Gram-Negative Bacilli and Coccobacilli: MacConkey Negative

CLINICAL BACTERIOLOGY Eikenella, Pasteurella, Moraxella GRAM NEGATIVE BACILLI AND COCCOBACILLI (MACCONKEY NEGATIVE, OXIDASE POSITIVE/VARIABLE) Eikenella Eikenella corrodens OUTLINE • Introduction • Eikenella, Pasteurella, Moraxella o Eikenella o Pasteurella o Moraxella o Summary of Biochemical Tests • Haemophilus spp. o Haemophilus influenzae o Haemophilus aegyptius o Haemophilus haemolyticus o Haemophilus parahaemolyticus o Haemophilus parainfluezae o Haemophilus ducreyi • • • • It is a normal flora of Human oral cavity The organism ia facultative anaerobe, non-motile, gram negative rod. It is an opportunitistic pathogen predominantly in immunocompromised patients, causes abscess and infections that may lead to death. Microscopic appearance o Gram negative rod o Non-motile o Facultative anaerobe INTRODUCITON • • The so-called HACEK group which consists of the following: Haemophilus, Aggregatibacter, Cardiobacterium and Capnocytophaga, Eikenella, and Kingenella are organisms that colonize the oropharynx; these are group of slow-growing gram-negative bacilli whose growth is enhanced under increased carbon dioxide. The HACEK bacteria are rare causes of bacterial endocarditis and peritonitis. Generally, infection occurs after the bacteria enter a normally sterile site. These infections are usually treated with beta lactam antibiotics with an amino glycoside. The genus Haemophilus on the other hand consists of tiny gram-negative coccobacilli that are non-motile. In clinical cultures, the organism may form long, threadlike or filamentous strands. The organisms are facultative anaerobes, reduce nitrates and utilize carbohydrates either fermentatively or by using oxygen as the final hydrogen ion acceptor. Most species are catalase and oxidase positive and prefer incubation at 35-37 degC with 5-10% Carbon dioxide. Haemophilus species are very sensitive to during and chilling; thus abrupt fluctuations in temperature must be avoided. Many species of haemophilus are non-pathogenic, or opportunistic pathogens. Haemophilus influenzae and Haemophilus ducreyi are the two most clinically significant pathogens in this genera. Haemophilius, which means blood loving, has special growth requirements, which include a group of iron-containing compounds known as hemin, hematin, or protoporphyrin X. These compounds, commonly known as X factors, are heat stable products that are released upon the breakdown of hemoglobin. Most Haemopholus species also require nicotinamide-adenine-dinucleotide (NAD) or con-enzyme I, also known as V factor, NAD is heat labile compound produced by certain bacteria and yeast. It can also be obtained through exogenous sources, such as potato or yeast extracts. • Colony appearance o Tiny at 24hrs but when the mature, colonies have moist, clear center. o The colonies pit/corrode the agar o Yellow in older culture o Bleach in odor. • Tests for identification o Catalase test – negative o Oxidase test – Positive o Indole test – negative o Urease test - Negative Clinical significance o Human bite wound infection o Head and neck infections o Can also cause endocarditis • Moraxella • • Moraxella organisms are from the family Moraxellaceae and often confused with Neisseria spp. because of its resemblance morphologically and phenotypically. Most Moraxella spp. are normal mucosal flora that rarely cause infections but still considered as potential contaminants. • • • • • o • o Moraxella species include: o Moraxella nonliquefaciens o Moraxella lacunata o Moraxella osloensis o Moraxella lincolnii o Moraxella canis o Moraxella atlantae Common characteristics of the Moraxella genus include a lack of colony pigmentation; Gram negative staining coccobacillus and Bacillus morphology except M. catarrhalis, which exhibits a coccoid morphology. Moraxella species have been isolated from the conjunctiva, upper respiratory tract, blood, inflammatory secretions of the middle ear, maxillary sinus, bronchial aspirate, nasal cavity, spleen, cerebrospinal fluid, genitourethral tract, joints and bursa of humans. Microscopic characteristics: o Gram negative that tends to resist decolorization o Coccobacilli,diplococci or rods of often very short and plump approaching a coccus o shape 1.0 - 1.5 x 1.5 - 2.5µm o Cells may be capsulated o Non-motile o Strictly aerobic o Asaccharolytic Test for Identification o Gram Stain Penicillin Disk Test- if you make a Gram stain smear from colonies growing around a penicillin disk (above), Moraxella spp. will form long filaments or spindle-shaped cells. • Biochemical Tests o Oxidase Test +ve DNase Test +ve for M. catarrhalis only Clinical Significance o Moraxella lacunata ▪ Conjunctivitis ▪ Keratitis ▪ Endocarditis o Moraxella oloensis ▪ Arthritis ▪ Meningitis o Moraxella nonliquefaciens ▪ Upper & Lower Respiratory Tract Infections o Moraxella catarrhalis ▪ Chronic Lung Disease o Moraxella canis ▪ Wound infection Pasteurella o Tributyrin Test +ve • General Characteristics and taxonomy o Small, gram - , non-motile, oxidase +, bacilli o Will not grow on MacConkey agar EPIDEMIOLOGY, SPECTRUM OF DISEASE, ANTI-MICROBIAL TESTING, SUSCEPTIBILITY TESTING, BIOCHEMICAL TESTING • • • • • • • • • • • • Both domestic and wild animal flora and are transmitted to human during close animal contact and bites Opportunistic pathogens: transmitted via wounds Commonly encountered species: P. multocida subs. multocida Virulence Factors: o Lipopolysaccharide o Cytotoxin o Antiphagocytic capsule o Surface antigen o Iron-acquisition protein Other manifestation: o Respiratory Disease o Endocarditis o Septicemia Susceptible: Penicillin; Resistant: Erythromycin Culture Media of choice: Tryptic Soy Agar supplemented with 5% Sheep’s Blood (Blood and Chocolate Agar) incubated at 35 degrees celsius or in an enriched environment 5% CO2 for 24 hours Habitat: Found in the naspharynx and GIT of wild and domestic animals Mode of transmission: Bite or scratch usually from veterinary host such as felines Colonial Appearance: Convex, smooth, grey, nonhaemolytic; rough and mucoid variants have musty or mushroom odor Haemophilus spp. Haemophilus influenzae • • Microscopic characteristics: o Gram Negative coccobacilli and a facultative anaerobic pathogen o Gram Staining - small, gram-negative, pleomorphic coccobacilli with polymorphonuclear cells o Non- Motile ; has both X and V factor o Capsule : Anti Phagocytic ; most common - Type B o Additional envelope factors allow its attachment to host cells. Test for Identification: o Colony appearance: Unencapsulated strains are small, smooth, and translucent at 24 hours; encapsulated strains form larger, more mucoid colonies; mouse nest odor; non hemolytic on rabbit or hors blood agar. o Positive for: ▪ X and V Factor ▪ Beta – Hemolytic on Rabbit Blood Agar ▪ Catalase ▪ Glucose ▪ Xylose o Negative for: ▪ Lactose ▪ Sucrose ▪ Mannose ▪ Beta - galactosidase Clinical Significance: o Encapsulated strains: ▪ Meningitis ▪ Epiglottitis ▪ Cellulitis with bacteremia ▪ Septic arthritis ▪ Pneumonia o Nonencapsulated strains: ▪ Localized infections ▪ Otitis media ▪ Sinusitis ▪ Conjunctivitis ▪ Immunocompromised patients: Chronic bronchitis, Pneumonia, Bacteremia Hamophilus influenzae on Gram stain • Haemophilus influenzae on Blood Agar • • Infection from a bite wound SUMMARY OF BIOCHEMICAL TESTS • • • • • • All are indole + except: M. haemolytica, P. aerogenes, P. caballi All reduces nitrate + except: S. Indologes All are catalase + except: P. bettyae, P. caballi All are mannitol - except: M. haemolytica, P. caballi All are sucrose fermenters + except: P. bettyae All are maltose fermenters + except: P. bettyae, P. Canis, P. canis, P. multocida, P. Stomatis Haemophilus aegypticus • • • Microscopic characteristics: o Non motile; Pleomorphic, spherical, oval, coccobacilli and rod-shaped; sometimes forming threads or filaments. Test for identification: o Colonial Appearance: resembles H. influenzae except colonies are smaller at 48 hours o Positive for: ▪ X and V Factor ▪ Beta – Hemolytic on Rabbit Blood Agar ▪ Catalase ▪ Glucose o Negative for: ▪ Lactose ▪ Xylose ▪ Sucrose ▪ Mannose ▪ Beta – galactosidase Clinical Significance: o Conjunctivitis o Brazilian purpuric fever Haemophilus parahaemolyticus • • Haemophilus haemolyticus • • • Microscopic characteristics: o Shape and type of bacteria: Small coccobacilli or short rods with occasional cells appearing as tangled filaments. o Appearance: Appearing like H. influenzae but except beta hemolytic on rabbit and horse agar. o Gram Staining: Gram negative o Motility: Non-motile o Pigment production: non pigment (buff or light tan) Test for identification: o Positive for: ▪ X and V Factor ▪ Beta – hemolytic on Rabbit Blood Agar ▪ Catalase ▪ Glucose o Negative for: ▪ Lactose ▪ Sucrose ▪ Mannose ▪ Beta – galactosidase ▪ Variable reaction to: ▪ Xylose Clinical Significance: o H. haemolyticus is a human commensal bacterium that colonizes the nasopharynx. o They are closely related to the human patogen H. influenzae but has rarely been reported to cause invasive disease. They may cause disease more as a opportunistic pathogen • Microscopic characteristics: o Shape and type of bacteria: Short to medium-length bacilli o Appearance: Resembles H. parainfluenzae; betahemolytic on rabbit or horse blood agar o Gram Staining: gram-negative o Motility: Non-motile o Pigment production: Non pigmented Test for Identification: o Positive for: ▪ V Factor ▪ Beta – hemolytic on Rabbit Blood Agar ▪ Glucose ▪ Sucrose o Negative for: ▪ X Factor ▪ Lactose ▪ Xylose ▪ Mannose o Variable reaction to: ▪ Catalase ▪ Beta – galactosidase Clinical Significance: o Associated with wide variety of infections similar to H. influenzae such as pharyngitis and, rarely, for subacute endocarditis. Haemophilus parainfluezae • • • Microscopic characteristics o Is a non motile, Gram Negative coccobacilli and a facultatively anaerobic pathogen. o It has V Factor. o Gram Staining - small, gram-negative, pleomorphic coccobacilli with polymorphonuclear cells Test for identification o Colony Appearance : Medium to large, smooth, and translucent; non-hemolytic on rabbit or horse blood agar. o Positive for: ▪ V Factor ▪ Glucose ▪ Sucrose ▪ Mannose o Negative for: ▪ X Factor ▪ Lactose ▪ Xylose o Variable reaction to: ▪ Beta – hemolytic on Rabbit Blood Agar ▪ Catalase ▪ Beta - galactosidase Clinical Significance: o Endocarditis o Meningitis o Bacteremia Haemophilus ducreyi • • • Microscopic Characteristics: o a gram-negative, rod-shaped, facultatively anaerobic, nonmotile, pathogenic bacillus Test for Identification: o Positive for: ▪ X factor o Negative for: ▪ V Factor o Variable reaction to: ▪ Carbon Dioxide ▪ Beta – hemolytic on Horse Blood Agar Clinical Significance: o Chancroid - STD