CLINICAL BACTERIOLOGY GRAM NEGATIVE BACILLI OPTIMALLY RECOVERED ON SPECIAL MEDIA OUTLINE • Brucella, Bordetella, Francisella o Brucella spp. o Bordetella spp. o Francisella spp. • Campylobacter, Helicobacter, Legionella o Campylobacter o Helicobacter o Legionella Brucella, Bordetella, Francisella Brucella spp. • General Characteristics o Small, non-motile, aerobic, gram-negative coccobacilli or short rods that stain poorly by conventional gram stain o Many isolates requires Carbon Dioxide (CO2) for growth o Brucella spp. are considered potential bioterrorism agents EPIDEMIOLOGY AND PATHOGENESIS • Brucellosis o Is a zoonosis and is recognized as a cause of economic losses among domestic livestock o Humans become affected in 3 primary routes ▪ Ingested of unpasteurized animal milk products (Most Common) ▪ Inhalation of infected aerosolized particles ▪ Direct contact with infected animal parts through ruptures of skin and mucous membranes o Brucellosis are facultative, intracellular parasites (able to exist in both extracellular and intracellular environments) o Following infection, brucellae are ingested by neutrophils within which they replicate resulting in cell lysis. o Brucellae exhibit a tendency to invade and persistin the human host through inhibiting apotosis o Brucella spp. can change from smooth to rough colonial morphology based on the composition of their cell wall lipopolysaccharide; those with smooth LPS are more resistant to intracellular killing by neutrophils than those with rough LPS Organism Preferred Animal Host Brucella abortus Cattle Brucella melitensis Sheep or Goats Brucella suis Swine Brucella canis Dogs SPECTRUM OF DISEASE • Brucellosis is a systematic infection than can involve any organ • Symptoms are nonspecific and includes fever, chills, weight loss, sweats, headaches, muscle aches, fatique and depression • Lymphadenopathy and splenomegaly are common physical findings • 2-3 weeks is the incubation period DIRECT DETECTION METHOD • Direct stains of clinical specimens are not particularly useful • Conventional and Real-time Polymerase Chain Reaction (PCR) assays indicate that these assays may prove to be reliable, sensitive and specific means directly to detect Brucella spp. IDENTIFICATION • On Gram Stain, organisms are small coccobacilli that resembles fine grains of sand • Most strains are oxidase-positive • Brucella spp. are non-motile, urease- and nitrate-positive, and strictly-aerobic • Brucella spp. are differentiated by the rapidity with which they hydrolyze urea, relative ability to produce H 2 S, requirements for CO 2 , and susceptibility to the aniline dyes and basic fuchsin • Most B. abortus do not grow in air but show growth in the candle jar OTHER INFORMATION • The species of this genus are important human and animal pathogens. • They are obligate aerobes and intracellular parasites. • They are non-motile, assacharolytic, and non-encapsulated; some species require an increased supply of CO2 for growth. • Preferred specimen for isolation: blood and bone marrow • Microscopy: small coccobacilli that are arranged singly, in pairs or in short chains, and which have “sandy appearance” • Culture: BAP – colonies are small, convex, translucent, yellowish and non-hemolytic; may become brownish in color with age. • Biochemical Test: (+) catalase and oxidase; rapid urease producer • Disease: Malta/Crimean/Mediterranean fever or undulant fever (Brucellosis) • Most virulent species are B. melitensis and B. suis
Bordetella spp. GENERAL CHARACTERISTICS • The species in this genus are obligatory aerobic, fastidious gram-negative coccobacilli. • Replicate on ciliated respiratory epithelial cells of humans. • Culture: Bordet-Gengou agar – colonies are smooth, glistening, and have silver color. • Biochemical test: (+) catalase, (-) indole • Growth factors: Nicotinic Acid, Cysteine, and methionine • Species: B. pertussis, B. parapertussis, B. bronchiseptica, B. avium Bordetella pertussis • Bordet-Gengou bacillus • It is the etiologic agent of whooping cough. • It only infects and causes disease in humans. • It does not survive well in outside the host. • Culture: Bordet-Gengou agar- colonies are small and shiny, resembles “mercury drops” • Preferred specimen for isolation: Nasopharyngeal swab • Related infection: Whooping cough (Pertussis) o It is highly contagious, acute infection of the upper respiratory tract (URT) and is a disease that primarily affects children. o Mode of acquisition: Inhalation of infected droplets. o Incubation period: 7 to 14 days • Three stages of whooping cough : o Catarrhal stage – it is a highly communicable stage that is characterized by mucous membrane inflammation and mild coughing with runny nose o Paroxysmal stage – it is associated with vomiting and “whooping” or hurried, deep respiration that may last for six weeks. Infected individuals experience severe coughing for at least 15 to 25 times in 24 hours. o Convalescent stage – this is the stage in which the symptoms slowly decline. This period may last for six months after infection. LABORATORY DIAGNOSIS • Specimens : Nasopharyngeal swab and Broncho alveolar lavage. • Gram stain : The use of two-minute safranin or 0.2% basic fuchsin as counter stain enhances its visibility. • Culture : o Culture media: Regan-Lowe agar, Bordet-Gengou potato infusion agar, modified Jones-Kendrick charcoal agar, and Casamino acid broth. o B. pertussis and B parapertussis have hemolytic reactions in Bordet-Gengou potato infusion agar. o The favorable transport and enrichment medium is Regan-Lowe agar. o The Casamino acid broth is used for transporting swab specimens. o The species cannot grow on MAC except: B. bronchiseptica because it is less fastidious. • Differential Tests for Bordetella spp. Francisella spp. GENERAL CHARACTERISTICS • Francisella spp. are facultative, intracellular pathogensthat requires cysteine and a source of iron for growth • Blood, Chocolate and MacConkey agars cannot be used for primary isolation of Fransicella spp. • Fransicella spp. are faintly staining, gram-negative coccobacilli that are nonmotile and obligately aerobic EPIDEMIOLOGY AND PATHOGENESIS • Francisella tularensis is the agent of human and animal tularemia • Humans become infected by handling the carcasses or skin of infected animals, through insect vectors, by being bitten by carnivores that have themselves eaten infected animals, or by inhalation
• Virulence factor of Francisella spp. is its capsule, allowing the organism to avoid immediate destruction by neutrophils • F. tularensis is an intracellular parasite that can survive in the cells of reticuloendothelial system, where it resides fter a bacteremic phase • Granulomatous lesions may develop in any organ • F. tularensis subsp. tularensis are the most virulent for humans with an infectious dose of less than 10 colony forming units • F. philomiragia has been isolated from several patients such as immunocompromised or were victims of near-drowning incidents • The organism is present in animal and ground water • F. tularensis is a Biosafety level 2 pathogen SPECTRUM OF DISEASE • Following inoculation o F. tularensis , a lesion appears at the site and progresses to ulcer; lymph nodes becomes enlarged and often necrotic • Once organism enters the bloodstream, patients become systematically ill with high temperature, chills, headache and generalized aching SPECIMEN COLLECTION • The most common specimens submitted to the laboratory are scrapings from infected ulcers, lymph nodes, biopsies and sputum • Isolation of F. tularensis from blood cultures might be considered as a potential bioterrorist attack DIRECT DETECTION METHODS • Fluorescent antibody staining is available for direct detection of organism in lesion smears • PCR assays have been developed to detect F. tularensis directly in clinical specimens CULTIVATION • Organism requires prolonged incubation in Chocolate agar • Oxidase-negative, weak or negative in catalase test • Requires cysteine and cystine for growth APPROACH TO IDENTIFICATION • Colonies are transparent, mucoid, and easily emulsified • F. philomiragia differs from F. tularensis biochemically; F. philomiragia is oxidase-positive and most strains produce hydrogen sulfide in Triple Sugar Iron agar medium, hydrokyzes gelatin, and grow in 6% sodium chloride (where F. tularensis are all negative) ANTIMICROBIAL SUSCEPTIBILITY TEST AND THERAPY • The organism is susceptible to aminoglycosides and streptomycin is the drug of choice • Gentamycin is a possible alternative drug; doxycycline and chlorampenicol also have been used, although these two agents have been associated with a higher rateof relapse after treatment • Fluoroquinolones appear promising for treatment of even severe tularemia Campylobacter, Helicobacter, Legionella Campylobacter • Faintly staining gram negative bacilli • Small • Curved or seagull-winged like • Darting motility TESTS FOR IDENTIFICATION • Most species of Campylobacter species require selective media, such as Campylobacter blood agar (Becton- Dickinson) • It contains a blood agar base with the antibiotic vancomycin, polymyxin, cephalothin, trimethoprim and amphotericin B added. • Catalase and Nitrate Test o Catalase test demonstrate the presence of catalase, an enzyme that catalyses the release of oxygen from hydrogen peroxide (H2O2). o Nitrate test is used to determine the ability of an organism to reduce nitrate (NO3) to nitrite (NO2) using the enzyme nitrate reductase o Most species of Campylobacter are positive such as C.jejuni subso. jejuni, C.coli and C. fetus subsp.fetus.
• Urease Test o The urease test identifies those organisms that are capable of hydrolyzing urea to produce ammonia and carbon dioxide. Campylobacter species are negative to this test • Hippurate Hydrolysis o It is used in the presumptive identification of Gardnerella vaginalis, Campylobacter jejuni, Listeria monocytogenes and group B streptococci, by detecting the ability of the organism to hydrolyze hippurate o C.jejuni subsp. jejuni is the only positive among Campylobacter species • Biochemical Results CLINICAL SIGNIFICANCE • C. conciscus, C. curvus, C. rectus, C. showae - periodontal disease; gastroenteritis • C. graciis - deep tissue infection of head, necl, and viscera; gingival crevices • C. coli - gastroenteritis, septicemia • C. Jejuni subsp. jejuni - gastroenteritis; septicemia, menigitis; proctitis • C. Jejuni subsp. doylei - gastroenteritis; septicemia, gastritis • C. lari - gastroenteritis; septicemia; prosthetic joint infection • C. Hyointestinalis subsp. hyoinstetinalis - gastroenteritis • C. upsaliensis - gastroenteritis • C. fetus subsp. fetus - gastroenteritis; septicemia; abortion; meningitis • C. fetus subsp. venerealis - septicemia • C. sputorum biovar sputorum - abscesses, gastroenteritis Helicobacter • Previously known as Campylobacter pylori • Gram negative, helically shaped, microaerophilic bacterium • Small, translucent, circular colonies • Affects the human stomach TESTS FOR IDENTIFICATION • For Cultivation o H. cenaedi & H. fennelliae are plated in selective media (CAMPY-CVA) o H. pylori is recovered from tissue biopsy such as gastric antral biopsies, chocolate agar, Brucella agar with 5% sheep blood (Non selective media). Selective agars in Skirrow’s and Modified Thayer Martin’s agar. o Cultivation up to 1 week in a moistened, microaerobic atmosphere at 35° to 37° C may be necessary before growth of human pathogen is observable. • For Serologic o Numerous EIAs for detection of IgA and IgG. • (+) results for oxidase, catalase, and rapid urease tests
CLINICAL SIGNIFICANCE • H. cinaedi - proctitis, enteritis, and sepsis (homosexual men); septicemia, cellulitis, meningitis (immunocompromised) • H. fennelliae - septic shock (non-HIV infected heterosexual immunocompromised patients) • H. pylori - peptic ulcer disease, gastritis, and gastric cancer Legionella • Gram negative bacilli which are faintly staining, thin, and motile • Requires medium containing Iron and L-Cysteine, buffered to pH 6.9 for optimum growth. TEST FOR IDENTIFICATION • Inoculated in two agar plates: o BCYE with charcoal, cysteine, yeast extract, a-ketoglutarase, and iron o BCYE base with polymyxin B, anisomycin, and cefamandole o Incubated in candle 35 to 37 C o Colonies should be visible at 3 - 4 days • Considered to be Biochemically Inert o Monoclonal Immunoflourescent Stain ▪ Used to identify P. pneumophila ▪ Emulsion of organism in 10% neutral formalin, diluted 1:100 and placed on slides for flourescent antibody staining. CLINICAL SIGNIFICANCE • Pneumonia o 10- 20% fatality rate. Also know as “Legionnaire’s Disease” o Legionnaire’s Disease ▪ Severe form of pneumonia. Also known as “Legionella” • Pontiac Fever o Self limited, non fatal respiratory infection • Other infections: Wound abscess, Encephalitis, Endocarditis INTRODUCTION • Legionella organisms are fastidious, narrow, gram negative, non-spore forming bacilli that are motile by one or more polar or subpolar flagella. The family Legionella contains a single genus, Legionella which includes the species L. pneumophila, the etiologic agent of Legionnaire's disease. Legionnaire's disease is an important cause of severe pneumonia, with majority of cases being sporadic. Bordetella organisms are aerobic, oxidative obligate parasites in animals and humans. The organisms bind to the ciliated epithelial cells in the mucous membranes of the respiratory tract. Except for B. avium and B. bronchiseptica, all seven species are capable of infecting humans. Human infection with B. bronchiseptica usually involves transmission from animals. The most important human species is B. pertussis, the agent of pertussis, is found only in humans. Brucella species are obligate parasites and can survive intracellularly in the reticuloendothelial system, bone , liver, and central nervous system of humans . The organism causes undulant fever/brucellosis, which is a chronic and recurring fever that causes rising and falling fevers, following a pattern that is described as a wave. Brucella is a biosafety level 3 biohazard, which includes common and unusual pathogens; the WHO identifies Brucella as a pathogen that presents a high risk to laboratory workers. Francisella tularensis, a small, non-motile pleomorphic gram negative bacillus, is the agent of tularemia, a disease of rodents, primarily rabbits. This zoonosis is acquired through direct contact with blood or through an animal bite or scratch. Infection also may result from the ingestion of contaminated water or food or inhalation of contaminated aerosols. It is highly infective when grown in culture, and extreme caution must be used in the clinical laboratory to avoid laboratory acquired infection.
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Clinical Bacteriology - 08 Gram Negative Bacilli on Special Media.pdf