Anaerobic bacteria are prokaryotic organisms. Anaerobic bacteria are growing in absence of oxygen by fermenting organic matter and produce value-added chemicals such as ethanol, acetic acid and lactic acid. They may be even died on exposure to oxygen or poison by oxygen (Zhe Ji et al., 2018). They are act as vital roles in the microbiota of human. Anaerobic bacteria also act as infectious agent. The microorganisms can cause pathological processes in both immunocompetent and immunocompromised patient such as elderly, diabetics, and HIV infection and using anticancer chemotherapy, immune suppressant drugs, or broad spectrum antibiotics. These infectious agents can be found in difference body sites and cause severe diseases to human being. Sometimes these severe diseases can cause human life threatening (Eva Maier et al., 2015).
Anaerobic bacteria have been classified into two important characteristics which are morphology and Gram staining. In morphology, anaerobic bacteria can be cocci or rods. In Gram staining, anaerobic bacteria can be Gram positive which stained in purple colour or Gram negative which stained in pink colour. Besides that the infections caused by anaerobic bacteria can be divided according to their origin microorganisms (Márió et al., 2017). (The summary of basic morphology, genera and pathogenesis of anaerobic bacteria will be shown as Table 1).
In Gram positive with endospore forming bacilli of anaerobic bacteria mainly caused exogenous anaerobic infection. The examples of Gram positive of endospore forming bacilli are Clostridium perfringens, Clostridium tetani and Clostridium botulinum. These bacteria are causative agent of severe infections to human beings like gas gangrene, tetanus and botulism.
In Gram positive with non-spore forming bacilli of anaerobic bacteria mainly caused based on the anatomical sites of human beings. The examples of Gram positive of non-spore forming bacilli are Actinomyces spp., Eubacterium spp., Propionibacterium spp. and Bifidobacterium spp. These bacteria are causative agent of infections of female genital tract, intra-abdominal infections, endocarditis, bacteraemia and brain abscesses.
In Gram negative with non-spore forming bacilli of anaerobic bacteria also caused based on anatomical sites of human beings. The examples Gram negative of non-spore forming bacilli are Bacteroides spp., Fusobacterium spp., Prevotella spp. and Leptotrichia spp. These bacteria are causative agent of infections of female genital tract, intra-abdominal and pleuroplumonary infection. Sometimes it also can found mixed infections in oral cavity or urogenital areas. However, the significance is not established.
In Gram positive and Gram negative with cocci shaped of anaerobic bacteria mainly caused based on invasion of damaged tissue by normal microbial flora. Most infections are polymicrobic. Example of Gram positive cocci shaped of anaerobic bacteria is Peptostreptococcus. Example of Gram negative cocci shaped of anaerobic bacteria is Veillonella. Both can infections of female genital tract, intra-abdominal, pleuroplumonary infection and bactereamia. Sometimes it also can found mixed infections in oral cavity and pleuroplumonary infection. However, the significance is not established.
Anaerobic bacteria are a normal flora of humans. Thus, there are multiple number of normal flora can be found in human body such as skin and gut. In additional, these normal floras act as important roles for physiological homeostasis of oral cavity and female genital tract. In colon, there is the largest number of normal flora found in human body. Most of the normal floras are anaerobic bacteria. These normal floras are mainly for maintaining our health and preventing for severe disease. If we are in low immune system, the normal flora may become pathogen which can cause severe disease to human being (Márió et al., 2017).
Besides that, bacteria are divided based on their oxygen requirement. Thus, prokaryotes can be separated into aerobes and anaerobes. Aerobic bacteria are required oxygen for growth. Aerobic bacteria are further divided into three groups which are obligate aerobes, facultative anaerobes and microaerophilic organisms (Surinder Kumar, 2012). (Basic classification of medically important bacteria had been shown in Table 2).
Obligate aerobes also known as strictly aerobes. They have an absolute or obligate requirement for oxygen (O_2 ) in order to grow. Thus, the obligate aerobes bacteria have no fermentative pathways. Example of Gram negative cocci bacteria is Neisseria. Example of Gram negative rods bacteria is Pseudomonas. Example of Gram negative spirochaetes bacteria is Leptospira.
In facultative anaerobes, they are ordinarily aerobic but can grow in the absence of oxygen. Thus, most bacteria of medical importance are facultative anaerobes. Example of Gram positive cocci bacteria are Staphylococcus, Streptococcus and Enterococcus. Example of Gram positive rods bacteria are Corynebacterium, Listeria, Lactobacillus, Nocardia, Mycobacterium and Bacillus. Example of Gram negative rods bacteria are Salmonella, Shigella, Klebsiella, Proteus, Escherichia, Yersinia, Bordetella, Haemophilus, Brucella, Pasteurella and Vibrio. Mostly facultative anaerobes are Enterobacteriaceae. Examples of Enterobacteriaceae are Salmonella, Shigella, Klebsiella, Proteus, Escherichia and Yersinia.
In microaerophilic organisms, they are growing best at low oxygen tension which around 5%. Example of Gram negative rods bacteria are Campylobacter and Helicobacter.
For anaerobic bacteria, they grow in absence of oxygen. It is mainly known as obligate anaerobes. In obligate anaerobes, they may even die on exposure to oxygen. It is also known as poison by oxygen. Example of Gram positive cocci bacteria is Peptostreptococcus. Example of Gram positive rods bacteria are Actinomyces and Clostridium. Example of Gram negative rods bacteria are Bacteroides, Fusobacterium and Prevotella. Example of Gram negative spirochaetes bacteria are Borrelia and Treponema.
Examples of Clinically Relevant Obligate Anaerobic Bacteria
Clostridium tetani are heterogeneous genera of saprophytic. Clostridium tetani is the causative agent of tetanus. It is produce neurotoxin exotoxin or tetanus toxin. Tetanus is a disease that is relatively rare in well-developed country. Tetanus had been found in early times. Thus, it was described by Hippocrates and Aretaeus. Clostridium tetani is a normal flora which found large amount in large intestines of human beings and animals (Surinder Kumar, 2012; Márió et al., 2017).
Morphology of Clostridium tetani, it is the Gram positive obligate anaerobic bacteria which can poison by oxygen. In Gram stain, it is stained in purple colour. The shape of Clostridium tetani is slender bacilli which in 2 to 5 × 0.4 to 1 µm with rounded ends. It is a spore forming bacteria. The spores are commonly found in soil, dust and air. The spores are in spherical, terminal and twice the diameter of vegetative cells giving them drumstick appearance. (Figure 2.1). However, in Gram stain, spores will stain as a colourless round structure. Clostridium tetani is a pleomorphic and sometimes may be in filamentous. It is the non-capsulated bacteria. They are in motile by using peritrichate flagella. Clostridium tetani can be gram variable when during cultures aged. During cultures aged, the bacteria also can stain in pink colour which known as Gram negative (Jonathan et al., 2017; Surinder Kumar, 2012; Stephen and Kathleen, 2012).
Pathogenesis of Clostridium tetani, it is caused by exotoxin (tetanospasmin) produce by bacteria. It can cause tetanus to human beings. The bacteria usually cause wound infection. This wound infection is trauma to host tissue and lower the oxidation reduction potential.
The spores of Clostridium tetani are ubiquitous which can found in the gastrointestinal tracts of human beings or animals. The spores are present in the soil especially in manured soil.
If there are any puncture wound in our skin. Tetanus can be develops due to accidental contamination of the wound with Clostridium tetani spores. Thus, it will introduce foreign bodies or small areas of cell killing create a nidus of devitalized material. Hence, tetanus spores will germinate and grow in the nidus of devitalized material. Tissue necrosis will formed after traumatic injuries or the injection of necrotizing substances. It will lower of the oxidation-reduction potential. Germination of spores may be due to the reduce oxygen tension which occur in devitalized tissues. After germination of the spores, the exotoxin produce by bacteria will be circulated through the central nervous system (CNS).
After bacteria exotoxin (tetanospasmin) circulated through the central nervous system. Tetanospasmin will block the release of neurotransmitters such as gamma-aminobutyric (GABA) and glycine. This tetanospasmin can inhibit the neurotransmitters for synapses to other neurotransmitters. Thus, it caused spastic paralysis. This is due to unregulated of excitatory synaptic activity of neurotransmitters. At the end, the exotoxin also can spread through other organs such as spinal cord, brain stem and peripheral nerves. The exotoxin only affects the relevant level of the spinal cord. It is known as local tetanus. However, the exotoxin affect the entire system is known as generalized tetanus (Surinder Kumar, 2012; Stephen and Kathleen, 2012).
Lab diagnosis of tetanus, the specimen will collected from wound exudates and tissues removed from the wound. In microscopy, it is demonstration of drumstick appearance of bacilli. The bacteria are stained in purple colour which is Gram positive. In biochemical test, the isolated specimen show positive result in hydrogen sulfide (H2S) production, DNase production and liquefied the gelatin. It shows negative result in nitrate reductase, amylase, lipase, and lecithinase activities (Márió et al., 2017; Hajra et al., 2015; Surinder Kumar, 2012).
In culture, the specimen will inoculate on one half of a blood agar. Clostridium tetani produces a swarming growth in blood agar after 1 to 2 days incubated at 30℃ in anaerobically. The specimen then inoculated into three tubes of cooked meat broth. First tube of cooked meat broth will heat at 80℃ for 15 minutes. Second tube of cooked meat broth also heat at 80℃ for 5 minutes. Third tube of cooked meat broth is unheated. The purpose of heating in different periods is used to kill vegetative bacteria while leaving undamaged tetanus spores. This is due to bacteria is heat resistance. The cooked meat tubes are incubated at 37℃ and subcultured on one half of blood agar daily for up to four days. It is used to identification and toxigenicity testing of tetanus antitoxin (Márió et al., 2017; Hajra et al., 2015; Surinder Kumar, 2012).
In toxigenicity test, it is best tested in animals. There are six mice were inoculated intramuscularly with tetanospasmin or tetanus toxin. Control mice were injected with tetanus antitoxin an hour before toxin inoculated to neutralize the effect of any tetanospasmin injected into the mouse. The signs of ascending tetanus develop in the mice which inoculated intramuscularly with tetanospasmin or tetanus toxin after several hours. The animals dies within 2 days but may be killed earlier as the appearance of ascending tetanus is diagnostic (Hajra et al., 2015; Surinder Kumar, 2012).
Treatment depends on debridement of infection wound. The patient is given 10,000 units of human tetanus immunoglobulin (HTIG) in saline by slow intravenous infusion. Penicillin and metronidazole is given to patients. This is used to ensure that the bacterial growth and toxin production will inhibit. The antitoxin and antibiotics will give immediately to patients before surgical excision. Vaccination with a series of three doses of tetanus toxoid and booster doses every 10 years will provide to patient. It is a highly effective in prevention of tetanus (Hajra et al., 2015; Surinder Kumar, 2012).
Bacteroides fragilis is the obligate anaerobic bacteria. It is the most important and prevalent of human opportunistic pathogens in gastrointestinal tract or gut (Mehrdad et al., 2017). It is the Gram negative bacilli bacteria which stained in pink colour. The common clinical infections caused by B. fragilis are intra-abdominal infection, female genital tract infection and bacteraemias. But rarely present in oral cavity and upper respiratory tract (Márió et al., 2017). The virulent factors of B. fragilis are expression of a capsular polysaccharide complex (CPC), adhesions and hydrolytic enzymes. Most virulent factor of B. fragilis is producing enterotoxin. It is known as B.fragilis toxin (BFT). B. fragilis enterotoxigenic (ETBF) is the strains which can cause acute diarrheal disease to human beings (Laís et al., 2015).
Morphology of B. fragilis, it is a pleomorphic. The size of bacteria will be around 1.5 to 6µm. It is the Gram negative bacilli and stained in pink colour. It is non-pigmented and non-motile. B. fragilis has surface pili and a capsule composed of a polymer of two polysaccharides. The capsule is help to prevent phagocytosis (Mehrdad et al., 2017).
Pathogenesis of B. fragilis, it is mainly caused by enterotoxin. The virulence factor B. fragilis mainly caused infection in intestinal tract (Zhang et al., 2017). The bacteria will attach on colon epithelial cell by helping of pili. The polysaccharides capsule which covered the bacteria can prevent phagocytosis and inhibition of macrophages migration. Thus, bacteria will trigger the T helper cell that causes severe infection. It can lead to abscess formation and cause inflammation (Thais et al., 2018). Some strain of B. fragilis produce enterotoxin also known as enterotoxin Bacteroides fragilis (ETBF). ETBF will produce enterotoxin which can causes enteric disease. The patient has associated with self-limited and watery diarrhea in children (Park et al., 2018).
In lab diagnosis, the watery feces sample and biopsy samples such as terminal ileum and colon is collected from infected patient (Park et al., 2018; Samin et al., 2017). In transmission electron microscopy, it is to analyse extracellular membrane vesicle that attach outer membrane of B. fragilis. It shows spherical vesicles with pleomorphic bilayer (Thais et al., 2018). In biochemical test, B. fragilis is catalase-positive and indole-negative (Márió et al., 2017). In culture, all biopsies were cultured in Bacteroides bile esculin agar (BBE) and incubated at 37℃ in anaerobic chamber for 3 days (Sami et al., 2017). B. fragilis growth in Brain Heart Infusion-pre-reduced anaerobically sterilized (BHI-PRAS) medium after incubated overnight at 37℃ in anaerobic chamber (Laís et al., 2015).
Treatment of drainage of abscesses, it can use penicillin. B. fragilis, resistant to vancomycin, kanamycin and colistin antibiotic disks, which can be used for their presumptive identification (Ho et al., 2017).