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Rochelle Overton

Bench E

23092016 ^[1]

Capnocytophaga gingivalis

Classification

Higher order taxa

Kingdom	Bacteria
Domain	Bacteroidetes
Phylum	Bacteroidetes
Class	Flavobacteriia
Order	Flavobacteriales
Family	Flavobacteriaceae
Genus	Capnocytophaga

Species

Capnocytophaga gingivalis ATCC 33624

Description and significance

Capnocytophaga gingivalis are a class of Flavobacteriia that inhabit the oral cavity of the mouth^[1], making up a large component of supragingival, and subgingival plaque^[2]. Microscopy has revealed that C. gingivalis are straight rod-shaped bacteria with a fusiform morphology, having a granulated outer surface. Capnocytophaga gingivalis are a motile facultative anaerobes, meaning that they can create ATP through aerobic respiration if oxygen is present^[3], but can also use fermentation in instances where oxygen is poor/absent^[1]^[4]. Gram staining produced a purple, gram negative cell, which was also able to be successfully cultured in laboratories, having a preference for environments with high carbon dioxide levels^[3].

Whilst present in the natural microbiome of the mouth, C. gingivalis can become pathogenic under certain conditions. Dental ailments such as periodontal infections, loss of teeth and supporting tissues as well as alveolar bone loss can occur. In severe cases, C. gingivalis can also spread to the eyes, brain, lungs, digestive tract, heart or muscular skeletal system, causing infection and disease. Although these diseases are treatable with antibiotics, there have been reports of resistant strains occurring since the mid 1980's, making it an important bacterium to study.

Genome structure

Capnocytophaga gingivalis strain ATCC 33624 contains approximately 5,000bp in its genome and 2,641 recorded genes^[5]. This microaerophilic organism contains a newly discovered bacterial Pnkp1-Rnl-Hen1 RNA repair mechanism^[6]. Studies have linked these heterohexamer proteins to the restoration of RNA and overall immunity. This can be seen as an adaptive defense mechanism against toxins as the organism is able to successfully excise ribotoxins, preventing infection and increasing longevity. In order for this process to occur, Hen1 must first methylate the RNA, marking the RNA for cleavage. Pnkp1 then attaches to the damaged RNA ends, followed by Rnl1 that ligates it, restoring the RNAs function. Once this process has been carried out, that strand of RNA will contain a form of immunity to that specific ribotoxin.

Cell structure and metabolism

Being a gram negative bacteria, the crystal violet stain used in gram staining does not stick to C. gingivalis as its cell wall contains a very small amount of peptidoglycan^[7]. The cell walls of gram negative bacteria are composed of a nuclear envelope, a thin layer of peptidoglycan and an outer membrane. Due to the presence of peptidoglycan in their cell walls, gram negative bacteria are still susceptible to lysozyme which catalyzes hydrolysis of the cell by weakening the glycosidic bonds, although often used in conjunction with ethylenediaminetetraacetic acid^[8].

A biofilm is the clustered formation of a thin layer of microorganisms which cling together to adhere to a hard surface, such as the tooth. It begins with the pellicle (saliva) providing large quantities of absorbed macromolecules directly to the teeth^[9]. Free-floating bacteria will then attach themselves to the saliva-coated tooth in order to feed, being the primary colonizers. Then the secondary colonizers, Capnocytophaga gingivalis, bind to the primary colonizers creating a second layer, proving increased strength and structure to the biofilm. When left over long periods of time, this can create instances of plaque which if left unchecked can result in periodontal infections. Throughout this micro-colony, each microorganism is exposed to different environmental conditions such as; physical proximity, oxygen and glucose requirements. This is why each C. gingivitis will exhibit different phenotype throughout the oral cavity^[10].

Capnocytophaga gingivalis are motile organisms, despite lacking both flagella and flagellates. They create longitudinal movement in a gliding motion. Although the exact mechanisms which control this form of movement are still unknown, it is hypothesized that the front of the bacteria extends and attaches to the surface before detaching at the rear creating a gliding motion^[11]^[12].

Aerobic metabolism is the preferred method of respiration used by Capnocytophaga gingivalis, although it is capable of reverting to fermentation if oxygen is absent. In order to aerobically respire, the bacteria will go through the process of glycolysis whereby the cell will uptake glucose, and oxidize it. This breaks the glucose apart into two pyruvates and two NAD molecules which are then put through the citric acid cycle. In this process the pyruvates are broken down into carbon dioxide and FADH molecules. The NAD becomes NADH which goes on to stimulate the production of ATP in the electron transport chain, performed by complexes I-IV. The oxidation of NADH and FADH2 are carried out in complexes I-II, creating the proton gradient. Capnocytophaga gingivalis is a flavobacteria, meaning that the electron transfer carrier will be either a cytochrome, electron-transfer quinone, or flavoprotein in the electron transport chain, using oxygen as the terminal acceptor. In cases of anaerobic respiration, the terminal acceptor can contain a variety of organic or inorganic compounds^[13].

Ecology

Capnocytophaga gingivalis is present in a standard human microbiome. Individuals with plaque were discovered to have a higher prevalence of the bacteria with children having a higher risk. When compared with physical ailments results concluded that patients diagnosed with leukeamia and oral diseases had 57% and 71% increase in the amount of C. gingivalis found in their oral microbiome respectively^[14].

When grown on Trypticase-soy-blood agar, Capnocytophaga gingivalis show consistent irregular colonization, staying isolated from other microorganisms^[15]. Due to their ability to translocate via gliding, the bacteria are confined by their surrounding cells and start clumping into mounds, creating halo zones. Their irregular populations comprise of smooth cell surfaces with small pioneer colonies on the outer edges of halo zones. This is in contrast to bacteria found within the human microbiome as they are forced to co-aggregate with other microorganisms in order to gain survival advantages such as adhesion^[16]. This agglutination of neighboring cells forms the basis for biofilms and in turn, dental plaque. Co-aggregation reacts to lectin-carbohydrate molecules which are recognized by each cell. Experiments conducted in alternate ecosystems concluded that this process was unique to the oral cavity, as predation or nutritional instability were common occurrences when grown elsewhere. This is definitive proof that C. gingivalis are opportunistic pathogens, as they will become predatory and pathogenic in environments where they have the selective advantage.

Commensalism is the relationship shared between the C. gingivalis and the oral cavity of humans, as the relationship is advantageous to the bacteria and has no benefit to the host^[17]. When C. gingivalis move into other substrates of the body they can soon become pathogenic^[18]. Capnocytophaga gingivalis has been shown to yield a much higher biomass when grown on rich brain-heart extracts^[4]. As a results of its preference for these high protein substrates, C. gingivalis have been known to invade and attack human tissues pertaining to the gums, brain, eyes, lungs, heart, and gastrointestinal tract under certain circumstances. Its ability to grow in such a vast array of environments stems from its ability to conduct respiration both aerobically and anaerobically through fermentation.

Pathology

Despite being commensalisms found in the plaque on our teeth, Capnocytophaga gingivalis can invade and attack the host tissue when the body's natural lines of defense are compromised^[14]. If given the opportunity, C. gingivalis will invade the oropharyngeal tract through cuts, abrasions, ulcers or other trauma in the oral cavity, spreading to induce infection^[15]. Supragingival plaque, present above the gum line is constantly exposed to glycoproteins contained within the pellicle^[16]. If left untended for extended periods of time, the plaque can start to creep below the surface of the gum creating gingivitis. The subgingival plaque, located below the gum line, will then become exposed to gingival crevicular fluid (GCF)^[19]. This inflammatory exudate is full of antibodies designed to keep the pathogen contained and prevent it from invading further down the tooth. This immune response creates inflammation of the gums, increasing the severity of the gingivitis as it becomes a periodontal infection. This invasion of bacteria below the gum lines can be enough to cause an imbalance in the microbiome of the oral cavity as the C. gingivalis proceeds to cause damage to the gums and surrounding teeth. If left unchecked this could result destruction of the cementum, bones and periodontal ligaments as well as creating large periodontal pockets caused by the bacteria eating away at the gum tissue^[20].

Studies have shown that in some cases C. gingivalis have been associated with numerous diseases throughout the body. Bronchoscopy of patients with pneumonia discovered high levels of C. gingivalis present in the lungs^[21]. When treated with beta-lactam antibodies, patients began showing signs of improvement over the first 24 hours of treatment, with the pneumonia settling down significantly over the following week. It was hypothesized that C. gingivalis could be the causality for the inflammation in the lungs, or leaving the patient immuno-compromised making it easier for another bacterium to infect the patient. Blood pathology examinations have also discovered the presence of Capnocytophaga gingivalis in the blood^[22]. This infection, also known as bacteraemia, has been tied with cancer, in particular leukeamia.

Application to biotechnology

There are many hypotheses surrounding how C. gingivalis first became resistant to antibiotics. The first stemming from long-term exposure to the gingival crevicular fluid present under the gums surface^[21]. A second hypothesis suggests horizontal gene transfer between its genus, Capnocytophaga, or closely related families such as prevotella or bacteroides^[14]. Alternatively, bacteraemia results in direct exposure to the hosts immune system in which the bacteria would be exposed to high selective pressures, forcing it to adapt in order to survive in the serous environment^[23].

A typical C. gingivalis cell shows susceptibility towards beta-lactam, whereas antibiotic resistant strains contain beta-lactamase within their chromosome or plasma. Through the development of antibiotics specific to C. gingivalis, cancer patients with bacteraemia can be treated for neutropenia with the use of broad-spectrum beta-lactam antibiotic therapy^[23].

Broad-spectrum Beta-lactam antibiotics target all antibodies which contain a Beta-lactam ring including; cephalosporins, imipenem, cefoxitin, and amoxicillin in C. gingivalis. When the antibiotics are released into the host, through natural or induced immunity, they will attach to their matching antibody. This prevents the bacterium from constructing its cell wall, as it is no longer able to link the beta-rings together. This will cause deterioration and lysis of the cell as a result. Due to eukaryotic cells lack of cell walls, they are not targeted by this antibiotic, being a safe treatment process for the host^[13].

Current research

A recent study published in August 2016 focused on the emergence of antibiotic resistant Capnocytophaga gingivalis and its prevalence in cardiovascular diseases^[24]. Not only was this strand of C. gingivalis resistant to cephalosporins, but was also discovered to have developed an immunity towards Macrolide-Lincosamid-Streptogramin (MLS) and fluoroquinolone (FQ). Their case study reported a patient presenting advanced periodontitis, chronic respiratory failure and a heightened white blood cell count all indicating signs of infection.

DNA exctraction through PCR gel electrophoresis confirmed the presence of a Capnocytophaga gingivalis, resistance to cephalosporine, MLS and fluoroquinolones was later confirmed through agar cultures. Treatment with amoxicillin/clavulanate (1000/125mg) and lebogloxacin three times a day for sixteen days resulted in successful termination of the C. gingivalis cultures.