Essential Microbiology for Dentistry. 5th ed.

Chapter 14. Neisseriaceae, Veillonella, parvobacteria and Capnocytophaga

Neisseriaceae

The Neisseriaceae family of bacteria includes the genera Neisseria and Moraxella. Two species of Neisseria are human pathogens:

 Neisseria gonorrhoeae (the gonococcus)

 Neisseria meningitidis (the meningococcus).

There are a number of non-pathogenic species, such as Neisseria sicca, Neisseria mucosa and Neisseria lactamica, which are members of the indigenous flora, including the oral mucosa. Hence it is important to differentiate these from the pathogenic species isolated from oral samples.

N. gonorrhoeae is the agent of gonorrhoea, the most frequently diagnosed venereal disease in Western Europe and the USA. Gonococci frequently cause pelvic inflammatory disease (PID) and sterility in women, in addition to arthritis and sometimes septicaemia. N. meningitidis is the aetiological agent of meningococcal meningitis, a highly contagious disease associated with a mortality rate approximating 80% when untreated.

General characteristics

Non-motile, Gram-negative cocci ranging from 0.6 to 1.0 μm in diameter. On microscopy, the cocci are seen as pairs with concave adjacent sides (bean-shaped); tetrads, short chains and clusters are occasionally seen but all show the characteristic pairing.

Pathogenic Neisseria species are nutritionally fastidious, especially on initial isolation from clinical specimens; the nonpathogenic species are less so. Though aerobic, most strains of N. gonorrhoeae are capnophilic (they require increased carbon dioxide for growth); haemolysed blood and solubilized starch enhance growth.

Members of this genus grow optimally at 36°-39°C, although the non-pathogenic species can grow at temperatures below 24°C.

Neisseria gonorrhoeae

Habitat and transmission

The human urogenital tract is the usual habitat; oral, nasopharyngeal and rectal carriage in healthy individuals is not uncommon. Spread is by both homosexual and heterosexual intercourse or intimate contact.

Characteristics

Non-motile, Gram-negative, non-capsulate diplococci.

Culture and identification

Specimens are usually inoculated onto an enriched medium (lysed blood or chocolate agar normally) and incubated under 5%-10% carbon dioxide (as the species is capnophilic). Small, grey, oxidase-positive colonies initially become large and opaque on prolonged incubation. Subsequent staining by fluorescent antibody techniques, and the production of acid from glucose but not from maltose or sucrose, confirms the identification. Gram-stained smears (of urethral exudate from men and the cervix in women) usually reveal Gram-negative, kidney-shaped intracellular cocci in pairs. Additionally, several reliable, and sensitive, nucleic acid amplification assays are available for direct detection of N. gonorrhoeae in genitourinary specimens.

Pathogenicity

Gonococci possess a number of virulent attributes:

 pili allow gonococci to adhere and colonize epithelial surfaces and thus cause infection

 immunoglobulin A (IgA) proteases produced by some gonococci break the heavy chain of IgA, thereby inactivating it (IgA is a major defence factor universally present on mucosal surfaces)

 some isolates of N. gonorrhoeae produce β-lactamase, which is plasmid mediated

 a tracheal cytotoxin damages the ciliated cells of the fallopian tube, leading to scarring and sterility.

Treatment and prevention

The majority of gonococci are resistant to β-lactam drugs and hence the choice is β-lactamase-stable cephalosporins. Prevention of gonorrhoea requires the practice of 'safe sex', health education and contact tracing.

Neisseria meningitidis

Habitat and transmission

The main reservoir is the nasopharynx in healthy individuals (10%-25%). Droplet spread is the most common transmission mode.

Characteristics

This organism resembles the gonococcus but N. meningitidis cells are capsulate.

Culture and identification

As for N. gonorrhoeae. Presumptive identification is made by observing Gram-negative cocci in pairs in nasopharyngeal discharge, cerebrospinal fluid or blood smears. Selective media are not required as the organism is found pure in cerebrospinal fluid. Identified by the carbohydrate utilization test: produces acid from the oxidation of glucose and maltose. Serology is useful.

Pathogenicity

In susceptible individuals, meningococci spread from the nasopharynx into the blood stream (septicaemia), and then to the meninges. Septicaemia is accompanied by a rash. Eventual death may be due to meningitis or adrenal haemorrhage (Waterhouse-Friderichsen syndrome). The antiphagocytic properties of the capsule help dissemination, whereas the toxic effects are mainly due to the meningococcal endotoxin.

Treatment and prevention

Penicillin or preferably ceftriaxone (or equivalent cephalosporin) due to wide prevalence of penicillin-resistant strains worldwide.

Commensal Neisseria species

Commensal Neisseria species are common in the oral cavity, nose and pharynx, and sometimes in the female genital tract. The taxonomy of the group is confused and the Human Oral Microbiome Database (HOMD) currently identifies 21 oral taxa. The three main species are Neisseria subflava, N. mucosa and N. sicca. The main difference between these and the pathogenic Neisseria species is the ability of the commensal species to grow on ordinary agar at room temperature in the absence of carbon dioxide supplements.

These organisms are essentially non-pathogenic and are almost always found in oral specimens contaminated with saliva or mucosa. Neisseria species are among the earliest colonizers of a clean tooth surface. They consume oxygen during the early plaque formation and facilitate subsequent growth of facultative and obligate anaerobic late colonizers.

Moraxella

Moraxella (formerly Branhamella) are Gram-negative cocci closely related to the non-pathogenic Neisseria species, but asaccharo- lytic and non-pigmented. They are commensals of the human respiratory tract and are recognized opportunistic pathogens causing meningitis, endocarditis, otitis media, maxillary sinusitis and chronic obstructive pulmonary disease. As the majority of strains produce P-lactamase, they may indirectly 'protect' other pathogens and thus complicate antibiotic therapy.

Veillonella

Veillonella species are obligate anaerobic, Gram-negative cocci frequently isolated from oral samples. Three oral species are recognized: Veillonella parvula (the type species), Veillonella dispar and Veillonella atypica.

Veillonella parvula

Gram-negative, small anaerobic cocci. Found in the human oral cavity, mostly in dental plaque, they are considered as 'benevolent organisms' in relation to dental caries as they metabolize the lactic acid produced by cariogenic bacteria into weaker acids (acetic and propionic) with a reduced ability to solubilize enamel. No known pathogenic potential.

Parvobacteria

Parvobacteria are so called because of their size (Latin parvus: small). They are a miscellaneous, heterogeneous group of small, Gram-negative bacilli that cause a number of different diseases. They include the following genera:

 Haemophilus

 Brucella

 Bordetella

 Pasteurella (includes Aggregatibacter species)

 Francisella

 Gardnerella

 Eikenella.

Of these, Haemophilus and Bordetella spp. are of particular interest, as the former causes significant morbidity in the general population and the latter is the agent of whooping cough. Additionally, Haemophilus spp. and Aggregatibacter spp. are common inhabitants of the oral cavity; the latter being an important periodontopathogen.

Haemophilus spp.

The genus Haemophilus is composed of tiny, non-motile, aerobic, Gram-negative coccobacilli; some are capsulated. One of its major distinguishing features is the requirement of two growth factors:

 X factor: haematin present in blood

 V factor: nicotinamide adenine dinucleotide (NAD) or NAD phosphate (NADP), a vitamin obtained from yeast and vegetable extracts or a metabolic product of most bacteria, including Staphylococcus aureus.

Table 14.1 Some characteristics of Haemophilus spp.a

Species

Factor

requirement

Diseases caused

 

X and V

Acute exacerbation of chronic bronchitis, epiglottitis, meningitis, sinusitis, otitis media, osteomyelitis, arthritis

H. parainfluenzae H. parahaemolyticus H. haemolyticus

V

V

X and V

Commensals of the oral cavity and upper respiratory tract; rarely cause disease

H. aegyptius

X and V

Conjunctivitis

H. ducreyi

X

Chancroid (a sexually transmitted disease; a soft sore)

aH. aphrophilus has been recently renamed as Aggregatibacter aphrophilus hence not included here, although it is a frequent oral commensal.

Fig. 14.1 Satellitism of Haemophilus influenzae (zigzag streak); enhanced growth adjacent to Staphylococcus aureus (vertical streak), which supplies the V factor.

Haemophilus species cause a variety of diseases, as shown in Table 14.1.

Haemophilus influenzae

Habitat and transmission

An upper respiratory tract commensal of humans and associated animals, Haemophilus influenzae is a major aetiological agent of upper respiratory tract infections and acute exacerbations of chronic bronchitis. Although not the cause, H. influenzae is a common secondary colonizer of the respiratory tract after a bout of influenza (the agent of which is the influenza virus).

Characteristics

Small, Gram-negative, non-sporing, non-motile rods; predominantly coccobacillary in nature with a few long bacilli and filamentous forms. Virulent strains (for instance, isolated from the cerebrospinal fluid in meningitis) are capsulated.

Culture and identification

Requires both V factor (NADP) and X factor (haematin) for growth on nutrient agar, but grows on blood-enriched media containing these nutrients. Typically forms large colonies around colonies of other organisms that secrete the V factor, a phenomenon called satellitism. For example, if a blood agar plate (containing the X factor) seeded with H. influenzae is streaked with S. aureus (which secretes the V factor) and incubated overnight at 37°C, the former will grow as large colonies adjacent to the streak of S. aureus (Fig. 14.1).

Pathogenicity

H. influenzae causes four major infections, often accompanied by septicaemia, especially in children and the elderly:

I. meningitis

2. acute epiglottitis

3. osteomyelitis

4. arthritis.

The most important virulence factor of H. influenzae is the polysaccharide capsule. An IgA protease and a factor that causes slowing and incoordination of (respiratory tract) ciliary beating are produced; the outer membrane and endotoxin may contribute to the pathogenesis; there are no known exotoxins. Non-capsulated strains cause exacerbation of chronic bronchitis.

Treatment and prevention

All strains are susceptible to the third-generation cephalosporins, and intramuscular cefotaxime gives excellent results. Prevention by haemophilus b conjugate vaccine against invasive H. influenzae type b infection is recommended in some countries. Close contacts of meningitis patients should be given rifampicin as a prophylactic measure.

Bordetella

There are three species in the genus Bordetella, of which Bordetella pertussis, the agent of whooping cough, is the most important.

Bordetella pertussis

Habitat and transmission

Found in the human respiratory tract in diseased individuals; healthy carriage is not known. Spread is by the airborne route.

Characteristics

Short, sometimes oval, Gram-negative rods; fresh isolates may be capsulated. Fastidious growth requirements.

Culture and identification

Requires a special enriched medium for growth, that is, Bordet-Gengou medium or blood-charcoal agar supplemented with antibiotics. On incubation for 3-5 days at 35°C, under high humidity, iridescent colonies resembling mercury drops appear on Bordet-Gengou medium. Identification is confirmed serologically. Currently, a polymerase chain reaction (PCR) based technique is the most sensitive and rapid method to confirm the identity.

Pathogenicity

Causes whooping cough, especially in preschool children (severe in those under 12 months). The characteristic symptom is the bout of paroxysmal coughs followed by the 'whoop' of rapid inhalation after coughing. Virulence factors identified are tracheal cytotoxin, fimbrial antigen and endotoxin.

Treatment and prevention

Erythromycin is the drug of choice for patients and close contacts but antibiotics have little effect on the course of infection, although they may reduce spread and minimize superinfection.

Prevention is by immunization with whole cell-inactivated vaccine, a component of the diphtheria-tetanus-pertussis (DTP) vaccine of childhood. New acellular, subunit vaccines appear effective.

Aggregatibacter actinomycetemcomitans

This relatively new genus Aggregatibacter (formerly called Actino- bacillus) includes species isolated from humans and mammals. (Latin aggregare: to come together, aggregate; bacter: bacterial rod; Aggregatibacter: rod-shaped bacterium that aggregates with others.)

The only species of this genus routinely isolated from the oral cavity is Aggregatibacter actinomycetemcomitans, so named because it is frequently isolated with Actinomyces spp. from actinomycotic lesions. The reason for this association is unknown. Multiple biotypes and up to six serotypes (a-e) have been described. This species is a major infective agent in particularly aggressive forms of periodontal disease in adolescents (localized aggressive periodontitis) and rapidly destructive periodontal disease in adults.

Habitat and transmission

Primary habitat is unknown but is likely to be subgingival sites of humans and mammals. Infection is endogenous.

Characteristics

Small, short (0.4-1 μm), straight or curved rods with rounded ends. Electron microscopic studies have revealed bleb-like structures on the cell surface, which appear to be released from the cells. Fresh isolates possess fimbriae (lost on subculture).

Culture and identification

Grows as white, translucent, smooth, non-haemolytic colonies on blood agar; grows best aerobically with 5%-10% carbon dioxide. Selective media are available for identification; the tryptone-soy-serum-bacitracin-vancomycin agar yields white, translucent colonies with a star-shaped or crossed cigar-shaped internal structure on first isolation, but this is not a consistent finding (Fig. 14.2). There are two phenotypes: smooth and rough. The latter phenotype is related to fimbriation and to the production of hexosamine-containing exopolysaccharide. Cells from rough colonies grow in broth as granular, autoaggregated cells that adhere to the glass and leave a clear broth. Identified by sugar fermentation and assimilation reactions and acid end products of carbohydrate metabolism.

Fig. 14.2 A star/crossed cigar-shaped colony of Aggregatibacter actinomycetemcomitans.

Pathogenicity

A number of virulence factors, including lipopolysaccharide (endotoxin), a leukotoxin, collagenase, cytolethal distending toxin (cdt), epitheliotoxin-bone resorption inducing factor and a protease-cleaving IgG, have all been isolated from Aggregatibacter actinomycetemcomitans. The leukotoxin, in particular, is thought to play a significant role in subverting the host immune response in the gingival crevice. It also has the potency to invade epithelial and vascular endothelial cells in vitro and buccal epithelial cells in vivo. Together with other coagents, Aggregatibacter actinomycetemcomitans is involved in localized aggressive periodontitis and destructive periodontal disease in adults. Also isolated from cases of infective endocarditis, and from brain and subcutaneous abscesses.

Treatment

This species is sensitive to tetracycline.

Eikenella

Members of the genus Eikenella resemble Haemophilus spp. and are commensals of the human oral cavity and the intestine. Although in the past their presence was linked to periodontal diseases, this has now been disproved. The type species is Eikenella corrodens. These organisms are capnophilic, Gramnegative, short coccobacillary forms that are asaccharolytic. When grown on non-selective media, they corrode the agar surface (hence the name corrodens). Human infection usually results from predisposing factors, such as trauma to a mucosal surface, which allow the organism access to the surrounding tissues; thus they may cause extraoral infections, including brain and abdominal abscesses, peritonitis, endocarditis, osteomyelitis and meningitis. Also associated with human bites or fist-fight injuries.

Capnocytophaga

The genus Capnocytophaga was created for fusiform species isolated from periodontal pockets, which, unlike Fusobacterium and Bacteroides spp., grow under capnophilic conditions. They have a characteristic ability to glide over routine blood agar (compare 'swarming' of Proteus spp.). Species recognized include Capnocytophaga ochracea (type species), Capnocytophaga sputigena, Capnocytophaga gingivalis, Capnocytophaga granulosa and Capnocytophaga haemolytica.

Habitat

The primary ecological niche is the subgingival area.

Characteristics

Long, thin fusiform organisms that demonstrate gliding motility seen on bright-field microscopy.

Culture and identification

Facultative anaerobes, but most strains require carbon dioxide for growth. Colonies spread over the agar surface with uneven edges and may be pink, yellow or white. Identification is by gliding characteristic, cell morphology, biochemical reactions and acid end products.

Pathogenicity

Opportunistic pathogens, sometimes associated with gingivitis and other systemic infections in immunocompromised patients; some strains produce an IgAl protease.

Key facts

 All Neisseria species are kidney shaped, Gram-negative

cocci usually arranged in pairs, and are oxidase-positive.

 Pathogenic Neisseria have fastidious growth requirements, unlike the non-pathogenic species, which are often part of the normal flora.

 Neisseria gonorrhoeae (the gonococcus) is the agent of the common sexually transmitted disease gonorrhoea and its complications.

 Neisseria meningitidis (the meningococcus) is an important cause of meningitis in children and young adults.

 Veillonella spp. present in plaque are considered benevolent organisms’ in relation to dental caries as they metabolize lactic acid produced by cariogenic bacteria into weaker acids.

 The generic name Haemophilus is derived from their requirement of blood or blood products to support growth.

Haemophilus influenzae causes meningitis, acute epiglottitis, osteomyelitis and arthritis, often accompanied by septicaemia, especially in children and the elderly. Bordetella pertussis is the agent of whooping cough (pertussis), prevented by the whole-cell vaccine incorporated in the childhood diphtheria-tetanus-pertussis (DTP) vaccination programme.

Aggregatibacter actinomycetemcomitans is a coagent of localized aggressive periodontitis (formerly localized juvenile periodontitis) and destructive periodontal disease in adults (also an agent of infective endocarditis, and brain and subcutaneous abscesses).

Eikenella and Capnocytophaga species are oral commensals and their role in oral disease is unclear.

Review questions (answers on p. 364 & p. 365)

Please indicate which answers are true, and which are false.

14.1 Which of the following statements on Neisseriaceae are true?

A. they possess a capsule

B. they are commensals of the oral cavity

C. they demonstrate motility

D. Neisseria gonorrhoeae causes syphilis

E. most gonococci are resistant to penicillin

14.2 Which of the following statements on Haemophilus are true?

A. it needs coagulation factors X and V for growth

B. it is a causative agent of periodontal disease

C. some are capsulated

D. it causes sexually transmitted diseases

E. it forms spores under harsh environmental conditions

14.3 Virulence factors of Haemophilus influenzae include:

A. the polysaccharide capsule

B. immunoglobulin A (IgA) protease

C. an exotoxin

D. an endotoxin

E. a pyrogenic factor causing influenza

14.4 Which of the following statements on Aggregatibacter actinomycetemcomitans are true?

A. it is a key pathogen in localized aggressive periodontitis

B. it possesses an IgG protease

C. it can cause deep-seated abscesses

D. it can be presumptively identified by star-shaped colonies in selective media

E. it is susceptible to tetracycline

14.5 Eikenella species:

A. are Gram-positive coccobacilli

B. are commensals of the oral cavity

C. are implicated in human bite (clench-fist) injuries

D. are known to cause endocarditis

E. are closely associated with severe periodontitis

14.6 Capnocytophaga spp.:

A. are isolated from periodontal pockets

B. are fusiform bacilli

C. frequently cause co-infections with Actinomyces spp.

D. require carbon dioxide for growth on blood agar

E. demonstrate gliding motility on agar media

Further reading

Brooks, J. F., Carroll, K. C., Butel, J. S., et al. (Eds.), (2013). The neisseriae (Chapter 20). In Jawetz, Melnick & Adelberg's medical microbiology (26th ed., pp. 285-293). New York: McGraw Hill. [e-Book].

Haffajee, A. D., & Sockransky, S. S. (1994). Microbial aetiological agents of destructive periodontal diseases. Periodontology 2000, 5, 78-111.

Kachlany, S. C. (2010). Aggregatibacter actinomycetemcomitans leukotoxin: from threat to therapy. Journal of Dental Research, 89, 561-570.

Periasamy, S., & Kolenbrander, P. E. (2010). Central role of the early colonizer Veillonella sp. in establishing multispecies biofilm communities with initial, middle, and late colonizers of enamel. Journal of Bacteriology, 192, 2965-2972.



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