Pseudomonas And Other Nonfermenters And Hsemophilus Bordetella Brucella(HBB) Notes

Nonfermenters

Nonfermenters utilize the sugars oxidatively. Important members are Pseudomonas, Burkholderia, and Acinetobacter.

Pseudomonas

Virulence Factors and Pathogenesis

1. Colonization: To colonize the host surface by pili or fimbria (the organ of attachment)
2. Toxin-mediated immune evasion and Tissue Injury:
   • Nondiffusible toxins (e.g. exotoxins S, U, T, and Y)
   • Diffusible toxins (e.g. exotoxin A, proteases, phospholipases, hemolysins, elastases, pyocyanin, etc.)
   • Exotoxin A is the most important virulence factor. It inhibits protein synthesis by inhibiting EF-2 (mechanism of action is similar to diphtheria toxin).
3. Host’s inflammatory response: Against lipid A of LPS and flagellin
4. Pigment production:
   • Pyocyanin (a blue-green pigment, produced only by P. aeruginosa)
   • Fluorescein (or pyoverdin): Gives greenish-yellow color, produced by all species
   • Pyorubin (imparts red color)
   • Pyomelanin (imparts brown black color).
5. Alginate coat: Mucoid strains of Pseudomonas have a slime layer or alginate layer which facilitates biofilm formation, thus helps in adhesion to purulent mucus.
6. Such strains can cause infections in patients with cystic fibrosis.
7. Capsular polysaccharide prevents the bacteria from phagocytosis.
8. Multi-drug resistance and Multi-disinfectant resistance.
9. Wide temperature range (5–45 °C).

Read And Learn More: Micro Biology And Immunology Notes

Clinical Manifestations

• Most of the infections are encountered in hospitalized patients.
• Pneumonia: (VAP or Ventilator-Associated Pneumonia).
  • Chronic respiratory tract infections: Occurs in patients with cystic fibrosis (in Caucasian populations), bronchiectasis or chronic panbronchiolitis (in Japan):
  • The mucoid strains (possessing alginate layer) of Pseudomonas commonly cause such infections.
  • Structural abnormalities of the airways result in mucus stasis.
  • Ear infections: Swimmer’s ear (among children) and malignant otitis externa (in elderly diabetic patients).
• Eye infections such as corneal ulcers (in contact lens wearers) and endophthalmitis.
• Shanghai fever: A mild febrile illness resembling typhoid fever.
• Skin and soft tissue infections:
• Burns patients: Pseudomonas is the most common organism to infect the burn wounds.
  • Ecthyma gangrenosum (is an acute necrotizing condition results from bacteremia), occurs more commonly in patients with febrile neutropenia and AIDS.
  • Pseudomonas dermatitis: Cause outbreaks in spas, and swimming pools.
  • Toe-web infections (in the tropics).
  • Green nail syndrome: It is a ‘paronychia’ results from prolonged submersion of the hands in water.

• Other infections:
  • Cellulitis (characterized by blue green pus)
  • Bone and joint infections such as osteomyelitis and septic arthritis
  • Meningitis (in postoperative or post-traumatic patients)
  • UTI (urinary tract infection) in catheterized patients.

Laboratory Diagnosis

Pseudomonas is nonfastidious, obligate aerobe and is motile with single polar flagellum:

• It produces large, opaque, irregular colonies with a metallic sheen (iridescence)
• Diffusible pigments: Blue green (pyocyanin) or yellow green (pyoverdin) pigmentation
• Pigment production can be enhanced in special media such as King’s media
• Most colonies have a characteristic sweet ether or alcohol-like fruity odor
• Blood agar: It produces β hemolytic colonies on blood agar
• MacConkey agar: Produce pale nonlactose fermenting colonies
  • Selective media-cetrimide agar
  • Oxidase and catalase positive
• Nonfermenter: It does not ferment any sugars, but utilizes sugars oxidatively.
• OF test (Hugh and Leifson oxidative fermentative test) shows oxidative pattern.

Treatment

Pseudomonas species are inherently resistant to most of the antibiotics. Only limited antipseudomonials are available:

• Penicillins: Piperacillin, mezlocillin, ticarcillin
• Cephalosporins: Ceftazidime, cefoperazone, and cefepime
• Carbapenems: Imipenem, meropenem
• Monobactam: Aztreonam
• Aminoglycoside: Tobramycin, gentamicin, amikacin
• Quinolones: Ciprofloxacin, levofloxacin
• Polymyxins: Polymyxin B, colistin.

Drug Resistance

• Pseudomonas possesses a number of drug resistant plasmids which confer multiple drug resistance.
• Many strains are producers of β lactmases such as ESBL (extended spectrum β lactamases), carbapenemases, and AmpC β lactamases.
• Many strains are resistant to aminoglycosides and quinolones.

Burkholderia

Burkholderia species are also oxidase positive nonfermenters; however they differ from

• Pseudomonas in being:
• Bipolar stained (safety pin appearance)
• Resistant to polymyxin B.

Burkholderia Pseudomallei (Melioidosis)

• B. pseudomallei is the causative agent of melioidosis.
• Habitat: B. pseudomallei is a saprophyte of soil and water and have large number of animal reservoirs.
• Mode of transmission: by inoculation, inhalation, aspiration or ingestion. Man to man transmission is very rare.
  • Virulence factors:
  • Polysaccharide capsule, type III secretion system,
  • LPS, toxins, enzymes and proteins (such as hemolysin, lipases and proteases),
  • Quorum sensing, type IV pili and siderophore for iron acquisition.
• Risk factors: Diabetes, renal failure and traumatic inolcutaion in children, weather (rainy season) and occupation (rice farmers).
• Incubation period: 2 days to many years. Has long latency; presented long time after the exposure; hence also known as ‘Vietnam time-bomb disease’.
• Clinical feature: Can present with an array of manifestations (hence called as ‘great mimicker’)
  • Acute, localized infection: Nodule, fever, general muscle aches
  • Sub-acute (Pulmonary) infection: bronchitis to severe tuberculosis-like pneumonia with cellulitis and lymphangitis
  • Acute bloodstream infection: Seen in patients with HIV, renal failure and diabetes and presents as septicemia
  • Chronic suppurative infection forming abscesses: Involves various organs such as joints, viscera, lymph nodes, skin, brain, liver, lung, bones, and spleen.
• Geographical distribution: 1.65 lakh new cases of melioidosis occur worldwide every year with mortality as high as 50%.
  • World: Endemic in Thailand, Australia, Singapore, Indian subcontinent and other Southeast Asian countries.
  • India: It has been reported mainly from South India such as Tamil Nadu, Karnataka, Puducherry and Kerala.
• Ashdown’s medium is used as a selective medium, where it produces wrinkled purple colonies.
  • Cultures can be confirmed by latex agglutination test using specific antisera.
  • Treatment of melioidosis consists of:
  • Intensive phase (2 weeks): Ceftazidime or a carbapenem is given
  • Maintenance phase (12 weeks): Oral cotrimoxazole is given to eradicate the bacilli and to prevent relapse.
  • Doxycycline or amoxicillin-clavulanate are the alternatives.

Burkholderia Mallei

• B. mallei is a pathogen of horses; causes glanders (nasal discharge and ulcers in the nasal septum) and farcy (skin lesions and lymph node involvement).
• Human infection is characterized by:
  • Local skin nodules and lymphadenitis (if transmitted by inoculation)
  • Pneumonia, ulceration of the trachea and sepsis (if transmitted by inhalation)
• B. mallei differs from B. pseudomallei in being:
  • Nonmotile and Oxidase negative
  • Inability to grow on MacConkey agar
  • Inoculation into Guinea pigs can cause testicular swelling (Strauss reaction).

Burkholderia Cepacia

• B. cepacia is currently the most commonly encountered Burkholderia species:
  B. cepacia inhabits moist environments, detergents and IV fluids.
• LPS of B. cepacia is among the most potent of all gram-negative bacteria.
• Cepacia syndrome characterized by a rapidly fatal respiratory infection and septicemia in cystic fibrosis patients.
  Nosocomial pathogen in ICU patients because as it is resistant to multiple antibiotics.

Acinetobacter

• Acinetobacter are saprophytic bacilli. However, it is recognized as a nosocomial pathogen:
• It can cause ventilator associated pneumonia, Central line associated bloodstream infection, Catheter associated UTI.
• Wound and soft tissue infections and infections in burn patients.
• A. baumannii is nonfermenter, but differs from Pseudomonas being Oxidase negative and Nonmotile.

Haemophilus

• Haemophilus species are oxidase positive, capsulated pleomorphic gram-negative bacilli. It
• (Pfeiffer’s bacillus) is blood loving organism; requires two accessory growth factors present in blood.
• Factor X- hemin present freely in blood Factor V is an NAD (present in side RBC)
• Virulence Factors and Typing
• Capsule-Based on Capsular polysaccharide, H. influenzae is typed into six serotypes (a to f):
  • H. influenzae serotype b (Hib) is the most virulent and accounts for most of the invasive infections.
  • Hib capsule has unique chemical structure, made up of polyribosylribitol phosphate (PRP) antigen.
  • It is strongly immunogenic, hence used for vaccination.
• Next to Hib, nontypeable strains are commonly isolated clinically. Other capsular serotypes are very rarely isolated.
• H. influenzae was the first free-living organism whose entire genome was sequenced.

Clinical Manifestations

• H. influenzae type b (Hib) is the most common and most invasive serotype.
• Central nervous system infections:
  • Pyogenic meningitis in < 2 years of age
  • Subdural effusion, MC CNS complication
• Epiglottitis: Seen in older children (2-7 years), absence among Navajo Indians and Alaskan Eskimos.
• Lobar Pneumonia in infants
• Less common invasive conditions seen in children include:
  • Cellulitis of neck and head region
  • Osteomyelitis, septic arthritis
  • Orbital cellulitis, endophthalmitis
  • Next to Hib, non-typeable strains are the commonest group clinically.
  • They are noninvasive, spread by contagious spread and usually affect adults.
• Their clinical manifestations include:
  • Childhood otitis media
  • Exacerbations of COPD: They are the MC bacterial cause for this condition.
  • Pneumonia in adults among patients with COPD or AIDS
  • Puerperal sepsis and neonatal bacteremia- by strains of biotype IV.
  • Sinusitis in adults and children.

Laboratory Diagnosis

• Specimen collection and transport:
  • CSF, blood, sputum, pus, aspirates from joints, middle ears or sinuses.
  • As it is highly sensitive to low temperature, the specimens should never be refrigerated.
  • Gram staining of CSF and other specimen shows pleomorphic gram-negative coccobacilli
• Capsule detection: By Quellung reaction or Latex aggl. test
• Culture: H. influenzae is largely aerobic, growth is enhanced by 5–10% CO2.
  • Blood agar with S. aureus streak line: Colonies of H. influenzae grow adjacent to S. aureus streak line (this property is called as satellitism).
  • This is due to release of V factor by lysis of  RBCs mediated by S.aureus.
• Chocolate agar: It grows well on chocolate agar but sparsely on blood agar.
  • Fildes agar and Levinthal’s agar.
  • Disk test for X and V requirement:
• Biotyping: It is done by IOU tests (indole, urease test and ornithine decarboxylase test).
  Slide agglutination test: Serotyping is carried out using type-specific antisera.

Treatment

• Invasive infection due to Hib: Cephalosporins are the drugs of choice.
• Nontypeable strains of H. influenzae are often resistant to β lactams [due to β-lactamase production (20–35% of strains) or rarely altered penicillin binding protein-3]. DOC is quinolones
• (levofloxacin) or macrolides (azithromycin).
• Chemoprophylaxis: Oral rifampin is indicated to household contacts or healthcare staff (if two or more cases occur within 60 days).

Hib Conjugate Vaccine

• The PRP capsular antigen of H. influenzae type b is used as vaccine.
• As capsular antigens are poorly immunogenic to children, they are conjugated with adjuvants such as diphtheria toxoid, tetanus toxoid.
• It also reduces the rates of pharyngeal colonization with Hib.
• Conjugate vaccines has dramatically reduced the incidence of Hib disease.

H. aegyptius

• Koch’s –Week’s bacillus
• Pink eye syndrome (Egyptian ophthalmia)
• Brazilian purpuric fever.
• H. ducreyi
  • Causes Chancroid/soft sore: Characterized by painful lymph node, tender non-indurated and bleeding genital ulcer
  • Chancroid increases both transmission and the degree of susceptibility to HIV infection
• In direct smear: Pleomorphic gram-negative coccobacilli that: Show bipolar staining
• Occurs in parallel chains called in ‘School of fish’ or ‘rail road track’ appearance
• Antigenically homogenous
• Culture Medium used:
  • Rabbit blood agar or Chocolate agar with 1% isovitalex, Vancomycin
  • Chorioallantoic membrane (CAM)
  • Drug of choice: Azithromycin (1 g oral; single dose), treatment of all sexual partners.
• Haemophilus aegyptius
  • It is also called as Koch-Weeks bacillus; closely resembles H. influenzae biotype III.
• It causes:
  • Brazilian purpuric fever: A fulminant condition, characterized by fever, purpura, hypotension and shock
  • Purulent contagious conjunctivitis (Egyptian ophthalmia).

HACEK Group

• HACEK organisms are a group of highly fastidious, gram-negative bacteria, normally residing in the oral cavity as commensal, but occasionally have been associated with local infections in the mouth and systemic infections, such as bacterial endocarditis:
• Haemophilus species: H. aphrophilus, H. paraphrophilus and H. parainfluenzae
• Aggregatibacter (formerly Actinobacillus) actinomycetemcomitans: Most common member
  Cardiobacterium hominis
• Eikenella corrodens: Produces twitching or jerky motility and pitting or corroded colonies on blood agar Kingella kingae

Treatment: Ceftriaxone (2 g/day) is the DOC except for Eikenella corrodens where ampicillin is indicated.

Bordetella

Bordetella is described first by Bordet and Gengou, causes a violent paroxysmal productive cough in children called as whooping cough or 100 days fever.

Virulence Factors

Toxins:

• Pertussis toxin (PT) expressed only by B.pertussis, similar to cholera toxin in its structure and function (↑ cAMP)
• Other toxins: Tracheal cytotoxin, adenylate cyclase toxin, dermonecrotic toxin and Endotoxin
• Adhesins: They play a role in bacterial attachment:
  • Filamentous hemagglutinin (FHA)
  • Pertactin, an outer-membrane protein
  • Fimbriae or pili or agglutinogens.

Clinical Manifestations

Whooping cough (or pertussis) passes through three stages following an IP of 7–10 days.
1. Catarrhal phase: It lasts for 1–2 weeks, is characterized by common cold like nonspecific

• symptoms. It is highly infectious stage and smear and cultures are likely to be positive.

2. Paroxysmal phase: It is characterized by specific symptoms such as:

• Whooping cough, post tussive vomiting
• In this stage, patient is less infectious; smear and culture become negative.

3. Convalescent stage: Severity decreases. Antibodies appear in serum.

Epidemiology

• Whooping cough is exclusively human disease. There is no animal reservoir:
• Mode of transmission is via inhalation of droplets or rarely through direct contact.
• Recent outbreaks: Washington epidemic in 2012 and California epidemic in 2014 Worldwide, the incidence of pertussis is declining.
• WHO estimated around 1,39,535 cases of pertussis in 2016 and 1,42,512 in 2015 with 89,000 deaths; (Mainly in unvaccinated children).
• WHO reported an estimated global vaccine coverage of 86% in 2016.
• There is no cross protection to B. parapertussis infection.

Laboratory Diagnosis

• Best Specimen: Nasopharyngeal secretions, obtained by nasopharyngeal aspiration (best method) or pernasal swab
• Type of swabs used: Alginate swabs are the best followed by dacron swabs for culture.
• However, for PCR, only dacron or rayon swabs are recommended.
• If delay is expected, then suitable charcoal-based transport medium (Amies or Stuart’s) can be used.
• Cough plate method and postnasal swabs used before are no longer recommended.
• Antigen detection: Direct fluorescent antibody tests of nasopharyngeal secretions
• Culture: Nasopharyngeal aspirate culture is the Gold standard method
• Media: Regan and Lowe medium, Bordet-Gengou glycerine-potato-blood agar
• Colonies: Mercury drops or bisected pearls appearance.
• Culture smear: Reveals small, ovoid coccobacilli arranged in thumb print appearance.
• Capsules and bipolar metachromatic granules may be seen occasionally.
• PCR: Most sensitive, gives quicker results, but yet to be standardized properly.
• The most common targeted genes are IS481 and the PT promoter region genes.
• Antibody detection: Enzyme immunoassays detecting IgA and IgG to pertussis toxin, filamentous hemagglutinin.

Treatment

• Antibiotics eliminates the bacteria from nasopharynx, but less useful for treatment as pertussis is toxin mediated.
• Macrolides are the drugs of choice (e.g. erythromycin for 7–14 days)
• Cotrimoxazole is recommended as an alternative in macrolide resistance.
• Chemoprophylaxis: Erythromycin is DOC.

Vaccine

• Whole-Cell Pertussis Vaccines
  • It is prepared by heating followed by chemical inactivation and purification of whole B.pertussis bacilli.
  • It is given along with DPT to children < 5 years age
  • Efficacy is good, average being 85%. Adverse effects
• Common: Fever, injection-site pain, erythema, swelling, and irritability.
• Rare: Neurological complications and hypotonic hyporesponsive syndrome
• WC vaccine is contraindicated in: Children > 5–6 years age
  • Associated progressive neurological conditions or family history of epilepsy
  • Hypersensitivity to previous dose.
• Acellular Pertussis Vaccine
  • It is composed of pertussis toxoid and ≥ 2 other bacterial components such as FHA, pertactin or fimbriae.
  • Though the efficacy is same as WC vaccine, it is associated with fewer side effects and safely given after 5–6 years.

Brucella

• Brucellosisis (also called undulant fever) primarily a zoonotic disease acquired from animals such as sheep, goat, or cattle.
• Nomen System of Classification
  • DNA hybridization reveals that Brucella are very closely related and probably represent variants of a single species.
  • However for the sake of convenience, these have been classified into nomen species.
  • Nomen species: Six nomen species identified so far, further classified into several biovars

Pathogenesis

• B.melitensis is most pathogenic followed by B. abortus andB. suis. Human infection with other species is extremely rare.
• Transmission—is usually from infected animals to man. There is no evidence of man to man transmission.
• Direct contact (MC mode) with the infected animal tissue > Ingestion of raw milk or dairy products > Air borne
• Organs affected: Brucellae are facultative intracellular pathogens, primarily infecting organs of reticuloendothelial system.
• Incubation period varies from 1 week to several months and the onset is often insidious.

Clinical Manifestations

• Classic triad: Fever with night sweats; arthralgia/arthritis and hepatosplenomegaly
• Typhoid-like illness: Overall, brucellosis resembles typhoid like illness except that,
• it is less acute, less severe with undulating pattern of fever (or Malta fever or Mediterranean fever) and more musculoskeletal symptoms.
• CNS (Depression and lethargy with meningitis or lymphocytic meningoencephalitis), CVS (Endocarditis rarely, affecting the aortic valve)
• Genitourinary manifestations (acute epididymo-orchitis, prostatitis, salpingitis and pyelonephritis) may also be seen.

Epidemiology

• Endemic area:
  • Human brucellosis is endemic in countries of Mediterranean zone,
  • Eastern Europe, Central Asia, Mexico and
  • South America and rare in most European countries, Australia and North America.
• Sources of infection:
  • Infected animals excreting the organisms in urine, milk, placenta or vaginal discharge and Contaminated animal food products (soft cheeses, milk, icecream)
  • People at higher risk are farmers, shepherds, goatherds, butchers and abattoir workers in endemic areas (occupationally exposed to infection).

Laboratory Diagnosis

• Culture and Identification
• Sample: Blood, bone marrow, CSF, joint fluid or other tissues.
• Cultural media: Biphasic blood culture bottles media (Castaneda’s) made up of Brain heart infusion (BHI) broth/agar
• Erythritol: Improves growth Automated techniques such as BACTEC and BacT/Alert systems.

Antibody Detection by Standard Agglutination Test (SAT)

• It remains the gold standard test serological test:
• It is a tube agglutination test detecting antibodies in serum by using standard strain of B.
• SAT detects IgM antibodies against antigens of smooth LPS: Hence useful for acute brucellosis

Other Tests

• Serological tests to detect IgG antibody—2ME (2-mercaptoethanol) test, CFT, ELISA.
• PCR using primers for rrs-rrl gene, Omp2 gene and IS711 insertion sequence.
• Brucellin skin test
• Guinea pig inoculation
• Tbilisi phage typing is done
• Diagnosis of brucellosis in animals
  • Isolation of brucellae from milk and dairy products.
  • Antibody detection in milk: By Milk ring test, Rose Bengal card test, and whey agglutination test.

Treatment

• Gold standard regimen in adults: Streptomycin plus doxycycline
• WHO regimen in adults: Rifampin plus doxycycline
• Relapse or treatment failure occurs in 5–10% of cases.
• For CNS involvement: Ceftriaxone is added to the regimen and treatment is prolonged for 3–6 months.