Lecture #12: The Gram Negative Coccobacilli (GNCB)

 

Gram–negative coccobacilli (GNCB)

1.     The HACEK Group

2.     Pasteurella Group

3.     Bordetella Group

4.     Francisella Group

5.     Brucella Group

 

THE HACEK GROUP

All the HACEK group are small, fastidious, pleomorphic GNCB and are commensals of the oropharyngeal/respiratory tract. They cause endocarditis and a range of other infections.

H	Haemophilus species
A	Aggregatibacter actinomycetemcomitans (formerly Actinobacillus actinomycetemcomitans)
	Aggregatibacter aphrophilus (formerly Haemophilus aphrophilus and Haemophilus paraphrophilus)
	Aggregatibacter segnis (formerly Haemophilus segnis)
C	Cardiobacterium valvarum, Cardiobacterium hominis
E	Eikenella corrodens
K	Kingella kingii, Kingella denitrificans and Kingella oralis

THE HAEMOPHILUS SPECIES

General characteristics

1.  They are gram negative, pleomorphic bacilli or coccobacilli, non–motile, non– sporeformers and they require factors present in blood for adequate growth.

a.  X Factor

(1)  A heat stable substance known as Protophorphyrin IX which is hemin associated with hemoglobin.

(2)  Necessary for synthesizing iron containing enzyme, cytochrome oxidase and catalase peroxidase.

b.  V Factor

(1) A heat labile substance known as coenzyme I or nicotinamide adenine dinucleotide (NAD) or nicotinamide adenine diphosphate which can be supplied by yeast, blood, potato extract in culture media.

(2) Responsible for “satellite phenomenon” produced by H. influenza.

2.  They are facultatively anaerobic.

3.  They are also known as hemophilic bacteria because of their affinity for the constituents of fresh blood.

Hemophilus sp.	Requirement		Hemolysis	Capsule
	X	V
H. influenza	+	+	–	+
H. aegypticus	+	+	–	+
H. haemolyticus	+	+	+	–
H. parahaemolyticus	–	+	+/–	–
H. parainfluenzae	–	+	+/–	+
H. ducreyi	+	–	+/–	–
H. aprophilus	+	–	–
H. paraprophilus	–	+	–
All species of Haemophilus require X factor for growth except H. parainfluenzae and all species requires V factor except H. ducreyi.
All species do not grow in the presence of CO2 except H. ducreyi. All species are non–hemolytic except H. hemolyticus and H. ducreyi.

HAEMOPHILUS INFLUENZAE

Characteristics:

a.  Young cultures are capsulated. Capsule swelling test is used for “typing” Haemophilus influenzae.

b.  Satellitism – luxuriant growth of Hemophilus near a source of incorporated V factor. Staphylococcal toxin lyses red blood cells and release hemoglobin for Hemophilus to use. The X factor is supplied by the blood.

Two types of strains of Haemophilus influenzae:

1.  Encapsulated Strains (Typeable strain)

Six different capsular serotypes (a to f) have been described. Haemophilus influenzae serotype b (Hib), whose capsule is a repeating polymer of polyribosyl–ribitol phosphate (PRP), has accounted for most invasive disease caused by Haemophilus influenzae.

a.  Invasive disease due to Haemophilus influenzae serotype a (Hia) was extremely rare, except among Indigenous populations in North America and Australia. In such populations, the clinical manifestations of invasive Hia disease were similar to Hib, typically causing meningitis and septicemia in children younger than 2 years, and adults with invasive Hia disease usually had underlying comorbidities and presented with pneumonia, septic arthritis, and osteomyelitis.

b.  Haemophilus influenza type b (Hib) is a bacterium responsible for severe pneumonia, meningitis, and other invasive diseases almost exclusively in children aged less than 5 years. It is transmitted through the respiratory tract from infected to susceptible individuals. Hib also causes potentially severe inflammatory infections of the face, mouth, blood, epiglottis, joints, heart, bones, peritoneum, and trachea.

c.   Haemophilus influenzae serotype c causes meningitis only occasionally in man.

d.  Serotype d strains are rarely associated with disease.

e.  Serotype e strains cause respiratory infections, conjunctivitis, vaginitis, abscess, cellulitis, peritoneal infection, sepsis, and meningitis.

f.  Haemophilus influenza type f (Hif) was the second most common capsular serotype causing invasive disease (e.g., meningitis).

2.  Unencapsulated Strains (Non–typeable strain)

They are small gram–negative coccobacilli, which neither produce nor have the genetic material to code for a polysaccharide capsule. They grow aerobically or as facultative anaerobes; require protoporphyrin IX or iron–containing protoheme, NAD, or NAD phosphate; and grow optimally between 35 and 37°C. They are chemoorganotrophic and occur as obligate parasites on the mucous membranes of humans.

In contrast to Hib, non–encapsulated or non–typeable Haemophilus influenzae (NTHi) are a genetically and antigenically diverse group of organisms consisting of numerous clonotypes.

Non–typeable strains cause otitis media, epiglottitis, acute exacerbations of chronic bronchitis, and other ailments.

Virulence Factor of NTHi
Factor	Activity
Lipopolysaccharide	Epithelial adhesin, interference with complement activation, impairs mucociliary escalator function
P2, P5	Promotes binding to mucus, adhesion to epithelium (P5)
Protein D	Impairs mucociliary escalator functions
HMW1, HMW2	Epithelial adhesin
Hia	Epithelial adhesin
Pili, Hap, Protein E	Promotes binding to the extracellular matrix, protects against the lytic action of complement (PE)

Cultural characteristics:

a.  Levinthal’s medium – large, opaque, flattened, and iridescent

b.  Filde’s medium – translucent and bluish

c.   Chocolate agar with isovitalex – colorless, transparent with “mousy” odor, “dew drop” colonies.

d.  Chocolate agar with Vancomycin, Bacitracin and Clindamycin

e.  Avery Oleate Hemoglobin Agar – enhances growth of H. influenza and inhibits growth of streptococcus and pneumococcus.

Other Haemophilus specie: 

1.  Haemophilus ducreyi (Ducrey’s bacillus)

a.  It requires X factor but not V factor.

b.  It is grown best from scrapings of the ulcer base on chocolate agar containing isovitalex and vancomycin in 10% CO₂. They can also be grown on chorioallantoic membrane of chick embryo.

c.   It gives a red color on Pappenheim’s stained urethral smear and ulcer secretion.

d.  It gives a positive result on Ito Reenstierna test. Killed Haemophilus ducreyi is injected intradermally and positive result is indicated by an induration of 7mm or more within 48 hours. The test remains positive permanently.

e.  Haemophilus ducreyi causes chancroid (soft chancre), a sexually transmitted disease consisting of ragged ulcer on the genitalia with marked swelling and tenderness. Regional lymph nodes are enlarged and painful.

2.  Haemophilus aegypticus (Koch–Weeks bacillus)

a.  It is the causative agent of pink eye conjunctivitis in children in hot climates as well as Brazilian purpuric fever.

3.  Haemophilus parainfluenzae – normal flora of mouth and nasopharynx

4.  Gardnerella vaginalis

a.  Formerly known as Hemophilus vaginalis or Corynebacterium vaginalis.

b.  They are non–motile, pleomorphic, non–encapsulated, gram-negative rods.

c.  It is associated with bacterial vaginosis (non–specific vaginosis)

d. The disease shows minimal or no signs of inflammation in the vagina and because of polymicrobial etiology.

e.  Clinical manifestations: malodor, leukorrhea, pruritus, excessive vaginal discharge, pH is more than 4.5, presence of clue cells (vaginal epithelial cells covered by small gram-negative rods).

f.  Common blood isolate is post–partum and post–abortal fever.

AGGREGATIBACTER ACTINOMYCETEMCOMITANS 

It causes a disease known as Juvenile periodontitis.

Serogroups:

Five serogroups of Aggregatibacter actinomycetemcomitans were classified by Taichman based on surface polysaccharide located on the O–side chain of lipopolysaccharide using tube agglutination studies:

Serotype	Surface Polysaccharide
Serotype A	consists of repeating disaccharide units of 0-acetyl-6-deoxy-D-talose and 6-deoxy–D–talose
Serotype B	repeating trisaccharide units of L–rhamnase, D–fucase and N–acetyl–D–galactosamine
Serotype C	repeating 0-acetyl–6–deoxy-L–talose and 6-deoxy–L–talose
Serotype D	repeating tetrasaccharide units of D–glucose, D–mannose and L–rhamnose
Serotype E	2–acetamide–2-deoxy–D glycose and L–rhamnose
Serotype F	2–acetamide–2–deoxy–D–galactose and L–rhamnose

Biochemical properties and Growth characteristics:

a.  Mesophilic and capnophilic require 5-10% carbon dioxide for good growth.

b.  Microaerophilic and a facultative anaerobe, nonmotile, nonhemolytic, non-sporing.

c.  Colonies on Chocolate Agar are small, with a diameter of 0.5mm after 24hr, but may exceed 1-2 mm after 48h. The colonies are translucent (or transparent) with irregular edges that appear smooth, circular & convex.  The colonial morphology of fresh isolates is distinctive with star shaped morphology form in the agar that gives Aggregatibacter actinomycetemcomitans its name. In addition to having a star shaped internal structure, colonies of fresh isolates are rough surfaced. Repeated subculture yields, two types of colonial variants: one is smooth–surfaced and transparent, smooth–surfaced, and opaque. The transparent smooth–surfaced variants appear to be an intermediate between the transparent rough–surfaced & opaque smooth-surfaced types. The colonial variation is associated with fimbriae.

d.  Exclusive growth of Aggregatibacter actinomycetemcomitans was found in a particular culture medium which contained TSBV (Trypticase Soy Serum with Bacitracin and Vancomycin) – it is an excellent primary selective medium in detecting microorganisms in levels as low as 20 viable cells per ml with spiramycin, fusidic acid and carbenicillin. Also grows on Dulbecco’s Modified Eagle Medium and the new selective medium called GCHB Medium.

e.  Aggregatibacter actinomycetemcomitans bind to collagen I, II, III and V but not IV. It also binds to fibronectin but not fibrinogen.

f.  X and V factors are not required.

g. Capable of producing acid from glucose, fructose, and maltose but not from arabinose, cellobiose, melibiose, melezitose, salicin and sorbitol.

h. Reduce nitrate and produce alkaline phosphatase but are negative for indole, urease, ornithine and lysine decarboxylases and arginine dihydrolase.

i.  Oxidase negative or weakly positive and catalase positive.

VIRULENCE FACTORS
Virulence Factors that Modulate Immune System	Leukotoxin of RTX (repeats in toxin) family
	Super antigen producing T cell apoptosis
	Cell cycle modulatory protein called as cytolethal distending toxin
	Fc binding protein termed as Omp34
	Monocyte/macrophage modulating protein and neutrophil chemotaxis inhibitor
Virulence Factors inducing Tissue Destruction	Lipopolysaccharide (LPS) present on the bacterial cell wall
	Secreted proteins like cell stress protein

 

AGGREGATIBACTER APROPHILUS

a.  It causes Infective Endocarditis.

b.  Haemophilus aphrophilus and Haemophilus paraphrophilus are now combined as the same species collectively known as Aggregatibacter aphrophilus.

c.  It is oxidase–weakly positive, catalase–negative coccobacillus, which was best grown on Chocolate Agar after 48 hours incubation in 5% CO₂. However, growth on blood agar was very slow and completely absent on MacConkey agar.

d. Infective endocarditis and cerebral abscesses are the most frequent invasive infections.

e.  Haemophilus paraphrophilus differs from Haemophilus aphrophilus because of its dependence on the V–factor. The V–factor is being considered as a variable phenotypic characteristic.

Characteristics	Aggregatibacter aphrophilus		Aggregatibacter actinomycetemcomitans
	H. aphrophilus	H. paraphrophilus
Catalase	–	–	+
Oxidase	v	v	v
X Requirement	–	–	–
V Requirement	–	+	–
ONPG	+	+	–
Indole	–	–	–
Ornithine decarboxylase	–	–	–
Urease	–	–	–
Acid from Lactose	+	+	–
Acid from Trehalose	+	+	–
v – variable ; ONPG – O-Nitrophenyl-β-D-galactopyranoside

 

AGGREGATIBACTER SEGNIS

a.  It is associated with skin and soft tissue infection (SSTI) and intra-abdominal infection as well as necrotic appendicitis but has been recognized as a rare cause of endocarditis.

b.  This bacterium is catalase positive, oxidase negative, requires V factor but not X factor to grow and xylose is not fermented. It can ferment lactose.

c.  Small amounts of acid result from the fermentation of glucose, fructose, galactose, sucrose, and maltose. Fermentation of sucrose is usually stronger than fermentation of glucose. Catalase and β-galactosidase (hydrolysis of ONPG) are variably present. They are negative for oxidase, indole, urease, and ornithine decarboxylase tests.

d.  Colonies on Chocolate Agar are smooth or granular, convex, greyish white and opaque, and may take 48 h to reach a diameter of 0.5 mm. Growth in broth and fermentation media is slow, and reactions are negative or weakly positive. CO₂ enhances growth for some strains.

CARDIOBACTERIUM HOMINIS AND

CARDIOBACTERIUM VALVARUM

a.  Cardiobacterium species are pleomorphic gram–negative rods; morphology varies considerably depending on culture conditions. They often have swelling of one or both ends and retain the crystal violet dye at the poles during the Gram stain procedure.

b.  Microscopically, organisms sometimes form rosettes, but short chains, teardrops, pairs, and clusters are also common.

c.  Colonies of Cardiobacterium valvarum are nonhemolytic; however, colonies of Cardiobacterium hominis produce slight α-hemolysis after 3 to 4 days of incubation and develop a rough appearance, with a serpentine pattern of growth from the edge to adjacent colonies.

d.  Cardiobacterium organisms are oxidase positive and catalase negative, and they produce indole (although positivity is weak in many strains of Cardiobacterium hominis and absent in some oral strains of Cardiobacterium valvarum).

EIKENELLA CORRODENS

a.  Eikenella corrodens is a microaerophilic gram–negative rod which is a normal inhabitant of human mucosal surfaces, particularly the oral cavity. Although an uncommon cause of infection, it is by no means rare.

b.  Eikenella corrodens usually require hemin (X factor) and carbon dioxide for primary isolation and may be misidentified as Haemophilus species.

c.  The colonies of Eikenella corrodens have a characteristic “bleach-like” odor and often “pit” the surface of the agar thus its name. Older cultures of Eikenella corrodens may appear yellow.

d.  The cells do not produce spores and are nonmotile. “Tremor-shaped movement” can be seen on the surface of the agar.

e.  Two different types of colonies can form on blood agar: invasive phenotype and noninvasive phenotype.

(1) The invasive strain forms on the surface of blood agar when conditions are 36°C, with 15% CO₂ and 100% humidity. Colony diameter ranges from 0.2 mm to 0.5 mm (after 24 h culture) or from 0.5 mm to 1.0 mm (after 48 h culture). Colonies are light yellow and opaque, and the center of the colony has a clear pearlescent ring. The edge of the colony is rough, refractive, and has a hair-like diffuse edge, and “tremor-shaped movement” can be seen on the surface of the agar.

(2) The noninvasive phenotype forms colonies with a diameter of 0.5–1 mm. The colonies are hemispherical, translucent, with no hair-like diffuse edge. They do not invade agar, show no adhesion to the agar, and have no “tremor–shaped movement.”

f.   Eikenella corrodens does not grow well in liquid media. Broth supplemented with 0.2% agar, cholesterol (10 mg/L), and 3% serum can promote its growth. Under aerobic conditions, 5–10% bile can inhibit growth. However, under anaerobic conditions, up to 10% bile can be tolerated.

g.  Eikenella corrodens is biochemically inactive. It does not ferment glucose and other carbohydrates or produce acid. It tests negative for catalase, urease, arginine dehydrogenase, and indole, but is positive for nitrate reduction, as well as oxidase and lysine decarboxylase.

h.  Evaluation of laboratory parameters showed that the ESR was a better indicator of Eikenella infection than was the WBC count.

i.   It has been linked to a variety of disease states including abscesses, endocarditis, meningitis, osteomyelitis, keratitis, conjunctivitis, and cellulitis.

j.   Virulence factors of Eikenella corrodens include a surface protein capable of binding to epithelial cells, pili, endotoxin, hemolysin, and B-lactamase.

THE KINGELLA SPECIES

a.  Kingella kingae is a common etiology of pediatric bacteremia and the leading agent of osteomyelitis and septic arthritis in children aged 6 to 36 months. Kingella kingae cells tend to resist decolorization, and thus the organism may be erroneously identified as gram–positive, but electron microscopic examination discloses a characteristic gram–negative cell wall structure. The bacterium is beta-hemolytic, nonmotile, and non–spore forming, exhibits negative catalase, urease, and indol tests, and, with rare exceptions, has oxidase activity.

b.  Kingella kingae produces acid from glucose and usually from maltose, hydrolyzes indoxyl phosphate and l-prolyl-β-naphthylamide, and exhibits positive alkaline and acid phosphatase reactions. The fatty acid content of the organism resembles that of Kingella denitrificans and comprises a high percentage of myristic acid and lesser concentrations of palmitic, lauric, palmitoleic, linoleic, oleic, 3–hydroxilauric, 3–hydroximyristic, and cis–vaccenic acids.

c.   Kingella kingae is a facultative anaerobic bacterium that grows on conventional Trypticase Soy Agar supplemented with 5% hemoglobin (blood agar medium), chocolate agar, Columbia-based blood agar.

d.  Kingella kingae appears as pairs or chains of 4 to 8 plump (0.6 to 1 μm by 1 to 3 μm) coccobacilli.

e.  Kingella kingae RTX toxin is a 100-kDa protein that appears to be secreted in the extracellular environment in a soluble form, as well as a component of outer membrane vesicles (OMVs) that are internalized by host's cells, suggesting that Kingella kingae utilizes multiple mechanisms for toxin release. RTX toxins have been divided into the following three categories based on cellular specificity:

(1) The hemolysins, which exhibit toxicity for a wide range of cell types (including erythrocytes)

(2) The cytotoxins which exhibit toxicity for a wide but defined range of cell types.

(3) The leukotoxins which exhibit very narrow cell types and species specificity.

f.   Kingella denitrificans, which has been implicated in cases of bacteremia, endocarditis, pleural empyema, pediatric vaginitis, chorioamnionitis, and granulomatous disease in AIDS patients. The microorganism is oxidase and catalase positive but negative on nitrate reduction test and has a colony morphology that is grossly indistinguishable from Neisseiria gonorrhea on Thayer Martin agar.

g.  Kingella oralis, which is a commensal dweller of the human buccal cavity and is associated with dental plaque and periodontitis.

PASTEURELLA GROUP

They are facultatively anaerobic, non–motile, gram–negative bacteria that may range morphologically from coccobacilli to long filamentous rods. They require organic nitrogen sources, oxidase, and catalase. It is isolated frequently from wounds resulting from animal bites or scratches.

Clinical manifestations are due to animal bites infection and associated with respiratory tract infection including sinusitis, pneumonia, bronchitis, and pulmonary abscess.

Pasteurellosis is the collective term that describes the infection caused by Pasteurella bacteria.

Species of Pasteurella:

1.  Pasteurella multocida

a.  The strains include Pasteurella multocida subspecies gallicida, Pasteurella multocida subspecies septica, Pasteurella multocida subspecies tigris and Pasteurella multocida subspecies multocida based on their ability to ferment sorbitol and dulcitol.

b.  The Carter Capsular Serogroup is grouped according to the structure of the lipopolysaccharide (LPS) antigens and designated as A, B, D, E and F. The capsular polysaccharide synthesized by serogroup A is hyaluronic acid, serogroup D is heparin and serogroup F is chondroitin. No structural information is available for serogroup B and E. Capsular types A and D produce a dermonecrotic toxin (PMT), encoded by the toxA gene, which modulates the immune response. The typing system includes Indirect Hemagglutination Asay, Hyaluronidase Decapsulation Test and Acriflavine reaction.

c.   The Heddleston Somatic Serovars is grouped based on somatic O antigen and designated 1 to 16. This was done through Gel Diffusion Precipitin Test.

d.  The Namioka System is based on a Tube Agglutination Test and can recognize 11 serovars.

e.  The selective medium used for the isolation of Pasteurella multocida is the CGT medium, containing Clindamycin, Gentamicin, Potassium Tellurite and Amphotericin B in 5% horse blood agar where they appear as small, discrete, round, smooth, convex, and opaque colonies.

f.   Another selective medium that is used in the isolation of Pasteurella multocida is the Mueller-Hinton blood agar that contains 2 mg of Amikacin, 4 mg of Vancomycin, and 4 mg of Amphotericin B where they appear as opaque and grayish colonies.

Other Pasteurella species affecting mostly animals:

1.  Pasteurella pneumotropica is differentiated from other Pasteurella species based on its ability to produce urease and its glucose oxidation profile. It differs from Pasteurella multocida because it does not form acid from mannitol or sorbitol. It rarely causes infection with only a few cases of septic arthritis or animal bites or licks to an open wound.

2.  Pasteurella hemolytica (Mannheimia haemolytica) – causes pneumonia in cattle but human infection is extremely rare. First isolated in human urine.

3.  Pasteurella ureae (Actinobacillus ureae) – normal human respiratory flora but rarely causes endocarditis and bacterial peritonitis.

BORDETELLA GROUP

Bordetella organisms express filamentous, polymeric protein cell-surface structures called FIM. Two serotypes of FIM, serotype 2 and serotype 3, are most observed on clinical strains.

BIOCHEMICAL IDENTIFICATION OF BORDETELLA SPECIES
Specie	Catalase	Oxidase	Nitrate	Urease	Motility	BAP	MAC
B. pertussis	+	+	–	–	–	–	–
B. parapertussis	+	–	–	+	–	+	+/– (d)
B. bronchiseptica	+	+	+	+	+	+	+
B. hinzii	+	+	–	+/–	+	+	+
B. holmesii	+	–	–	–	–	+	+ (d)
B. trematum	+	–	+/–	–	+	+	+
B. avium	+	+	–	–	+	+	+
(d) – delayed

BORDETALLA PERTUSSIS

            Characteristics:

a.  They are short, gram–negative coccobacilli with bipolar metachromatic granules with toluidine blue stain, capsulated and non–motile.

b.  They are non–sporeformers, hemolytic organisms.

c.  It does not require X and V factors for growth.

Antigenic structures and biologically active substance:

a.  Pertussis toxin (PTX) – a major virulence factor, responsible for the paroxysmal coughing characteristics of the disease. It is an adenosine diphosphate (ADP)–ribosylating protein toxin that inhibits signaling through a subset of G protein–coupled receptors (GPCRs) in mammalian cells. PTX expresses a wide variety of different biological activities. They include the induction of morphological changes of target cells, of exocrine secretion, of the stimulation of lipolysis, the activation of pancreatic islet cells, histamine sensitization, the induction of lymphocytosis and many others. Therefore, PTX was originally given different names, such as islet–activating protein, histamine sensitization factor, and lymphocytosis promoting factor. It was only in the early 1980s, through the work of Munoz on very pure, crystallin PTX that all these different activities were related to a single molecule, named pertussigen, and now referred to as PTX. PTX has been shown to inhibit the ability of monocytes to internalize bacteria, suggesting that the inhibition of monocyte phagocytosis by PTX may represent an immune evasion mechanism of Bordetella pertussis.

b.  Filamentous hemagglutinin (FHA) – enables the attachment of bacteria to the epithelium of the upper respiratory tract. FHA mediates bacterial adherence to epithelial cells and macrophages in vitro and is absolutely required for tracheal colonization in vivo.

c.   Adenylate Cyclase Toxin (ACT) – was originally identified as hemolysin because it causes lysis of blood cells. This enzyme in fact is responsible for producing zone of hemolysis in colonies on blood agar. ACT can upregulate MHC class II and costimulatory molecules on dendritic cells, inducing a semimature state that decreases proinflammatory cytokine production. This effect was due to the enzymatic activity of the toxin, while cytotoxic effects of ACT on these cells were associated with the pore-forming activity.

d.  Heat Labile Toxin (HLT) – induces hemorrhagic lesion. This toxin produces strong vasoconstrictive effects, which are probably important during the initial phase of pertussis by its action on the respiratory tract. The activity of the toxin is detoxified by heat inactivation at 56^oC for 30 minutes.

e.  Tracheal cytotoxin (TCT) – is a cell wall peptidoglycan fragment and cannot be classified as an exotoxin or an endotoxin. The toxin kills ciliated epithelial cells in the respiratory tract and stimulates the release of interleukin 1 (IL-1), which acts as an endogenous pyrogen. It also increases the production of nitric oxide, which diffuses into ciliated cells, causing cell death.

f.   Pertactin (PRN) – is an autotransporter protein located on the surface of Bordetella pertussis and is highly immunogenic. Antibodies to it are found after natural disease and immunization with vaccines containing this protein. It was reported to be essential for resisting neutrophil–mediated clearance and possesses additional immunomodulatory abilities that aid Bordetella pertussis in suppressing the production of proinflammatory cytokines.

g.  Dermonecrotic Toxin (DNT) – the toxin possesses novel transglutaminase activity that catalyzes polyamination or deamidation of the small GTPases of the Rho family. The modified GTPases lose their GTP hydrolyzing activity, function as a constitutive active molecule, and continuously transduce signals to downstream effectors, which mediate the consequent phenotypes of cells intoxicated by DNT.

Pathogenicity:

It is responsible for whooping cough or pertussis or tusperina and is characterized by three stages:

a.  Catarrhal stage – appears after an incubation period of two weeks, the patient is highly infectious but not very ill. Characterized by upper respiratory tract infections, fever, fatigue, rhinorrhea, lacrimation, and cough.

b.  Paroxysmal stage – manifested by continuous coughing that ends in an inspiratory whoop. Leukocytosis and lymphocytosis were highest at this stage of the disease, and the highest levels of leukocytosis were observed in children with fatal complications. It usually lasts up to six weeks.

c.   Convalescent stage – lasts from two to three weeks characterized by residual cough. The patient is susceptible to respiratory tract infections.

Laboratory Tests:

a.  Culture 

(1)   Stuart’s medium, Mishulow’s charcoal agar – transport media

(2)   Bordet–Gengou media (Potato Blood Glycerol Agar) – colonies are pearl–like clusters resembling mercury droplets surrounded by a zone of hemolysis.

(3)   Jones Kendrick Charcoal Agar with Cephalexin – used both as a Transport and Isolation media. The colonies are tiny, smooth, transparent, glistening, and domed.

(4)   Regan–Lowe medium – colonies appear opaque and black in color.

(5)   Stainer–Scholte Medium with heptakis – used primarily for vaccine production. The key ingredients of the medium are sodium glutamate and proline. One of the disadvantages of this medium is it leads to the formation of large amounts of ammonium and poly-hydroxybutyrate, which can influence virulence factor production.

b.  Direct Fluorescent Antibody (DFA) Test

c.  Polymerase Chain Reaction (PCR) using Nasopharyngeal Swab

BORDETELLA PARAPERTUSSIS

It produces a disease similar to Bordetella pertussis. It grows rapidly and produces large colonies as compared with Bordetella pertussis. It is non–motile, urease positive but oxidase negative. Produces a brown pigment called melanin which is one of its virulence factors.

BORDETELLA BRONCHISEPTICA

It inhabits the respiratory tract of carriers in which it may cause “kernel cough” and pneumonitis. They are motile (peritrichous), urease and oxidase positive. It’s infection mimics that of Pneumocystis jiroveci.

FRANCISELLA GROUP

Francisella are nonmotile, encapsulated, gram–negative coccobacilli and are facultative intracellular pathogens of humans and many animals. The genus consists of three recognized species: Francisella tularensis, Francisella novicida, and Francisella philomiragia. 

Francisella bacteria are commonly transmitted to vertebrates via bites by ticks or mosquitoes. The three tick species that are most important for human transmission include Dermacentor andersoni, Dermacentor variabilis, and Amblyomma americanum.

FRANCISELLA TULARENSIS

            Characteristics

a.  Francisella tularensis is a small zoonotic, non–motile, bipolar staining gram–negative coccobacillus. It is an obligate aerobe and grows poorly on ordinary culture media. Its growth is stimulated by the presence of cysteine in the medium.

b.  Common hosts associated with Francisella tularensis are rodents, ground squirrels, wild rabbits, semi-aquatic rodents, hares, ticks, tabanid flies, and mosquitoes.

c.  It can survive and grow within Acanthamoeba castellanii. The infection process begins when trophozoites engulf Francisella tularensis cells, which replicate and grow in vacuole structures inside the trophozoites.

d.  It was classified by the CDC in 2002 as a biological weapon category A because of its high virulence and the possibility of inducing fatal pneumonia by aerosol diffusion. Indeed, 10 CFU of this bacterium can cause infection in humans. No tularemia vaccine is currently authorized for human use.

e.  It causes tularemia, a necrotizing bronchopneumonia that leads to sepsis and death.

Francisella tularensis Infection:

Infection by Francisella tularensis occurs primarily after inadvertent exposure to infected wildlife species, most frequently rodents, hares, and rabbits. Transmission to humans occurs via direct contact, through arthropod or insect vectors, by ingestion of contaminated material(s), or by inhalation of aerosolized organisms. Regardless of the entry route, Francisella tularensis can disseminate from the initial infection site to the lungs where it can cause respiratory tularemia, the most severe form of the disease.

Tularemia usually manifests in humans by a flu–like syndrome occurring on average 3 to 5 days after infection, with a maximum incubation period of two weeks. Then, the disease classically progresses to one of the six clinical forms (sometimes combined), depending on the route of contamination.

a.  Ulceroglandular form manifests by a skin lesion at the inoculation site of bacteria and regional lymphadenopathy.

b.  Glandular form manifests by regional lymphadenopathy without skin lesions.

c.   Oculoglandular form corresponds to the bacterium inoculation through the conjunctiva. It corresponds to conjunctivitis with satellite lymphadenopathy.

d.  Oropharyngeal form occurs after oral contamination. It corresponds to pharyngitis with submandibular or cervical lymphadenopathy.

e.  Pneumonic form is triggered by the bacteria’s inhalation and corresponds to acute, subacute, or even chronic pneumonia.

f.  Typhoidal form manifests by severe sepsis usually associated with neurological signs (confusion) and bacteremia. The typhoidal form is a severe form of tularemia without the skin or lymph node symptoms, but with gastrointestinal and pulmonary symptoms.

These two last systemic infections are the most severe forms of tularemia.

Tularemia disease has two different cycles:

a.  Terrestrial cycle – the main vectors of the terrestrial cycle are wild rabbits, ticks, and certain fly species. The agent of the terrestrial cycle is Francisella tularensis subspecies tularensis.

b.  Water cycle – beaver, muskrat, and other rat species play a role in the water cycle. The agent of the water cycle is Francisella tularensis subspecies holarctica.

Francisella tularensis Serotypes:

a.  Jellison Type A strain (Francisella tularensis tularensis) – the more virulent form; infection through tick bite or direct contact will lead to tularemia. This strain is also known as “Francisella tularensis nearctica” as used in USSR in 1970.

b.  Jellison Type B strain (Francisella tularensis holarctica) – have been tightly associated with transmission by mosquitoes. This strain is also known as "Francisella tularensis palearctica" as used in USSR in 1970. This strain is being used as live attenuated vaccine strain (LVS) and is currently the only effective vaccine against tularemia. The vaccine is approved only for use in clinical trials, and its future availability is undetermined because the protective response offered by the vaccine has not been well characterized, the basis of its attenuation is unknown, and the LVS strain is fully virulent when delivered intraperitoneally in mice. The fact that the LVS provides protective immunity suggests that efforts to create a defined attenuated mutant may be fruitful, however such strains may not be available for some time.

Both tularensis and the holarctica subspecies require Level 3 biocontainment.

Laboratory Tests:

a.  On Glucose Cysteine Blood Agar (GCBA) colonies are surrounded by a characteristic green discoloration not associated with true hemolysis. The colonies reach maximal size after 2 to 4 days incubation.

b.  Maltose fermentation is commonly seen with Francisella tularensis and sucrose fermentation is a distinguishing feature of Francisella novicida.

c.  The Foshay Skin Test for tularemia is based on a delayed hypersensitivity reaction. Results will be positive for patients with current infection of at least 7 days or for up to 5 years after recovery.

d.  Microagglutination Test (MAT) is still considered a reference serological method. This test has often been used as a gold standard to evaluate the performances of newly developed serological methods.

e.  ELISA methods have several advantages compared to older methods. Several studies have shown that significant antibody titers are usually detected earlier with ELISA than with TAT, MAT, and IFA tests. ELISA methods generally make it possible to detect specific antibodies as early as the second week of the evolution of the disease. In contrast, TAT, MAT, and IFA methods usually detect antibodies only during the third week.

Other Pathogenic Francisella species:

1.     Francisella novicida

a.  Francisella novicida is “officially a separate species” but often considered an “unofficial fourth subspecies” thus you will see in several literature naming it as Francisella tularensis subspecies novicida.

b.  In the 2010 publication proposing reclassification of Francisella novicida, 11 metabolic traits unique to Francisella novicida were compared to Francisella tularensis were identified.

c.   Francisella tularensis is a classic vector-borne zoonotic pathogen, whereas Francisella novicida is not.

d.  The sole source of Francisella novicida isolates to date has been salt water.

e.  Francisella novicida is unable to efficiently evade the host innate immune response and is recognized by the inflammasome upon escape from the phagosome and entry into the host cell cytoplasm. In contrast, Francisella tularensis successfully escapes inflammasome activation early in infection (~12 h) via a mechanism involving suppression of TLR2 signaling.

f.   Francisella novicida cases occurred in patients who were immunocompromised or had underlying health problems.

g.  Francisella novicida infection in humans is exceedingly rare and therefore often difficult to diagnose accurately.

2.  Francisella philomiragia

a.  It was previously known as Yersinia philomiragia.

b.  It is cysteine–independent and yields white colonies. In contrast, Francisella tularensis is fastidious, with colonies typically appearing gray.

c.   Neither animal nor arthropod vectors are implicated for Francisella philomiragia and although initial isolates of the organism came from freshwater, human cases have often been associated with saltwater exposure.

d.  Hydrogen sulfide production in a Triple Sugar Iron slant is one of the key features in the identification of Francisella philomiragia.

BRUCELLA GROUP

General characteristics

1.  Short, coccobacillary forms predominate. They are aerobic, non–motile, non– sporeforming and capsulated.

2.  Their nutritional requirements are complex requiring amino acids, vitamins, salts, and glucose. H₂S is produced by many strains and nitrates are reduced.

3.  Five to ten percent of CO₂ is required for their growth.

4.  They are moderately sensitive to heat and acidity. They are killed in milk by pasteurization.

5.  They are obligate parasites of animals and humans and characteristically located intracellularly.

6.  They are relatively inactive metabolically.

7.  To be a successful infectious agent, Brucella requires four steps: adherence, invasion, establishment, and dissemination within the host.

8.  The Brucella lifecycle contains two phases: (a) chronic infection of phagocytic macrophage leading to Brucella survival and replication, and (b) acute infection of non-phagocytic epithelial cells leading to reproductive tract pathology and abortion.

9.  Brucella strains may occur as either smooth LPS (S-LPS) or rough LPS (R-LPS) as major surface antigen. This bacterium possesses an unconventional non-endotoxic lipopolysaccharide that confers resistance to anti-microbial attacks and modulates the host immune response. The strains that are pathogenic for humans (Brucella abortus, Brucella suis, Brucella melitensis) carry a smooth LPS involved in the virulence of these bacteria. The LPS O-chain protects the bacteria from cellular cationic peptides, oxygen metabolites and complement-mediated lysis and it is a key molecule for Brucella survival and replication in the host.

BIOVAR DIFFERENTIATION OF BRUCELLA SPECIES
Species	Colony	Biovar	CO2	H2S	Urease	Oxidase	Growth on Dyes		Agglutination in serum
							Basic Fuchsin (20 μg)	Thionin  (40 μg)	A	M	R
Brucella melitensis	S	1	–	–	+	+	+	+/v	–	+	–
		2	–	–	+	+	+	+	+	–	–
		3	–	–	+	+	+	+	+	+	–
Brucella abortus	S	1	+/v	+	+	+	+	–	+	–	–
		2	+/v	+	+	+	–	–	+	–	–
		3	+/v	+	+	–/v	+	+	+	–	–
		4	+/v	+	+	+	+/v	–	–	+	–
		5	–	–	+	+	+	+	–	+	–
		6	–	–	+	+	+	+	+	–	–
		9	+/–	+	+	+	+	+	–	+	–
Brucella suis	S	1	–	+	+	+	–/v	+	+	–	–
		2	–	–	+	+	–	+	+	–	–
		3	–	–	+	+	+	+	+	–	–
		4	–	–	+	+	–/v	+	+	+	–
		5	–	–	+	+	–	+	–	+	–
Brucella canis	R	none	–	–	+	+	–/v	+	–	–	+
(+) positive; (–) negative; –/v or +/v, variable; A – monospecific Brucella abortus antiserum; M – monospecific Brucella melitensis antiserum; R – antirough Brucella serum

Species of Brucella

1.     Brucella abortus (Bang’s bacillus) – causes epidemic abortion in cattle

2.     Brucella suis – epidemic abortion of swine

3.     Brucella canis – affects dogs

4.     Brucella melitensis – affecting sheeps and goats

Routes of infection

1.     Intestinal tract – ingestion of infected milk

2.     Mucous membranes – droplets

3.     Skin – contact with infected tissues of animals.

It causes Brucellosis or Undulant Fever or Malta Fever or Mediterranean Fever or Goat Milk Fever which is transferred to man through infected milk of goats. It is characterized by an acute bacteremic phase followed by a chronic stage that may extend over many years and involve many tissues.

It is called Undulant Fever because of the irregular temperature curve seen during the illness, often there are several weeks of high temperature followed by several days of abatement. The disease was earlier called Gastric Remittent Fever.

Laboratory Tests:

1.  Bacterial culture

a.  Growth is often enhanced by the presence of 5 – 10% CO_2.

b.  Brucella broth, Trypticase Soy Broth (TSB) which is soybean casein digest agar – used for qualitative cultivation.

c.  Castaneda technique – a biphasic bottle for blood cultures

d.  Farrell’s medium – composed of serum dextrose agar with added quantities of antibiotic: Polymyxin B sulphate (5mg), Bacitracin (25mg), Natamycin (50mg), Nalidixic Acid (5mg), Vancomycin (20mg), Nystatin (100,000 IU).

2.     Abortus Bang Ring Probe Test (ABR)

a.  One drop of hematoxylin–stained organism into a tube of 1 ml of milk and thoroughly shaken.

b.  Incubate at 37^oC for 50 minutes.

c.   The agglutinated bacteria are carried to the surface with fat globules to form a colored ring (Schern–Gorli Reaction). 

3.  Rose Bengal Test is used as a screening test and positive results are confirmed by the serum agglutination tests. The Rose Bengal test (RBT) is a rapid slide-type agglutination assay performed with a stained Brucella abortus suspension at pH 3.6–3.7 and plain serum. Because of its simplicity, it is often used as a screening test in human brucellosis and would be optimal for small laboratories with limited means.

4.  Coomb’s Test is the most suitable and sensitive test for confirmation in relapsing patients with persisting disease, but it is complex and demands technique.