2024 HACEK Exam Notes PDF

FASTIDIOUS G R A M - N E G AT I V E B A C I L L I : THE HACEK GROUP R A J K U M A R R A J E N D R A N , D C L S , M L S ( A S C P )CM ADAPTED FROM POWERPOINT BY JANET ENDERLE, PHD, MLS(ASCP) CM OBJECTIVES Upon completion of this lecture, the student will accomplish the followin...

FASTIDIOUS G R A M - N E G AT I V E B A C I L L I : THE HACEK GROUP R A J K U M A R R A J E N D R A N , D C L S , M L S ( A S C P )CM ADAPTED FROM POWERPOINT BY JANET ENDERLE, PHD, MLS(ASCP) CM OBJECTIVES Upon completion of this lecture, the student will accomplish the following at the 70% (BS/cat) or 80% (MS) level: 1. Define the acronym HACEK using the correct bacterial names. 2. Describe incubation conditions and appropriate media for growth, isolation and identification of the HACEK bacteria. 3. Differentiate between the HACEK organisms based on Gram’s stain, colony morphology and biochemical reactions. 4. Distinguish between the HACEK organisms and other GNB including Enterobacteriaceae and N. gonorrhoeae based on Gram’s stain, colony morphology and biochemical reactions. 5. Discuss the clinical significance of each organism, including most common disease states and associations with other infectious agents. OBJECTIVES 1. Explain “X factor” and “V factor.” 2. Identify the components of agars used for the growth of Haemophilus species and justify the presence of each for bacterial identification. 3. Illustrate satellitism as it applies to growing Haemophilus. 4. Define “typeable” and “non-typeable” as they apply to H. influenzae. 5. Distinguish the virulence factors associated with H. influenzae infection. 6. Assess the major human disease states caused by Haemophilus and correlate the symptoms with the species. 7. Evaluate the laboratory identification of Haemophilus to include: – Growth characteristics and requirements of major species – Biochemical tests that differentiate species and biotypes 8. List common antibiotics to which Haemophilus spp are susceptible and those to which Haemophilus are often resistant. HACEK GROUP Haemophilus spp Cardiobacterium spp – H. influenzae group – C. hominis – H. parainfluenzae group – C. valvarum – H. ducreyi Eikenella corrodens Aggregatibacter spp – A. aphrophilus Kingella spp – A. paraphrophilus – K. kingae – A. segnis – K. denitrificans – A. actinomycetemcomitans HACEK GROUP Fastidious gram-negative bacilli Require at least 2 to 3 days for growth Normal flora of oropharyngeal and – Some may require up to 2 weeks urogenital tracts Grow best at 35-37°C in increased CO2 environment Pathogenic Growth on SBA (hemolysis) and CHOC – Endocarditis agar (V factor) – Bacteremia No growth on MAC or other selective – Mixed-flora wound infections enteric media GENERAL CHARACTERISTICS OF HAEMOPHILUS Gram negative, small, coccoid- to rod-shaped X-factor-requiring Haemophilus spp will grow on Nonmotile SBA but… Facultative anaerobes V-factor-requiring Haemophilus spp will not grow Form nitrites from nitrates Agar of choice for isolation of Haemophilus is CHOC agar Oxidase positive – RBCs are lysed by heating which releases X and V Catalase positive factors Normal flora of human upper respiratory tract – Enzymes (NADases) that hydrolyze V factors are Requirement for X and/or V factor, with or without inactivated hemolysis Also grows on 5% horse or rabbit blood agar – Patterns of hemolysis may be present which may aid in identification – Some species are ß-hemolytic  X & V FACTORS X FAC TO R : H E M I N & H E M AT I N V FAC TO R : N A D & N A D P Hemin is the deep red, ferrous, Coenzymes involved in cellular processes chloridated form of heme of all living cells: glycolysis and Krebs cycle (NAD), lipid and nucleic acid synthesis Hematin is the blue-blackish brown, (NADP) ferric, hydrolyzed form of heme Found in RBCs but unable to be utilized Found in RBCs and directly available by Haemophilus spp that require it for to Haemophilus spp that require it for growth because RBCs remain intact and growth SBA contains NADases that hydrolyze V factor (NAD) HAEMOPHILUS SPP H. influenzae group – H. haemolyticus – H. influenzae biogroup aegyptius H. parainfluenzae group – H. parainfluenzae – H. parahaemolyticus H. ducreyi Most species inhabit the upper respiratory tract of humans as normal flora but may occasionally act as pathogens H. INFLUENZAE – NOT THE “FLU” Influenza – acute inflammation of upper airways leading to headache, bronchitis and myalgias – is caused by Orthomyxoviridae (a virus) The bacillus H. influenzae frequently isolated from nasopharynx of influenza patients and postmortem lung cultures during influenza pandemic 1889-1890, Popular Science, May 1919 thus leading to its name Later development of viral culture techniques showed the culprit of influenza was actually a virus H. influenzae was determined to be a secondary (opportunistic) invader PATHOGENIC HAEMOPHILUS SPP H. influenzae – Typeable strains are encapsulated Cause invasive disease: meningitis, epiglottitis, pneumonia, septicemia – Nontypeable strains do not produce a capsule and usually are part of the normal flora of upper respiratory tract Only pathogenic if they gain access to normally sterile sites Localized infection near respiratory tract: conjunctivitis, sinusitis, otitis media Can be responsible for invasive disease (bronchitis, pneumonia) in older patients Localized infection can enter the CNS directly through infected sinuses, otitis media and head trauma H. ducreyi – Cause of chancroid, a sexually transmitted disease H. INFLUENZAE Virulence factors – Capsular polysaccharide production Helps bacteria to resist phagocytosis – Pili Aid in attachment of organism to epithelial cells Found on nonencapsulated strains – Outer membrane proteins Contribute to adhesion and invasion of host cells – IgA1 protease Specifically cleaves heavy chain of IgA1 cells H. INFLUENZAE Typeable strains are based upon capsular Type b is most common serotype found in antigens present serious infections – Types a, b, c, d, e and f – Type b is the only one with a polyribosyl- ribitol-phosphate (PRP) capsule – Systemic and life-threatening Most commonly affects children age 2 months to 5 years – Majority of infections occur before age of 2 years Differences in this complex are basis for – Conjugate vaccines (PRP is bound to a protein serotyping of this species carrier) to H. influenzae type b have dramatically lowered infection rate of children Type b is the only one with a polyribosyl- ribitol-phosphate (PRP) capsule in this age group Still leading cause of bacterial pneumonia deaths and meningitis in children in developing countries HAEMOPHILUS DISEASE STATES Meningitis in children and adults Conjunctivitis Epiglottitis Urogenital, maternal and perinatal Otitis media infection Sinusitis Chancroid Upper respiratory tract infections Endocarditis Tracheobronchitis Bacteremia Pneumonia Brazilian purpuric fever HAEMOPHILUS DISEASE STATES Meningitis – Highly contagious when H. influenzae is causative agent All individuals exposed should be treated prophylactically with rifampin – regardless of their immunization status – Easily transmitted – Nosocomial infections CSF shunt complications May take several months to years after shunt placement for infection to become apparent BACTERIAL CAUSES OF MENINGITIS – CSF LEFT: Gram stain of N. meningitidis in CSF with associated PMNs. N. meningitidis may occur intracellularly or extracellularly in PMN leukocytes and will appear as GN, coffee-bean shaped diplococci. RIGHT: N. meningitidis on BAP Approx. half of cases of acute bacterial meningitis in U.S. attributed to H. influenzae LEFT: Gram stain of H. influenzae: H. influenzae are small, pleomorphic GN rods or coccobacilli with random arrangements. RIGHT: H. influenzae on CHOC LEFT: Gram stain of S. pneumoniae with WBCs S. pneumoniae may occur intracellularly or extracellularly and will appear as gram-positive, lanceolate diplococci, sometimes occurring in short chains. RIGHT: S. pneumoniae on BAP  HAEMOPHILUS DISEASE STATES MENINGITIS IN CHILDREN MENINGITIS IN ADULTS Clinically, the symptoms of meningitis caused by H. influenzae resemble those of meningococcal Predisposition to infection by meningitis this organism can be found in Sudden high fever, nuchal rigidity, altered mental patient with suppressed immune status systems or with other Colonization of the upper respiratory tract of a susceptible host leads to invasion of the debilitating diseases bloodstream and infection of the meninges Frequently results from bacteria Most cases occur in infants and children under age of 5 gaining access to a sterile site via H. influenzae type b most common cause of trauma or as a result of bacterial meningitis in children between ages of 3 secondary infection months and 5 years (before Hib vaccine) – Both encapsulated and H. influenzae and N. meningitidis cause childhood meningitis at about the same rate nonencapsulated forms are found between ages of 2 years and 5 years equally in cases of adult meningitis  HAEMOPHILUS DISEASE STATES Epiglottitis – Second most common disease process associated with H. influenzae infections – Presents mostly in children ages 2-4 years, rarely in adults – Rapid onset with severe symptoms of laryngeal edema that can obstruct the airway, requiring emergency tracheostomy Otitis media – S. pneumoniae (#1) and H. influenzae (#2) are most common causes of acute ear infections – Mostly affects children age 6 months to 2 years – Most H. influenzae-caused infections are nontypeable form – Type b-associated otitis media frequently presents with bacteremia or meningitis HAEMOPHILUS DISEASE STATES Sinusitis Upper respiratory tract infection (URI) – H. influenzae is frequently cause of sinusitis in – History of previous influenza virus associated both children and adults with higher incidence of H. influenzae-related – Haemophilus and S. pneumoniae are most URI commonly isolated organisms if needle aspirate – Usually cause pharyngitis but can spread to is performed cause systemic disease – Bacterial sinusitis usually considered secondary – Most children with H. influenzae meningitis to viral sinus infection show symptoms of URI and otitis media Usually stems from nontypeable form of H. influenzae – Anaerobic infections cause more severe symptoms than Haemophilus or S. pneumoniae HAEMOPHILUS DISEASE STATES Tracheobronchitis Pneumonia – Initial presentation with chronic bronchitis but – H. influenzae type b most often causative agent then causes purulent sputum and acute febrile – Nontypeable or typeable forms other than tracheobronchitis – inflammation of trachea type b usually associated with pneumonia in and bronchi elderly patients with underlying respiratory – Most common causes of this disease are S. conditions aureus, group A Streptococcus, and nontypeable – Usually a complication of respiratory tract H. influenzae infections and bronchitis – Can eventually develop into pneumonia and – Symptoms resemble pneumococcal rarely causes airway stenosis pneumonia HAEMOPHILUS DISEASE STATES Conjunctivitis – Also known as “pink eye” – H. influenzae biogroup aegyptius is most frequently isolated organism in this condition (identified in 50% of cases) – Inflammation of the conjunctiva – Bacterial conjunctivitis occurs in preschool children Causes a purulent (pus) discharge May be associated with otitis media – Viral conjunctivitis occurs in older school-age children Causes a watery discharge from eye More often associated with a URI Bilateral H. influenzae conjunctivitis (culture positive) in a 3-year-old child HAEMOPHILUS DISEASE STATES Urogenital, maternal and perinatal infections Chancroid – Nontypeable H. influenzae and H. parainfluenzae – Caused by H. ducreyi can cause – Sexually transmitted disease that presents with Nongonococcal urethritis genital and perianal ulcers with tender inguinal Infections of the female genital tract lymphadenopathy (buboes) Postpartum bacteremia – Termed “soft chancroid” because the ulcers look Neonatal sepsis that may or may not include different than those caused by syphilis (chancre) meningitis Gray to yellowish exudate – Usually behave as opportunistic agents, causing Painful problems only in association with pre-existing – Enlarged lymph nodes (buboes) can drain through conditions or in the presence of IUDs the skin – Asymptomatic in women but initial lesion presents in the male approximately one week post transmission  HAEMOPHILUS DISEASE STATES Endocarditis – Most commonly caused by HACEK organisms Normal upper respiratory tract flora Rarely caused by H. influenzae Aggregatibacter aphrophilus – Probable entry is through upper respiratory tract and/or poor dental hygiene – Usually seen in young to middle-aged adults HAEMOPHILUS DISEASE STATES BACTEREMIA BRAZILIAN PURPURIC FEVER Common manifestation in early stages of High fever, abdominal pain, vomiting and meningitis development of petechiae within 3 days Occur in context of maternal-fetal or Eventual vascular collapse, hypotensive maternal-perinatal transmission shock and death Most often caused by H. influenzae type b Caused by one biogroup of H. influenzae biotype aegyptius This strain has only been seen in Sao Paulo, Brazil Hippelates flies in face and eyes of a child during a conjunctivitis epidemic in Maracaju, Mato Grosso do Sul State, which also had concomitant BPF cases LAB ID OF HAEMOPHILUS Gram stain usually shows small GN Haemophilus will grow on rabbit or horse coccobacilli blood agar Frequently very few organisms present in – Lacks the enzymes that inactivate V factor specimen, especially CSF – Addition of bacitracin to inhibit normal – Additional testing should be performed even if oropharyngeal flora Gram stain is negative – Observation of hemolysis Extremely fastidious Chocolate agar for optimal primary – Proper collection and transport isolation – Appropriate media – X and V factors available Capnophilic – Prefers a moist environment with increased CO2 (candle jar) Does not grow well on SBA – V factor inactivated in SBA  HAEMOPHILUS SPECIES ID: X AND V H. influenzae and H. haemolyticus require both X and V Quad plate: Mueller-Hinton agar with H. haemolyticus causes hemolysis on horse blood agar X factor (HBA) V factor X and V factors H. parainfluenzae and H. parahaemolyticus require only V Horse blood factor H. parahaemolyticus causes hemolysis on HBA H. ducreyi requires only X factor Quadrants and Factors Present I (X) II (V) III (XV) IV (Growth/ Hemolysis) H. influenzae --- --- + +/--- H. haemolyticus --- --- + +/+ H. parainfluenzae --- + + +/--- H. parahaemolyticus --- + + +/+ H. ducreyi + --- + +/---  LAB ID OF HAEMOPHILUS Staphylococcal streak technique – Several species of bacteria and yeast secrete NAD during growth on media – A lawn of Haemophilus organisms is plated on SBA and a single streak of S. aureus is made down the middle of the plate – X factor is released because the RBCs are hemolyzed and V factor is secreted by S. aureus as it grows – Haemophilus spp grow as tiny satellite colonies around the staphylococcal streak LAB ID OF HAEMOPHILUS ALA-porphyrin test – Delta-aminolevulinic acid (ALA)-porphyrin test allows for more reliable determination of X factor requirement – Determines ability of organism to synthesize protoporphyrin intermediates from ALA Uses enzymes of the biosynthetic pathway of hemin production – Strains that need X factor cannot synthesize protoporphyrin and are negative – Strains that do not require X factor are positive LAB ID OF H. DUCREYI GN coccobacillus Fastidious nature leads to difficulty in Catalase negative culture and identification, even with X and V factor strips Oxidase negative No sugar utilization Usually requires specialized media Indole, urea and ornithine negative – Mueller-Hinton-based chocolate agar supplemented with IsoVitaleX (X factor, vitamins, minerals) and vancomycin – Heart infusion-based agar supplemented with fetal bovine serum (FBS) and vancomycin LAB ID OF H. DUCREYI Can be identified with RapID-NH or similar Biochemical tests system – Species differentiation can be made based on acid production from carbohydrate utilization in addition to X and V factor requirements – Other tests that can be used Catalase Oxidase Indole Urea Nitrate Ornithine decarboxylase Porphyrin Molecular methods available HAEMOPHILUS INFECTION TREATMENT PREVENTION Antimicrobial susceptibility Vaccines – Preferred – Several protein-polysaccharide conjugate vaccines available for H. Amoxicillin (non-beta-lactamase influenzae type b producing) – Vaccination begins at 2 months of age 2nd or 3rd generation cephalosporin or amoxicillin- clavulanate (beta-lactamase producing) AGGREGATIBACTER APHROPHILUS Normal flora in oral cavity and upper respiratory tract Small colonies on CHOC after 24 hrs – Dental plaque – 0.5 to 1 mm in diameter, convex, granular, faint yellowish pigment; “grade school paste” odor at 48-72 hrs Numerous associated adverse outcomes – Endocarditis Distinguished from other HACEK organisms In context of valvular heart disease – ONPG positive – Brain abscess – Catalase negative – Meningitis – Oxidase positive – Osteomyelitis – Acid production from GLU, MAL, SUC, LAC – Soft tissue infection Some strains of A. aphrophilus require V factor These occur as a result of – Will not grow on SBA V-factor-inactivating enzymes are present in sheep blood – Dental disease – Require CHOC for V factor found in rabbit or horse blood – Dog bites – Piercings AGGREGATIBACTER SEGNIS Dental plaque and upper respiratory tract 0.5 mm diameter, greyish-white to opaque colonies on CHOC at 48 hrs. – No growth on SBA – requires V factor Endocarditis Periodontal infections Catalase +/- (V) GN, pleomorphic rods, often with filamentous Indole neg. forms ALA-porphyrin test pos. GAL, GLU, MALT, FRU, SUC weakly positive AGGREGATIBACTER ACTINOMYCETEMCOMITANS Formerly known as Actinobacillus Normal flora of oral microbiota Very small GN, nonmotile coccobacillus Portals of entry – Oral lesions – Skin abrasions – Pulmonary infections – Thoracotomy – Urinary tract instrumentation (catheters, cystoscopes) Virulence factors – Production of potent leukotoxin that kills neutrophils, monocytes and T-lymphocytes by causing damage to chromosomal DNA Inhibits the immune response – Collagenase that is toxic to PMNs and monocytes AGGREGATIBACTER ACTINOMYCETEMCOMITANS Growth on SBA, CHOC and BHI agar in 48-72 hrs – Star-shaped areas form on colonies after 1 wk Oxidase negative Urease negative Indole negative Does not require X or V factor Lysine, ornithine and arginine negative Glucose, fructose and mannose fermentation Catalase positive Nitrate reduction SBA at 24 hrs with CO2 CHOC agar at 8 days with CO2 AGGREGATIBACTER ACTINOMYCETEMCOMITANS Abscesses – Frequently co-isolated with Actinomyces Subacute bacterial endocarditis – Previous history of valvular disease Localized juvenile periodontitis of older Khan SZ, et al. Detection of subgingival periodonto- children/young adults pathogenic microorganisms around a one-stage implant supported prosthesis. J. Dent Implant 2011;1:26-8 – Rapid degeneration of alveolar bones supporting molars and incisors Implicated in Papillon-Lefevre syndrome – Inherited disorder that produces hyperkeratosis of palms and soles with periodontal destruction Janjua, Shahbaz A MD; & Khachemoune, Localized juvenile periodontitis Amor MD. (2004). Papillon-Lefèvre syndrome: Case report and review of the literature. Dermatology Online Journal, 10(1). CARDIOBACTERIUM SPP C. hominis May not see on Gram stain unless centrifuged C. valvarum – Gram stain variable – Tends to stain positive at the poles Clinical significance – May appear as teardrop or rosette structures – Normal flora of upper respiratory tract C. hominis on – Causes endocarditis almost exclusively phase microscopy Dental disease or procedures – Usually infects damaged/previously diseased heart valves BACTEC system can detect growth in 3-5 days – Prenotification by clinician of organism suspected usually necessary because of slow growth  CARDIOBACTERIUM SPP Culture characteristics Biochemical characteristics – Growth on SBA or CHOC agar in – Oxidase positive 48-72 hrs in increased CO2 – Catalase negative C. valvarum grows slower than C. hominis – Nitrate negative – Some strains cause pitting of the – Urease negative agar – Indole – C. hominis positive, C. More evident on CHOC valvarum pos/neg – Frequent subcultures are necessary Heart infusion or tryptone broth because rarely causes any visible requires heavy inoculation and 48-hr change in media incubation SUTTONELLA SPP Family Cardiobacteriaceae Growth on SBA and CHOC agar – Previously Kingella indologenes – Can show multiple colony types – S. indologenes – Can pit agar – Similar 16S rRNA sequence as C. hominis Oxidase positive Normal flora of upper respiratory tract Catalase negative Prosthetic valve endocarditis Indole positive Plump GN rods in pairs or short chains – Only C. hominis is indole pos in HACEK group GLU, FRU, SUC, LAC pos. MNTL, SBTL neg. – C. hominis pos. EIKENELLA CORRODENS Family Neisseriaceae Possible virulence factors Normal flora of mouth and upper respiratory – Pili formation tract – Slime layer that prevents phagocytosis Clinical significance – Proteins that promote adherence to epithelial – Hand infections from human bite cells and RBC agglutination – Head and neck infections – Outer membrane proteins that trigger release of lysosomal enzymes from macrophages Can disseminate and cause meningitis – Immunosuppressed individuals Pulmonary infections Bacteremia Endocarditis – Bacteremia seen in IV drug users due to dissolving the drug in saliva or licking the skin to clean it EIKENELLA CORRODENS Slender GN rod or coccobacillus Oxidase positive Catalase negative Culture characteristics Indole negative – Growth on SBA and CHOC agar in 48 hrs Urease negative – 50% of strains cause pitting of agar Nitrate positive – Pale yellow pigment produced Does not require X or V factors – Bleach odor X (hemin) needed if grown aerobically so sometimes mistaken for Haemophilus if non-pitting Asaccharolytic – does not produce acid from carbohydrates – Different from other HACEK organisms Lysine and ornithine positive KINGELLA SPP Family Neisseriaceae Transmitted to blood from oral mucosa in people with poor oral hygiene Kingella denitrificans Kingella kingae Infection occurs in those with underlying disorders Normal flora of upper respiratory tract and genitourinary (GU) tract Clinical conditions – Bacteremia – Endocarditis Infections of – Septic arthritis – Cardiac tissue – Osteomyelitis – Valvular tissue – Skeletal tissue – Joint spaces KINGELLA KINGAE Normal flora in throats of young children Plump GN rods in pairs or chains Transmitted child to child Growth on SBA and CHOC agar – Day care settings – Can show multiple colony types Anderson de la Llana, et al. Oropharyngeal K. kingae carriage in children: characteristics and correlation with osteoarticular infections, Usually preceding illness of stomatitis or URI β-hemolytic on SBA 95% of cases reported in ages 6 mo to 4 yrs Can pit agar – Colonization occurs at highest rates in this age group Oxidase positive Older children with underlying medical condition Catalase negative K. DENITRIFICANS Septicemia Endocarditis (native or prosthetic valve) May be confused with N. gonorrheae – Growth on Neisseria selective medium – Coccobacillary but may appear diplococcoid similar to Neisseria spp Both produce acid from glucose Both are colistin resistant Both are indole positive HACEK ANTIBIOTIC SUSCEPTIBILITY Generally susceptible to penicillin and ampicillin Some resistant strains, incl. rare β-lactamase-producing strains Drugs of choice are third and fourth-generation cephalosporins for serious infections (endocarditis) WRAP-UP √ X factor = hemin √ H. influenzae √ V factor = NAD Typeable – encapsulated, invasive disease √ H. influenzae and H. haemolyticus Non-typeable – nonencapsulated, localized disease require both X and V √ Virulence factors √ H. parainfluenzae and H. Capsule parahaemolyticus require only V factor Pili √ H. haemolyticus and H. parahaemolyticus OMPs show hemolysis on HBA IGA1 protease √ H. ducreyi requires only X factor WRAP-UP √ H. influenzae type b – bacterial meningitis √ Small GN coccobacillus (rounded ends) children between ages of 1 mo and 2 yrs √ Chocolate agar is preferred isolation medium √ H. influenzae and N. meningitidis – cause √ Quad plate childhood meningitis at about the same √ Satellite streak technique with S. aureus for rate between ages of 2 yrs and 5 yrs Haemophilus growth √ Bacterial conjunctivitis in children √ Other methods for Haemophilus identification under 2 yrs of age – Antigen/antibody – latex agglutination, ELISA √ Viral conjunctivitis in children ages 2 to – Biochemical tests 7 yrs of age – Serotyping √ H. ducreyi causes soft chancroid, asymptomatic in women √ Susceptible amoxicillin +/- clavulanate, 2nd/3rd gen cephalosporins √ Brazilian purpuric fever caused by H. influenzae biotype aegyptius √ Resistant to ampicillin, chloramphenicol √ Hib vaccine