram-negative coccobacillus. Eight species are currently recognized, with four species responsible for human disease Bordetella pertussis , the agent responsible for pertussis or whooping cough; Bordetella parapertussis, responsible for a milder form of pertussis; Bordetella bronchiseptica, responsible for respiratory disease in dogs, swine, laboratory animals, and occasionally respiratory disease in humans; and Bordetella holmesii, an uncommon cause of sepsis. Bordetella pertussis • After a 7 - to 10 -day incubation period, disease is characterized by the catarrhal stage (resembles the common cold), progressing to the paroxysmal stage (repetitive coughs followed by inspiratory whoops), then the convalescence stage (diminishing paroxysms and secondary complications) Bordetella parapertussis • Produces a milder form of pertussis Bordetella bronchiseptica • Primarily a respiratory disease of animals but can cause bronchopneumonia in humans Bordetella holmesii • Uncommon cause of sepsis

• A direct fluorescent antibody procedure using either monoclonal or polyclonal antibodies can be used to examine specimens. • In this method, the aspirated specimen is smeared onto a microscopic slide, air-dried and heat fixed, and then stained with fluorescein-labeled antibodies directed against B. pertussis. • Antibodies against B. parapertussis should also be used to detect mild forms of pertussis caused by this organism. The direct fluorescent antibody test results are positive in about half of the patients with pertussis, but false-positive results can occur as a result of cross-reactions with other bacteria. Because of the sensitivity and specificity problems with this test, PCR and/or culture should also be performed. Nucleic acid amplification methods such as polymerase chain reaction (PCR) are the most sensitive diagnostic test available for pertussis. •

• These methods have replaced microscopy and culture in most laboratories that offer laboratory testing. • At the present time, culture is generally offered by laboratories unable to perform nucleic-acid-based assays or in conjunction with these assays. The sensitivity of culture is affected by patient factors (i. e. , stage of illness, use of antibiotics), the quality of the specimen, transport conditions, and culture methods. • The traditional use of Bordet-Gengou medium has been replaced by Regan-Lowe charcoal medium supplemented with glycerol, peptones, and horse blood. The media should be incubated in air at 35° C and in a humidified chamber. • Prolonged incubation (e. g. , 7 to 12 days) is necessary. Because the quality of the media dramatically affects the success of culture, laboratories that infrequently culture specimens for Bordetella should arrange for the state public health department to process these specimens. Despite use of optimized culture conditions, fewer than half the infected patients have positive cultures

• Francisella and Brucella are important zoonotic pathogens that occasionally cause human disease. • These organisms have also gained notoriety as potential agents of bioterrorism. Although the organisms have some common properties (e. g. , very small coccobacilli, fastidious and slow growth requirements, always pathogenic in humans), they are taxonomically unrelated.

• The genus Francisella consists of two species, Francisella tularensis and Francisella philomiragia. F. tularensis is the causative agent of tularemia (also called glandular fever, rabbit fever, tick fever, and deer fly fever) in animals and humans. F. tularensis is subdivided into four subspecies that are subdivided based on their biochemical properties. F. tularensis is a very small (0. 2 × 0. 2 to 0. 7 μm), faintly staining, gram-negative coccobacillus. The organism is nonmotile, has a thin lipid capsule, and has fastidious growth requirements (i. e. , most strains require cysteine for growth). It is strictly aerobic and requires 3 or more days before growth is detected in culture.

Francisella tularensis Biology, Virulence, and Disease • Very small, gram-negative coccobacilli (0. 2 × 0. 2 to 0. 7 μm) • Strict aerobe; nonfermenter • Requires specialized media and prolonged incubation for growth in culture • Antiphagocytic capsule • Intracellular pathogen resistant to killing in serum and by phagocytes • Clinical symptoms and prognosis determined by route of infection: ulceroglandular, oculoglandular, typhoidal, oropharyngeal, gastrointestinal, pneumonic Epidemiology • Wild mammals, domestic animals, birds, fish, and blood-sucking arthropods are reservoirs; rabbits, cats, hard ticks, and biting flies are most commonly associated with human disease; humans are accidental hosts • Worldwide distribution; most common in United States in Oklahoma, Missouri, and Arkansas • Approximately 100 cases seen in United States, although the actual number may be much higher • The infectious dose is small when exposure is by arthropod, through skin, or by inhalation; large numbers of organisms must be ingested for infection by this route

Diagnosis • Microscopy is insensitive • Culture on cysteine-supplemented media (e. g. , chocolate agar, BCYE agar) is sensitive and specific if prolonged incubation is used • Serology can be used to confirm the clinical diagnosis; fourfold increase in titer or single titer >1: 160; high titers can persist for months to years; cross-reactive with Brucella Treatment, Prevention, and Control • Gentamicin is the antibiotic of choice; fluoroquinolones (e. g. , ciprofloxacin) and doxycycline have good activity; penicillins and some cephalosporins are ineffective • Disease prevented by avoiding reservoirs and vectors of infection; clothing and gloves are protective • Live attenuated vaccine available but rarely used in human disease

Brucella Brucellosis: initial nonspecific symptoms of malaise, chills, sweats, fatigue, myalgias, weight loss, arthralgias, and fever; can be intermittent (undulant fever); can progress to systemic involvement (gastrointestinal tract, bones or joints, respiratory tract, other organs) • Brucella melitensis: severe, acute disease with complications common • Brucella abortus: mild disease with suppurative complications • Brucella suis: chronic, suppurative, destructive disease • Brucella canis: mild disease with suppurative complications Francisella • Ulceroglandular tularemia: painful papule develops at site of inoculation and progresses to ulceration; localized lymphadenopathy • Oculoglandular tularemia: following inoculation into the eye (e. g. , rubbing eye with a contaminated finger), painful conjunctivitis develops, with regional lymphadenopathy • Pneumonic tularemia: pneumonitis with signs of sepsis develops rapidly after exposure to contaminated aerosols; high mortality unless promptly diagnosed and treated

• Ulceroglandular tularemia is the most common manifestation. The skin lesion, which starts as a painful papule, develops at the site of the tick bite or direct inoculation of the organism into the skin (e. g. , a laboratory accident). • The papule then ulcerates and has a necrotic center and raised border. Localized lymphadenopathy and bacteremia are also typically present (although bacteremia may be difficult to document). • Oculoglandular tularemia is a specialized form of the disease and results from direct contamination of the eye. The organism can be introduced into the eyes, for example, by contaminated fingers or through exposure to water or aerosols. Affected patients have a painful conjunctivitis and regional lymphadenopathy. • Pneumonic tularemia results from inhalation of infectious aerosols and is associated with high morbidity and mortality unless the organism is recovered rapidly in blood cultures (it is generally difficult to detect in respiratory cultures). • There is also concern that F. tularensis could be used as a biologic weapon. As such, creation of an infectious aerosol would be the most likely method of dispersal.

Laboratory Diagnosis • • • The collection and processing of specimens for the isolation of F. tularensis are hazardous for both the physician and the laboratory worker. The organism, by virtue of its small size, can penetrate through unbroken skin and mucous membranes during collection of the sample, or it can be inhaled if aerosols are produced (a particular concern during processing of specimens in the laboratory). Although tularemia is rare, laboratory-acquired infections are disproportionately common. Gloves should be worn during collection of the specimen (e. g. , aspiration of an ulcer or lymph node), and all laboratory work (both initial processing and identification tests) should be performed in a biohazard hood. • Detection of F. tularensis in Gram-stained aspirates from infected nodes or ulcers is almost always unsuccessful, because the organism is extremely small and stains faintly. A more sensitive and specific approach is direct staining of the clinical specimen with fluorescein-labeled antibodies directed against the organism. • Polymerase chain reaction (PCR)-based assays are under development but are not widely available at this time. This may change rapidly with the increased interest in the development of diagnostic tests for this organism in the event of a bioterrorist attack. • It has been stated that F. tularensis cannot be reliably recovered on common laboratory media because the organism requires sulfhydryl-containing substances (e. g. , cysteine) for growth. However, F. tularensis can grow on chocolate agar or buffered charcoal yeast extract (BCYE) agar, media supplemented with cysteine that are used in most laboratories. • Cultures of respiratory specimens will be positive if appropriate selective media are used to suppress the more rapidly growing bacteria from the upper respiratory tract. F. tularensis also grows on the selective media used for Legionella (e. g. , BCYE agar). Aspirates of lymph nodes or draining sinuses are usually positive if the cultures are incubated for 3 days or longer.

To prevent infection, • people should avoid the reservoirs and vectors of infection (e. g. , rabbits, ticks, biting insects), but this is often difficult. • At a minimum, people should not handle ill-appearing rabbits and should wear gloves when skinning and eviscerating animals. • Because the organism is present in the arthropod's feces and not saliva, the tick must feed for a prolonged time before the infection is transmitted. Prompt removal of the tick can therefore prevent infection. Wearing protective clothing and using insect repellents reduce the risk of exposure. Persons who have a high risk of exposure (e. g. , exposure to an infectious aerosol) should be treated with prophylactic antibiotics. • • Live attenuated vaccines are not completely effective in preventing disease but can lessen the severity of the disease. These are recommended for people at a significantly increased risk of exposure to the organism. Inactivated vaccines do not elicit protective cellular immunity.

Brucella Biology, Virulence, and Disease • • • Very small, gram-negative coccobacilli (0. 5 × 0. 6 to 1. 5 μm) Strict aerobe; nonfermenter Requires complex media and prolonged incubation for in vitro growth Intracellular pathogen that is resistant to killing in serum and by phagocytes Smooth colonies associated with virulence Epidemiology • • • Animal reservoirs are goats and sheep (Brucella melitensis); cattle and American bison (Brucella abortus); swine, reindeer, and caribou (Brucella suis); and dogs, foxes, and coyotes (Brucella canis) Infects animal tissues rich in erythritol (e. g. , breast, uterus, placenta, epididymis) Worldwide distribution, particularly in Latin America, Africa, the Mediterranean basin, Middle East, and Western Asia Vaccination of herds has controlled disease in the United States Most disease in the United States is reported in California and Texas in travelers from Mexico Individuals at greatest risk for disease are people who consume unpasteurized dairy products, people in direct contact with infected animals, and laboratory workers

Diagnosis • Microscopy is insensitive • Culture (blood, bone marrow, infected tissue if localized infection) is sensitive and specific if prolonged incubation is used (minimum of 3 days to 2 weeks) • Serology can be used to confirm the clinical diagnosis; fourfold increase in titer or single titer >1: 160; high titers can persist for months to years; cross-reactive with other bacteria Treatment, Prevention, and Control • Recommended treatment is doxycycline combined with rifampin for a minimum of 6 weeks for nonpregnant adults; trimethoprim-sulfamethoxazole for pregnant women and for children younger than 8 years • Human disease is controlled by eradication of the disease in the animal reservoir through vaccination and serologic monitoring of the animals for evidence of disease; pasteurization of dairy products; and use of proper safety techniques in clinical laboratories working with this organism

Laboratory Diagnosis Specimen Collection • Several blood samples should be collected for culture and serologic testing. Bone marrow cultures and cultures of infected tissues may also be useful. To ensure safe handling of the specimen, the laboratory should be notified if brucellosis is suspected. Microscopy • Brucella organisms are readily stained using conventional techniques, but their intracellular location and small size make them difficult to detect in clinical specimens. Currently, specific immunofluorescent antibody tests are not available. Culture • Brucella organisms grow slowly during primary isolation. The organisms can grow on most enriched blood agars and occasionally on Mac. Conkey agar; however, incubation for 3 or more days may be required. Blood cultures should be incubated for 2 weeks before they are considered negative. • Preliminary identification of Brucella is based on the isolate's microscopic and colonial morphology, positive oxidase and urease reactions, and reactivity with antibodies directed against B. abortus and B. melitensis, B. abortus, and B. suis will react with antisera prepared against B. abortus or B. melitensis

Antibody Detection • Subclinical brucellosis and many cases of acute and chronic diseases are identified by a specific antibody response in the infected patient. Antibodies are detected in virtually all patients. • Immunoglobulin M (Ig. M) response is initially observed, after which both Ig. G and Ig. A antibodies are produced. Antibodies can persist for many months or years; thus a significant increase in the antibody titer is required to provide definitive serologic evidence of current disease. • A presumptive diagnosis can be made if there is a fourfold increase in the titer or a single titer is greater than or equal to 1: 160. High antibody titers (1: 160 or more) are noted in 5% to 10% of the population living in endemic areas; thus serologic tests should be used to confirm the clinical diagnosis of brucellosis and not to form the basis of the diagnosis. • The antigen used in the Brucella serum agglutination test (SAT) is from B. abortus. Antibodies directed against B. melitensis or B. suis cross-react with this antigen; however, there is no cross-reactivity with B. canis.

Control of human brucellosis is accomplished through control of the disease in livestock. This requires • systematic identification (by serologic testing) • elimination of infected herds • animal vaccination (currently with the rough strain of B. abortus strain RB 51). • The avoidance of unpasteurized dairy products, • the observance of appropriate safety procedures in the clinical laboratory, • the wearing of protective clothing by abattoir workers are further ways to prevent brucellosis. • The live attenuated B. abortus and B. melitensis vaccines have been used successfully to prevent infection in animal herds. • Vaccines have not been developed against B. suis or B. canis, and the existing vaccines cannot be used in humans because they produce symptomatic disease. The lack of an effective human vaccine is of concern, because Brucella (as well as Francisella) could be used as an agent of bioterrorism.

LEGIONELLA Biology, Virulence, and Disease • Slender, pleomorphic, nonfermentative, gram-negative rods • Stains poorly with common reagents • Nutritionally fastidious with requirement for l-cysteine and enhanced growth with iron salts • Capable of replication in alveolar macrophages (and amoebae in nature) • Prevents phagolysosome fusion • Responsible for legionnaires disease and Pontiac fever Epidemiology • Capable of sporadic, epidemic, and nosocomial infections • Commonly found in natural bodies of water, cooling towers, condensers, and water systems (including hospital systems) • Estimated to be between 10, 000 and 20, 000 cases of infection in United States annually • Patients at high risk for symptomatic disease include patients with compromised pulmonary function and patients with decreased cellular immunity (particularly transplant patients)

Diagnosis • Microscopy is insensitive • Antigen tests are sensitive for L. pneumophila serogroup 1 but have poor sensitivity for other serogroups and species • Culture on buffered charcoal yeast extract (BCYE) agar is the diagnostic test of choice • Seroconversion must be demonstrated; this can take as long as 6 months to develop; positive serology may persist for months • Nucleic acid amplification assays are as sensitive and specific as culture Treatment, Control, and Prevention • Newer macrolides (e. g. , azithromycin, clarithromycin) or fluoroquinolones (e. g. , ciprofloxacin, levofloxacin) are the treatment of choice • Decrease environmental exposure to reduce risk of disease • For environmental sources associated with disease, treat with hyperchlorination, superheating, or copper-silver ionization

• The bacteria are commonly present • • in natural bodies of water such as lakes and streams in air conditioning cooling towers and condensers and in water systems (e. g. , showers, hot tubs). • Human infections are most commonly associated with exposure to contaminated aerosols (e. g. , air conditioning cooling towers, whirlpool spas, shower heads, or water misters). • The organisms can survive in moist environments for a long time, at relatively high temperatures, and in the presence of disinfectants such as chlorine. • One reason for their survival is that the bacteria can parasitize amoebae in the water and replicate in this protected environment (similar to their replication in human macrophages). • The bacteria also survive in biofilms that develop in the pipes of water systems.

• Asymptomatic Legionella infections are believed to be relatively common. Symptomatic infections primarily affect the lungs and present in one of two forms • an influenza-like illness (referred to as Pontiac fever) • a severe form of pneumonia (i. e. , legionnaires disease). Pontiac Fever • L. pneumophila was responsible for causing a self-limited, febrile illness in people working at the Pontiac, Michigan, Public Health Department in 1968. • Fever, chills, myalgia, malaise, and headache, without clinical evidence of pneumonia, are characteristic of the disease. • The symptoms developed over 12 hours, persisted for 2 to 5 days, and then resolved spontaneously without antibiotic treatment and with minimal morbidity and no deaths. • The precise pathogenesis of this syndrome is unknown, although it is believed that the pathology of this disease is caused by a hypersensitivity reaction to bacterial toxin (e. g. , endotoxin).

Legionnaires disease (legionellosis) • is characteristically more severe than Pontiac fever and if untreated, promptly causes considerable morbidity, often leading to death in 15% of previously healthy individuals and up to 75% in immunocompromised patients. • • After an incubation period of 2 to 10 days, systemic signs of an acute illness appear abruptly (e. g. , fever and chills, a dry, nonproductive cough, headache). Multiorgan disease involving the gastrointestinal tract, central nervous system, liver, and kidneys is common. Pulmonary function steadily deteriorates in susceptible patients with untreated disease. The clinical presentation of pneumonia caused by Legionella is not unique, so laboratory tests are required to confirm the diagnosis.

Laboratory Diagnosis Microscopy • Legionellae in clinical specimens stain poorly with Gram stain and the small, intracellular bacteria are rarely recognized. The organism will stain with nonspecific methods, such as Dieterle silver stain, but this stain is used with tissue specimens and not sputum. • The most sensitive way of detecting legionellae microscopically in clinical specimens is to use the direct fluorescent antibody (DFA) test, in which fluorescein-labeled monoclonal or polyclonal antibodies directed against Legionella species are used. Because of the problems with the DFA test, antigen detection tests are now used in most laboratories. • Urinary Antigen Tests • Immunoassays are used to detect soluble Legionella serogroup 1 -specific lipopolysaccharide antigens excreted in the urine of infected patients. The sensitivity of these assays for L. pneumophila serogroup 1 is relatively high (range, 60% to 90%), particularly with concentrated urines, but the assays cannot be used to detect other serogroups or Legionella species. • • • This is an important distinction because L. pneumophila serogroup 1 is responsible for 80% to 90% of community-acquired infections but is responsible for fewer than 50% of hospital-acquired infections. Antigens persist in the urine of treated patients, with almost 50% of patients remaining positive at 1 month and 25% at 2 months. Persistence is particularly common with immunosuppressed patients, in whom antigens can persist for up to 1 year.

Nucleic Acid Amplification Assays • Nucleic acid amplification (NAA) assays are highly specific and have a sensitivity equivalent to culture for the detection of Legionella species in respiratory secretions (i. e. , bronchial alveolar lavage [BAL] fluid). • • • Culture Although legionellae were difficult to grow initially, commercially available media now make culture easy (test sensitivity 80% to >90%). The medium most commonly used for the isolation of legionellae is buffered charcoal yeast extract (BCYE) agar.

Prevention of legionellosis requires identification of the environmental source of the organism and reduction of the microbial burden: • Hyperchlorination of the water supply • the maintenance of elevated water temperatures • Elimination of Legionella organisms from a water supply is often difficult or impossible to achieve. • Hospitals with patients at high risk for disease should monitor their water supply on a regular basis for the presence of Legionella and their hospital population for disease. If hyperchlorination or superheating of the water does not eliminate disease (complete elimination of the organisms in the water supply is probably not possible), continuous copper-silver ionization of the water supply . may be necessary