Oral Microbiology - C - Gram negative - cocci groups

Oral Microbiology Lecture no. 3



C - Gram negative - cocci groups


1- Veillonella sp.
2- Neisseria sp.
3- Moroxella sp.


1- Veillonella sp.

Scientific classification

Kingdom: Bacteria

Phylum: Firmicutes

Class: Negativicutes

Order: Vellionellales

Family: Veillonellaceae

Genus: Veillonella
The genus Veillonella was first isolated by Veillon and Zuber in 1898; Prevot further described these bacteria in 1933 and suggested the present taxonomy. Of 6 species of anaerobic gram-negative cocci—Veillonella parvula, Veillonella alcalescens, Veillonella atypica, Veillonella dispar, Acidaminococcus fermentans, and Megasphaera elsdenii—only V. parvula and V. alcalscens have been isolated from clinical specimens. V. parvula colonizes in coaggregation with other aerobic and anaerobic bacteria. Veillonella are commensals in the oropharynx, gastrointestinal tract and most surface of oral cavity, including tongue, saliva, and plaque biofilms. Strains detected in oral cavities include Veillonella parvula, Veillonella atypic, and Veillonella dispar have no association with disease. This bacteria rarely implicated in cases of osteomylitis and endocrditis especially Veillonella parvula.

Morphology and Cultural characteristics
Veillonella parvula cells is a gram-negative, strict anaerobic, spherical and often arranged diplococci or short chain, small in size (0.3 -0.5) mm, non – motile, non- sporing, require strictly anaerobic conditions, forming small gray-white colonies on the surface of BHI blood agar as enrichment medium while Rogosa vancomycin agar is considered as a selective medium. They can be rapidly identified because of their red fluorescence under ultraviolet light in specific growth media. Veillonella sp. lack glucokinase and fructokinase and therefore, unable to metabolize carbohydrates. Although they are unable to use carbohydrates or amino acids, they actively ferment organic acids, and they produce a highly endotoxic lipopolysaccharide. Instead, they utilize several intermediary metabolites, in particular lactate, as energy sources produced by Streptococcus mutans to raise the pH of plaque, in addition to lactic acid is the strongest acid produced by oral bacteria and is implicated in dissolution of enamel so, Veillonella can reduce the potentially harmful effect of lactic acid by converting it to weaker acids predominantly propionic acid, thus, they considered as beneficial bacteria in dental plaques.

3 lactate acetate + 2propionate + H2O +CO2

Biochemical characters
The cells are relatively biochemically inactive when tested using classical biochemical tests. They are unable to ferment carbohydrate to produce acid and do not produce indole. They appear negative with the catalase test but are able to reduce nitrate to nitrite.


2- Neisseria sp.
Neisseria are gram-negative diplococci, aerobic belonging to the family Neisseriaceae, which mainly colonize the human oral cavity and nasopharynx. Most Neisseria are members of the normal microflora of the human body and are usually non pathogenic like N. sicca and N. subflava. Neisseria are less nutrition-demanding aerobic bacteria that can grow easily on agar medium. Most Neisseria cells are spherical, but occasionally short rods are observed with a diameter of 0.6–1.0 ?m. Many are arranged in pairs with a flat adjacent surface. Neisseria cells may have a capsule and pili, but no endospores and flagella

3- Moraxella
Moraxella, Neisseria, and Kingella all belong to the family Neisseriaceae.
M. catarrhalis are gram- negative cocci, belonging to the genus Moraxella found in oral cavity and upper respiratory tract. They were previously known as Neisseria catarrhalis and Branhamella catarrhalis. M. catarrhalis are aerobic and can grow better on blood agar. After 48 hr. incubation, smooth, opaque, relatively flat, gray colonies form with a diameter of about 2 mm.

D - Gram negative - bacilli group

There are characterized into groups:

1- Gram negative bacilli – facultative anaerobic and capnophilic (carbon dioxide – dependent) genera.
2- Gram negative bacilli – obligate anaerobic genera.



1- Gram negative bacilli – facultative anaerobic and capnophilic ( carbon dioxide – dependent) including the fallowing genera:

A- Eikenella sp.
B- Capnocytophaga sp.
C- Aggregatibacter sp.
D- Haemophilus sp.
E- E.coli


A- Eikenella

Scientific classification
Kingdom: Bacteria

Phylum: Proteobacteria

Class: Beta Proteobacteria

Order: Neisseriales

Family: Neisseriaceae

Genus: Eikenella
Species: corrodens

Neisseriaceae
The Neisseriaceae are a family of Proteobacteria, within the Neisseriales order. While many organisms in the family are mammalian commensals or part of the normal flora, the genus Neisseria includes two important human pathogens, specifically those responsible for gonorrhea (caused by N. gonorrhoeae) and many cases of meningitis ("meningococcal meningitis", caused by N. meningitidis). As a group, the Neisseriaceae are strictly aerobic and Gram-negative, occur mainly in pairs (diplococci), and typically do not have flagella

Eikenella
Eikenella is a genus of gram-negative facultative anaerobic bacteria that do not produce spores. E. corrodens is the only species in the genus was thus named because it produces typical colonies that can erode agar. It is also known as Bacteroides corrodens. E. corrodens is a member of the normal flora in the human oral cavity and intestinal tract. As an opportunistic pathogen, it is often associated with other bacterial pathogens to cause mixed bacterial infections, especially in the mouth and respiratory tract. Its detection rate is higher in lesions of active adult periodontitis and specimens of dry socket after tooth extraction.

Morphology and Cultural characteristics
E. corrodens cells are stain gram-negative coccobacilli, facultative anaerobe, the primary cultures require anaerobic conditions with 5–10% CO2. However, under anaerobic conditions, up to 10% bile can be tolerated. It is essential to add hemin (5–25 mg/L) or x- factor dependent to the culture. The optimum growth temperature is from 35 °C to 37 °C, the optimum pH is 7.3, and cultures require sufficient humidity. E. 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.
Colonies of E. corrodens on BHI blood agar, a pearlescent ring can be observed at the center of colonies. Colonies of E. corrodens have a bleach-like smell, do not produce hemolytic reactions on blood agar, but a light green ring can be seen around colonies. Two different types of colonies can form on blood agar: invasive phenotype and noninvasive phenotype. 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 cultures. Colonies are light yellow, 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.
The noninvasive phenotype forms colonies with a diameter of 0.5–1mm. 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.”


Biochemical characters
E. 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.



B- Capnocytophaga sp.

Scientific classification
Kingdom: Bacteria

Phylum: Bacteroidetes

Class: Flavobacteria

Order: Flavobacteriales

Family: Flavobacteriaceae

Genus: Capnocytophaga

Capnocytophaga
Gram-negative bacilli, facultative anaerobic bacteria. They were the earliest bacteria to be isolated and named from the human subgingival plaque. They are common oral bacteria and can be obtained from various parts of the oral cavity, including plaque, gingival sulcus, and saliva specimens. These bacteria are often detected in mixed bacterial infections, such as juvenile periodontitis, infected root canal, and dry socket after tooth extraction, oral ulcers, and other clinical specimens as opportunistic pathogen. Common Capnocytophaga species are: Capnocytophaga ochracer, C. sputigena, C. gingivalis, C. granulose and C. heamolytica.

Morphology and Cultural characteristics
Capnocytophaga are fusiform Gram negative bacilli, and are part of the oral commensal flora. Microscopic observation revealed a high degree of polymorphism, Bacilli and shaped like bent rods or filaments, usually with rounded or slightly pointed ends with a variation in size measure 0.42–0.6 ?m × 2.5–2.7 ?m and appearance depending on the strain and culture conditions. In liquid culture, cells are polymorphic or filamentous morphology, and tight clumps can be observed. This polymorphism is also reflected in the observation of colonies (orange pigmented colonies, spreading on agar). The bacteria have no capsule, spore and flagella. Capnocytophaga are capnophilic bacteria; they can live only in environments where the concentration of carbon dioxide is greater than that of the atmosphere (at least 5% CO2). They can also grow anaerobically. They require enriched media, type blood agar, incubated at 37 °C. The isolation of strains of Capnocytophaga from polymicrobial samples is also possible on selective media containing antibiotics.
Species in this genus often form colonies of wet, thin, flat, diffuse growth with ragged edges on BHI blood agar. After 24 h incubation at 35–37 °C at pH 7.3, the size of colonies is like pinpricks. After incubation for 48–96 h, colonies become 2–4 mm in diameter and take on the appearance of bumps. Some colonies may become recessed into the agar. Aside from hemolytic Capnocytophaga (which produces ?-hemolysis), other species are not hemolytic on blood agar. The concentration of agar in the medium affects the force of sliding motility. Capnocytophaga cultures can produce a special smell, similar to caramel or a bitter almond flavor. Colonies on the agar surface can produce white to pink or orange-yellow pigmentation.

Biochemical characters
Capnocytophaga can ferment glucose, lactose, maltose, mannose, sucrose to produce acid, and do not ferment mannitol and xylose. They can hydrolyze esculin and test negative for catalase, oxidase and do not produce indole, while testing positive for ONPG and benzidine. Nitrate reduction, dextran hydrolysis, starch or gelatin hydrolysis and other biochemical tests can help identify this genus of bacteria.

Pathogenicity
Capnocytophaga is a commensally species considered as an opportunistic pathogen. This bacterium is involved in different types of infections the severity of which depends on the immune status of the patient, cases were reported in immunocompromised patients, these bacteria belong to the oral bacterial community responsible for periodontal infections affecting and destroying the supporting tissues of teeth (periodontal tissue). Capnocytophaga strains are often isolated from periodontal pockets but also from apical and periodontal abscesses, in association with other bacterial parodontal species. This condition increase alveolar bone loss, attachment loss, tooth mobility, and finally tooth loss. It can cause other diseases such as bacteremia (potentially complicated by septic shock), infections of the musculoskeletal system (osteomyelitis, arthritis), lung (empyema, lung abscess), digestive (peritonitis), maternal- fetal (ovarian abscess, chorioamnionitis), eye (conjunctivitis), heart (endocartitis) or brain (meningitis). Capnocytophaga is clinically important in Pediatric Oncology and Hematology.

C- Aggregatibacter sp.

Scientific classification

Domain: Bacteria

Phylum: Proteobacteria

Class: Gammaproteobacteria

Order: Pasteurellales

Family: Pasteurellaceae

Genus: Aggregatibact

Pasteurellaceae
Pasteurellaceae comprise a large family of Gram-negative bacteria. Most members live as commensals on mucosal surfaces of birds and mammals, especially in the upper respiratory tract. Pasteurellaceae are typically rod-shaped and facultative anaerobes. Their biochemical characteristics can be distinguished from the related Enterobacteriaceae by the presence of oxidase, and from most other similar bacteria by the absence of flagella. Bacteria in the family Pasteurellaceae have been classified into a number of genera based on metabolic properties, but these classifications are not generally accurate reflections of the evolutionary relationships between different species.
Haemophilus influenzae was the first organism to have its genome sequenced and has been studied intensively by genetic and molecular methodologies. The genus Haemophilus is a notorious human pathogen associated with bacteremia, pneumonia, meningitis and chancroid. Other pathogenic members belonging to the family Pasteurellaceae include Aggregatibacter, Pasteurella, and Actinobacillus species.
Aggregatibact sp.

The Aggregatibacter actinomycetemcomitans formerly classified as Actinobacillus actinomycetemcomitans, subsequently named Haemophilus actinomycetemcomitans, and is now known as Aggregatibacter actinomycetemcomitans. It was detected both in normal oral bacteria and the main colonization site of this species is subgingival plaque, also in lesions of juvenile periodontitis patient’s at higher detection rate. Therefore, A. actinomycetemcomitans is considered as a major pathogen produces a range of virulence factors including a powerful leukotoxin, collagenase, immunosuppressive factors and proteases capable of cleaving IgG; in addition, strains can be invasive for epithelial cells. A. actinomycetemcomitans is also an opportunistic pathogen, being isolated from cases of endocarditis, brain and subcutaneous abscesses, osteomyelitis, and periodontal disease.

Morphology and Cultural characteristics

Aggregatibacter actinomycetemcomitans are gram- negative coccobacilli bacteria, facultative anaerobe grows well in the microaerophilic environment of 5–10% CO2. Its optimum growth at temperature is 37 °C and it does not grow at 22 °C and pH is 7.3. Freshly isolated strains possess fimbriae, although these can be lost on sub-culture. Cells of A. actinomycetemcomitans are 0.5–0.8 ?m - 0.6–1.4 ?m in size. Cells are spherical, club-shaped, or rod-shaped. Rod-shaped cells are common in agar cultures. The cells arrange as single cells, in pairs, or in piles. They produce no spores, are nonmotile, and do not form capsules.
Aggregatibacter actinomycetemcomitans colonies (BHI blood agar) forms small colonies, with a diameter of approximately 0.5–1.0 mm. Primary cultures are often difficult to lift off the agar surface. Typical colonies are star-shaped or shaped like crossed cigars, with irregular edges. In broth culture, the growth shows small particle-like opacity and often sticks to the flask wall. However, some strains grow into a homogeneously turbid culture after repeated cultures.


Biochemical characters
Aggregatibacter cells are ferment fructose, glucose, maltose, and mannose to produce acid, but do not ferment sucrose, trehalose, lactose, raffinose, melibiose, and arabinose. A. actinomycetemcomitans tests positive for oxidase and catalase, can reduce nitrate, does not hydrolyze esculin and hippurate sodium, and does not produce H2S and indole.


D- Haemophilus sp.
Haemophilus are gram-negative bacilli, facultative anaerobic, is named Haemophilus due to their requirement for blood during growth. Most bacteria of this genus are part of the normal microflora of the oral cavity. The main colonization site within the oral cavity is dental plaque, followed by saliva, and soft palate. Occasionally, they can cause mixed infections such as periodontal abscess or jaw infection as conditional pathogens. Species detected in the oral cavity include H. actinomycetemcomitans, H. influenzae, H. parainfluenzae, H. aphrophilus, H. paraphrophilus, H. parahaemolyticus, H. paraprohaemolyticus, and H. segnis. In the latest classification, H. actinomycetemcomitans, H. aphrophilus, and H. segnis were classified into a new genus: Aggregatibacter, and renamed A. actinomycetemcomitans, A. aphrophilus and A. segnis, respectively.


E- Escherichia coli
Commonly known as E. coli this is the most common bacteria belonging to the family Enterobacteriaceae found in human body. E. coli is the first gram-negative bacillus that was detected in the neonatal oral cavity, and is thought to be transmitted from the mother during vaginal birth. E. coli is a facultative anaerobe with low nutritional requirement, which can grow well under aerobic conditions. E. coli occasionally can cause maxillofacial infections.