Table of Contents
The most important corynebacteria is C. diphtheriae which produces the diphtheria toxin and causes a serious, life-threatening disease called diphtheria. C. ulcerans and C. pseudotuberculosis are 2 other important species that are potentially toxigenic and can cause diphtheria. Recently, many non-toxigenic corynebacteria infections have been implicated as opportunistic infections in immunosuppressed patients.
Morphology of Corynebacteria
Corynebacteria are small prokaryotes that owe their name to the swollen end of their cell. On microscopy, this swollen end resembles a club (coryne = old Greek for ‘club’). Nevertheless, corynebacteria are pleomorphic which means they can assume other shapes during their growth and appear more coccoid.
Another important attribute of Corynebacteria is the presence of a V-shaped connection between the mother and daughter cells after cell division. This form appears due to the snapping post-fission movement. This snap division occurs because only the inner cell wall of corynebacteria participates in the cell division.
The outer wall surrounds the mother and daughter cell afterward and splits on one side as a result of cell growth. Both cells drift apart there but are still connected on one side so that the V-shape appears. This is also known as an array taking the shape of Chinese letters.
Laboratory diagnosis of Corynebacteria
Corynebacteria are aerobes and amastigotes, and, for this reason, are immobile rod cells that are unable to form spores or capsules. Polar bodies that include polyphosphate and calcium can be found in the cytoplasm of the bacteria. These polar bodies are barely visible on Gram stain so the Neisser stain is used to identify them.
After staining, the bacteria are colored yellow-to-rose, while the polar bodies are dark blue-to-black. This kind of staining is still widely used in the detection of C. diphtheriae and C. pseudodiphtheriticum. Culture takes place on blood agar and can be made with sputum, gastric juice, laryngeal smear, urine, and even ejaculate or menstrual blood.
By placing a fosfomycin platelet onto the blood agar, the growth of the normal pharyngeal flora can be inhibited. Corynebacteria are visible as greyish colonies with a slight flare of hemolysis. Tinsdale agar (sodium tellurite agar or Clauberg agar) works as an indicator medium. Because of the reduction of the included tellurium, Corynebacteria grow black with a blue flare on this medium. The tellurite also suppresses the growth of some bacteria of the pharyngeal flora.
It takes about 18–24 hours before the colonies become visible macroscopically. Afterward, pathogenic and non-pathogenic corynebacteria are differentiated by biochemical reactions. Pathogenic species are characterized by their positive catalase reaction, negative urease reaction, the fermentative breakdown of glucose (but not of saccharose), and nitrate reduction.
By their different colony morphology, hemolysis behavior, and ability to break down dextrin and glucose, the 3 biovars of C. diphtheriae: mitis, intermedius, and gravis can be distinguished. Different in vitro methods like gel immunodiffusion or PCR and in vivo tests, like the Draize test where the serum of the patient or culture solution is put onto the shaved skin of a rabbit, are used to detect the diphtheria toxin.
The Elek test is a particular type of gel immunodiffusion test. In this test, a stripe of filtering paper drained with antitoxin is applied. Additionally, the stem to be analyzed is applied crosswise. If the stem includes the diphtheria toxin, antigen-antibody complexes occur which can be seen as S-shaped lines. Nevertheless, the diagnosis of diphtheria is primarily clinical, with the laboratory diagnosis only for identifying specifics.
Epidemiology and incidence of Corynebacteria
Infection with C. diphtheriae is exclusively human-to-human via droplet infection, other secretions, or direct contact. Due to the good vaccination coverage in western countries, contamination often takes place in subtropical areas where diphtheria is endemic. The incidence of diphtheria in Middle Europe is about 0.001/100,000 per year. Dogs and cats are a natural reservoir for toxigenic strains of C. ulcerans. Non-pathogenic species are part of the normal flora of the skin and mucosa.
Pathomechanism of Corynebacteria
Only Corynebacteria that are infected with a specific phage trigger the disease. This phage carries genes for the exotoxin that inhibits the translation during the phase of elongation in the protein biosynthesis. First, the B-part of the bacterium docks to the host cell, afterward the A-toxin is absorbed into the cell in a vacuole. The diphtheria toxin A inhibits elongation factor 2 by the ADP ribosylation of a specific amino acid residue (diphthamide).
Moreover, some stems of C. ulcerans carry these phages and are, consequently, pathogenic. With the destruction of the infected cells, the typical pseudomembrane appears, consisting of necrotic cell components.
Clinical features 0f Corynebacteria infection
Two to 6 days after infection, symptoms like fatigue, sickness, and painful swallowing first occur. With the typical pharyngeal diphtheria, a yellowish-white fur and musty-sweet mouth odor emerge. Infants are often affected with diphtheria up to the nose so that a nose discharge characterized by blood pus occurs.
Laryngeal diphtheria is a dreaded condition as stridor or even asphyxiation can occur. A resulting ‘diphtheritic croup’ or ‘true croup’ could occur with a symptom triad of barking cough, hoarseness, and aphonia.
With other corynebacteria, clinical features like wound infections, otitis, and urinary tract infections can also result.
Therapy for Corynebacteria infection
The early administration of antitoxin is of priority in diphtheria. Simultaneously, antibiotic therapy with penicillin G should be started. Persons who have contacted infected people and asymptomatic carriers should receive penicillin V or clarithromycin prophylactically. Other Corynebacteria are often multidrug-resistant and are only sensitive to vancomycin, teicoplanin, and linezolid. Immunization with diphtheria toxin is also helpful. Suspicion of disease, infection, and death from diphtheria are reportable.
Listeria monocytogenes is an opportunistic pathogen that causes foodborne infectious disease. Infection is common in immunocompromised individuals and in vulnerable groups, such as pregnant women, neonates, and the elderly, with a high mortality rate of up to 20–30% of clinical cases. Infection may cause mild and self-limiting febrile gastroenteritis in healthy people.
Attributes of Listeria
Listeriae are Gram-positive rod cells that are mastigotes at low temperatures under 20°C (68°F) and have a typical end-to-end movement. The fermentative catalase-positive listeria can grow aerobically and anaerobically but do not form spores.
L. monocytogenes secretes a pore-forming toxin called listeriolysin O. On culture with blood-containing agars, this toxin causes ß-hemolysis; this characteristic allows the differentiation of virulent and avirulent strains. The toxin is analogous to streptolysin O from group A streptococci and pneumolysin from Pneumococci.
Listeria is very resistant to outer influences as it can even survive the pasteurizing of milk. This special attribute is used in diagnostics in the form of cold enrichment. Moreover, in the body, Listeriae protect themselves from the host’s immune defense by residing intracellularly.
In infected cells, listeria causes an evagination of the host cell by increasing in size. Due to this evagination, the bacteria can enter neighboring cells without any contact with the extracellular defense.
Incidence and epidemiology of Listeria
Listeria can be found in the intestine of domestic and wild animals and in humans. It also occurs in soil, water, and waste. Very often it can be isolated from milk and milk products.
Typically, occupationally exposed persons like butchers, farmers, or veterinary medicals are infected. Moreover, pregnant women and unborn children, who have relative immunosuppression, are at risk of listeria infection. About one-third of listeriosis affects pregnant women and newborns. Next to rubella and toxoplasmosis, listeriosis is the most frequent prenatal infection in Germany. Due to contaminated food, local outbreaks can occur. In Germany, the prevalence is 1–4 cases per 1,000,000 inhabitants per year.
Pathomechanism of listeriosis
L. monocytogenes mostly infects the host organism via the intestine over the M cells of the Peyer plaques and from there into local lymph nodes. From there, further spread takes place over the thoracic duct into the blood. Afterward, the bacteria are absorbed from macrophages and leave the phagosome via pore-forming listeriolysin.
In the cytoplasm of the macrophages, they can reproduce unhindered, leading to a release of chemotactic factors and monocytosis. The specific immune defense leads to the formation of granulomas.
In pregnant women, symptom-poor bacteremia usually occurs, which causes severe sepsis by placental transmission to the unborn child (granulomatosis infantiseptica). Typical symptoms in the fetus are infections of the liver, lungs, kidneys, and brain. Infections with Listeria can occur in every stage of pregnancy but are most common in the 3rd trimester.
Laboratory diagnosis for Listeria
Amniotic fluid, blood, and tissue samples can be used as testing material. The cold resistance of listeria is useful for isolation because only a few other bacteria can grow at 4°C (39.2°F) (known as cold enrichment). The propagation takes place on blood agar or in trypticase-soy-bouillon.
The colonies are small and white; in virulent stems, they are additionally surrounded by a flare of ß-hemolysis. In liquid culture mediums, they show a typical somersault-like movement. In microscopy, they often appear coccoid.
Therapy for listeriosis
The drug of choice is ampicillin in combination with an aminoglycoside.
Attributes of bacilli
Bacilli are big aerobic, immobile, spore-forming rod cells that are mostly Gram-positive but also slightly gram-variable. The species Bacillus anthracis, cereus, and subtilis are relevant to humans.
The rod cells of B. anthracis are stringed together like a chain and feature a capsule made of D-glutamic acid. This capsule is only formed on nutrient agar if increased CO2 tension is present. Under unfavorable culture conditions, endospores are formed, which are very environmentally resistant.
During the Second World War, some islands in the Atlantic were exposed to anthrax during biological weapon tests and are still uninhabitable. B. anthracis is a biological agent that can be used in bioterrorism attacks. It is ubiquitous in the soil. The plasmid-coded exotoxin, also known as anthrax toxin, consists of 3 parts: the edema factor, the protective antigen, and the lethal factor.
Anthrax is a zoonosis. The human is infected by direct contact with sick or deceased grazers or indirectly by animal products like wool, bone meal, or saddle cloth. That is why anthrax is also called Woolsorter’s disease. Through skin lesions, the spores enter the human body and develop into the vegetative, toxin-building form.
Pathomechanism of Bacillus anthracis
The edema factor and lethal factor invade leukocytes under protection by their protective antigen. In the leukocytes, they increase the concentration of cAMP, which inhibits the phagocytosis of the infected cells. Since leukocytes are infected, the infected tissue typically seems non-different. The lethal factor induces the necrosis of granulocytes and a lot of anthrax bacteria are released. The uninhibited reproduction of bacilli and proliferation via lymph vessels and bloodstream follows.
Clinical picture of infections with Bacillus anthracis
- Skin anthrax: The most common localizations are the hands, forearms, and face. After an incubation time of 2–5 days, a painless but itchy papule (pustula maligna) with an edematous edge appears at the infection site. After a short period, necrosis of the center with black coloration occurs. At the edge, serous cysts emerge. Without treatment, the infection is fatal in about 20% of cases due to toxemia and bacteremia. After a survived infection, humoral immunity of unknown duration occurs.
- Pulmonary anthrax: After initial symptoms, massive edemas appear in the neck and thorax regions. The patients suffer from dyspnea and fever. Pulmonary anthrax is the most dangerous clinical form and is notably difficult to treat.
- Intestinal anthrax: Severe enteritis can occur by the ingestion of anthrax spores. This ends in death in most cases due to the toxemia.
All 3 forms of anthrax can lead to sepsis with fatal outcomes.
Laboratory diagnosis of anthrax
The content of serous cysts in skin anthrax, sputum in pulmonary anthrax, and feces in intestinal anthrax can be used as test material depending on the kind of infection. Tests must only take place in S3 laboratories. Simple culture mediums with aerobic conditions are used.
B. anthracis colonies have curly branches at the edges, which are also called the ‘head of Medusa’. In microscopy, the bacteria appear as box-like rod cells with central endospores which are arranged in long chains (‘bamboo stick’).
Suspicion of illness, disease, and death by anthrax is subject to mandatory reporting. Moreover, reporting is mandatory in case of the detection of the pathogen.
Therapy for anthrax infections
Upon suspicion of anthrax, high-dose antibiotic therapy with penicillin G, ciprofloxacin, or tetracyclines should be started. In bioterrorist assaults, ciprofloxacin is preferred because resistance can be transferred from B. cereus to B. anthracis in the laboratory.
New antibiotics found to be effective against anthrax include levofloxacin, daptomycin, gatifloxacin, and dalbavancin. Newer therapies like peptides, bacteriophages enzymes, monoclonal antibodies, etc. are being evaluated
Farm animals should be vaccinated against anthrax. Exposed persons can profit from new vaccines that are based on the blockade of the protective antigen. Carcasses have to be burned.
In humans, B. cereus causes invasive local infections and self-limiting food intoxications.
Food intoxications from Bacillus cereus
B. cereus produces 3 toxins: the emetic toxin and 2 enterotoxins. The enterotoxins are heat-labile and are inactivated via proteolysis, while the emetic toxin is heat-stable and cannot be inactivated by proteolysis.
The enterotoxins are often absorbed from cooked rice or meat. About 1–6 hours after the intake of contaminated food, vomiting occurs. After 10–12 hours, stomach ache and watery diarrhea with tenesmus and vomiting appear. The symptoms last for about 24 hours, and the intoxication is self-limiting. Two forms of intestinal illness, diarrheal and emetic, have been described and are attributed to different toxins. Treatment is symptomatic and most patients recover within 24 hours after symptom onset.
Local infections with Bacillus cereus
Since B. cereus produces numerous tissue-destructing virulence factors, the wound infection leads to gas gangrene-like myonecrosis that is only superficial. Therapy can be surgical and/or antibiotic. In contrast to B. anthracis, B. cereus often forms ß-lactamases; therefore, antibiosis with vancomycin or clindamycin is preferable.
Laboratory diagnosis of Bacillus cereus
Feces and food can be investigated. The most important difference with regard to B. anthracis is the motility of B. cereus.
Attributes of Propionibacteria
Propionibacteria are anaerobic, oxygen-tolerant, non-spore-forming, immobile, Gram-positive rod cells. They are characterized by their slow growth and high culture requirements. They owe their name to propionic acid fermentation where carbohydrates like glucose and fructose, in particular, serve as a substrate for the main product—propionic acid. On blood agar, ß-hemolysis can be detected. Some propionibacteria have probiotic properties and are being used in dairy probiotic products.
Clinical picture of pathogenic Propionibacteria
Non-pathogenic propionibacteria mostly live on the skin as normal flora and commensals. Propionibacterium acnes can be found primarily in the sebum of hair follicles where it can cause inflammation by chemokine induction and cytokine production. The leukocytes are attracted by this, leading to the formation of pus-filled pustules.
In the hair follicles, optimal conditions for proliferation are provided because there is an anaerobic milieu and the propionibacteria have a lipase which they can use to harvest the fat contained in sebum for energy production.
Furthermore, propionibacteria have been implicated in endocarditis and spondylodiscitis. P. acnes can additionally lead to the development of circulating immune complexes which can be deposited on bones and joints. This clinical picture is especially relevant to young adults and is called the SAPHO syndrome (synovitis, acne, pustulosis, hyperostosis, and osteitis).
Attributes and detection of actinomycetes
For humans, the anaerobic, fermentative actinomycetes are particularly important. They are characterized by a positive Gram stain and their elongated, branched form. They are immobile and do not form spores. Actinomycetes can often be found as pathogens or commensals on the mucosa of endotherms and metabolize carbohydrates and organic acids in the mucosa.
The radial-filamentous branches of actinomycetes are visible under the microscope. They are called ray fungus and owe their name to their appearance (Greek aktis for ray and mykes for fungus). Moreover, macroscopically, a radial structure shows in the druses formed by the actinomycetes. Druses are nodular conglomerates surrounded by a wall of lymphocytes where the bacteria hyphae spread radially on the inner side.
Culture is via Columbia agar but this usually takes several days or weeks. The colonies appear yellowish and dry with small streaks.
The main cause of the actinomycosis is Actinomyces israelii. This bacterium belongs to the normal oral flora but can intrude into deeper layers in mucosal injuries and lead to the formation of fistulas and granulation tissue. Via these fistulas, actinomycetes can reach the blood circulation. Actinomycosis may mimic malignancy at various sites.
In 95% of the cases, only cervicofacial actinomycosis occurs where inviscid pus with sulfur-yellow druses drains from the fistulas. Hormonal factors likely play a role in the emergence of actinomycosis because men are distinctly more often affected, in contrast to children who are never affected.
Therapy should be a combination of surgery and antibiotics. Because of the ß-lactamase-forming collateral flora, a combination of amoxicillin and clavulanic acid has been established as standard therapy.
Other clinical features of Actinomycetes
An important ophthalmologic differential diagnosis is the lacrimal canaliculitis caused by A. israelii.
Furthermore, actinomycetes can have sensitizing properties and, by the formation of immune complexes, can cause a type III allergy. In this context, they are a typical cause of farmer’s lung, which is a form of exogenous allergic alveolitis.
Clostridia are obligately anaerobic, spore-forming rod cells. This species is ubiquitous in nature and can often be found in the intestinal tract of humans. The endospores formed under unfavorable living conditions are resistant to heat and exsiccosis and are able to survive in an aerobic milieu.
This bacterium is the main (but not the only) cause of gas gangrene, also called clostridial myonecrosis. This infection frequently occurs, especially in times of war, because C. perfringens spores are ubiquitous in the soil and can infect wounds that contact the soil.
Attributes of Clostridium perfringens
C. perfringens is a box-shaped, Gram-positive, immobile rod cell without granulocytes. It is frequently referred to as having a clay brick-shape. Additionally, in most isolates, a polysaccharide capsule can be found. Because of its toxins, C. perfringens is divided into types A–E.
Pathophysiological mechanism of gas gangrene
C. perfringens type A strains are the predominant cause of gas gangrene. The alpha-toxin is a lecithinase that destroys cell membranes by splitting lecithin.
A decrease in the redox potential of the infected tissue is required for the sprouting of Clostridia spores. For example, this is the case in circulatory disorders or necroses. This is why the cell destruction and occurrence of edemas by additionally occurring toxins from C. perfringens can be seen as a vicious cycle because it facilitates its own growth. Due to fermentation, the eponymous gas is formed.
Clinical picture of gas gangrene
After an incubation period of about 2 days, a swelling and brownish-livid discoloration of the extremely painful infection area occurs. Crepitations due to the CO2 gasification can be detected by palpation. A serous and (due to volatile fatty acids) foul-smelling fluid drains from the wounds. The muscles undergo necrotic changes. Toxin-induced shock in gas gangrene can lead to death within hours.
Diagnosis of clostridial myonecrosis
The diagnosis has to be made clinically based on symptoms and radiography. In the radiograph, a typical pinnation of the affected muscles shows. For microscopic examination, wound secretions and muscle excisions are suitable.
The detection of the alpha-toxin takes place with the help of Nagler’s test. In this test, Clostridia are cultured on agar with egg yolk where the formation of lecithinase can be seen as a haze around the bacteria colony.
Blood-containing agar or nutritious bouillons like liver bouillon could also be used. C. perfringens grow relatively fast at 45°C (113°F).
Suspected clostridial infection and gastroenteritis caused by C. perfringens are subject to mandatory reporting.
Therapy for infections with Clostridium perfringens
Surgical wound revision with eventual amputation of the infected extremity is essential because antibiotic therapy often does not reach the necrotic tissue. A combination of penicillin G and metronidazole is often used because in most cases, it is a combined infection with other anaerobic bacteria.
Furthermore, hyperbaric oxygenation in hyperbaric oxygen chambers is the standard solution for gas gangrene infections in order to eliminate these obligately anaerobic bacteria.
Attributes of Clostridium tetani
The structure of C. tetani is similar to those of other Clostridia. The tennis racket shape is characteristic.
Clinical picture of tetanus
Newborns are most likely to be affected by this bacterium, where C. tetani leads to the so-called disease of the 8th day (or tetanus neonatorum). This is an infection of the umbilicus since the umbilicus provides an optimal anaerobic milieu.
At the portal of entry, C. tetani proliferates and forms tetanospasmin which is released by autolysis and retrogradely spreads down the nerves up to the anterior horn cells in the spinal cord. There, it proteolytically divides into synaptobrevins. The synaptobrevins are involved in the release of GABA into the synaptic cleft. Thus, the neutralization of the inhibitory effect takes place.
Because of the excessive activity of the spinal motoneurons, spasticity with strychnine-like, tonic-clonic convulsions occurs.
The first symptoms appear after an incubation time of a few days to 3 weeks. Tension of the masticatory muscles (trismus) and a permanent contraction of the facial muscles (risus sardonicus) are typical.
Laboratory diagnosis of Clostridium tetani
Since culture often fails, toxin detection is important. For this detection, an animal experiment is necessary. In most cases, mice or guinea pigs are vaccinated with different amounts of the patient’s serum. The experiment is rated as positive when the animals die in the seal position, which means with catalepsy of the rear legs.
Control animals are simultaneously vaccinated with the patient’s serum and antitoxin. These animals should survive a positive mice protection experiment.
Therapy for tetanus infections
In addition to the administration of anti-tetanus toxin antibodies, the therapy consists of anticonvulsant drugs and artificial respiration to prevent the paralysis of the respiratory muscles.
Because of the markedly lethal nature of tetanus, active vaccination with formalinized toxins (toxoids) is crucial.
The atypical mycobacteria which are potentially pathogenic for humans are often summarized as MOTT (mycobacteria other than tuberculosis).
Attributes of atypical mycobacteria
The structure of MOTT do not differ from those of other mycobacteria. MOTT also have an acid-proof capsule and are extremely resistant to environmental influences, heat, and many disinfectants. They ubiquitously occur in the soil and in water samples. Some of them occur only in particular regions, for example, Mycobacteria ulcerans which is only prevalent in Africa.
MOTT are classified into 4 groups. Slow-growing mycobacteria that only produce a pigment during light exposure, belong to group I. The scotochromogenic mycobacteria of group II which produce pigment in the dark. Slowly growing MOTT that do not produce pigment belong to group III, whereas all fast-growing MOTT belong to group IV.
Diseases due to atypical mycobacteria
As opportunistic pathogens, the atypical mycobacteria especially cause infections in immunosuppressed patients.
Pulmonary infections that do not differ clinically from tuberculosis frequently occur. Further, granulomatous infections of the skin and lymphangitis of the cervical lymph nodes are frequent. M. scrofulaceum causes ‘scrofula’, which is granulomatous cervical adenitis that is usually seen in children.
M. marinum is the cause of swimming pool granuloma. After bathing in contaminated water, and with an incubation time of 2–3 weeks, granulomas that ulcerate appear at entry portals like the elbows and knees. M. ulcerans causes ‘Buruli ulcer’, primarily affecting the lower extremities.
Laboratory diagnosis of atypical mycobacteria
As with M. tuberculosis, culture is usually successful in Löwenstein-Jensen agar. Fast-growing mycobacteria develop colonies after 3–4 days, while other mycobacteria develop colonies only after several days to weeks. Differentiation of MOTT organisms especially depends on the growth rate, culture morphology, and pigment formation. The differentiation is complemented with restriction fragment length polymorphism.
In contrast to tuberculosis, it is not obligatory to report the disease.
Therapy for atypical mycobacteria
Most MOTT are highly resistant to tuberculostatics which is why therapy can be very difficult. In infection with M. avium or M. intracellulare, a combination of clarithromycin, ethambutol, and rifabutin is recommended. In other atypical mycobacteriosis, up to 6 antibiotics may be necessary.