MacCallum et al. 1950
Mycobacterium ulcerans is a slow-growing mycobacterium that classically infects the skin and subcutaneous tissues, giving rise to indolent nonulcerated (nodules, plaques) and ulcerated lesions. After tuberculosis and leprosy, Buruli ulcer is the third most common mycobacteriosis of humans. M. ulcerans grows optimally on routine mycobacteriologic media at 33 °C and elaborates a necrotizing immunosuppressive cytotoxin (mycolactone). The bacteria are considered microaerophilic. Large ulcers almost certainly caused by M. ulcerans were first observed by Cook in Uganda in 1897; however, the etiologic agent was not isolated and characterized until 1948 in Australia by MacCallum and associates.
Lesions of M. ulcerans disease have several synonyms (e.g. Bairnsdale or Searle's ulcer). The name Buruli is probably most appropriate for historic reasons, as it is a county of Uganda where important foci of the disease were studied.
The source(s) of M. ulcerans in nature is becoming clearer from epidemiologic data and from molecular biologic findings. Because all major endemic foci are in wetlands of tropical or subtropical countries, environmental factors must play an essential role in the survival of the etiologic agent. Koalas and possums are naturally infected animals in Australia. The disease is rarely transmitted from patient to patient. Trauma is probably the most frequent means by which M. ulcerans is introduced into the skin from surface contamination. Individuals of all ages are affected, but the highest frequencies of infection are in children under 15 years of age (Debacker et al. accepted for publication).
In many areas, M. ulcerans infection has only occurred after significant environmental disturbance. In the original paper describing the disease, published in 1948, the first patients presented in 1939 in the Bairnsdale District of Victoria, Australia.In December 1935, there had been terrible floods in the district, when all road and rail links had been cut and there had been considerable destruction of property. In Uganda, Barker examined cases of M. ulcerans infection (Buruli ulcer disease) occurring in the Busoga region on the east side of the Victoria Nile, north of Lake Victoria. Although cases were known in the other parts of the country, cases were unknown in the district before 1965. Barker postulated that the outbreak was related to the unprecedented flooding of the lakes of Uganda between 1962 and 1964 as a result of heavy rainfall.
In Nigeria, cases have occurred among Caucasians living on the campus of University of Ibadan only after 1965,when a small stream flowing through the campus was dammed to make an artificial lake. The first case reported in Côte d'Ivoire was a French boy of seven years who lived with his parents beside Lake Kossou, an artificial lake in the center of the country. In Liberia, cases have been reported in the north of the country following the introduction of swamp rice to replace upland rice. This introduction has been associated with construction of dams on the Mayor river and extended wetlands. In Papua New Guinea, the infection occurs mainly in relation to the Sepik and Kumusi rivers; in the latter areas, the disease is known as the "Kumusi ulcer". The disease occurred after flooding and devastation, which followed the eruption of Mount Lamington in 1951. Reid described how older people living in the villages blamed the volcano for the disease. The recent outbreak of the disease on Philip Island, Victoria, was initially associated with the building of a roadway, inadvertently forming marshlands at the headwaters of an estuary, which was divided by the construction. Again in Australia, the increase in the number of cases between 1991 and 1994 in Victoria was associated with the use of recycled waste-water to irrigate a golf course.
Despite significant environmental disturbances due to mining operations in the headwaters of the Fly River, the largest river in Papua New Guinea, no cases were identified. It would consequently seem that additional factors could be responsible besides simple disturbances. One of these could be the formation of new water areas where the water is stagnant or slow-moving. A delay between one or three years is said to occur between environmental changes and the first patients appearing.
A series of epidemiological studies show the existence of seasonal variation in the appearance of Buruli ulcer cases. It seems that the number of cases augments during dry periods or after inundations.These conditions are probably favorable for the development of M. ulcerans, because of the concentration of possible vectors in areas that are frequently visited by humans.
The major virulence determinant in M. ulcerans is a polyketide-derived macrolide: mycolactone. Mycolactone was originally isolated from M. ulcerans 1615, a Malaysian isolate, as a mixture of cis/trans isomers designated mycolactone A and mycolactone B. Identical molecules were also found to be present in two M. ulcerans isolates from the Democratic Republic of Congo.More recent evidence shows that M. ulcerans 1615 produces a family of mycolactone congeners which differ primarily in the number of hydroxyl groups and double bonds.
Mycolactone appears to play a key role in the pathogenesis of Buruli ulcer. In vivo studies using a guinea pig model of infection suggest that mycolactone is responsible for both the extensive tissue damage and immunosuppression which accompanies Buruli ulcer.The activity of mycolactone on cultured fibroblasts and macrophage cell lines produces a distinct cytopathic phenotype. The earliest effect is cell rounding, which occurs within 10 h after addition of mycolactone to cultured cells. At 36 h, treated cells are arrested in G1 of the cell cycle, and by 72 h, cells begin to die via apoptosis.
Bacterial macrolides are produced as secondary metabolites by soil bacteria, particularly bacteria such as Streptomyces and Saccharopolyspora species in the order Actinomycetales.A number of related macrolides or congeners are often produced by a single bacterial isolate.
According to the traditional methods, mycobacteria are preliminarily identified by growth rate and pigmentation.This preliminary grouping may provide presumptive identification of the organism and directs the selection of key biochemical tests to characterize an unknown mycobacterium.
Because M. ulcerans infection is associated with nonspecific clinical manifestations and indolent course, it is important to consider every nodule or ulcer in an endemic area as a suspected M. ulcerans infection until proven otherwise. A nodule is firm and painless. In the absence of superinfection(s) an ulcer is painless or minimally painful, has the characteristic undermined edge and a whitish-yellow necrotic base. Previous residence in an endemic area should raise the suspicion of M. ulcerans infection.
Appropriately selected tissue specimen that include necrotic subcutaneous tissue and the undermined edge of ulcerated lesions are frequently diagnostic. Specimens from skin and subcutaneous tissue from nonulcerated lesions are likewise often diagnostic.
Buruli ulcer is often diagnosed late, when treatment can be very difficult and frustrating. Confirmation by culture takes 6–8 weeks. Rapid diagnostic methods for M. ulcerans infection, as well as methods of rapid identification of the organism in clinical and environmental specimens would be a significant advance in the management of M. ulcerans infection. Screening to detect early infection could guide early intervention.
There are several polymerase chain reaction PCR methods available that could increase the speed of diagnosis of M. ulcerans infection.PCR is relatively expensive compared to microscopy, and is notorious for producing false-positive results in laboratories that lack experience with PCR. In high-prevalence regions such as West Africa, PCR may not be any more rapid than an accurate clinical case definition combined with a smear that shows acid-fast bacilli. In countries such as Australia, where the incidence is low, the great majority of patients who have nodules, papules or skin ulcers do not have M. ulcerans disease. In this situation, PCR is a quicker way of making the diagnosis with a high degree of confidence. The main advantage of PCR is that M. ulcerans disease can be diagnosed within 24 hours. PCR usefulness for mycobacterial infections is generally limited, however, and at present it is recommended that PCR is used as a rapid ancillary test, not as a replacement for culture and histology.
The PCR method developed by Stinear et al.targets a DNA insertion sequence in M. ulcerans. When genomic M. ulcerans DNA is digested with the restriction enzyme AluI, many 1109 base-pair fragments were obtained. These AluI fragments have been shown to be part of a larger 1293 base-pair repeated sequence that, by chance, happened to contain two AluI restriction sites. The sequence has been named IS2404 (Genbank accession number AF003002) It has been recently discovered that IS2404 copies are also present in a large circular plasmid. The total number of IS copies is thus 220. It has been identified in all isolates of M. ulcerans tested to date and has not been found in at least 45 other mycobacterial species, including M. marinum, M. leprae and M. tuberculosis. Recent publications have however demonstrated the presence of IS2404 in M. marinum-like bacteria (Trott et al. accepted for publication).
PCR methods that have been developed are based on the 16S rRNA gene,the hsp65 gene, or the insertion sequence IS2404. In 1999, Guimaraes-Peres et al. evaluated two nested PCRs: the nested IS2404-based PCR and the nested 16S rRNA gene-based PCR. IS2404-based PCR was positive only with M. ulcerans isolates and the closely related M. shinshuense. The 16S rRNA gene-based PCR was positive not only for these two strains but also for M. marinum. The use of IS2404-based PCR as a detection method for M. ulcerans showed better sensitivity and specificity, required less time, and was less costly than the 16S rRNA gene-based PCR.
To date, it has been established that PCR has a specificity of 100% and a sensitivity of 96% compared with culture.
Additionally kits that utilise qPCR detection by probes to confirm the bacterium are as accurate as regular PCR but is quicker, cheaper and requires less skill. Several manufacturers have kits available.
M. ulcerans belongs to the group of occasional pathogens. Most species belonging to this group are found almost everywhere in nature, and may become pathogenic under special circumstances. Some of them have rarely (e.g. M. malmoense) or never (M. ulcerans) been isolated from the environment. The epidemiological profiles of the diseases they cause, however, suggest that they are present in nature.Recently, M. ulcerans has been detected by molecular biological techniques in water samples collected in Australia and in bugs collected from roots of aquatic plants in swamps in endemic regions of Benin and Ghana. M. ulcerans was, however, not recovered by culture from these environmental samples.
PCR is not inhibited by the presence of culturable organisms. Unfortunately, PCR is exquisitely sensitive to inhibition by many compounds such as humic and fulvic acids, which are ubiquitous in the environment and are not removed by standard DNA extraction protocols. The first confirmation that M. ulcerans was present in environmental water samples was obtained in 1997,by combining the highly sensitive and specific IS2404 PCR with a method that separated sample DNA from naturally occurring inhibitors of PCR.
Three different strategies have now been used to overcome inhibition in environmental samples from M. ulcerans endemic regions. The first of these is gel chromatography. Environmental water samples are concentrated and subjected to homogenization with glass beads, followed by heat and alkaline lysis to release DNA. Total extracted DNA is then run through gel chromatography columns that separate DNA from contaminants on the basis of size.Although relatively simple, the method is cumbersome and time-consuming. The second method uses paramagnetic beads linked to M. ulcerans antibodies to capture whole cells and separate them from contaminants in a magnetic field (immunomagnetic separation). Antibodies are raised in laboratory animals. Captured cells are washed to remove inhibitors and then DNA is released by standard methods prior to PCR. The third approach also uses paramagnetic beads, but here the beads are linked to M. ulcerans-specific oligonucleotide probes, which capture IS2404 DNA that has been released from M. ulcerans by homogenization and alkaline lysis. The immobilized DNA is washed to remove inhibitors and used directly as a template for IS2404 PCR. The latter two methods each have limitations and advantages, but offer superior detection sensitivity and are less time-consuming than gel chromatography.
Molecular typing methods may be categorized into three broad groups on the basis of the type of macromolecules targeted for sub-typing, i.e. methods based on fatty acids, proteins and nucleic acids. Actually, the genotypic typing methods (DNA fingerprinting) that evaluate differences at the DNA level are used more commonly and have emerged as revolutionary tools for epidemiological studies.
The use of DNA fingerprinting for the identification of M. tuberculosis has greatly improved understanding of the epidemiology of tuberculosis: transmission routes of different strains have been recognized;outbreaks of multidrug-resistant strains have been detected early; and the relative importance of reinfection versus reactivation can now be elucidated.
Various molecular methods for fingerprinting of M. ulcerans are now being developed to facilitate studies on the epidemiology of Buruli ulcer. So far, 12 genotypes, spread over the world, have been discriminated, based on a variable number of tandem repeats and mycobacterial interspersed repetitive units. Next-Generation Sequencing will soon dramatically ameliorate subtyping and genotype differentiation.
Direct comparison of some genomic DNA sequences of bacterial strains is the best means of quantitatively determining whether two strains are similar or different. Portaels et al. have analyzed the 3’-terminal region of the 16S rRNA gene sequence of 17 strains of M. ulcerans from Africa, Australia and America.This analysis has revealed three subgroups that vary according to the continent of origin. Later, a fourth subgroup was discovered in China and Japan confirming the existence of an Asian type.
Insertion sequences (IS) are mobile genetic elements that are usually present in numerous copies within a bacterial genome. These elements can be used as probes, and because the number and location of IS elements vary, each strain will have a unique banding pattern. Molecular analysis of M. ulcerans has revealed two insertion sequences: IS2404 and IS2606.Southern blot analysis to detect IS2404 and IS2606 shows inconclusive RFLP patterns between different strains. Due to the high number of copies of both elements, the banding patterns are difficult to interpret, limiting the value of the Southern blot method to type M. ulcerans isolates.
Jackson et al. have used pTBN12, a well-defined plasmid, as a probe with AluI restriction fragments.The probe was able to distinguish 11 RFLP patterns.
PFGE permits the generation of simplified chromosomal restriction fragment patterns without having to resort to probe hybridization methods. In this method, restriction enzymes that cut DNA infrequently are used to generate large fragments of chromosomal DNA, which are then separated by special electrophoretic procedures. Preliminary results showed that M. ulcerans genomes produce three different profiles according to the three geographical origins of the strains (Type I: Africa, Type II: Australia and Type III: North America)
The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA.This technique involves three steps: restriction of DNA and ligation of oligonucleotides and adaptors; selective amplification of sets of restriction fragments; and gel analysis of the amplification fragments. Typically 50–100 restriction fragments are amplified and detected on denaturing polyacrylamide gel. AFLP typing results in a clear distinction of M. marinum from M. ulcerans, but interspecies differentiation is not trustworthy
PCR is another molecular method that has become increasingly important for epidemiological studies. The technique detects and amplifies small amounts of DNA; 10–100 copies of the templates are enough to perform DNA amplification. Thus, PCR can be used to type organisms that grow slowly on laboratory media, such as M. tuberculosis.PCR also can be used to detect and type pathogens in patients whose culture are negative because they have been treated. Moreover, PCR can be used to amplify the DNA from organisms that are present in tissues preserved in formalin and from non-cultivable organisms (e.g. M. leprae).
Rep-PCR is a modification of the PCR technique that is more suitable for epidemiological purposes than conventional PCR. In this case, the primers are directed towards repetitive chromosomal elements such as IS6110 in M. tuberculosis and the ERIC sequence in other bacteria.In M. ulcerans, the genomic sequence between the IS2404 elements has been amplified. The profiles produced by this technique categorized the strains into three subgroups related to the three different endemic regions (Africa, Australia and North America).
Ribotyping: This method involves amplification of a known sequence cut by restriction enzymes, and compares restriction fragments of amplified DNA from different strains. Using this technique, the M. ulcerans genome has been found to produce three different restriction profiles related to the origin of the strains.
Mycobacterium tuberculosis is a species of pathogenic bacteria in the family Mycobacteriaceae and the causative agent of tuberculosis. First discovered in 1882 by Robert Koch, M. tuberculosis has an unusual, waxy coating on its cell surface primarily due to the presence of mycolic acid. This coating makes the cells impervious to Gram staining, and as a result, M. tuberculosis can appear either Gram-negative or Gram-positive. Acid-fast stains such as Ziehl-Neelsen, or fluorescent stains such as auramine are used instead to identify M. tuberculosis with a microscope. The physiology of M. tuberculosis is highly aerobic and requires high levels of oxygen. Primarily a pathogen of the mammalian respiratory system, it infects the lungs. The most frequently used diagnostic methods for tuberculosis are the tuberculin skin test, acid-fast stain, culture, and polymerase chain reaction.
Mycobacterium is a genus of Actinobacteria, given its own family, the Mycobacteriaceae. Over 190 species are recognized in this genus. This genus includes pathogens known to cause serious diseases in mammals, including tuberculosis and leprosy in humans. The Greek prefix myco- means "fungus," alluding to the way mycobacteria have been observed to grow in a mold-like fashion on the surface of cultures. It is acid fast and cannot be stained by the Gram stain procedure.
In molecular biology, an amplicon is a piece of DNA or RNA that is the source and/or product of amplification or replication events. It can be formed artificially, using various methods including polymerase chain reactions (PCR) or ligase chain reactions (LCR), or naturally through gene duplication. In this context, amplification refers to the production of one or more copies of a genetic fragment or target sequence, specifically the amplicon. As it refers to the product of an amplification reaction, amplicon is used interchangeably with common laboratory terms, such as "PCR product."
Nontuberculous mycobacteria (NTM), also known as environmental mycobacteria, atypical mycobacteria and mycobacteria other than tuberculosis (MOTT), are mycobacteria which do not cause tuberculosis or leprosy. NTM do cause pulmonary diseases that resemble tuberculosis. Mycobacteriosis is any of these illnesses, usually meant to exclude tuberculosis. They occur in many animals, including humans.
Buruli ulcer is an infectious disease caused by Mycobacterium ulcerans. The early stage of the infection is characterised by a painless nodule or area of swelling. This nodule can turn into an ulcer. The ulcer may be larger inside than at the surface of the skin, and can be surrounded by swelling. As the disease worsens, bone can be infected. Buruli ulcers most commonly affect the arms or legs; fever is uncommon.
Mycobacterium smegmatis is an acid-fast bacterial species in the phylum Actinobacteria and the genus Mycobacterium. It is 3.0 to 5.0 µm long with a bacillus shape and can be stained by Ziehl-Neelsen method and the auramine-rhodamine fluorescent method. It was first reported in November 1884 by Lustgarten, who found a bacillus with the staining appearance of tubercle bacilli in syphilitic chancres. Subsequent to this, Alvarez and Tavel found organisms similar to that described by Lustgarten also in normal genital secretions (smegma). This organism was later named M. smegmatis.
AFLP-PCR or just AFLP is a PCR-based tool used in genetics research, DNA fingerprinting, and in the practice of genetic engineering. Developed in the early 1990s by Keygene, AFLP uses restriction enzymes to digest genomic DNA, followed by ligation of adaptors to the sticky ends of the restriction fragments. A subset of the restriction fragments is then selected to be amplified. This selection is achieved by using primers complementary to the adaptor sequence, the restriction site sequence and a few nucleotides inside the restriction site fragments. The amplified fragments are separated and visualized on denaturing on agarose gel electrophoresis, either through autoradiography or fluorescence methodologies, or via automated capillary sequencing instruments.
Bartonellosis is an infectious disease produced by bacteria of the genus Bartonella. Bartonella species cause diseases such as Carrión's disease, trench fever, cat-scratch disease, bacillary angiomatosis, peliosis hepatis, chronic bacteremia, endocarditis, chronic lymphadenopathy, and neurological disorders.
A real-time polymerase chain reaction, also known as quantitative polymerase chain reaction (qPCR), is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR). It monitors the amplification of a targeted DNA molecule during the PCR, not at its end, as in conventional PCR. Real-time PCR can be used quantitatively and semi-quantitatively.
A mycobacteriophage is a member of a group of bacteriophages known to have mycobacteria as host bacterial species. While originally isolated from the bacterial species Mycobacterium smegmatis and Mycobacterium tuberculosis, the causative agent of tuberculosis, more than 4,200 mycobacteriophage have since been isolated from various environmental and clinical sources. Almost 1400 have been completely sequenced. Mycobacteriophages have served as examples of viral lysogeny and of the divergent morphology and genetic arrangement characteristic of many phage types.
Mycobacterium pseudoshottsii, a slowly growing chromogenic species was isolated from Chesapeake Bay striped bass during an epizootic of mycobacteriosis.
Mycobacterium ulcerans liflandii has been isolated from Xenopus tropicalis and Xenopus laevis in a laboratory in the US and causes a Mycobacterium ulcerans-like disease in anurans. The strain was unofficially titled under its own species name until it was renamed to be an ecovariation of Mycobacterium ulcerans.
Terminal restriction fragment length polymorphism is a molecular biology technique for profiling of microbial communities based on the position of a restriction site closest to a labelled end of an amplified gene. The method is based on digesting a mixture of PCR amplified variants of a single gene using one or more restriction enzymes and detecting the size of each of the individual resulting terminal fragments using a DNA sequencer. The result is a graph image where the x-axis represents the sizes of the fragment and the y-axis represents their fluorescence intensity.
Mycobacterium marinum is a free-living bacterium, which causes opportunistic infections in humans. M. marinum sometimes causes a rare disease known as aquarium granuloma, which typically affects individuals who work with fish or keep home aquariums.
Mycolactone is a polyketide-derived macrolide produced and secreted by a group of very closely related pathogenic Mycobacteria species that have been assigned a variety of names including, M. ulcerans, M. liflandii, M. pseudoshottsii, and some strains of M. marinum. These mycobacteria are collectively referred to as mycolactone-producing mycobacteria or MPM.
Loop-mediated isothermal amplification (LAMP) is a single tube technique for the amplification of DNA. This may be of use in the future as a low cost alternative to detect certain diseases. It may be combined with a reverse transcription step to allow the detection of RNA.
PCR inhibitors are any factor which prevent the amplification of nucleic acids through the polymerase chain reaction (PCR). PCR inhibition is the most common cause of amplification failure when sufficient copies of DNA are present. PCR inhibitors usually affect PCR through interaction with DNA or interference with the DNA polymerase. Inhibitors can escape removal during the DNA purification procedure by binding directly to single or double-stranded DNA. Alternatively, by reducing the availability of cofactors (such as Mg2+) or otherwise interfering with their interaction with the DNA polymerase, PCR is inhibited.
Amplified rDNA Restriction Analysis is the extension of the technique of RFLP to the gene encoding the small (16s) ribosomal subunit of bacteria. The technique involves an enzymatic amplification using primers directed at the conserved regions at the ends of the 16s gene, followed by digestion using tetracutter Restriction enzymes. The pattern obtained is said to be representative of the species analysed. Patterns obtained from several restriction enzymes can be used to phylogenetically characterize cultured isolates and 16s genes obtained through cloning from community DNA
Wayne Marvin Meyers was an American physician, microbiologist, chemist, humanitarian, and medical missionary. He pioneered new medical techniques, discovered new infectious agents, and trained countless researchers and scientists. Meyers was particularly well known for his work with Hansen's disease (Leprosy), Buruli ulcer, and filarial diseases.