Oral microbiology - Gram positive bacilli groups

Oral microbiology Lecture no. 2


B- Gram positive bacilli groups

1- Lactobacillus sp.
2- Rothia sp.
3- Bifidobacterium sp.
4- Eubacterium sp.
5- Propionibacterium sp.
6- Actinomyces sp.


1- Lactobacillus sp.
Domain: Bacteria

Phylum: Firmicutes

Class: Bacilli

Order: Lactobacillales

Family: Lactobacillaceae

Genus: Lactobacillus


Order: Lactobacillales

Lactic acid bacteria (LAB) are an order of gram-positive, low-GC, acid-tolerant, generally non sporulating, either rod-shaped (bacilli) or spherical (cocci) bacteria that share common metabolic and physiological characteristics. These bacteria, usually found in decomposing plants and milk products, produce lactic acid as the major metabolic end product of carbohydrate fermentation. This trait has, throughout history, linked LAB with food fermentations, as acidification inhibits the growth of spoilage agents. The genera that comprise the LAB are at its core Lactobacillus, Streptococcus, Aerococcus, Enterococcus.

Lactobacillus

Lactobacillus is a group of gram-positive bacilli facultative anaerobic usually form a part of human oral flora cavity. Bacteria of this genus are commonly isolated from dental plaque and the tongue; although they are usually comprise less than 1 % of total oral flora. However, their proportion and prevalence increase in advanced caries lesions both of the enamel and of the root surface.



This genus is cariogenic, can promote the development of tooth decay, as it detection as significantly increased in deep caries material. It’s thought the level of salivary lactobacilli number correlate well with intake of dietary carbohydrates.

Lactobacillus species
This genus includes 44 species according to Bergey’s Manual of Systematic Bacteriology and also contains seven subspecies. The common species found in the oral cavity include Lactobacillus acidophilus, Lactobacillus salivavius, Lactobacillus plantarum, Lactobacillus fermentum, Lactobacillus brevis, and Lactobacillus casei.
The taxonomy of Lactobacillus is complex, they are characterized into two groups, homofermenters which produce lactic acid from glucose fermentation like Lactobacillus acidophilus and L. casei, the other group are heterofermenters, which produce lactic acid as well as acetate, ethanol and carbon dioxide from glucose fermentation like Lactobacillus fermentum.

Cultural characteristics
The shapes and sizes of Lactobacillus cells can vary greatly measure 0.3–0.4 ?m × 1.5–4.0 ?m in size and arranged as single cells, in pairs, or as short chains.
Lactobacilli grow under microaerophilic conditions in the presence of 5–10% carbon dioxide within 15–45 °C range, which promotes bacterial growth. Its acidogenic organisms and acid tolerant the optimal pH for growth is 5.5–6.2.

Media enriched with glucose or blood promotes growth. A special selective medium, tomato juice agar (pH 5.0), promotes the growth of lactobacilli while
suppressing other bacteria. Rogosa agar is the culture medium of choice for many strains of Lactobacillus. The colonies are round, white or gray, transparent or
non transparent with a diameter from pinprick-sized to 2 mm on the agar surface. Smooth colonies are soft, raised, and lustrous, and the edge of the colony is neat. The surface of rough colonies is dry, flat, and lackluster, and the edge is not neat. The bacteria normally do not produce pigment.

Biochemical characters
Lactobacilli can ferment glucose to produce acid, and are negative for catalase test, urease. They cannot reduce nitrate, cannot hydrolyze gelatin, and cannot produce H2S. Sugar fermentation test and arginine hydrolysis test can help identify the genus of bacteria.

Pathogenicity
Lactobacilli are frequently isolated from deep carious lesions where the pH tends to be acidic. Indeed, early workers believed that lactobacilli were the main cariogenic because:


1- Their high numbers of lactobacilli in most carious lesions affecting enamel 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.
2- Their ability to grow in low pH environment less than 5 and to produce lactic acid.
3-Their ability to synthesis both extracellular and intracellular polysaccharides from sucrose.
4-They are the pioneer organisms in the advancing front of carious process, especially in dentine.


2- Rothia sp.

Scientific classification

Kingdom: Bacteria

Phylum: Actinobacteria

Order: Actinomycetales

Family: Actinomycetaceae

Genus: Rothia

Species: R. dentocariosa

Actinomycetales
Actinomycetales are generally gram-positive and anaerobic and have mycelium in a filamentous and branching growth pattern. Some actinobacteria can form rod- or coccoid-shaped forms, while others can form spores on aerial hyphae. Actinomycetales bacteria can be infected by bacteriophages, which are called actinophages. Actinomycetales can range from harmless bacteria to pathogens with resistance to antibiotics. Actinomycetales have 2 main forms of reproduction; spore formation and hyphae fragmentation. Many species of Actinomycetes produce antimicrobial compounds under certain conditions and growth media. Streptomycin, actinomycin, and streptothricin are all medically important antibiotics isolated from Actinomycetes bacteria




Rothia dentocariosa
Rothia dentocariosa (previously known as Stomatococcus mucilaginosus) is a species of Gram-positive, round- to rod-shaped bacteria variable or pleomorphic in shape and similar to Actinomyces and Nocardia, facultative anaerobic . Rothia was only defined as a genus in 1967 first isolated from dental caries, Rothia dentocariosa is largely benign, but does very rarely cause disease.. it is a part of normal community of microbes residing in the mouth and respiratory tract. Do not produce spores or capsule, non motile and are not acid tolerant. R. dentocariose are detected in the human oral cavity, its main sites of colonization are saliva and subgingival plaque. They are non pathogenic members of the human oral microflora and have no confirmed relationship to oral infections. As an opportunistic pathogen, it has been detected from endocarditis samples typically in people with underlying heart valve disorders and other clinical infected specimens.
Cultural characteristics
R. dentocariose is a G+ve irregular in shape spherical or filamentous. The cell diameter is generally 1.0 ?m, as the apical ends can reach a diameter of 5.0 ?m.
R. dentocariose are facultative anaerobes, they grow well in an aerobic environment, although primary cultures require incubation under anaerobic conditions (80% N2, 10% H2, 10% CO2). The optimal growth temperature is 35–37 °C. When cultured for 18–24 hr. under anaerobic conditions, young colonies are always filamentous and appear as spider-like colonies. When incubated under aerobic conditions, young colonies can reach diameter 1 mm the colony surface is smooth or grainy and show an umbrella edge. After 2 days culture, mature colonies can reach a diameter of 2–6 mm, with a milky, glossy, and smooth appearance. Smooth colonies and rough colonies can exist simultaneously on the same agar plate.


Biochemical characters
R. dentocariose can ferment glucose, maltose, sucrose, trehalose, fructose, and salicylate to produce acid, mainly lactic acid. R. dentocariose does not produce propionic acid when inoculated into Peptone yeast glucose broth (PYG). Cells test positive for catalase but do not produce indole. It is positive for urease activity

and can produce H2S in triple sugar iron agar. Rothia dentocariosa, like several other species of oral bacteria, is able to reduce nitrate to nitrite, and one study found it in 3% of isolates of nitrate-reducing bacteria from the mouth.

3-Bifidobacterium sp.
Domain: Bacteria

Phylum: Actinobacteria

Class: Actinobacteria

Order: Bifidobacteriales

Family: Bifidobacteriaceae

Genus: Bifidobacterium

Bifidobacterium is G +ve bacilli anaerobic organisms, were first isolated from infant feces and attracted attention because of their important physiological significance to the host organism. The species isolated from the oral cavity of dental caries patients belonging to the Bifidobacterium spp. include mainly Bifidobacterium dentium, Bifidobacterium breve, Bifidobacterium inopinatum, and Bifidobacterium denticolenu. B. dentium can cause tooth decay in human due to it’s tolerance to acidic environment reach pH 4.5 causing harm to tooth and braks dawn to sugar.

Cultural characteristics
Bifidobacterium cells are gram-positive irregular bacilli with various forms, arranged as single cells, chains, polymer-shaped, V-shaped, or palisade-shaped, non motile and non- sporulating. The cells are anaerobic; some strains are resistant to oxygen in the presence of CO2. The optimal temperature for the growth of this bacterium is 37–41°C, and the optimal pH value ranges from 6.5 to 7.0. Trypticase yeast – extract cystine agar (TPY) culture medium supplemented with neomycin, kanamycin, and various salt solutions is commonly used as the selective culture medium. Colonies formed on agar plates are convex, creamy or white, glossy, smooth, neat-edged, sticky.

Biochemical characters
B. dentium is biochemically active; it can ferment d-ribose, l-arabinose, lactose, sucrose, cellobiose, trehalose, raffinose, melibiose, mannitol, salicin, starch, galactose, maltose, fructose, xylose, mannose, and glucose to produce acid, but cannot ferment sorbitol and inulin. They test positive using the catalase test. The main terminal acid products in liquid culture medium Brain heart infusion (BHI) containing glucose are acetic acid and lactic acid, but a few species also make formic acid and succinic acid.

Clinical uses
Adding bifidobacterium as a probiotic to conventional treatment of ulcerative colitis has been shown to be associated with improved rates of remission and improved maintenance of remission. Some Bifidobacterium strains are considered as important probiotics and used in the food industry. Different species and/or strains of bifidobacteria may exert a range of beneficial health effects, including the regulation of intestinal microbial homeostasis, the inhibition of pathogens and harmful bacteria that colonize and/or infect the gut mucosa, the modulation of local and systemic immune responses, the repression of procarcinogenic enzymatic activities within the microbiota, the production of vitamins, and the bioconversion of a number of dietary compounds into bioactive molecules.[4] Bifidobacteria improve the gut mucosal barrier and lower levels of lipopolysaccharide in the intestine. Naturally occurring Bifidobacterium spp. may discourage the growth of Gram-negative pathogens in infants. Mother s milk contains high concentrations of lactose and lower quantities of phosphate (pH buffer). Therefore, when mother s milk is fermented by lactic acid bacteria (including bifidobacteria) in the infant s gastrointestinal tract, the pH may be reduced, making it more difficult for Gram-negative bacteria to grow.

3- Eubacterium sp.
Domain: Bacteria

Phylum: Firmicutes

Class: Clostridia

Order: Clostridiales

Family: Eubacteriaceae

Genus: Eubacterium

Eubacterium is a genus of gram-positive anaerobic bacilli, non sporulating. The name of the genus is still disputed, Bergey’s Manual of Systematic Bacteriology vol. 2 (1986) points out that the Greek prefix eu means good, useful, rather than true. Therefore, the author believes that Eubacterium is the more appropriate name. Eubacteria mainly colonize the saliva and plaque as a member of the normal oral microflora. Eubacterium lentum and E. limosum can be detected in the oral cavity. Eubacterium nodatum, E. brachy, E. timidum, E. saphenus, and E. minutum are new species isolated from subgingival plaque of patients with periodontitis and are considered as potential periodontal pathogens.




Cultural characteristics
Eubacteria is a gram-positive anaerobic bacilli homogeneous or polymorphous rod shape, culturing cells can be difficult due to this bacteria’s strict anaerobic demands. The optimum growth temperature is 37 °C, while the optimum pH is 7.0. Eubacteria are chemoheterotrophs and produce energy from mixed organic acids which produced by carbohydrates or protein metabolism. Eubacteria sp. on blood agar forms surface colonies are 0.5 – 2 mm in diameter, rounded, convex, low, dim and dark or lustrous, translucent or opaque, smooth, wedge- shaped or neat –edge.


Biochemical characters
Most Eubacteria from the oral cavity are relatively biochemically inactive, in most cases cells test negative for catalase and do not hydrolyze hippurate. Carbohydrate fermentation, indole production, nitrate reduction, esculin hydrolysis, and other biochemical tests can help differentiate the different species in the genus.



4- Propionibacterium sp.
Kingdom: Bacteria

Phylum: Actinobacteria

Class: Actinobacteria

Order: Propionibacteriales

Family: Propionibacteriaceae

Genus: Propionibacterium

Propionibacterium is Gram positive bacilli non sporulate, strict anaerobic bacteria that are found in root surface canal and plaque biofilm. The genus can be divided into two groups: one that lives on the skin, including inside the oral and intestinal tracts, named P. acnes; and another that lives in dairy products named P. propionicus. Propionibacterium acidifaciens was isolated from a human oral cavity in London in England.


Cultural characteristics
Cells of Propionibacterium acnes gram positive irregular bacilli range 0.5–0.8 ?m in diameter and 1–5 ?m in length. Members of this genus either anaerobic or microaerophilic bacteria, the highest rate of growth takes place 48h after inoculation at tempreture 37 C?. The Propionibacteria are chemoheterotrophic bacteria that need a complex nutritional medium such as Brain heart infusion


(BHI) blood agar in order to be cultured. Most species can grow in dextrose broth containing 20% bile salt or 6.5% NaCl.
Propionibacterium acnes colonies on the surface of agar can produce colorful pigmentation including white, gray, pink, red, or yellow. The main acid metabolites are propionic acid and acetic acid when cultured in Peptone yeast glucose broth (PYG) it is enrichment and non selective media and final pH 4.5 - 5.




Biochemical characters
The production of a significant quantity of propionic acid is the identifying feature of this genus when attempting to differentiate them from other gram-positive non sporulating anaerobic bacteria; they can also produce some isovaleric acid, formic acid, succinic acid, and lactic acid. All species in this genus can use glucose to produce acid and ferment of sucrose, maltose, and mannitol. Species of Propionibacterium are distinguished using positive test of catalase, indole production, nitrate test, esculin hydrolysis, gelatin liquefication, and other biochemical tests.