Fastidious Gram-Negative Bacilli

General Characteristics

• Organisms covered are miscellaneous and fastidious
• These organisms are pleomorphic (many shapes)
• Gram-negative bacilli.
• Isolation and identification require special nutrients

Organism Classifications - Family

• Haemophilus classification
• Actinobacillus classification
• Pasteurella classification
• Aggregatibacter classification

Organism Classifications - HACEK Group

• Haemophilus classification
• Aggregatibacter classification
• Cardiobacterium classification
• Eikenella classification
• Kingella classification

Other Fastidious Gram-Negative Bacilli

• Capnocytophaga
• Brucella
• Francisella

Haemophilus

• Haemophilus displays a gram-negative pleomorphic coccobacilli or rods
• Coccobacilli appear in direct smears
• Rods can appear as long filamentous rods from colony growth
• Haemophilus is nonmotile, oxidase positive, and catalase positive
• Haemophilus reduces nitrate and ferments carbohydrates.

Haemophilus - Location

• Resides as obligate parasites on mucous membranes

Haemophilus - Species

• Approximately 13 species exist
• Eight species are associated with humans: H. influenzae, H. parainfluenzae, H. haemolyticus, H. parahaemolyticus, H. pittmaniae, H. aegyptius, H. ducreyi
• H. segnis, H. aphrophilus, and H. paraphrophilus moved to the genus Aggregatibacter.

Haemophilus - Pathogens

• Most species are nonpathogenic or cause opportunistic infections
• Major pathogenic species include H. influenza, H. aegyptius, and H. ducreyi.

Haemophilus - Definition and Growth Factors

• Its definition means “blood lover” in Greek
• Organisms prefer growth factors in blood and require X and V factors
• The X factor is hemin, hematin
• V factor (for Vitamin) is nicotinamide-adenine dinucleotide (NAD).
• Species with the prefix Para require only V factor
• H. parainfluenzae produces X factor and requires V factor.

Differential Characteristics

• Hemolysis occurs on 5% horse or rabbit blood agar by H. haemolyticus species and sometimes H. ducreyi
• Sheep blood agar (SBA) has X factor but lacks V factor
• Some won't grow without added V factor because NADases in blood agar plate (BAP) destroy V factor
• Chocolate (CHOC) agar releases X and V factors and deactivates NADases.

Satellitism

• Fastidious organisms grow around other bacteria that release necessary growth factors or break down toxic products
• S. aureus, S. pneumoniae, and Neisseria species cause hemolysis, releasing factors or naturally producing V factor
• A clinically significant exception to growth patterns is H. ducreyi.

Upper Respiratory Tract Presence

• Haemophilus species make up about 10% of normal flora in the adult upper respiratory tract
• In youngsters, they constitute 2% to 6% from birth through childhood, with higher percentages in daycare centers
• As they mature, they switch from encapsulated to non-encapsulated strains

H. influenzae - Historical Perspective

• Erroneously named during the influenza worldwide pandemic (1889–1890)
• It was frequently isolated from patients with influenza and postmortem lung cultures
• Viral culture techniques revealed that influenza is caused by a virus
• H. influenzae serves as a secondary (opportunistic) invader

H. influenzae - Virulence Factors

• Capsule presence
• Immunoglobulin A (IgA) proteases present
• Adherence by fimbriae and structures
• Outer membrane proteins and lipopolysaccharide (LPS)

H. influenzae - Capsule and Serotypes

• Classification depends on capsule serotype
• Possible antigenic types include a, b, c, d, e, and f
• The differences in the capsular polysaccharide determines the type
• Serotype b(Hib) consists of ribose, ribitol, and phosphate.
• Antiphagocytic and anticomplement activity is suggested by evidence
• Hib vaccine lowers disease incidence

Nontypable H. influenzae

• Some strains do not have a capsule
• Referred to as nontypable strains
• These invade the respiratory tract and tissues in the same area
• They cause localised infections

H. influenzae - Other Virulence Factors

• Immunoglobulin A (IgA) protease cleaves IgA on mucous membranes
• Adherence mechanisms are not well defined.
• Most NTHi strains attach to human epithelial cells while most Hibs do not
• This may explain the tendency for NTHi strains to cause systemic infections
• Outer membrane components are not well defined
• Antibodies against antigens may play a significant role in immunity

H. influenzae - Two Patterns of Disease

• Encapsulated strains cause invasive disease, examples are septicemia, meningitis, arthritis, epiglottitis, tracheitis, and pneumonia
• A localized infection is due to Hib vaccination by the contiguous spread of NTHI
• Examples are conjunctivitis, sinusitis, and Otitis media with effusion (middle ear infection)

Clinical Manifestations - Meningitis

• Prior to the Hib vaccine, most meningitis presented in children ages 3 months to 6 years
• Bloodstream invasion and bacteremic spread happens after colonization, invasion, and organism replication in the respiratory mucous membranes

Clinical Manifestations - Epiglottitis

• Epiglottitis involves acute inflammation and swelling, causing airway obstruction
• It affects children 2 to 4 years old
• Emergency tracheostomy was often necessary

Clinical Manifestations - Bacterial Tracheitis

• A life-threatening disease that affects young children
• It arises after an acute, viral respiratory infection
• Mild to moderate illness lasts for 2 to 7 days and progresses rapidly
• Broad-spectrum antibiotics are imperative since thick secretions can occlude the trachea
• A key differential diagnosis is from epiglottitis

Infections Associated with H. influenzae biogroup aegyptius

• Both H. aegyptius and H. influenzae biogroup aegyptius can cause conjunctivitis
• H. influenzae biogroup aegyptius causes Brazilian purpuric fever (BPF)
• BPF occurs in warm tropical climates
• Presents with recurrent conjunctivitis, high fever, vomiting, petechiae, purpura, septicemia, shock, and vascular collapse
• Has high mortality, as high as 70% within 48 hours after onset

Infections Associated with H. ducreyi

• A sexually transmitted infection causes genital ulcer disease (GUD) called Chancroid
• Incubates in 4 to 14 days
• Results in a painful lesion with an irregular edge
• Genital and perianal areas involved are the penis and labia or vagina
• Causes Enlarged and draining lymph nodes (Buboes)

Infections Associated with H. parainfluenzae

• Has a low incidence of pathogenicity
• Can cause otitis media and acute sinusitis
• May cause Endocarditis though it is rare to do so
• Symptoms include insidious onset
• First symptoms appear about 1 month after routine dental procedures
• infection occurs at the mitral valve
• Absence of other pathogens means the organism may cause pharyngitis

Specimen Processing and Isolation

• Organisms known to disappear rapidly
• Plating must occur within 10 minutes for maximum recovery for H. ducreyi
• Clean site with sterile saline and sterile gauze
• Swab should be moistened with sterile phosphate-buffered saline and plated immediately
• Pus aspiration from the buboes is possible

Media Selection

• H. influenzae needs CHOC agar with bacitracin (300mg/L) to inhibit other organisms, especially respiratory flora
• H. aegyptius needs CHOC agar supplemented with 1% IsoVitaleX
• H. ducreyi needs GC agar (hemoglobin, fetal bovine serum [FBS], vancomycin to reduce genital flora), enriched chocolate, or Mueller-Hinton agar with 5% chocolatized lysed horse blood

Growth Requirements

• Species grow on in a 5% to 10% CO2 and place culture media into atmosphere with high humidity to prevent drying at 35-37° C
• H. ducreyi grows at 33° C with 5% to 10% carbon dioxide (CO2) for 7 days before reporting a negative result
• H. aegyptius follows similar methods with a 4 day incubation

Colony Morphology - H. influenzae

• Translucent, tannish, moist, smooth, and convex
• May produce a mousy or bleach-like odor
• Encapsulated strains appear more mucoid

Microscopic Morphology

• H. influenzae - Gram: small gram-negative coccobacilli or small rods to long filaments; clear nonstaining areas (halos) may be seen.
• H. ducreyi - Gram: coccobacilli arranged in groups resembling "school of fish,” “railroad tracks,” or “fingerprints"

Laboratory ID Initial Findings

• Gram-negative pleomorphic coccobacilli on CHOC agar indicate Haemophilus
• Absence of growth on SBA or MacConkey (MAC) agar is a clue

X and V Factor

• Take colonies from an initial isolation of Haemophilus, without picking up growth media
• Place the colonies in nutrient broth and mix.
• Plate inoculated nutrient broth on a nutrient agar plate or a Mueller-Hinton agar plate.
• Add X and V strips to the media, then incubate at 35 to 37° C in 5% to 10% CO2 18 to 24 hours and read plates

Porphyrin Test

• Differentiates heme-producing Haemophilus species
• Porphyrin positive if they don't require X factor as they can make it
• δ-aminolevulinic acid (ALA) converts to porphyrins or protoporphyrins
• Use Kovacs reagent
• Red color, produced in the lower tube part tests positive if porphobilinogen is present
• A Wood ultraviolet (UV) lamp can be used as it produces a reddish-orange fluorescence under UV light

CLSI Guidelines for ID of Bacteria

• Clinical and Laboratory Standards Institute (CLSI) has guidelines
• From respiratory tract or cerebrospinal fluid (CSF) specimens
• Gram-negative bacilli or coccobacilli exhibit colonies bigger than 1 mm on CHOC agar
• No growth or satellitism on SBA
• Negative porphyrin
• Can identify as H. influenzae

Treatment - H. influenzae

• Hospitalisation is requires for Invasive strains of H. influenzae often
• Cefotaxime or ceftriaxone are recommended for life-threatening illness
• Alternative choices include trimethoprim sulfamethoxazole, imipenem, ciprofloxacin, and chloramphenicol with ampicillin
• Azithromycin, ceftriaxone, ciprofloxacin, or erythromycin are used. with H. ducreyi

Treatment - β-Lactamase Production

• Designed in response to increased ampicillin resistance of Haemophilus spp.
• A disk impregnated with nitrocefin is moistened
• Smear several colonies onto disk and ẞ-lactam ring will break and a red color develops on the disk within 5 minutes
• A test strip contains benzylpenicillin and bromocresol purple.
• Strip is moistened with 1 or 2 drops of sterile distilled water and it will have a color change from purple (negative) to yellow (positive) within 5 to 10 minutes as penicilloic acid develops in pH

HACEK Group

• Includes Haemophilus, Aggregatibacter, Cardiobacterium, Eikenella, and Kingella
• Haemophilus spp are now Aggregatibacter aphrophilus
• Aggregatibacter actinomycetemcomitans was formerly Actinobacillus actinomycetemcomitans.

Haemophilus - General Characteristics

• Gram-negative bacilli
• Fastidious nutritional needs
• increased CO2 environment (5%-10%)
• cause endocarditis
• Usual flora of the oral cavity causes septicemia from wounds on human bites

Defining characteristics of A. aphrophilus.

• The Greek words aphros and philia for foam loving or desiring high CO2 concentrations
• Discovered in dental plaque and gingival scrapings
• Yellow, granular, convex colonies with opaque zone near centers form on CHOC agar

Defining characteristics of A. actinomycetemcomitans

• Primarily an animal pathogen, but been found in oral cavity of humans
• Human tissue infections occur
• Human infections are from cattle, sheep, and swine
• The 6 serotypes are a-f
• a, b, c are types that are common
• A major contributor to periodontitis is also a characteristic

Growth requirements and morphology forA. actinomycetemcomitans

• Growth takes 24 to 48 hours
• CO2 promotes better growth
• Distinctive star formation marks the center of the colonies
• Best viewed at ×100 magnification on clear medium with a light microscope or with a stereomicroscope at the best possible magnification

C. hominis Description

• Pleomorphic, nonmotile, and gram-negative bacillus
• Has false positive on Gram stain
• Display rosettes, swellings, and long filaments
• Flora consists of the nose, mouth, and throat
• Gastrointestinal (GI) tract

Key reactions and management for A. actinomycetemcomitans

• Carbohydrates in serum are fermented
• Lactose or sucrose
• Has positive Catalase, variable oxidase
• Does not grow on MAC plates
• X and V growth factors
• Negative for urease, indole, esculin, and citrate
• Aminoglycosides, 3rd generation cephalosporins, quinolones, chloramphenicol, and tetracycline serves as treatment

Defining characteristics C. hominis

• Related to endocarditis
• Increases on the heart and the valve also has an added resistance to antibiotics
• Needs CO2

Biochemical reactions and therapy for C. hominis

• Requires Oxidase
• Not Catalase
• Requires Indole
• Negative for Urease, nitrate, gelatin, and esculln
• Sucrose can be Fermented
• Therapy used include Penicillin and aminoglycoside

Defining characteristics of E. corrodens

• Oral and bowel colonies
• Wound fights
• Meningitis, pneumonia, and osteomyelitis form as infections from the oral and bower colonies
• Capnophilic environment
• Hemin (x factor)
• Yellow pigmentation
• Bleach-like odour

E. corrodens biochemical tests

• Non-motile, non-fermenter
• Ornithine decarboxylase positive
• Arginine dihydrolase negative
• Lysine decarboxylase positives

Kingella Species Members

• Four are in the genus
• K. kingae
• K. denitrificans
• K. Oralis
• K. potus
• Commonly seen in young pediatric patients bones and joints
• Results in Osteoarthritis

Kingellas Clinical Importance

• K. kingae is a type of oropharynx microbiota that is an agent of the HACEK species
• K. denitrificans is a agent of the disease from the heart and can be seen through poor hygiene

Kingella general type

• short bacilli ends on a chain

Characteristics: Bordetella Species

• Eight major species exist
• Key pathogens in this discussion are B. pertussis and parapertussis, whom both causes the same symptoms, cough is the main indication
• Others include; B. bronchiseptica, avium, etc..

General Characteristics

• Tiny gram-bacteria
• Aerobic, grown in 35-37 degrees celcius
• Doesn’t ferment
• oxidises
• Media

Tracheal Factors for Bordetella pertussis -

- Hemagglutinin
-PT Interferes, with signal process
- cAMP toxins; leads to Air flow,

Key points for Pertussis

= 1/3 week window after cold.

• Paroxysmal phases of whooping
• Vomiting can be the result of coughing episodes

What to take note for Pertussis and how it impacts the infected individual

• No animal reservoirs known at all. Can just be with the specific human
• Direct contact is always the result for transmission or transport
• Highly communicable
• Vax available

Pertussis: Identification/Treat

• Isolate B. or PCR Transport is key for the specimen taken

What you need

• NP Swabs that are collected
• Culture and trans
• No wires are needed
• BAP and Mac can work wonders but also CHOC agar for the isolation
• 3 weeks to see the effects of growth and the best way to see cultures are through a microscope

What to expect and remember with a Pertussis related cough

• A silver silver pinpoint/mercury colonies is a sign, also note; It'll get grey with time
• Increase safranin when checking gram-neg
• Test the Bord as well, Gengou is another agent that can help the process