Leprae Strains Research Articles

Optimized Protocols for <em>Mycobacterium leprae</em> Strain Management: Frozen Stock Preservation and Maintenance in Athymic Nude Mice

Leprosy, caused by Mycobacterium leprae, is an important infectious disease that is still endemic in many countries around the world, including Brazil. There are currently no known methods for growing M. leprae in vitro, presenting a major obstacle in the study of this pathogen in the laboratory. Therefore, the maintenance and growth of M. leprae strains are preferably performed in athymic nude mice (NU-Foxn1nu). The laboratory conditions for using mice are readily available, easy to perform, and allow standardization and development of protocols for achieving reproducible results. In the present report, we describe a simple protocol for purification of bacilli from nude mouse footpads using trypsin, which yields a suspension with minimum cell debris and with high bacterial viability index, as determined by fluorescent microscopy. A modification to the standard method for bacillary counting by Ziehl-Neelsen staining and light microscopy is also demonstrated. Additionally, we describe a protocol for freezing and thawing bacillary stocks as an alternative protocol for maintenance and storage of M. leprae strains.

On the Age of Leprosy

Leprosy is a chronic infection of the skin and nerves caused by Mycobacterium leprae and the newly discovered Mycobacterium lepromatosis. Human leprosy has been documented for millennia in ancient cultures. Recent genomic studies of worldwide M. leprae strains have further traced it along global human dispersals during the past ∼100,000 years. Because leprosy bacilli are strictly intracellular, we wonder how long humans have been affected by this disease-causing parasite. Based on recently published data on M. leprae genomes, M. lepromatosis discovery, leprosy bacilli evolution, and human evolution, it is most likely that the leprosy bacilli started parasitic evolution in humans or early hominids millions of years ago. This makes leprosy the oldest human-specific infection. The unique adaptive evolution has likely molded the indolent growth and evasion from human immune defense that may explain leprosy pathogenesis. Accordingly, leprosy can be viewed as a natural consequence of a long parasitism. The burden of leprosy may have affected minor selection on human genetic polymorphisms.

Update on Genetics of Leprosy

Leprosy is a chronic granulomatous infectious disease caused by Mycobacterium leprae that mainly infects skin macrophages and Schwann cells in peripheral nerves. Genetic and environmental factors play an important role in leprosy. It is estimated that approximately 90% of the population develop protective immunity in infection, and, therefore, do not get sick. Other individuals, however, show clinical susceptibility to a wide spectrum of pathogens associated to changes in immune response. Data observed in various populations show that some aspects related to disease progression are due to host genetic factors that influence control of the initial infection and the host immune response to that infection. Nevertheless, individual genetic factors have a strong influence on the acquisition of or protection against leprosy, since the limited genetic diversity observed among M. leprae strains is not a significant factor for the development and the clinical course of disease.

Mycobacterium leprae genomes from a British medieval leprosy hospital: towards understanding an ancient epidemic.

BackgroundLeprosy has afflicted humankind throughout history leaving evidence in both early texts and the archaeological record. In Britain, leprosy was widespread throughout the Middle Ages until its gradual and unexplained decline between the 14th and 16th centuries. The nature of this ancient endemic leprosy and its relationship to modern strains is only partly understood. Modern leprosy strains are currently divided into 5 phylogenetic groups, types 0 to 4, each with strong geographical links. Until recently, European strains, both ancient and modern, were thought to be exclusively type 3 strains. However, evidence for type 2 strains, a group normally associated with Central Asia and the Middle East, has recently been found in archaeological samples in Scandinavia and from two skeletons from the medieval leprosy hospital (or leprosarium) of St Mary Magdalen, near Winchester, England.ResultsHere we report the genotypic analysis and whole genome sequencing of two further ancient M. leprae genomes extracted from the remains of two individuals, Sk14 and Sk27, that were excavated from 10th-12th century burials at the leprosarium of St Mary Magdalen. DNA was extracted from the surfaces of bones showing osteological signs of leprosy. Known M. leprae polymorphisms were PCR amplified and Sanger sequenced, while draft genomes were generated by enriching for M. leprae DNA, and Illumina sequencing. SNP-typing and phylogenetic analysis of the draft genomes placed both of these ancient strains in the conserved type 2 group, with very few novel SNPs compared to other ancient or modern strains.ConclusionsThe genomes of the two newly sequenced M. leprae strains group firmly with other type 2F strains. Moreover, the M. leprae strain most closely related to one of the strains, Sk14, in the worldwide phylogeny is a contemporaneous ancient St Magdalen skeleton, vividly illustrating the epidemic and clonal nature of leprosy at this site. The prevalence of these type 2 strains indicates that type 2F strains, in contrast to later European and associated North American type 3 isolates, may have been the co-dominant or even the predominant genotype at this location during the 11th century.

Characteristic mutations found in the ML0411 gene of Mycobacterium leprae isolated in Northeast Asian countries

Genome analysis of Mycobacterium leprae strain Kyoto-2 in this study revealed characteristic nucleotide substitutions in gene ML0411, compared to the reference genome M. leprae strain TN. The ML0411 gene of Kyoto-2 had six SNPs compared to that of TN. All SNPs in ML0411 were non-synonymous mutations that result in amino acid replacements. In addition, a seventh SNP was found 41bp upstream of the start codon in the regulatory region. The seven SNP sites in the ML0411 region were investigated by sequencing in 36 M. leprae isolates from the Leprosy Research Center in Japan. The SNP pattern in 14 of the 36 isolates showed similarity to that of Kyoto-2. Determination of the standard SNP types within the 36 stocked isolates revealed that almost all of the Japanese strains belonged to SNP type III, with nucleotide substitutions at position 14676, 164275, and 2935685 of the M. leprae TN genome. The geographical distribution pattern of east Asian M. leprae isolates by discrimination of ML0411 SNPs was investigated and interestingly turned out to be similar to that of tandem repeat numbers of GACATC in the rpoT gene (3 copies or 4 copies), which has been established as a tool for M. leprae genotyping. All seven Korean M. leprae isolates examined in this study, as well as those derived from Honshu Island of Japan, showed 4 copies of the 6-base tandem repeat plus the ML0411 SNPs observed in M. leprae Kyoto-2. They are termed Northeast Asian (NA) strain of M. leprae. On the other hand, many of isolates derived from the Okinawa Islands of Japan and from the Philippines showed 3 copies of the 6-base tandem repeat in addition to the M. leprae TN ML0411 type of SNPs. These results demonstrate the existence of M. leprae strains in Northeast Asian region having characteristic SNP patterns.

In vitro and in vivo Activity of K-130, a Dihydrofolate Reductase inhibitor, against Mycobacterium leprae

The antimicrobial effects of a new dihydrofolate reductase inhibitor, K-130 (2,4-diaminodiphenyl sulfone substituted 2,4-diamino-5-benzylpyrimidine), alone and in combination with dapsone (CAS 80-08-0) against both dapsone-sensitive and dapsone-resistant strains of Mycobacterium leprae were evaluated in vitro, in cell-free culture system, and in vivo, in mouse foot pads. The minimal inhibitory concentration of K-130 against dapsone-sensitive as well as dapsone resistant strains of M, leprae was 0.03 microgram/ml, and the activity was bactericidal in both cases. However, when combined with dapsone, K-130 exhibited synergism in case of dapsone-sensitive M. leprae, while in case of dapsone-resistant M. leprae, the effect was merely additive. Similar synergistic effects were also observed in the mouse foot pad system for both types of M. leprae strains.

Probable Zoonotic Leprosy in the Southern United States

In the southern region of the United States, such as in Louisiana and Texas, there are autochthonous cases of leprosy among native-born Americans with no history of foreign exposure. In the same region, as well as in Mexico, wild armadillos are infected with Mycobacterium leprae. Whole-genome resequencing of M. leprae from one wild armadillo and three U.S. patients with leprosy revealed that the infective strains were essentially identical. Comparative genomic analysis of these strains and M. leprae strains from Asia and Brazil identified 51 single-nucleotide polymorphisms and an 11-bp insertion-deletion. We genotyped these polymorphic sites, in combination with 10 variable-number tandem repeats, in M. leprae strains obtained from 33 wild armadillos from five southern states, 50 U.S. outpatients seen at a clinic in Louisiana, and 64 Venezuelan patients, as well as in four foreign reference strains. The M. leprae genotype of patients with foreign exposure generally reflected their country of origin or travel history. However, a unique M. leprae genotype (3I-2-v1) was found in 28 of the 33 wild armadillos and 25 of the 39 U.S. patients who resided in areas where exposure to armadillo-borne M. leprae was possible. This genotype has not been reported elsewhere in the world. Wild armadillos and many patients with leprosy in the southern United States are infected with the same strain of M. leprae. Armadillos are a large natural reservoir for M. leprae, and leprosy may be a zoonosis in the region. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Transmission of leprosy in Qiubei County, Yunnan, China: Insights from an 8-year molecular epidemiology investigation

Leprosy continues to be endemic in parts of China. To track the occurrence of leprosy and determine at risk communities, molecular strain typing based on variable number of tandem repeats (VNTRs) was applied in Qiubei County, Wenshan Prefecture, Yunnan Province of the People's Republic of China, a multiethnic region that is home to four predominant ethnic minorities. A previous study, conducted between 2002 and 2005, provided the first descriptions of Mycobacterium leprae strains in the region. M. leprae strains in Qiubei are highly conserved, so only sufficiently polymorphic loci can distinguish strains. A balance between mutation rate and loci stability is needed, so that secondary transmissions can be identified as genotypic matches. The long incubation period of leprosy necessitated an extension of the study to assess the validity of VNTR typing and observe allelic shifts in the same multiethnic population. From 2006 to early 2010 the extension was performed to yield a cumulative total of 164 enrolled patients and 130 skin samples suitable for VNTR typing. Patient demographic information revealed that the case detection rate among certain minority populations in the county is considerably higher than the national rate. Cluster analysis of allele frequencies showed similar strain types within family groups and neighboring townships. Allele frequencies were not found to significantly differ between genders or clinical presentations. The percentage of cases showing near-matching genotypes varied with geography; showing a considerably higher rate in the northern townships. The northern townships continue to show strain types falling into the groups previously defined. Southern genotypes were distinct from those in the north, but clonal genetic relationships were indiscernible in the south. Social interactions and the physical, residential and occupational environments may be more conducive to transmission of community strains in the north.

Journeys of a Pathogen

Microbiology Leprosy is caused by Mycobacterium leprae , which infects the neurons and mucosa of humans. Strikingly, its genome has decayed extensively, which probably accounts for its slow rate of growth; global variation between strains is limited to four SNP clusters, each having a distinctive geographical prevalence that reflects past human migrations. Monot et al. have sequenced M. leprae strains from Brazil, Thailand, and the United States, and made comparisons with archaeological samples. Genotyping the pathogen can be used to pinpoint a person's origins quite precisely. For example, samples from an Egyptian burial revealed that the corpse had been infected with a European strain of leprosy and was more probably a Roman legionary than an African. Their updated map of leprosy strains supports the out-of-Africa theory for modern humans and indicates that leprosy crossed to Asia twice: once via a southern route into India and through Indonesia to the Philippines, and also via a northern route along the Silk Road in countercurrent fashion to the Black Death. Subsequently, leprosy arrived in South America via European immigrants rather than by pre-Columbian colonization via the Bering Strait. Nat. Genet. 41 , 10.1038/ng.477 (2009).

Single Nucleotide Polymorphism Analysis of European Archaeological M. leprae DNA

BackgroundLeprosy was common in Europe eight to twelve centuries ago but molecular confirmation of this has been lacking. We have extracted M. leprae ancient DNA (aDNA) from medieval bones and single nucleotide polymorphism (SNP) typed the DNA, this provides insight into the pattern of leprosy transmission in Europe and may assist in the understanding of M. leprae evolution.Methods and FindingsSkeletons have been exhumed from 3 European countries (the United Kingdom, Denmark and Croatia) and are dated around the medieval period (476 to 1350 A.D.). we tested for the presence of 3 previously identified single nucleotide polymorphisms (SNPs) in 10 aDNA extractions. M. leprae aDNA was extracted from 6 of the 10 bone samples. SNP analysis of these 6 extractions were compared to previously analysed European SNP data using the same PCR assays and were found to be the same. Testing for the presence of SNPs in M. leprae DNA extracted from ancient bone samples is a novel approach to analysing European M. leprae DNA and the findings concur with the previously published data that European M. leprae strains fall in to one group (SNP group 3).ConclusionsThese findings support the suggestion that the M. leprae genome is extremely stable and show that archaeological M. leprae DNA can be analysed to gain detailed information about the genotypic make-up of European leprosy, which may assist in the understanding of leprosy transmission worldwide.

Molecular epidemiology of leprosy based on VNTR typing in Thailand

Recently about 500 new cases of leprosy have been reported each year in Thailand. In addition to a steady rate of new case detection, Thailand is in Southeast Asia where leprosy is endemic in neighbouring countries; therefore, strain differentiation could be useful in tracing origins and routes of infection, and general leprosy surveillance. To identify suitable markers for differentiation of M. leprae strains in different global geographic regions and to determine the applicability of a systematic genotyping method for tracing leprosy transmission, variable nucleotide tandem repeats (VNTRs) of 14 loci were evaluated using DNA extracts from a total of 97 skin biopsies and slit skin smear samples. The alleles per locus ranged from 2-26 providing adequate strain differentiation. Microsatellite loci (GAA)21, (AT)17 are highly polymorphic followed by (GTA)9, (AC)8a, (AC)8b, and (AC)9. The minisatellites 6-7, 21-3 and 27-5 exhibited a limited number of alleles. The repeat of 23-3 showed no polymorphism. Overall, the strain types can be divided into two distinct Thai groups, according to the alleles at the (GGT)5 and 21-3 loci. However, there are no obvious geographical patterns of distribution of VNTR strain types. Closely matched VNTR profiles found in household members of two multi-case families suggested infection through a common source.

A NewMycobacteriumSpecies Causing Diffuse Lepromatous Leprosy

Mycobacterium leprae causes leprosy. M leprae strains collected worldwide have been genetically clonal, which poorly explains the varying severity and clinical features of the disease. We discovered a new Mycobacterium species from 2 patients who died of diffuse lepromatous leprosy (DLL). The Mycobacterium was purified from heavily infected, freshly frozen autopsy liver tissue followed by DNA extraction in 1 case. Paraffin-embedded skin tissue was used for DNA extraction in another case. Six genes of the organism were amplified by polymerase chain reaction, sequenced on cloning or from amplicons, and analyzed. Significant genetic differences with M leprae were found, including a 2.1% divergence of the 16S ribosomal RNA (rRNA) gene, a highly conserved marker of bacterial evolution, and 6% to 14% mismatches among 5 less conserved genes. Phylogenetic analyses of the genes of 16S rRNA, rpoB, and hsp65 indicated that the 2 most related organisms evolved from a common ancestor that had branched from other mycobacteria. These results and the unique clinicopathologic features of DLL led us to propose Mycobacterium lepromatosis sp nov. This species may account for some of the clinical and geographic variability of leprosy. This finding may have implications for the research and diagnosis of leprosy.

Are all the DNA gyrase mutations found in Mycobacterium leprae clinical strains involved in resistance to fluoroquinolones?

Mycobacterium leprae DNA gyrases carrying various mutations, previously described in clinical strains, were investigated for quinolone susceptibility by inhibition of supercoiling and DNA cleavage promotion. We demonstrated that the gyrA mutations leading to G89C or A91V confer fluoroquinolone resistance whereas the gyrB mutation leading to D205N does not.

Identification and distribution of Mycobacterium leprae genotypes in a region of high leprosy prevalence in China: a 3-year molecular epidemiological study.

Multiple-locus variable-number tandem-repeat (VNTR) analysis (MLVA) has been proposed as a means of strain typing for tracking the transmission of leprosy. However, empirical data for a defined population are lacking. To this end, a study was initiated to assess the diversity and distribution of prevalent Mycobacterium leprae strains in Qiubei County, Yunnan Province, People's Republic of China, where the annual detection rate of leprosy is 10-fold higher than the national average rate. Sixty-eight newly diagnosed leprosy patients were included in the study. MLVA at eight M. leprae loci was applied using DNA extracts from skin biopsies. The number of alleles per locus ranged from 4 to 24, providing adequate strain discrimination. MLVA strain typing identified several clusters of patients whose M. leprae specimens shared similar VNTR profiles. Two of these clusters were comprised of patients who resided predominantly in the north and northwest parts of Qiubei County. Furthermore, it was found that multicase families are common in this county: 23 of the 68 patients were from 11 families. Intrafamilial VNTR profiles closely matched within six families, although they were different between the families. Moreover, VNTR patterns related to those found in some multicase families were also detected in patients in the same or adjacent townships, indicating the utility of VNTR strain typing to identify and detect short-range transmission events. Social contact through village markets is proposed as a means of transmission.

Variable numbers of TTC repeats in Mycobacterium leprae DNA from leprosy patients and use in strain differentiation.

Strain differentiation of Mycobacterium leprae would be of great value for epidemiological investigation to identify the infectious sources of leprosy, to understand transmission patterns, and to distinguish between relapse and reinfection. From the M. leprae genome sequence database, TTC DNA repeats were identified. Primer sets designed to amplify the region flanking TTC repeats revealed PCR products of different sizes, indicating that the number of repeats at each locus may be variable among M. leprae strains. The TTC repeats were not found in Mycobacterium tuberculosis, Mycobacterium avium, Mycobacterium marinum, or human tissues, which indicated their specificity to M. leprae. Sequence analysis of the TTC repeat region in each of the M. leprae strains showed a variation of 10 to 37 repeats. In the M. leprae strains of 34 multibacillary patients at Cebu, Philippines, M. leprae with 24 and 25 TTC repeats was most common, and this was followed by strains with 14, 15, 20, 21, and 28 repeats. This study thus indicates that there are variable numbers of TTC repeats in a noncoding region of M. leprae strains and that the TTC region may be useful for strain differentiation for epidemiological investigations of leprosy.

Diaminodiphenylsulfone resistance of Mycobacterium leprae due to mutations in the dihydropteroate synthase gene

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.

Diaminodiphenylsulfone resistance ofMycobacterium lepraedue to mutations in the dihydropteroate synthase gene

The nucleotide sequence analysis of the dihydropteroate synthase (DHPS) gene of six diaminodiphenylsulfone-resistant Mycobacterium leprae strains revealed that the mutation was limited at highly conserved amino acid residues 53 or 55. Though the mutation at amino acid residue 55 or its homologous site has been reported in other bacteria, the mutation at residue 53 is the first case in bacteria. This is the first paper which links the mutations in DHPS and sulfonamide resistance in M. leprae. This finding is medically and socially relevant, since leprosy is still a big problem in certain regions.

Molecular basis of rifampin resistance in Mycobacterium leprae

Rifampin is currently the most potent drug used in leprosy control programs. We show that the rifampin resistance which emerged in nine patients with lepromatous leprosy, who had received rifampin monotherapy, stemmed from mutations in the rpoB gene, which encodes the beta subunit of RNA polymerase of Mycobacterium leprae. In eight cases missense mutations were found to affect a serine residue, Ser-425, while in the remaining mutant a small insertion was found close to this site. These findings will be of use for the development of a rapid screening procedure, involving the polymerase chain reaction, for monitoring the emergence of rifampin-resistant M. leprae strains.

Genetic relationships among Mycobacterium leprae, Mycobacterium tuberculosis, and candidate leprosy vaccine strains determined by DNA hybridization: identification of an M. leprae-specific repetitive sequence

Comparative DNA hybridization studies of genomic DNA indicated that, while different isolates of armadillo-derived Mycobacterium leprae have a high degree of homology, binding of M. leprae genomic DNA to DNA of other species of mycobacteria or to corynebacteria was low, establishing that M. leprae is only remotely genetically related to any of the species examined. Several candidate leprosy vaccine mycobacterial strains were similarly found to have little genetic similarity to M. leprae. In contrast, the DNAs of the slow-growing mycobacteria M. tuberculosis, M. africanum, M. bovis, and M. microti were found to be very closely related. In the course of these studies, an M. leprae-specific repetitive sequence, greater than 15-fold per genome equivalent, was identified that might be useful for diagnostic and epidemiological studies.

Antileprotic action of macromolecular immunomodifiers in the mouse foot-pad model

Infectious drug resistance denotes the transmission of drug resistance from one bacterium (so far only Gram-negatives) to another of the same or different strain, species, or genus. This is accomplished by conjugation that leads directly to a new recipient strain with a comparable level of resistance. The surprising facet of this phenomenon is the simultaneous transfer of resistance to an entire series of chemically and biochemically unrelated antimicrobials; most commonly in sulfanilamides, tetracyclines, streptomycin, and chloramphenicol, but also more recently penicillins, kanamycin, neomycin, nalidixic acid, and nitrofurans. Resistant bacteria can be stripped of infectious drug resistance with various acridines in vitro, albeit with low frequency; usually the method applies to all the resistance factors, but some segregation has been achieved. Several sulfanilamides and other synthetic antibacterials as well as the antibiotics are involved with infectious drug resistance. No new antibacterial screening methods are reported. Recent success in experimental animals in vitro with growing the leprosy bacillus has led to significant and promising additions in this field. Over 100 Myco leprae strains from all over the world and from the several clinical types of leprosy have now produced mouse foot-pad infections. The relationship of bacterial morphology, administration of antileprotic agents, BCG vaccination, and immune-response suppression to production of increased infectivity in mice has been studied.