Haemophilus: Fastidious Bacteria

Fastidious Bacteria

• Difficult to grow in the laboratory because of complex or restricted nutritional and/or environmental requirements.
• Genera include Hemophilus, Bordetella, and Brucella.
• Parvobacteriaceae are small bacteria.

Genus Haemophilus

• Classification:
  • Domain: Bacteria
  • Phylum: Pseudomonadota
  • Class: Gammaproteobacteria
  • Order: Pasteurellales
  • Family: Pasteurellaceae
  • Genus: Haemophilus
• Species include: H. influenzae, H. para influenza, H. aphrophilus, H. para aphrophilus, H. ducreyi, H. aegypticus.
• General characteristics:
  • Small, pleomorphic, gram-negative rods or coccobacilli
  • Non-motile
  • Mostly oxidase positive
  • Facultative anaerobes and fermentative
  • Requires enriched media for isolation
  • Grows better in carbon dioxide-enriched conditions
  • Some species require blood factors for growth, specifically X factor (hemin) and V factor (nicotinamide adenine dinucleotide or NAD)

Haemophilus influenzae

• Growth requires both hemin (X factor) for synthesizing cytochrome C and NAD (V factor) for oxidation-reduction processes in cell metabolism.
• Both factors are present in blood-enriched media; sheep blood agar must be gently heated at 70-80°C to destroy inhibitors of V factor (NADase).
• Capsule:
  • Surface covered by polysaccharide capsule that has six antigenic serotypes (a-f) in many, but not all, strains.
  • Type b is clinically the most virulent, containing polyribitol phosphate [PRP] in its capsule.
• Antibodies:
  • Directed against the capsule greatly stimulate bacterial phagocytosis and complement-mediated bactericidal activity.
  • Develop due to natural infection, vaccination with purified PRP, or passive transfer of maternal antibodies.
• Epidemiology:
  • Isolated exclusively from humans, mainly the respiratory tract.
  • Has no animal or environmental reservoir.
  • Non-capsulated strains are present in the nasopharynx or throat of 25-80% of normal people; capsulated strains (type b) in 5-10%.
  • Type b remains the most significant pediatric pathogen in many countries worldwide.
  • Outbreaks of type b infection occur, especially in nurseries and childcare centers.
  • Causes an estimated 3 million cases of serious disease with up to 700,000 child fatalities worldwide yearly.
• Virulence factors:
  • Pili and non-pilus adhesins mediate colonization of the Haemophilus influenzae in the oropharynx.
  • Cell wall components like lipopolysaccharide and low molecular-weight glycopeptide impair ciliary function, damaging the respiratory epithelium.
  • The antiphagocytic polysaccharide capsule is the major virulence factor in type b strains and contains PRP.
  • Lipopolysaccharide (lipid A) induces meningeal inflammation.
  • Immunoglobulin IgA1 proteases are produced by both encapsulated and non-encapsulated strains and may facilitate colonization on mucosal surfaces by interfering with humoral immunity.
• Pathogenesis:
  • Transmitted via respiratory droplets or direct contact with contaminated secretions.
  • It enters the body through the upper respiratory tract, resulting in asymptomatic colonization or infections like sinusitis, otitis media, or pneumonia.
  • Organism produces IgA protease, which degrades secretory IgA, facilitating its attachment to the respiratory mucosa.
  • Encapsulated organisms can penetrate the epithelium of the nasopharynx, invade blood capillaries, and reach the meninges directly.
• Virulence:
  • The presence of an antiphagocytic polysaccharide capsule is a major factor in virulence.
  • Non-typable strains are less invasive, but they, as well as typable strains, induce an inflammatory response that causes disease.
• Clinical Diseases:
  • Invasive infections include Meningitis, Epiglottitis, Pneumonia, Cellulitis, Septic arthritis and Osteomyelitis
• Non-invasive infections include Otitis media, Sinusitis, Acute exacerbation of chronic obstructive pulmonary disease, and Conjunctivitis
• Invasive Infections:
  • Risk is significantly greater in patients with no anti-PRP antibodies, depletion of complement, undergone splenectomy, immunosuppression, or hypogammaglobuliemia.
  • Children around 1 year are vulnerable due to a decrease in maternal IgG and the inability to form sufficient anti-capsular antibodies until the age of 2 years.
• Meningitis:
  • H. influenzae type b caused many cases of pediatric meningitis
  • Peak incidence occurs around 1 year of age (3 to 18 months)
  • More common in winter season
  • Cases increase with household contacts under 5 years of age, especially if under 2 years
  • Outbreaks can occur in nursery schools
  • Mortality rate is at 5%
  • Complications may occur in 10-20% of cases that includes hearing loss, convulsions, and intellectual impairment
  • Contacts should be traced and given prophylaxis
• Epiglottitis:
  • Life-threatening emergency or pediatric disease
  • Peak incidence is between 2-4 years of age (pre-vaccine era)
  • Symptoms include pharyngitis, fever, difficulty breathing (progresses rapidly to airway obstruction), and even death
  • A sign of this is the swollen epiglottis, called the "thumb sign"
• Cellulitis:
  • Presents with fever and reddish-blue patches on the cheeks or periorbital areas
  • Rates decreased with vaccination
  • Can result in primary Pneumonia
  • Can affect patients with an underlying disease such as chronic pulmonary disease or malignancy.
• Laboratory Diagnosis:
  • Specimens: CSF, Sputum, Blood, Joint, and Pleural fluid
  • Meningitis: uses both blood and CSF
  • Epiglottitis, cellulitis and arthritis: uses blood cultures
• Collecting samples: not collect from the posterior pharynx in patients with suspected epiglottitis because this may stimulate coughing and obstruct the airway
• Microscopy: sensitive and specific, detected in more than 80% of CSF specimens from patients with untreated Haemophilus meningitis.
• Antigen detection: Rapid latex agglutination test
  • Rapid and sensitive
  • Can detect less than 1ng/ml of PRP capsular Ag in clinical specimens such as CSF and urine
  • Only applies to Haemophilus influenzae type b strains
  • Should be used when the specimen taken after antibiotic treatment, but some serotypes of S. pneumoniae and E. coli may share similar antigens
• Culture:
  • Requires chocolate agar; V factor is destroyed if agar is overheated.
  • Incubated in an aerobic atmosphere enriched with 5-10% CO2.
  • Forms 1-2mm, smooth, opaque colonies after 24 hours, with a "mousy" or "bleach-like" odor
• Blood cultures:
  • Delayed
  • Requires X & V factors and inhibitors of V factor not be supplemented with optimum concentrations
  • Grows better in anaerobically, does not require X factor
• Identification on nutrient agar with V, X, XV factors:
  • H. influenzae requires both X and V factors
  • H. parainfluenzae requires V factor alone
  • H. ducreyi require X factor alone
• The Quellung reaction or capsule swelling test: Antibodies bind to the capsule, allowing visualization under a microscopeIf the reaction is positive, the capsule becomes opaque and appears enlargedHaemophilus influenza type bSatellite phenomenon can occur, growing around colonies of S. aureus on Blood agar that releases X factor and excreting V factor
• PCR techniques are use to identify Hemophilus species in clinical specimens and as confirmatory test on isolate
• Capsular type of Haemophilus influenzae isolates is determined by slide agglutination or PCR
• Treatment: Mortality rate in patients with untreated meningitis or epiglottitis is approximately 100%
• Ceftriaxone are used for third generation cephalosporins in treating meningitis & acute epiglottitis Less severe infections, such as sinusitis and otitis can be treated with oral antibiotics that include Azithromycin, Amoxicillin, and Clarithromycin
• Prevention and control: Purified capsular or PRP (conjugated) vaccines with only for H. influenzae type b and given at 2, 3, and 4 months in Libya, 2,4,6 pentavalent
• Effective for reducing the incidences of Haemophilus influenzae type b disease and colonization, and recommended for children and adults with splenic dysfunction

Antibiotic Prophylaxis

• Rifampin given orally once daily for 4 days eradicates H. influenzae carriage.
• Household members of someone with a serious Hib infection are at increased risk of Hib based on age (< 2 years), vaccination status, and/or immunocompromising conditions.
• Indicated in daycare centers.

Haemophilus para influenzae, Haemophilus aphrophilus, H. para aphrophilus

• Are occasionally implicated in human disease that includes endocarditis, dental infection, brain abscess, and lung abscess that can be treated successfully with ampicillin and gentamycin

Haemophilus ducreyi

• Causes genital ulcers (chancroid), soft chancres, sexually transmitted disease (STD).
• Found in tropical regions of Africa and Asia.
• 5-7 days after exposure, a tender papule with an erythematous base will develop on the genitalia/perianal area.
• Within 2 days, the lesion ulcerates, becomes painful, and inguinal lymphadenopathy can be present and can facilitate for transmission of HIV.
• Diagnosis: specimens from the base/margin of the ulcer, and/or performing gram stain of smear from ulcer streptobacillary chains, special fastidious culture techniques on GC media, cultures should be incubated at 33 C° in 5-10% CO2 for 7 days or more, to confirm chancroid, other causes of genital ulcers such as herpes/syphilis should be ruled out, and/or performing multiplex PCR
• Treatment: most isolates are successfully treated with erythromycin, can also give Ceftriaxone in a single dose therapy

H. Influenza aegyptius

• Presents as koch-weeks bacillus, biotype III and can be tested via PCR
• Associated with acute purulent conjunctivitis (epidemic) and in warm months
• Associated with Brazilian purpuric fever, Brazilian purpuric fever, 1st in Brazil 1984 conjunctivitis leading to fulminant septicemia
• Can be cultured on chocolate agar supplemented with 1% IsoVitaleX, incubating in CO2 atmosphere for 2 to 4 days and can be treated with ampicillin and chloramphenicol

Genus Bordetella

• Bordetella pertussis:
  • Extremely small, Gram-negative coccobacillus.
  • Strictly aerobic and Non-fermentative can oxidize amino acids as an energy source
  • Requires prolonged incubation and media supplemented with charcoal, starch, blood, or albumin to absorb toxic substances
  • Human pathogens
  • Distributed worldwide, periodic epidemics every 2-5 years, droplet transmission
• No maternal protection
• Infection or vaccination are not lifelong
• Virulence factors and pathogenesis of Bordetella pertussis:
  • Attachment factors, pertactin, filamentous hemagglutinin, fimbrae and dermonecrotic toxin
  • Attachment of the organisms to ciliated epithelial cells mediated by protein adhesins.
  • Localized tissue damage mediated by dermonecrotic toxin.
  • Tracheal cytotoxin inhibits cilia movement - Also known ass systemic toxicity pertussis toxin, inactivates protein that controls adenylate cyclase activity and characteristic of the paroxysmal stage of pertussis

Pertussis (Whooping Cough)

• Spreads directly from person to person by infectious aerosols
• IP: 3-12 days
• Stage are the catarrhal stage, the paroxysmal stage/acute and then the convalescent phase
• The catarrhal stage:
  • Common cold symptoms and low grade fever or anorexia
  • Peak number of bacteria for detection and transmission

Parosysmal Stage

• The classic whooping cough with 40 to 50 paroxysms
• Severe cough with inspiratory loud whoop. Infants less than 6 are apneic.
• Thick production in respiratory tract
• Leads to exhaustion and vomiting
• Risk of death with severe increased lymphocytosis, risk of head bleeds, and/or prolapsed rectum
• Late convalescent stages result in complications or permanent respiratory damage

Bordetella pertussis testing/therapy

• Can test with deep nasopharyngeal aspirate (or a pernasal/flexible swab from nasopharynx)
• Should be collected during the catarrhal and/or early stage and should be treated on transport plates. Microscopic evaluation is considered insensitive to bacteria
• The standard test is culture on: Blood charcoal Bordet-Gengou agar (blood and starch), Regan-Lowe agar (blood and charcoal), and/or CCBA contain antibiotic for selectivity, but requires more intensive treatment and incubations
• ELISA to Detect toxins to confirm diagnosis from samples of Pertussis toxin, Filamentous hemagglutinin or by conducting acid PCR
• Antibiotics are effective, but needs to be coupled with supportive therapy and the course is more effective early on, where macrolides are most commonly given; prophylaxis to decrease infections with azithromycins is common
• Therapy can be supported from vaccines contain inactivated pertussis toxins, filamentous hemagglutinin, pertactin that are given across 3 doses, between 2 and 6 months, and repeated in older ages.

Other Bordetella Species

• B. parapertussis causes mild form of pertussis in 10-20% cases.
• B. bronchiseptica causes respiratory disease primarily in laboratory animals (dogs & swine) and also associated with human respiratory tract colonization and bronchopulmonary disease.

Differentiation of Bordetella Species

• B. pertussis has negative Urease and Motility with positive Oxidase.
• B. parapertussis has positive Urease with negative Oxidase and Motility.
• B. bronchiseptica has positive Urease, Oxidase and Motility.

Genus Brucella

• Key Identification points of species:
• Small, gram-negative coccobacilli (rod shape)
• Non- motile, non-capsulated and are strictly aerobic, with few strains only requiring only CO2 Are oxidase, watalase and Urease positive
• Brucella is considered fastidious and it grows slowing, often being categorized as a zoonotic disease Classification:
  • Domain: Bacteria
  • Phylum: Pseudomonadota
  • Class: Alphaproteobacteria
  • Order: Hyphomicrobiales
  • Family: Brucellaceae
  • Genus: Brucella
• Species include: B. melitensis (goat), B. abortus (cattle), B. suis (pigs), B. canis (dogs)

Epidemiology

• Brucella strains are more common in undeveloped nations or in areas without domesticated vaccination protocols/policies that include African nations or throughout the Mediterranean and middle east
• Key Host factors and modes of transmission
  • Each strain has a particular or dominate host species, that exhibits mild to asymptomatic disease
  • Br melitensis goes to goats and sheep, B. abortus goes to Cattle, B suis (pigs) and B. canis goes to dogs
  • Brucella strains are readily shed or released from animals through milk, urine and/or birth products
• In humans, Brucella infections can result in brucellosis, through contact or inhalation, bio weaponized agents The sites of recognized outbreaks include Undulant fever, Malta fever, Mediterranean remittent fever, Rock fever of Gibraltar, County fever of Constantinople, Fever of Crete

Specific Properties

• Brucella Evades macrophages using monophosphates and adenine, interfering myeloperoxidase-peroxide production or can use oxygen radicals
• Smooth LSP strain has a distinct virulence factor, where reversion of smooth versions correlates with reduced virulence, and forms granuloma in organs of the RES system

Pathogenesis

• Brucella organisms can survive with intracellular bacteria via facultative intracellular pathogen means, where bacteria are transferred through macrophages
• Infection process can be summarized by. Intial infection, bacteria replicates-> granulomas form=>replicate -> leads to relapsing fever with inflammation and more severe outcomes and potentially more chronic infections

Clincial Presentation

• Often with acute 1-3 week duration that presents with inconsistent symptoms or indicators, or potential coughing depending factors
• The majority of patients recovers within 3 months, but it can impact other systemic functions

Advanced Disease presentation

• Often involves GI problems in more than 70% patients, bone issues can be prevalent, respiratory, cutaneous (lesions), neurological and cardiovascular issues

Chronic infection facts

• Chronic cases are often results of inconsistent treatment and or from antibiotic use that is only given in shorter courses that often lead to relapse

Relapse facts

• Can be associated with persistent infections in bones or other organs, these infection may display a high resistance to standard antibiotic types

Other sequel

• Hepatitis , low Uveisis, anemia and meningitis are all types of sequela

Key Disease Factors

• Strains can demonstrate different complications, in the cases if B canis and arbos strains, infections often are more mild, with long term/severe complications and chronic infections from B suis

Diagnosis and Testing

• Sample: blood, bonemarrow or potential fluid to localized zones
• Microscopy is often insensitive
• Culture: specific Testing is sensitive and prolonged incubation is a key process of cultures and requires significant blood and sample collection Requires slow growth or requires enriched agars and needs prolonged incubation with specific honey droplets like colonies

Brucella Test

• Testing often required microscopic confirmation such as CO2, or agglutination tests
• Key tests include: microscopic sample analysis followed up with agglutination test, and requires for Brucella abortus a single titer 160 ratio Livers can be biopsied and a molecular acid test to detect infections

Treatment and Prevention

• tetracyclines are the first line for detection and are effective against both B strains and B suis, however the potential for re-infection is possible To test, most often its measured through antibody testing WHO guidelines, to pair tetracyclines with rifampin to get rid of Brucella stains and to prevent relapsing Treatments during pregnancy might impact the fetus because of the nature of doxy and trymethoprim, so that is often reserved for sever cases and other steps should be taken before and after infection, otherwise patients are treated prophylactically Vaccinations are effective in live attenuated brucella strains and prevents infections