Lecture 8 – Gram-Negative Coccobacilli PDF

BIOL 2010 Lecture 7 – Gram Negative coccobacilli BAILEY AND SCOT T’S – CHAPTER 28, 29, 30,31 DIAGNOSTIC MICROBIOLOGY – CHAPTER 18 In this lecture Explain the characteristics and pathogenesis of commonly encountered clinically significant microorganisms, including the most frequently isolated sp...

BIOL 2010 Lecture 7 – Gram Negative coccobacilli BAILEY AND SCOT T’S – CHAPTER 28, 29, 30,31 DIAGNOSTIC MICROBIOLOGY – CHAPTER 18 In this lecture Explain the characteristics and pathogenesis of commonly encountered clinically significant microorganisms, including the most frequently isolated species by body system Gram negative coccobacilli (All do NOT grow on MAC) Haemophilus influenzae *** Most common organism out of this list Related organisms: HACEK organisms, Capnocytophaga, Pasteurella Discuss bacterial virulence, as related to these organisms  Haemophilus Haemophilus species isolated in Humans H. influenzae H. parainfluenzae H. haemolyticus H. parahaemolyticus H. aphrophilus H. paraaphrophilus H. segnis H. ducreyi H. aegyptius (H. influenzae biogroup) Haemophilus – General Characteristics Facultative Anaerobes Haemophilus = Greek word meaning “blood lover” Most species are part of the normal flora of the upper respiratory tract Pathogenic species: Haemophilus influenzae H. influenzae biogroup aegyptius H. ducreyi (genital tract pathogen) Optimal Growth for most is in 5-10% CO2 at 35+/- 2 degrees Celsius H. aphrophilus requires CO2 Haemophilus – Nutritional requirement Fastidious Bacteria Will NOT grow on routine media ex. BAP, TSA, MAC Require Accessory Growth Factors which are found inside RBC’s X factor (hemin/hematin) V factor (nicotine adenine dinucleotide or NAD) or Both X and V ** Species with the prefix Para require only V factor Haemophilus – Nutritional requirement Media with Horse or rabbit blood Hemolysis of 5% horse or rabbit blood agar will provide the necessary growth factors and can be used for differentiation BAP – made with sheep blood Sheep blood contains X factor that is readily available however the V factor must be supplied Sheep RBC’s contain X and V factors along with enzymes (NADase) that hydrolyze the V factor Needs a source for V factor Haemophilus – Nutritional requirement BAP is routine media so it would be helpful to be able to grow Haemophilus on it On BAP, we can add an additional source of V factor ex. Other organisms When adding a staph streak, fastidious organisms grow as satellites Growth of fastidious organisms around other bacteria that release the necessary growth factors or break down toxic products S. aureus (commonly used – called “Staph Streak’)”, S. pneumoniae, and Neisseria spp. that naturally produce V factor Organisms, which require V factor for growth, will grow adjacent to the V factor supplying organism Haemophilus – Nutritional requirement A common way of supplying the V factor is to place a “staph streak” on the blood agar plate after inoculation The Staphylococcus will supply the NAD (V factor) Organisms that require the V factor will grow close to the staph streak on the blood agar plate producing the phenomenon known as “satellitism” This is a KEY identifying feature of Haemophilus influenzae Haemophilus – Nutritional requirement CHOC agar supplies both X and V factors Recall CHOC is an enriched, non-selective media CHOC includes Nutritional Base with heated RBC’s or other supplements added – meant for fastidious organisms The RBC’s are lysed (during the heating process) which release the factors while deactivating the NADases Therefore, Haemophilus will grow happily in CHOC Haemophilus influenzae Was erroneously named during the influenza worldwide pandemic (1889– 1890) Frequently isolated in infected patients with influenza and from postmortem lung cultures After viral culture techniques were developed, it became clear that influenza was caused by a virus and not the bacteria H. influenzae was actually a secondary (opportunistic) invader H. influenzae is the main pathogen within the genus Multiple strains of H. influenzae possible Characterized by capsule production (typeable strain) or no capsule production (Non- typeable strain) Haemophilus influenzae – virulence factors Capsule Only in typeable strains Six types, based on polysaccharide capsule - Named a, b, c, d, e, f Type B is most common before vaccination (Called Hib) Implicated in meningitis in children Can be typed, if required Haemophilus influenzae – virulence factors Immunoglobulin A (IgA) proteases Destroys sIgA Adherence by fimbriae and other structures Mostly in non-typeable strains Allows organisms to adhere to one are and cause localized damage This is missing in HiB – hence it can cause systemic infections Outer membrane proteins and lipopolysaccharide (LPS) Haemophilus influenzae – Infection Infections are most common in children Mode of Infection Organism enters the body through the nasopharynx Colonize the nasopharynx and may remain only transiently for several months in the absence of symptoms (asymptomatic carrier) Organism can enter the bloodstream and lead to an invasive infection Two Patterns of Disease Invasive disease caused by encapsulated strains A more localized infection caused by non-encapsulated strains Haemophilus influenzae – Infection INVASIVE DISEASE LOCALIZED INFECTION Bacteremia, septicemia Occurs in respiratory tract due to Meningitis vaccination Arthritis Conjunctivitis Epiglottitis** Sinusitis Tracheitis Otitis media pneumonia Can occasionally cause invasive disease ex. meningitis, Haemophilus influenzae – Specimens Specimens of Choice Blood CSF Sputum Conjunctival Swab Transport specimen at room temperature and without delay Organisms known to die rapidly Haemophilus influenzae – Colonial morphology On CHOC agar (top image) Colonies are large, translucent, tannish, moist, smooth and convex Distinct Pungent Odor (mousey or bleach like) Encapsulated strains appear more mucoid On BAP NO staph streak – NO growth With Staph Streak -Satelliting colonies Note: this will serve as first clues when Identifying Haemophilus influenzae – Cellular morphology Pleomorphic – filaments, singles, varied appearance Cells are tiny Gram Negative Cocco-Bacilli (Coccobacilli) Clear nonstaining areas (halos) may be seen - capsules Faint staining – can easily miss! Ensure your microscope is clean  Haemophilus influenzae – ID Remember First clues: TINY Gram-negative pleomorphic coccobacilli on Only grows on CHOC agar NG on MAC, or BAP without staph streak BAP with staph streak = satellites Oxidase - Positive X and V Factor Test Growth around X+V Disk Only No growth around X or V disk Porphyrin Test (ALA)- Negative Catalase - Positive Latex Agglutination and Coagglutination kits used to detect Hib antigen Chromogenic tests – API NH Haemophilus influenzae – ID (Staph Streak) Set up a staph streak test if not already done so – this is a KEY test Premise: BAP consists of X factor but NO V factor Staph streak provides V factor, hence H. influenzae only grow around the Staphylococcus i.e. satellite positive Haemophilus influenzae – ID (X and V Factor) Purpose Identify growth requirements of Haemophilus spp. for speciation Principle A clear agar missing X and V factor is inoculated with target organism Ex. Mueller Hinton and TSA 3 nutrients are added separately (available as discs or filter paper) X factor V factor X and V factor Plate is incubated and observe growth around EACH factor – growth pattern is indicative Haemophilus influenzae – ID (X and V Factor) Results: Growth around X/V factor only: H. influenzae (Right image) Growth around V and X/V factor only: Haemophilus parainfluenzae Growth around X and XV factor: Haemophilus ducreyi Note: Growth will often be a haze and can be easily missed  Haemophilus influenzae – ID (ALA test) Purpose Differentiation of Haemophilus spp. Principle Test determines if an isolate requires X factor Organisms that don’t require X factor are able to convert aminolevulinic acid (ALA) to hemin (ie. produce their own hemin) A tube of ALA or an impregnated disk, agar or broth is inoculated with organism. View under UV light to observe for fluorescence Results: Positive: fluorescence under UV light Negative: NO fluorescence under UV light Quality Control: Positive: Haemophilus parainfluenzae Negative: Haemophilus influenzae Haemophilus influenzae – ID (ALA test) Different variations of ALA tests Haemophilus influenzae – Treatment More than 35% of strains produce β-lactamase resulting in resistance to ampicillin, the drug of choice for treatment of meningitis and bacteremia B lactamase testing is common Antimicrobial treatment for H. influenzae infections that are β-lactamase positive (resistant to the penicillins) include: Recommended Cefotaxime, ceftriaxone and cefuroxime Alternate trimethoprim/sulfamethoxole (SXT), Imipenem, Ciprofloxacin (in conjunction with ampicillin) Haemophilus influenzae – Vaccination Hib vaccination recommended for all children younger than 5 years Children need multiple (3 or 4) shots of a Hib vaccine Pre-Vaccination Era Hib was leading cause of invasive disease in children (under 2 yrs) Most common cause of acute bacterial meningitis in children until mid 1980’s (when vaccination became available) Haemophilus influenzae biogroup aegyptus This is a subgroup of H. influenzae - very closely related to H. influenzae aegyptus Very difficult to separate the two Infections: Acute, contagious conjunctivitis (Pink eye!) Brazilian purpuric fever Recurrent conjunctivitis accompanied by sudden onset of high fever, rash, septicemia, shock, vascular damage Mortality rate is as high as 70% within 48 hours Small outbreaks in South America Haemophilus ducreyi – Infection NOT normal flora Infection: Genital Ulcer disease (GUD) or Chancroid or “soft chancre” A highly contagious sexually transmitted disease Painful, suppurative ulcers or buboes (swollen lymph nodes in the groin) Men have symptoms related to the inguinal tenderness and genital lesions, whereas most women are asymptomatic More common in Latin Africa, Africa, Asia Specimen of Choice Material from the ulcer Aspirate of the infected lymph node (pus) **Genital specimens** Haemophilus ducreyi – Cellular Morphology Cellular morphology – Tiny GNCB, arranged as “school of fish” Haemophilus ducreyi – Culture and ID Requires Enriched CHOC Specimens inoculated immediately Incubated in high humidity in CO2 at 35° C for up to 7 days ID X and V Factors – Growth around X Factor Only Catalase – Negative Oxidase – Negative Porphyrin (ALA) Test - Negative Other Haemophilus species - Infections H. parainfluenzae, H. hemolyticus, H. parahaemolyticus are all members of the normal flora of the upper respiratory tract H. aphrophilus and H. paraphrophilus are found normally in the oral cavity Haemophilus species (Other than H. influenza and H. ducreyi) are typically non-pathogenic but have been associated with infections; Endocarditis Respiratory Tract Infections Abscesses (brain) Bacteremia Urethritis Pneumonia  Haemophilus species Identification ORGANISM X FACTOR V FACTOR BETA PORPHY HEMOLYSIS RIN H. 0 + NEG POS parainfluenzae H. haemolyticus + + POS NEG H. 0 + POS POS parahaemolyticu s *** H. aphrophilus – May require X Factor on primary isolation but NOT after subcultures HACEK - General Characteristics Gram-negative bacilli Do NOT grow on MAC Fastidious nutritional requirements Require an increased CO2 (5%–10%) environment Significant cause of endocarditis Usual flora of the oral cavity Thus are pathogens associated with human bite wounds, causing septicemia and subacute endocarditis Opportunists in immunocompromised hosts HACEK Group H—Haemophilus spp. Now Aggregatibacter aphrophilus A—Aggregatibacter actinomycetemcomitans Formerly Actinobacillus actinomycetemcomitans C—Cardiobacterium hominis E—Eikenella corrodens K—Kingella spp. HACEK ID Summary A. aphrophilus Recall previously under Haemophilus From the Greek words aphros and philia for foam loving or desiring high concentrations of CO2 Found in dental plaque and gingival scrapings Colony morphology on CHOC and BAP agar Yellow, granular, convex colonies with opaque zone near centers Non hemolytic on BAP A. actinomycetemcomitans Primarily an animal pathogen but has been found in human oral cavity Human tissue infections are attributed to cattle, sheep, pig, and horse bites or through contact with infected animals. Six serotypes a–f a, b, c are most common Major contributor to periodontitis A. actinomycetemcomitans – Cultural characteristics 24 to 48 hours to grow Grows better with increased CO2 Distinctive star formation in the center of the colonies Best viewed under ×100 magnification under a light microscope when grown on clear medium or via stereomicroscope at the highest magnification available A. actinomycetemcomitans – ID and treatment Key reactions Ferments carbohydrates in the presence of serum Not lactose or sucrose Catalase positive, Oxidase variable Negative for X and V growth factors Negative for urease, indole, esculin, and citrate Treatment Aminoglycosides, third-generation cephalosporins, quinolones, chloramphenicol, tetracycline C. hominis – General Characteristics Normal flora of the nose, mouth, and throat Can be present in gastrointestinal (GI) tract Infections: Endocarditis Grows on the heart valve and is resistant to antibiotics, so valve must be replaced C. hominis – Cellular morphology Cellular morphology Pleomorphic, nonmotile, gram-negative bacillus Sometimes appear false positive on Gram stain Show rosettes (image), swellings, or long filaments C. hominis – ID and treatment Grows slowly in BAP and CHOC Biochemical reactions Oxidase positive Catalase negative Indole positive Negative for Urease, nitrate, gelatin, and esculin Ferments sucrose Usual therapy Penicillin and an aminoglycoside E. corrodens - Infections Normal flora of oral and bowel cavities Fight and bite wounds Infections Meningitis Pneumonia Osteomyelitis Arthritis Postoperative tissue infections usually from bacteremia associated with wounds. Cellulitis Inoculation with needles “licked clean” E. corrodens – cellular morphology Gram-negative coccobacilli E. corrodens – colonial morphology Flat, pitted colonies on BAP and CHOC Non-hemolytic on BAP Bleach-like odor with yellow colonies pitting (hence the name corrodens) the agar (about 45% of isolates) Often yellow pigment present E. corrodens Growth and ID Grow in capnophilic environment ID Nonmotile Nonfermenter Oxidase positive and catalase negative Lysine decarboxylase positive Ornithine decarboxylase positive Arginine dihydrolase negative Require hemin (X factor) and CO2 Kingella Species Four species in genus K. kingae Member of normal oropharynx microbiota Associated with HACEK endocarditis - Particularly in immunocompromised patients Isolates have been found in blood, bone, joint fluid, urine, and wounds. K. denitrificans Associated with endocarditis, poor dental hygiene, and oral surgery K. oralis K. potus Important in pediatric population with predilection for bones and joints Causes osteoarthritis infection in children less than 4 years old Kingella - General Characteristics Gram stain appearance Coccobacilli or short bacilli with square ends in pairs or chains. Resist decolorization in the Gram stain Biochemical reactions Nonmotile, oxidase positive, catalase negative; sugar fermenters without gas production Usually susceptible to most agents Kingella kingae – Colonial morphology Grows on BAP, CHOC, Neisseria media Can get confusing but gram stain will help differentiate Neisseria from Kingella Colony types Spreading, corroding colony (image) OR Smooth, convex, and β-hemolytic colony Hemolysis may appear beneath colony or in close proximity after 24 hours. Hemolysis more prevalent after 48 hour Kingella kingae - CLSI ID Requirements Strains that meet the following CLSI abbreviated test requirements may be identified as K. kingae. Gram-negative short coccoid bacilli forming large white to beige β-hemolytic colonies on SBA No growth on MAC Catalase negative Oxidase positive Capnocytophaga Group of fastidious, facultatively anaerobic, gram negative bacilli Normal flora in oral cavity Infections: Septicemia Neutropenic patients – oral ulcers, soft tissue infections, peritonitis, endocarditis Capnocytophaga CELLULAR MORPHOLOGY COLONIAL MORPHOLOGY Thin, pointed ends GNB Pasteurella - General Characteristics Many species, most clinically relevant: Pasteurella multocida Has similar characteristics to Haemophilus Infection: Causes pasteurellosis A disease acquired from human exposure to infected animals or products may from infected animals = zoonotic disease Animal bites (often those as result of cat bites) are the most common clinical Organisms live in the respiratory tract and oral cavity of birds and mammals. Pasteurella - Infection Most common presentation: Soft tissue infection Injuries can be aggressive, with skin manifestations typically appearing within 24 hours following a bite Wounds can exhibit a rapidly progressive soft- tissue inflammation Soft tissue infections can progress into Deep tissue infections Ex. Septic arthritis, osteomyelitis, tenosynovitis Rarer presentation: Systemic infections Ex. Endocarditis, septicemia, meningitis, pneumonia Pasteurella – cellular morphology Can be easily mistaken with Haemophilus Gram-negative coccobacilli Ovoid, filamentous, or as bacilli Bipolar staining Look like safety pins End of bacilli retains more stain Pasteurella – colonial morphology NOT fastidious – Grows on BAP, CHOC Growth on BAP differentiates between Haemopihlus and Pasteurella Tends to have a “musty” smell similar to wet dog smell Growth on BAP Nonhemolytic, grey colonies Older colonies – mucoid and green/brown halo  Pasteurella – ID Nonmotile Catalase positive Oxidase positive (most isolates) Nitrate positive Indole Positive Ornithine Positive Urease negative Glucose fermentation Weak acid production Differential Characterist ics of Pasteurella Species Haemophilus vs Pasteurella HAEMOPHILUS PASTEURELLA NG on MAC or BAP NG on MAC Growth on CHOC – mousy odor Growth on BAP and CHOC – Small GNCB musty odor Small GNB – Bipolar staining Infections: Respiratory, can become systemic Infections: Wounds, rarely respiratory or systemic Knowledge Check Which of these are NOT defining characteristics of Haemophilus influenzae? A. Growth around staph streak on Blood agar plate B. Growth on XV disc but not X or V disc C. Satelliting colonies on CHOC agar D. Gram negative coccobacilli Knowledge Check Which Haemophilus species causes soft chancres and demonstrates a school of fish cellular morphology? A. H. aegyptus B. H. influenzae C. H. parainfluenzae D. H. ducreyi Knowledge Check Which Haemophilus species causes brazillian purpuric fever? A. H. influenzae B. H. aegyptus C. H. parainfluenzae D. H. ducreyi Knowledge Check Which of these is NOT a HACEK organism? A. Aggregatibacter aphrophilus B. Aggregatibacter actinomycetemcomitans C. Cardiobacterium hominis D. Haemophilus influenzae Knowledge Check Which of these should be suspected with soft tissue infections following animal bites? A. Haemophilus aegyptus B. Kingella kingae C. Pasteurella multocida D. Cardiobacterium hominis Knowledge Check Which of these is NOT a HACEK organism? A. Haemophilus influenzae B. Aggregatibacter aphrophilus C. Aggregatibacter actinomycetemcomitans D. Cardiobacterium hominis Knowledge Check Which of these is a differentiating characteristic between Haemophilus influenzae and Pasteurella multocida? A. Musty odor B. Growth on MacConkey Agar C. Gram stain D. Growth on Blood Agar