Hanseniaspora menglaensis f.a., sp. nov., a novel apiculate yeast species isolated from rotting wood.

Two apiculate strains (NYNU 181072 and NYNU 181083) of a bipolar budding yeast species were isolated from rotting wood samples collected in Xishuangbanna Tropical Rainforest in Yunnan Province, southwest PR China. On the basis of phenotypic characteristics and the results of phylogenetic analysis of the D1/D2 domain of the large subunit (LSU) rRNA, internal transcribed spacer (ITS) region and the actin (ACT1) gene, the two strains were found to represent a single novel species of the genus Hanseniaspora, for which the name Hanseniaspora menglaensis f.a., sp. nov. (holotype CICC 33364T; MycoBank MB 847437) is proposed. In the phylogenetic tree, H. menglaensis sp. nov. showed a close relationship with Hanseniaspora lindneri, Hanseniaspora mollemarum, Hanseniaspora smithiae and Hanseniaspora valbyensis. H. menglaensis sp. nov. differed from H. lindneri, the most closely related known species, by 1.2 % substitutions in the D1/D2 domain, 2.5 % substitutions in the ITS region and 5.4 % substitutions in the ACT1 gene, respectively. Physiologically, H. menglaensis sp. nov. can also be distinguished from H. lindneri by its ability to assimilate d-gluconate.

Phylogenomic and comparative analyses support the reclassification of several Komagataeibacter species as novel members of the Novacetimonas gen. nov. and bring new insights into the evolution of cellulose synthase genes.

The genus Komagataeibacter harbours bacteria presenting the ability to produce increased levels of crystalline nanocellulose, as well as strains used in the industrial production of fermented products and beverages. Still, most of the studies of this biotechnologically relevant genus were conducted based on limited phenotypic methodologies and taxonomical classifications. In this work, a detailed analysis of the currently described genus Komagataeibacter was conducted based on phylogenomic analysis, unveiling the phylogenomic relationships within the genus and allowing a detailed phylogenetic analysis of biotechnologically important genes such as those involved in cellulose biosynthesis (bcs genes). Phylogenomic and comparative genomic analysis revealed that several type strains formed an independent genomic group from those of other Komagataeibacter, prompting their reclassification as members of a novel genus, hereby termed Novacetimonas gen. nov. The results support the reclassification of Komagataeibacter hansenii, Komagataeibacter cocois, Komagataeibacter maltaceti and Komagataeibacter pomaceti as novel members of the genus Novacetimonas. The Novacetimonas hansenii species is the proposed representative of the novel genus. Importantly, phylogenetic analysis based on cellulose biosynthesis genes (bcsABCD, bcsAB2XYC2, bcsAB3C3, bcsAB4), showed that the evolutionary history of these genes is closely related to the strain's phylogenomic/taxonomic classification. Hence, the robust taxonomic classification of these bacteria will allow the better characterization and selection of strains for biotechnological applications.

Veronia nyctiphanis gen. nov., sp. nov., Isolated from the Stomach of the Euphausiid Nyctiphanes simplex (Hansen, 1911) in the Gulf of California, and Reclassification of Enterovibrio pacificus as Veronia pacifica comb. nov.

The bacterial strain 42Xb2 T was isolated from a female adult krill Nyctiphanes simplex infected with the apostome parasitoid ciliate Pseudocollinia brintoni in January 2007 in the Gulf of California. The strain has the morphological, phenotypic, and molecular characteristics of the bacteria of the family Vibrionaceae. The 16S rRNA gene sequence has a similarity of 97.7% with Enterovibrio pacificus SW014 T and 96.1% similarity with Enterovibrio norvegicus LMG 19839 T. A phylogenomic and a multilocus sequence analyses placed this strain close to the genera Enterovibrio, Grimontia, and Salinivibrio, but clearly forming a separate branch from these bacterial genera. Genomic analyses presented further support this result. A novel genus Veronia gen. nov. and a species Veronia nyctiphanis sp. nov. is here described with CAIM 600 T (= DSM 24592 T = CECT 7578 T) as the type strain. Morphological, physiological, and genetic evidence presented here support the unification of Enterovibrio pacificus and Veronia nyctiphanis in the new genus Veronia. Enterovibrio pacificus is reclassified as Veronia pacifica. V. pacifica is assigned as the type species of the new genus Veronia.Genome Sequencing Data The GenBank/EMBL/DDBJ accession numbers for the genome sequence of Veronia nyctiphanis CAIM 600 T is PEIB01 and of Enterovibrio pacificus CAIM 1920 T is LYBM01. The 16S rRNA gene sequence of V. nyctiphanis CAIM 600 T is JX129353.

Blautia argi sp. nov., a new anaerobic bacterium isolated from dog faeces.

Two isolates of a Gram-positive, non-motile, coccoid or oval-shaped anaerobic bacterium, designated strains N6H1-15T and YH1_16, were isolated from faecal samples obtained from a mature dog. Analysis of 16S rRNA gene sequences indicated that the isolates belonged to the Blautia coccoidesrRNA gene group (cluster XIVa) and were closely related to Blautia hansenii KCTC 5951T, Blautia stercoris KCTC 5981T, Blautia producta producta KCTC 3695T and B. coccoides DSM 15327T, with 96.7, 94.4, 94.2 and 93.9 % sequence similarity, respectively. The two isolates contained m-diaminopimelic acid within their peptidoglycans. The major polar lipids were diphosphatidylglycerol and phosphatidylglycerol, and the major fatty acids were C16 : 0 (18.5 %), C16 : 0 (18.0 %) and C18 : 1cis 9 (16.2 %). The predominant metabolic end products of glucose fermentation were acetic and lactic acids, and the G+C content was 44.2 mol%. Thus, the polyphasic data suggest that the two new isolates represent a new species, proposed as Blautia argi sp. nov. The type strain is N6H1-15T (=KCTC 15426=JCM 31394).

Genome sequences and description of novel exopolysaccharides producing species Komagataeibacter pomaceti sp. nov. and reclassification of Komagataeibacter kombuchae (Dutta and Gachhui 2007) Yamada et al., 2013 as a later heterotypic synonym of Komagataeibacter hansenii (Gosselé et al. 1983) Yamada et al., 2013.

Strains T5K1 and AV446 isolated from apple cider vinegars during a submerged vinegar production in two separate vinegar facilities showed 94% 16S rRNA gene similarity to its closest neighbors Komagataeibacter maltaceti LMG 1529T and Gluconacetobacter entanii LTH 4560T. Further phylogenetic and phenotypic characterizations indicated that the isolates belonged to a novel species of the Komagataeibacter genus. Comparison based on 16S-23S rRNA gene ITS sequences and concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, grouped both strains to a single phylogenetic cluster well separated from the other species of the Komagataeibacter genus. Average nucleotide identity of T5K1 and AV446 draft genome sequences compared to other Komagataeibacter type strains was below 94% and at the same time, in-silico DNA-DNA hybridization was below 70%. Both strains on the other hand showed approximately 98% (average nucleotide identity) and 87% (in silico DNA-DNA hybridization) similarity to each other. Strains T5K1 and AV446 can be differentiated from other Komagataeibacter type strains based on their ability to produce 2-keto-d-gluconic acid and at the same time inability to produce 5-keto-d-gluconic acid. Furthermore, strains of the new species do not grow on Asai medium supplemented with d-glucose or d-mannitol. The growth is also absent (T5K1) or weak (AV446) on Hoyer-Frateur medium supplemented with afore mentioned sugars. Both strains produce cellulose. In addition, draft genome analysis revealed that strains T5K1 and AV446 possess genes involved in the synthesis of acetan-like extracellular heteropolysaccharide. We propose the name Komagataeibacter pomaceti sp. nov. for the new species with LMG 30150T [=CCM 8723T=ZIM B1029T] as the type strain. Data collected in this study and in a previous study also revealed that Komagataeibacter kombuchae is a later heterotypic synonym of Komagataeibacter hansenii.

Unraveling microbial ecology of industrial-scale Kombucha fermentations by metabarcoding and culture-based methods.

Kombucha, historically an Asian tea-based fermented drink, has recently become trendy in Western countries. Producers claim it bears health-enhancing properties that may come from the tea or metabolites produced by its microbiome. Despite its long history of production, microbial richness and dynamics have not been fully unraveled, especially at an industrial scale. Moreover, the impact of tea type (green or black) on microbial ecology was not studied. Here, we compared microbial communities from industrial-scale black and green tea fermentations, still traditionally carried out by a microbial biofilm, using culture-dependent and metabarcoding approaches. Dominant bacterial species belonged to Acetobacteraceae and to a lesser extent Lactobacteriaceae, while the main identified yeasts corresponded to Dekkera, Hanseniaspora and Zygosaccharomyces during all fermentations. Species richness decreased over the 8-day fermentation. Among acetic acid bacteria, Gluconacetobacter europaeus, Gluconobacter oxydans, G. saccharivorans and Acetobacter peroxydans emerged as dominant species. The main lactic acid bacteria, Oenococcus oeni, was strongly associated with green tea fermentations. Tea type did not influence yeast community, with Dekkera bruxellensis, D. anomala, Zygosaccharomyces bailii and Hanseniaspora valbyensis as most dominant. This study unraveled a distinctive core microbial community which is essential for fermentation control and could lead to Kombucha quality standardization.

High-throughput identification of the microbial biodiversity of cocoa bean fermentation by MALDI-TOF MS.

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a powerful biotyping tool increasingly used for high-throughput identification of clinical microbial isolates, however, in food fermentation research this approach is still not well established. This study examines the microbial biodiversity of cocoa bean fermentation based on the isolation of micro-organisms in cocoa-producing regions, followed by MALDI-TOF MS in Switzerland. A preceding 6-week storage test to mimic lengthy transport of microbial samples from cocoa-producing regions to Switzerland was performed with strains of Lactobacillus plantarum, Acetobacter pasteurianus and Saccharomyces cerevisiae. Weekly MALDI-TOF MS analysis was able to successfully identify microbiota to the species level after storing live cultures on slant agar at mild temperatures (7°C) and/or in 75% aqueous ethanol at differing temperatures (-20, 7 and 30°C). The efficacy of this method was confirmed by on-site recording of the microbial biodiversity in cocoa bean fermentation in Bolivia and Brazil, with a total of 1126 randomly selected isolates. MALDI-TOF MS analyses revealed known dominant cocoa bean fermentation species with Lact. plantarum and Lactobacillus fermentum in the lactic acid bacteria taxon, Hanseniaspora opuntiae and S. cerevisiae in the yeast taxon, and Acet. pasteurianus, Acetobacter fabarum, Acetobacter ghanensis and Acetobacter senegalensis in the acetic acid bacteria taxon. SIGNIFICANCE AND IMPACT OF THE STUDY: Microbial identification with MALDI-TOF MS has increased the number of samples that can be analysed in a given time, a prerequisite for high-throughput methods. This method is already widely used for the identification of clinical microbial isolates, whereas in food fermentation research, including cocoa bean fermentation, microbiota is mostly identified by time-consuming, biochemical-based phenotyping and molecular approaches. This study presents the use of MALDI-TOF MS for characterizing the microbial biodiversity of cocoa bean fermentation. The feasibility of MALDI-TOF MS identification of cocoa-specific microbiota has been shown with samples collected during on-site studies in two countries of origin, Bolivia and Brazil.

Rhodovulum algae sp. nov., isolated from an algal mat.

A reddish-brown-pigmented, phototrophic bacterium, designated strain JA877T, was isolated from a brown algae mat sample collected from Jalandhar beach, Gujarat, India. On the basis of the 16S rRNA gene sequence, strain JA877T belongs to the class Alphaproteobacteria and is closely related to the type strains Rhodovulum viride JA756T (99.0 %), Rhodovulum sulfidophilum Hansen W4T (98.9 %), Rhodovulumvisakhapatnamense JA181T (98.8 %),Rhodovulum kholense JA297T (97.5 %) and Rhodovulum salis JA746T (97.0). However, strain JA877T showed only 20-45 % relatedness with its phylogenetic neighbours and had a ∆Tm between 5.8 and 7.0 °C. The major respiratory quinone was ubiquinone-10 (Q10), and the polar lipid profile was composed of the major components phosphatidylglycerol, phosphatidylethanolamine, an unidentified phospholipid, two unidentified sulfolipids and five unidentified lipids. The major fatty acids were C18 : 1ω5c, C18 : 1ω7c/C18 : 1ω6c, C16 : 0 and C18 : 0. The DNA G+C content was 64.5 mol%. On the basis of 16S rRNA gene sequence analysis, physiological data, and chemotaxonomic and molecular differences, strain JA877T is significantly different from other species of the genus Rhodovulum and represents a novel species, for which the name Rhodovulum algae sp. nov. is proposed. The type strain is JA877T (=LMG 29228T= KCTC 42963T).

The discovery, function and development of the variable number tandem repeats in different Mycobacterium species.

The method of genotyping by variable number tandem repeats (VNTRs) facilitates the epidemiological studies of different Mycobacterium species worldwide. Until now, the VNTR method is not fully understood, for example, its discovery, function and classification. The inconsistent nomenclature and terminology of VNTR is especially confusing. In this review, we first describe in detail the VNTRs in Mycobacterium tuberculosis (M. tuberculosis), as this pathogen resulted in more deaths than any other microbial pathogen as well as for which extensive studies of VNTRs were carried out, and then we outline the recent progress of the VNTR-related epidemiological research in several other Mycobacterium species, such as M. abscessus, M. africanum, M. avium, M. bovis, M. canettii, M. caprae, M. intracellulare, M. leprae, M. marinum, M. microti, M. pinnipedii and M. ulcerans from different countries and regions. This article is aimed mainly at the practical notes of VNTR to help the scientists in better understanding and performing this method.

Ancient DNA analysis - An established technique in charting the evolution of tuberculosis and leprosy.

Many tuberculosis and leprosy infections are latent or paucibacillary, suggesting a long time-scale for host and pathogen co-existence. Palaeopathology enables recognition of archaeological cases and PCR detects pathogen ancient DNA (aDNA). Mycobacterium tuberculosis and Mycobacterium leprae cell wall lipids are more stable than aDNA and restrict permeability, thereby possibly aiding long-term persistence of pathogen aDNA. Amplification of aDNA, using specific PCR primers designed for short fragments and linked to fluorescent probes, gives good results, especially when designed to target multi-copy loci. Such studies have confirmed tuberculosis and leprosy, including co-infections. Many tuberculosis cases have non-specific or no visible skeletal pathology, consistent with the natural history of this disease. M. tuberculosis and M. leprae are obligate parasites, closely associated with their human host following recent clonal distribution. Therefore genotyping based on single nucleotide polymorphisms (SNPs) can indicate their origins, spread and phylogeny. Knowledge of extant genetic lineages at particular times in past human populations can be obtained from well-preserved specimens where molecular typing is possible, using deletion analysis, microsatellite analysis and whole genome sequencing. Such studies have identified non-bovine tuberculosis from a Pleistocene bison from 17,500 years BP, human tuberculosis from 9000 years ago and leprosy from over 2000 years ago.

Phenotypic, molecular and antimicrobial susceptibility assessment in isolates from chronic ulcers of cured leprosy patients: a case study in Southern Brazil.

BACKGROUND: One of the most stigmatizing physical sequelae of leprosy in cured patients is the development of chronic lower extremity ulcers. The bacterial diversity present in ulcers is considered one of the factors that can delay the healing process, as well as serve as a focus for severe secondary infections. OBJECTIVE: To identify the microbiota and antimicrobial resistance profile of bacteria isolated from skin ulcers in patients cured of leprosy. METHODS: After obtaining informed consent, material was collected from ulcers of 16 patients treated at the Outpatient Public Health Dermatology Clinic of Rio Grande do Sul and Hospital Colônia Itapuã. Samples were collected during dressing, and the material sent to the Microbiology Laboratory of the Federal University of Health Sciences of Porto Alegre for microbiological culture. Methicillin-resistant Staphylococcus aureus (MRSA) was characterized by two molecular methods, including detection of the mecA gene by PCR and SCCmecgene typing. RESULTS: Cultures revealed microorganisms in all ulcers: Gram-negative bacilli in 80%, Gram-positive cocci in 63%, and mixed microflora in 36%. Staphylococcus aureus and Pseudomonas aeruginosa were the most prevalent bacteria. Assessment of the antimicrobial resistance profile was notable for the presence of MRSA. Molecular analysis of this isolate revealed presence of the mecA gene contained in a type IV staphylococcal cassette chromosome mec (SCCmec). CONCLUSIONS: In patients with leprosy, laboratory culture of skin ulcers is essential for correct antibiotic selection and to control emerging pathogens, such as MRSA carrying SCCmec type IV.

Phenotypic, molecular and antimicrobial susceptibility assessment in isolates from chronic ulcers of cured leprosy patients: a case study in Southern Brazil

BACKGROUND: One of the most stigmatizing physical sequelaeof leprosy in cured patients is the development of chronic lower extremity ulcers. The bacterial diversity present in ulcers is considered one of the factors that can delay the healing process, as well as serve as a focus for severe secondary infections. OBJECTIVE: To identify the microbiota and antimicrobial resistance profile of bacteria isolated from skin ulcers in patients cured of leprosy. METHODS: After obtaining informed consent, material was collected from ulcers of 16 patients treated at the Outpatient Public Health Dermatology Clinic of Rio Grande do Sul and Hospital Colônia Itapuã. Sampleswere collected during dressing, and the material sent to the Microbiology Laboratory of the Federal University of Health Sciences of Porto Alegre for microbiological culture. Methicillin-resistant Staphylococcus aureus (MRSA) was characterized by two molecular methods, including detection of the mecA gene by PCR and SCCmecgene typing. RESULTS: Cultures revealed microorganisms in all ulcers: Gram-negative bacilli in 80%, Gram-positive cocci in 63%, and mixed microflora in 36%. Staphylococcus aureus and Pseudomonas aeruginosa were the most prevalent bacteria. Assessment of the antimicrobial resistance profile was notable for the presence of MRSA. Molecular analysis of this isolate revealed presence of the mecA gene contained in a type IV staphylococcal cassette chromosome mec (SCCmec). CONCLUSIONS: In patients with leprosy, laboratory culture of skin ulcers is essential for correct antibiotic selection and to control emerging pathogens, such as MRSA carrying SCCmec type IV. .

Isolation and characterization of an efficient bacterial cellulose producer strain in agitated culture: Gluconacetobacter hansenii P2A.

In this study, typical niches of acetic acid bacteria were screened for isolation of cellulose producer strains. Hestrin Schramm broth was used as enrichment and production media. Only nine out of 329 isolates formed thick biofilms on liquid surface and were identified as potential cellulose producers. Physiological and biochemical tests proved that all cellulose producers belonged to Gluconacetobacter genus. Most productive and mutation-resistant strain was subjected to 16S rRNA sequence analysis and identified as Gluconacetobacter hansenii P2A due to 99.8 % sequence similarity. X-ray diffraction analysis proved that the biofilm conformed to Cellulose I crystal structure, rich in Iα mass fraction. Static cultivation of G. hansenii P2A in HS medium resulted with 1.89 ± 0.08 g/l of bacterial cellulose production corresponding to 12.0 ± 0.3 % yield in terms of substrate consumption. Shaking and agitation at 120 rpm aided in enhancement of the amount and yield of produced cellulose. Productivity and yield reached up to 3.25 ± 0.11 g/l and 17.20 ± 0.14 % in agitated culture while a slight decrease from 78.7 % to 77.3 % was observed in the crystallinity index.

Characterization of the MSMEG_2631 gene (mmp) encoding a multidrug and toxic compound extrusion (MATE) family protein in Mycobacterium smegmatis and exploration of its polyspecific nature using biolog phenotype microarray.

In Mycobacterium, multidrug efflux pumps can be associated with intrinsic drug resistance. Comparison of putative mycobacterial transport genes revealed a single annotated open reading frame (ORF) for a multidrug and toxic compound extrusion (MATE) family efflux pump in all sequenced mycobacteria except Mycobacterium leprae. Since MATE efflux pumps function as multidrug efflux pumps by conferring resistance to structurally diverse antibiotics and DNA-damaging chemicals, we studied this gene (MSMEG_2631) in M. smegmatis mc(2)155 and determined that it encodes a MATE efflux system that contributes to intrinsic resistance of Mycobacterium. We propose that the MSMEG_2631 gene be named mmp, for mycobacterial MATE protein. Biolog Phenotype MicroArray data indicated that mmp deletion increased susceptibility for phleomycin, bleomycin, capreomycin, amikacin, kanamycin, cetylpyridinium chloride, and several sulfa drugs. MSMEG_2619 (efpA) and MSMEG_3563 mask the effect of mmp deletion due to overlapping efflux capabilities. We present evidence that mmp is a part of an MSMEG_2626-2628-2629-2630-2631 operon regulated by a strong constitutive promoter, initiated from a single transcription start site. All together, our results show that M. smegmatis constitutively encodes an Na(+)-dependent MATE multidrug efflux pump from mmp in an operon with putative genes encoding proteins for apparently unrelated functions.

Gluconacetobacter maltaceti sp. nov., a novel vinegar producing acetic acid bacterium.

Comparison of HaeIII- and HpaII-restriction profiles of PCR-amplified 16S-23S rDNA ITS regions of Gluconacetobacter sp. LMG 1529(T) and SKU 1109 with restriction profiles of reference strains of acetic acid bacteria described by Trcek and Teuber [34] revealed the same but unique restriction profiles for LMG 1529(T) and SKU 1109. Further analyses of nearly complete 16S rRNA gene sequences, nearly complete 16S-23S rDNA ITS sequences, as well as concatenated partial sequences of the housekeeping genes dnaK, groEL and rpoB, allocated both strains to a single phylogenetic cluster well separated from the other species of the genus Gluconacetobacter. DNA-DNA hybridizations confirmed their novel species identity by 73% DNA-DNA relatedness between both strains, and values below the species level (<70%) between SKU 1109 and the type strains of the closest phylogenetic neighbors. The classification of strains LMG 1529(T) and SKU 1109 into a single novel species was confirmed also by AFLP and (GTG)(5)-PCR DNA fingerprinting data, as well as by phenotypic data. Strains LMG 1529(T) and SKU 1109 can be differentiated from their closely related Gluconacetobacter species, Gluconacetobacter entanii and Gluconacetobacter hansenii, by their ability to form 2-keto-d-gluconic acid from d-glucose, their ability to use d-mannitol, d-gluconate and glycerol as carbon source and form acid from d-fructose, and their ability to grow without acetic acid. The major fatty acid of LMG 1529(T) and SKU 1109 is C(18:1ω7c) (60.2-64.8%). The DNA G+C content of LMG 1529(T) and SKU 1109 is 62.5 and 63.3mol% respectively. The name Gluconacetobacter maltaceti sp. nov. is proposed. The type strain is LMG 1529(T) (=NBRC 14815(T)=NCIMB 8752(T)).

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.

SNP genotypes of Mycobacterium leprae isolates in Thailand and their combination with rpoT and TTC genotyping for analysis of leprosy distribution and transmission.

Based on the discovery of three single nucleotide polymorphisms (SNPs) in Mycobacterium leprae, it has been previously reported that there are four major SNP types associated with different geographic regions around the world. Another typing system for global differentiation of M. leprae is the analysis of the variable number of short tandem repeats within the rpoT gene. To expand the analysis of geographic distribution of M. leprae, classified by SNP and rpoT gene polymorphisms, we studied 85 clinical isolates from Thai patients and compared the findings with those reported from Asian isolates. SNP genotyping by PCR amplification and sequencing revealed that all strains like those in Myanmar were SNP type 1 and 3, with the former being predominant, while in Japan, Korea, and Indonesia, the SNP type 3 was found to be more frequent. The pattern of M. leprae distribution in Thailand and Myanmar is quite similar, except that SNP type 2 was not found in Thailand. In addition, the 3-copy hexamer genotype in the rpoT gene is shared among the isolates from these two neighboring countries. On the basis of these two markers, we postulate that M. leprae in leprosy patients from Myanmar and Thailand has a common historical origin. Further differentiation among Thai isolates was possible by assessing copy numbers of the TTC sequence, a more polymorphic microsatellite locus.

Dynamics of Mycobacterium leprae transmission in environmental context: deciphering the role of environment as a potential reservoir.

Leprosy is a disease caused by Mycobacterium leprae. Various modes of transmission have been suggested for this disease. Transmission and risk of the infection is perhaps related to presence of the infectious cases and is controlled by environmental factors. Evidence suggests that humidity may favor survival of M. leprae in the environment. Several reports show that non-human sources like 'naturally' infected armadillos or monkeys could act as reservoir for M. leprae. Inanimate objects or fomites like articles used by infectious patients may theoretically spread infection. However, it is only through detailed knowledge of the biodiversity and ecology that the importance of this mode of transmission can be fully assessed. Our study focuses here to decipher the role of environment in the transmission of the disease. Two hundred and seven soil samples were collected from a village in endemic area where active cases also resided at the time of sample collection. Slit skin smears were collected from 13 multibacillary (MB) leprosy patients and 12 household contacts of the patients suspected to be hidden cases. DNA and RNA of M. leprae were extracted and amplified using M. leprae specific primers. Seventy-one soil samples showed presence of M. leprae DNA whereas 16S rRNA could be detected in twenty-eight of these samples. Samples, both from the environment and the patients, exhibited the same genotype when tested by single nucleotide polymorphism (SNP) typing. Genotype of M. leprae found in the soil and the patients residing in the same area could help in understanding the transmission link in leprosy.

Genotyping of Mycobacterium leprae present on Ziehl-Neelsen-stained microscopic slides and in skin biopsy samples from leprosy patients in different geographic regions of Brazil.

We analysed 16 variable number tandem repeats (VNTR) and three single-nucleotide polymorphisms (SNP) in Mycobacterium leprae present on 115 Ziehl-Neelsen (Z-N)-stained slides and in 51 skin biopsy samples derived from leprosy patients from Ceará (n = 23), Pernambuco (n = 41), Rio de Janeiro (n = 22) and Rondônia (RO) (n = 78). All skin biopsies yielded SNP-based genotypes, while 48 of the samples (94.1%) yielded complete VNTR genotypes. We evaluated two procedures for extracting M. leprae DNA from Z-N-stained slides: the first including Chelex and the other combining proteinase and sodium dodecyl sulfate. Of the 76 samples processed using the first procedure, 30.2% were positive for 16 or 15 VNTRs, whereas of the 39 samples processed using the second procedure, 28.2% yielded genotypes defined by at least 10 VNTRs. Combined VNTR and SNP analysis revealed large variability in genotypes, but a high prevalence of SNP genotype 4 in the Northeast Region of Brazil. Our observation of two samples from RO with an identical genotype and seven groups with similar genotypes, including four derived from residents of the same state or region, suggest a tendency to form groups according to the origin of the isolates. This study demonstrates the existence of geographically related M. leprae genotypes and that Z-N-stained slides are an alternative source for M. leprae genotyping.