Lactobacilli
Lactobacilli are saprophytes in vegetable and animal material (e.g., milk). Some species are common animal and human commensals inhabiting the oral cavity and other parts of the body. They have the ability to tolerate acidic environments and hence are believed to be associated with human and animal caries.
The taxonomy of lactobacilli is complex. They are characterized into two main groups: homofermenters, which produce mainly lactic acid (65%) from glucose fermentation (e.g., Lactobacillus casei), and heterofermenters, which produce lactic acid as well as acetate, ethanol and carbon dioxide (e.g., Lactobacillus fermentum). L. casei and Lactobacillus rhamnosus, Lactobacillus acidophilus and the newly described species, Lactobacillus oris, are common in the oral cavity. It should be noted that the taxonomy of lactobacilli is under constant revision.
Habitat and transmission
Lactobacilli are found in the oral cavity, gastrointestinal tract and female genital tract. In the oral cavity, they constitute less than 1% of the total flora. Transmission routes are unknown.
Characteristics
Gram-positive coccobacillary forms (mostly bacillary), a- or non-haemolytic, are facultative anaerobes. These organisms ferment carbohydrates to form acids (i.e., they are acidogenic) and can survive well in acidic milieu (they are aciduric); they may be homofermentative or heterofermentative. The question as to whether they are present in carious lesions because they prefer the acidic environment, or whether they generate an acidic milieu and destroy the tooth enamel, has been debated for years (the classic 'chicken and egg' argument). Lactobacilli are also major constituents of the vaginal flora and help maintain its low pH equilibrium.
The beneficial role of lactobacilli in maintaining the homoeostasis of the intestinal microbiome has been recognized, and 'lactobacillus-laced' food items, sold as probiotics, have gained popularity among the health-conscious public (see Chapter 26).
Culture and identification
Lactobacilli grow under microaerophilic conditions in the presence of carbon dioxide and at acidic pH (6.0). Media enriched with glucose or blood promote growth. A special selective medium, tomato juice agar (pH 5.0), promotes the growth of lactobacilli while suppressing other bacteria. Identification is by biochemical reactions.
Pathogenicity
Lactobacilli are frequently isolated from deep carious lesions where the pH tends to be acidic, and they are commonly isolated from the advancing front of the dentinal caries lesions. Indeed, early workers believed that lactobacilli were the main cariogenic agent (a theory that has been disproved), so much so that the number of lactobacilli in saliva (the lactobacillus count) was taken as an indication of an individual's caries activity. Although this test is not very reliable, it is useful for monitoring the dietary profile of a patient because the level of lactobacilli correlates well with the intake of dietary carbohydrate.
Corynebacteria
The genus Corynebacterium contains many species that are widely distributed in nature. These Gram-positive bacilli demonstrate pleomorphism (i.e., coccobacillary appearance) and are non-sporing, non-capsulate and non-motile. In common with Mycobacterium and Nocardia spp., they have a cell wall structure containing mycolic acid. A number of species are important human pathogens and commensals. The sometimes fatal upper respiratory tract infection of childhood diphtheria is caused by Corynebacterium diphtheriae. It is important to distinguish this, and other pathogens within the genus, from commensal corynebacteria.
Corynebacterium diphtheriae
Habitat and transmission
Human throat and nose, occasionally skin; patients carry toxigenic organisms up to 3 months after infection. Transmission is via respiratory droplets.
Characteristics
Pleomorphic, Gram-positive, club-shaped (tapered at one end) bacilli, 2-5 μm in length, arranged in palisades. They divide by 'snapping fission' and hence are arranged at angles to each other, resembling Chinese characters. The rods have a beaded appearance, with the beads comprising an intracellular store of polymerized phosphate. The granules stain metachromatically with special stains such as Neisser methylene blue stain (i.e., the cells are stained with blue and the granules in red).
Culture and identification
A non-fastidious, facultative anaerobe that grows well at 37°C. Grows on blood agar but selective media are helpful for isolation from clinical specimens. In blood tellurite agar, commonly used for this purpose, corynebacteria produce distinctive grey-black colonies after 48-h incubation at 35°C. Preliminary identification is helped by the shape and size of the colonies on tellurite agar. Specific identification is by biochemical reactions and demonstration of toxin production.
The test for toxin production is important as some corynebacteria are non-toxigenic (and hence non-virulent) and are normal skin or throat commensals.
Toxin production
The exotoxin responsible for virulence can be demonstrated by the gel precipitation test, which uses the Elek plate. In this test, a filter paper soaked in diphtheria antitoxin is incorporated into serum agar before it has set; the test strain of C. diphtheriae under investigation is then streaked on to the agar at right angles to the filter-paper strip and incubated at 37°C. After 24 h, white lines of precipitation will be visible as a result of the combination of the antitoxin and the antigen (i.e., the toxin) if the strain is a toxigenic isolate (Fig. 12.1). Although this is the traditional method for toxin detection, enzyme-linked immunosorbent assays (ELISAs) and immunochromatographic strips are now available for quick detection of the exotoxin from the cultured isolates.
A rapid diagnostic test based on polymerase chain reaction for the toxin gene (tox) is another new direct assay of patient specimens, prior to culture and isolation of the organism.
Diphtheria toxin
This exotoxin—produced by strains carrying bacteriophages with the tox gene—inhibits protein biosynthesis in all eukaryotic cells. The toxin has two components: subunit A, which has the adenosine diphosphate ribosylating activity, and subunit B, which binds the toxin to cell surface receptors. Essentially, the toxin blocks protein synthesis of host cells by inactivating an elongation factor.
Macroscopically, its action on the respiratory mucosa results in the production of a grey, adherent pseudomembrane comprising bacteria, fibrin and epithelial and phagocytic cells. This may obstruct the airway, and the patient may die of asphyxiation. When the toxin permeates into the bloodstream, it acts systemically, affecting motor nerves of the myocardium and the nervous system.
Fig. 12.1 Elek test for toxin-producing Corynebacterium diphtheriae. A filter paper impregnated with diphtheria antitoxin is incorporated into agar and the unknown (test) and the known (control) toxin-producing C. diphtheriae are streaked at right angles; after 24-h incubation, white lines of precipitation are produced due to the combination of the antigen (toxin) and the antibody. (Only the test inoculum is shown.)
The toxin can be converted to a toxoid (i.e., made nontoxic but still antigenic) by treatment with formaldehyde; the toxoid can then be used for prophylactic immunization: the first component of the diphtheria-tetanus-pertussis (DTP) vaccine.
Antitoxin, produced by injecting the toxin into horses, neutralizes the toxin (see below).
Pathogenicity
C. diphtheriae is the agent of diphtheria; it usually affects the mucosa of the upper respiratory tract, and sometimes the skin. Cutaneous infections are especially seen in the tropics and are usually mixed infections with Staphylococcus aureus and/ or Streptococcus pyogenes. Serious systemic manifestations are the result of the absorption of the exotoxin.
Treatment and prevention
In the acute phase, supportive therapy to maintain the airway is critical. Antitoxin is given to neutralize the toxin and penicillin to kill the organisms. Antibiotics have little effect once the toxin has spread, but will eliminate the toxigenic focus of bacteria. In epidemic outbreaks, carriers are given either penicillin or erythromycin.
Immunization is highly effective in preventing diphtheria. A special test (the Schick test) is used to demonstrate immunity. Here, the circulating level of antibody after immunization (or clinical/subclinical infection) is assessed by inoculating a standardized dose of the toxin.
Other corynebacteria
Corynebacterium ulcerans is responsible for diphtheria-like throat lesions, but it does not cause toxaemia.
Corynebacterium (formerly Bacterionema) matruchotti is the only true coryneform organism in the oral cavity. It resembles a whip (whip-handle cell), with a short, fat body and a long filament at one end.
Diphtheroids
Bacilli that morphologically resemble diphtheria bacilli are called diphtheroids (e.g., Corynebacterium hofmannii, Corynebacterium xerosis). They are normal inhabitants of the skin and conjunctiva and are occasional opportunistic pathogens in compromised patients (e.g., endocarditis in prosthetic valves).
Propionibacteria
Propionibacteria are obligate anaerobic, Gram-positive rods, sometimes called 'diphtheroids' for the aforementioned reasons. Propionibacterium acnes is part of the normal skin flora and may also be isolated from dental plaque. The pathogenesis of facial acne is closely related to the lipases produced by P. acnes, hence the name.
A new member of this genus is Propionibacterium propionicum (formerly Arachnia propionica), morphologically similar to Actinomyces israelii (except for the production of propionic acid from glucose by the former).
Key facts
• Lactobacilli are acidogenic and aciduria
• Lactobacilli are common constituents of the oral flora and are regular isolates from dentinal caries lesions.
• The numbers of lactobacilli in saliva correlate positively with caries activity.
• Toxigenic strains of Corynebacterium diphtheriae are responsible for diphtheria, the sometimes fatal upper respiratory tract infection of childhood.
The diphtheria toxin is toxoidable and is a component of the triple (diphtheria-tetanus-pertussis or DTP) vaccine. Propionibacterium acnes (loosely termed ‘diphtheroids’) is a significant component of the normal skin flora.
Review questions (answers on p. 364)
Please indicate which answers are true, and which are false.
12.1 Lactobacilli:
A. are saprophytic
B. are mostly homofermenters
C. are aciduric and acidogenic
D. are best grown in strict anaerobic conditions
E. count in a saliva sample is by far the best indicator of cariogenic activity
12.2 Corynebacterium diphtheriae:
A. are Gram-positive club-shaped spore-bearing rods
B. contains metachromatic granules
C. produces a toxin that is similar to endotoxin
D. causes pharyngeal and skin infections
E. is transmitted by airborne droplets
12.3 Toxin produced by C. diphtheriae:
A. is mediated by a lysogenic phage
B. is similar to endotoxin
C. is a polypeptide
D. inhibits protein synthesis
E. causes neurological symptoms
12.4 Propionibacteria:
A. are Gram-negative coccobacilli
B. are the members of 'diphtheroids'
C. are facultatively anaerobic
D. are exclusive to the oral cavity
E. are frequently associated with dental caries
Further reading
Brooks, J. F., Carroll, K. C., Butel, J. S., et al. (Eds.), (2013). Aerobic non-spore-forming Gram-positive bacilli: Corynebacterium, Listeria, Erysipelothrix, Actinomycetes, and related pathogens. In Jawetz, Melnick & Adelberg's Medical microbiology (26th ed., pp. 187-195). New York: McGraw Hill. Chapter 12.
Grange, J. M. (2003). Streprococci and Staphylococci. In D. Greenwood, R. Slack, & J. Peutherer (Eds.), Medical microbiology (16th ed.). Edinburgh: Churchill Livingstone. Chapter 18.