Differentiation of the Mycobacterium tuberculosis W-Beijing family strains from the Russian Federation by the VNTR-typing

Recent phylogenetic studies allowed the Mycobacterium tuberculosis complex to be divided into a number of the strain families. The W-Beijing family is one of most widespread M. tuberculosis variants frequently causing epidemic outbreaks. This family is genetically homogenous and conserved so that ETR A, B, C, D, E - typing is insufficient for the W-Beijing differentiation. All W-Beijing isolates have common profile (42435). This led to the false clustering in the molecular epidemiology study, especially in the region of predominance of the W-Beijing family. In this investigation we searched for the VNTR loci with high evolution rate, which were polymorphic in the W-Beijing genome. Eleven VNTR-loci were assayed in the DNA panel of 99 M. tuberculosis isolates from the tuberculosis patients in North-West and West-Siberian regions of Russia during the period from 2000 to 2001. Ninety nine strains of M. tuberculosis were divided into 74 VNTR-types, 51 isolates of the W-Beijing family were subdivided into 30 VNTR-types. The Hunter-Gudson index (HGDI) for all studied loci (ETR-A, ETR-C, ETR-E, V, V2, V3, V4, V5, V6, V10, V11) was close to one of the IS6110 RFLP indices being "the gold standard" of the M. tuberculosis complex genotyping. The V2, V3 loci located in the sequences of the PPE gene family, were highly polymorphic and more discriminative then others (HGDI is about 0.8). The congruence between the IS6110 RFLP-typing and 11 loci VNTR-typing was measured during genotyping for 23 isolates of the W-Beijing family. The isolates were divided into 9 genotypes by the IS6110 RFLP and into 13 variants by the VNTR-typing. The profiles correlation coefficient was 0.767689 that reflected the differences in the rate and type of the given genome target evolution.     

Identification of unique genetic markers in Rv0927c among Mycobacterium tuberculosis W-Beijing strains

Fifty-six clinical isolates of Mycobacterium tuberculosis were analyzed by spoligotyping to determine the prevalence of W-Beijing strains. Forty-nine of the 56 isolates belonged to W-Beijing strains and 7 isolates were non-Beijing strains. Comparative two-dimensional gel electrophoresis analysis of protein patterns between the W-Beijing and non-Beijing strains identified a unique protein Rv0927c that is absent in the former but present in the latter and the reference strain M. tuberculosis H37Rv. Compared with 7 non-Beijing clinical isolates and H37Rv, all 49 W-Beijing strains had two characteristic mutations, a deletion of AGC at nucleotide position 421 of Rv0927c gene encoding a putative short dehydrogenase/reductase, causing deletion of serine codon at amino acid position 141 and a -127 G-->A mutation in Rv0927c-pstS3 intergenic region, resulting in failure to express Rv0927c. Western blot analysis indicated that polyclonal antibody raised against H37Rv Rv0927c overexpressed in Escherichia coli reacted with non-Beijing strains and H37Rv but not W-Beijing strains. Characteristic mutations of Rv0927c that are present in W-Beijing strains can be used as a novel genetic marker for rapid molecular typing of M. tuberculosis W-Beijing strains.     

Fitness of Mycobacterium tuberculosis Strains of the W-Beijing and Non-W-Beijing Genotype

Background

Multidrug resistant tuberculosis (MDR-TB) is a major threat for global tuberculosis control. The W-Beijing Mycobacterium tuberculosis genotype has been associated with drug resistance. Elucidation of the mechanisms underlying this epidemiological finding may have an important role in the control of MDR-TB. The aim of this study was to evaluate the fitness of drug-susceptible and MDR M. tuberculosis strains of the W-Beijing genotype compared with that of Non-W-Beijing strains.

Methodology/Principal Findings

Fitness of M. tuberculosis strains was determined by evaluating the difference in the growth curves obtained in the MGIT960 automated system and assessing the competitive growth capacity between W-Beijing and non-W-Beijing strains. The W-Beijing MDR strains had a significant longer lag phase duration compared to the other strains but did not present a significant fitness cost. When grown in competition they had an advantage only in medium containing 0.1% Tween 80.

Conclusions/Significance

It was not possible to confirm a selective advantage of W-Beijing strains to grow, except for differences in their resistance to Tween 80. Further studies are needed to elucidate the putative advantage of W-Beijing strains compared to other genotypes.     

A rapid and simple method for identifying Mycobacterium tuberculosis W-Beijing strains based on detection of a unique mutation in Rv0927c by PCR-SSCP

Recently we have found that W-Beijing Mycobacterium tuberculosis strains have a unique in-frame trinucleotide (AGC) deletion at position 421 of Rv0927c and a −127G → A mutation in Rv0927c-pstS3 intergenic region. Based on detecting the 421 trinucleotide deletion of these two mutations which can alter the ssDNA conformation more extensively than the other, we developed a PCR-SSCP method for rapid identification of W-Beijing strains among non-Beijing strains. Altogether, 104 clinical isolates were analyzed, including 68 W-Beijing strains and 36 non-Beijing strains. We found that PCR-SSCP successfully differentiated all the W-Beijing strains from the non-Beijing strains. In addition, we unexpectedly discovered that SDS-PAGE protein gels had better resolving power than conventional TBE polyacrylamide gel in detecting the AGC deletion mutation in the SSCP analysis.     

Impact of drug resistance on fitness of Mycobacterium tuberculosis strains of the W-Beijing genotype

Mycobacterium tuberculosis strains of the W-Beijing genotype became a common cause of tuberculosis during the past years and they are often associated with drug resistance. The biological factors facilitating the selection and wide dissemination of these strains are not known. To determine how acquisition of drug resistance affected growth of strains of the W-Beijing genotype, the growth of 55 M. tuberculosis isolates were studied using the BBL MGIT Mycobacteria Growth Indicator Tube and the BACTEC MGIT 960 System. Susceptible strains of non-Beijing genotypes were found to be the most fit strains. Drug-resistant strains of non-Beijing genotypes were more likely to grow slower than susceptible strains (P=0.001). Drug-resistant strains of the W-Beijing genotype had two tendencies of growth: some of them showed reduced growth compared to susceptible strains, while others did not show loss of fitness measured as growth.     

A non-sense mutation in the putative anti-mutator gene <Emphasis Type="Italic">ada/alkA</Emphasis> of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> and <Emphasis Type="Italic">M. bovis</Emphasis> isolates suggests convergent evolution

Background  

Previous studies have suggested that variations in DNA repair genes of W-Beijing strains may have led to transient mutator phenotypes which in turn may have contributed to host adaptation of this strain family. Single nucleotide polymorphism (SNP) in the DNA repair gene mutT1 was identified in MDR-prone strains from the Central African Republic. A Mycobacteriumtuberculosis H37Rv mutant inactivated in two DNA repair genes, namely ada/alkA and ogt, was shown to display a hypermutator phenotype. We then looked for polymorphisms in these genes in Central African Republic strains (CAR).     

Baseline predictors of sputum culture conversion in pulmonary tuberculosis: importance of cavities, smoking, time to detection and W-Beijing genotype

Background

Time to detection (TTD) on automated liquid mycobacterial cultures is an emerging biomarker of tuberculosis outcomes. The M. tuberculosis W-Beijing genotype is spreading globally, indicating a selective advantage. There is a paucity of data on the association between baseline TTD and W-Beijing genotype and tuberculosis outcomes.

Aim

To assess baseline predictors of failure of sputum culture conversion, within the first 2 months of antitubercular therapy, in participants with pulmonary tuberculosis.

Design

Between May 2005 and August 2008 we conducted a prospective cohort study of time to sputum culture conversion in ambulatory participants with first episodes of smear and culture positive pulmonary tuberculosis attending two primary care clinics in Cape Town, South Africa. Rifampicin resistance (diagnosed on phenotypic susceptibility testing) was an exclusion criterion. Sputum was collected weekly for 8 weeks for mycobacterial culture on liquid media (BACTEC MGIT 960). Due to missing data, multiple imputation was performed. Time to sputum culture conversion was analysed using a Cox-proportional hazards model. Bayesian model averaging determined the posterior effect probability for each variable.

Results

113 participants were enrolled (30.1% female, 10.5% HIV-infected, 44.2% W-Beijing genotype, and 89% cavities). On Kaplan Meier analysis 50.4% of participants underwent sputum culture conversion by 8 weeks. The following baseline factors were associated with slower sputum culture conversion: TTD (adjusted hazard ratio (aHR) = 1.11, 95% CI 1.02; 1.2), lung cavities (aHR = 0.13, 95% CI 0.02; 0.95), ever smoking (aHR = 0.32, 95% CI 0.1; 1.02) and the W-Beijing genotype (aHR = 0.51, 95% CI 0.25; 1.07). On Bayesian model averaging, posterior probability effects were strong for TTD, lung cavitation and smoking and moderate for W-Beijing genotype.

Conclusion

We found that baseline TTD, smoking, cavities and W-Beijing genotype were associated with delayed 2 month sputum culture. Larger studies are needed to confirm the relationship between the W-Beijing genotype and sputum culture conversion.     

Unexpected Role for IL-17 in Protective Immunity against Hypervirulent Mycobacterium tuberculosis HN878 Infection

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis (TB), infects one third of the world''s population. Among these infections, clinical isolates belonging to the W-Beijing appear to be emerging, representing about 50% of Mtb isolates in East Asia, and about 13% of all Mtb isolates worldwide. In animal models, infection with W-Beijing strain, Mtb HN878, is considered “hypervirulent” as it results in increased mortality and causes exacerbated immunopathology in infected animals. We had previously shown the Interleukin (IL) -17 pathway is dispensable for primary immunity against infection with the lab adapted Mtb H37Rv strain. However, it is not known whether IL-17 has any role to play in protective immunity against infection with clinical Mtb isolates. We report here that lab adapted Mtb strains, such as H37Rv, or less virulent Mtb clinical isolates, such as Mtb CDC1551, do not require IL-17 for protective immunity against infection while infection with Mtb HN878 requires IL-17 for early protective immunity. Unexpectedly, Mtb HN878 induces robust production of IL-1β through a TLR-2-dependent mechanism, which supports potent IL-17 responses. We also show that the role for IL-17 in mediating protective immunity against Mtb HN878 is through IL-17 Receptor signaling in non-hematopoietic cells, mediating the induction of the chemokine, CXCL-13, which is required for localization of T cells within lung lymphoid follicles. Correct T cell localization within lymphoid follicles in the lung is required for maximal macrophage activation and Mtb control. Since IL-17 has a critical role in vaccine-induced immunity against TB, our results have far reaching implications for the design of vaccines and therapies to prevent and treat emerging Mtb strains. In addition, our data changes the existing paradigm that IL-17 is dispensable for primary immunity against Mtb infection, and instead suggests a differential role for IL-17 in early protective immunity against emerging Mtb strains.     

Genotypes of Helicobacter pylori in Polish population

Here we have studied the genetic diversity of Helicobacter pylori strains recovered from 64 individual patients, 5 family members and 13 unsuccessfully treated patients. The recovered bacteria were finger-printed by the PCR-RFLP and RAPD methods and virulence associated loci (cagPAI, vacA) were PCR studied. Unique differentiation of every independently isolated strain from not-related persons was possible by RAPD technique. In PCR-RFLP technique several profile groups (7 and 15) for particular endonuclease tested were found. Eleven patients carried strains of the same gene profile (PCR-RFLP) and the same overall genotype (RAPD) before and after therapy. In the family studies, essentially the same strain was found in different relatives in three cases, and different strains were found in the other two cases. Island of cagPAI was present in 79% of all strains tested, half and one-fifth of all strains tested presented, s1am2 and s1m1 alleles of vacA gene, respectively. Independently from identity or diversity of pre- and post-treatment strains and strains recovered from the family members we have been observed identical cagPAI/vacA genotypes. These results suggest that H. pylori infections in Poland can be mixed, although just one strain may often predominate, and that inter-family transmission may be significant even in this high risk society. The genetic feature of virulence-associated loci are similar to those seen elsewhere in Europe, although strains that carry the cagPAI and the potentially more toxigenic alleles of the vacA gene are more common. RAPD technique is proven as most differentiating, however PCR-RFLP allows for easy recognition of mixed infection with two or more different strains. Molecular typing study in case of children therapy may allow reduce rate of relapses by reduction of possible transmission from family source.     

Analysis of copy-number variation,insertional polymorphism,and methylation status of the tiniest class I (TRIM) and class II (MITE) transposable element families in various rice strains

Transposable elements (TEs) dominate the genetic capacity of most eukaryotes, especially plants, where they may compose up to 90% of the genome. Many studies, both in plants and animals reported that in fact non-autonomous elements that have lost their protein-coding sequences and became miniature elements were highly associated with genes, and showed a high level of transpositional activity such as mPing family in rice. In this study, we have investigated in detail the copy number, insertional polymorphism and the methylation status of the tiniest LTR retrotransposon family, termed TRIM, in nine rice strains, in comparison with mPing. While TRIM showed similar copy numbers (average of 79 insertions) in all the nine rice strains, the copy number of mPing varied dramatically (ranging from 6 to 203 insertions) in the same strains. Site-specific PCR analysis revealed that ~58% of the TRIM elements have identical insertion sites among the nine rice strains, while none of the mPing elements (100% polymorphism) have identical insertion sites in the same strains. Finally, over 65% of the TRIM insertion sites were cytosine methylated in all nine rice strains, while the level of the methylated mPing insertion sites ranged between 43 and 81.5%. The findings of this study indicate that unlike mPing, TRIM is most probably a fossil TE family in rice. In addition, the data shows that there might be a strong correlation between TE methylation and copy number.     

Distribution and evolution of the xylanase genes xynA and xynB and their homologues in strains of Butyrivibrio fibrisolvens.

The ruminal bacterium Butyrivibrio fibrisolvens is being engineered by the introduction of heterologous xylanase genes in an attempt to improve the utilization of plant material in ruminants. However, relatively little is known about the diversity and distribution of the native xylanase genes in strains of B. fibrisolvens. In order to identify the most appropriate hosts for such modifications, the xylanase genotypes of 28 strains from the three 16S ribosomal DNA (rDNA) subgroups of Butyrivibrio fibrisolvens have been investigated. Only 4 of the 20 strains from 16S rDNA group 2 contained homologues of the strain Bu49 xynA gene. However, these four xynA-containing strains, and two other group 2 strains, contained members of a second xylanase gene family clearly related to xynA (subfamily I). Homologues of xynB, a second previously described xylanase gene from B. fibrisolvens, were identified only in three of the seven group 1 strains and not in the group 2 and 3 strains. However, six of the group 1 strains contained one or more members of the two subfamilies of homologues of xynA. The distribution of genes and the nucleotide sequence relationships between the members of the two xynA subfamilies are consistent with the progenitor of all strains of B. fibrisolvens having contained a xynA subfamily I gene. Since many xylanolytic strains of B. fibrisolvens did not contain members of either of the xynA subfamilies or of the xynB family, at least one additional xylanase gene family remains to be identified in B. fibrisolvens.     

Identification of an Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein with roles in adhesion and cytotoxicity

Entamoeba histolytica is a leading cause of parasitic death globally. However, the molecular framework regulating pathogenesis is poorly understood. We have previously used expression profiling to identify Entamoeba genes whose expressions were strictly associated with virulent strains (R. C. MacFarlane and U. Singh, Infect. Immun. 74:340-351, 2006). One gene, which we have named EhSTIRP (Entamoeba histolytica serine-, threonine-, and isoleucine-rich protein), was exclusively expressed in virulent but not in nonvirulent Entamoeba strains. EhSTIRP is predicted to be a transmembrane protein and is encoded by a multigene family. In order to characterize its function in amebic biology, we used a double-stranded RNA-based approach and were able to selectively down-regulate expression of this gene family. Upon EhSTIRP down-regulation, we were able to ascribe cytotoxic and adhesive properties to the protein family using lactate dehydrogenase release and Chinese hamster ovary cell adhesion assays. EhSTIRP thus likely represents a novel determinant of virulence in Entamoeba histolytica. This work validates the fact that genes expressed exclusively in virulent strains may represent virulence determinants and highlights the need for further functional analyses of other genes with similar expression profiles.     

The Diversity of Alleles at the Hsd Locus in Natural Populations of Escherichia Coli

In enteric bacteria three discrete families of type I restriction and modification systems (IA, IB and ID) are encoded by alleles of the serB-linked hsd locus. Probes specific for each of the three familes were used to monitor the distribution of related systems in 37 of the 72 wild-type Escherichia coli strains comprising the ECOR collection. All 25 members of group A in this collection were screened; 12 were probe-positive, nine have hsd genes in the IA family, two in the IB and one in the ID. Twelve strains, representing all groups other than A, were screened; five were probe-positive, one has hsd genes in the IA family, one in the IB and three in the ID. The type ID genes are the first representatives of this family in E. coli, the probe-negative strains could have alternative families of hsd genes. The type IA and IB systems added at least five new specificites to the five already identified in natural isolates of E. coli. The distribution of alleles is inconsistent with the dendrogram of the bacterial strains derived from other criteria. This discrepancy and the dissimilar coding sequences of allelic hsd genes both imply lateral transfer of hsd genes.     

Genome Comparison and Phylogenetic Analysis of Orientia tsutsugamushi Strains

Orientia tsutsugamushi (OT) is an obligate intracellular bacterium belonging to the family Rickettsiaceae and is the causative agent of scrub typhus, or Tsutsugamushi disease. The complete genome sequences of two OT strains (Boryong and Ikeda) have recently been determined. In the present study, we performed a fine genome sequence comparison of these strains. Our results indicate that although the core gene set of the family Rickettsiaceae is highly conserved between the two strains, a common set of repetitive sequences have been explosively amplified in both genomes. These amplified repetitive sequences have induced extensive genome shuffling and duplications and deletions of many genes. On the basis of the results of the genome sequence comparison, we selected 11 housekeeping genes and carried out multilocus sequence analysis of OT strains using the nucleotide sequences of these genes. This analysis revealed for the first time the phylogenetic relationships of representative OT strains. Furthermore, the results suggest the presence of an OT lineage with higher potential for virulence, which may explain the clinical and epidemiological differences between ‘classic’ and ‘new’ types of Tsutsugamushi disease in Japan.     

A Novel Method of Identifying Mycobacterium tuberculosis Beijing Strains by Detecting SNPs in Rv0444c and Rv2629

A particular genotype of tuberculosis, named Beijing strain, is strongly associated with drug resistance and high virulence. Therefore, rapid prospective identification of Mycobacterium tuberculosis Beijing strains is very important for identifying and controlling tuberculosis of Beijing genotype. In the present study, we found that the co-mutation, A191C in Rv2629 and G243C in Rv0444c, is closely related to Beijing genotype. Gene Rv2629 and Rv0444c of 139 clinical isolates of M. tuberculosis were analyzed by PCR amplification and sequencing. Among 99 Beijing strains, 86 % (n = 85) isolates had the mutation G243C in Rv0444c and 92.93 % (n = 92) isolates had the mutation A191C in Rv2629. Among 40 non-Beijing isolates, only six isolates carried the mutation G243C in Rv0444c and eight isolates carried the mutation A191C in Rv2629. The co-mutation existed in 84.85 % (n = 84) of 99 clinical genome samples of W-Beijing strains and in only 12.5 % (n = 5) of the 40 non-Beijing strains, and the positive predictive value of 94.38 %, obtained in our experiment with a designed ratio of Beijing isolates, is similar to that in China at present. This result suggested that the detection method of the co-mutation, A191C in Rv2629 and G243C in Rv0444c, proposed in this study was a rapid, reliable, and sensitive one for identifying tuberculosis with Beijing genotype.     

Genetic Diversity and Transmission Characteristics of Beijing Family Strains of Mycobacterium tuberculosis in Peru

Beijing family strains of Mycobacterium tuberculosis have attracted worldwide attention because of their wide geographical distribution and global emergence. Peru, which has a historical relationship with East Asia, is considered to be a hotspot for Beijing family strains in South America. We aimed to unveil the genetic diversity and transmission characteristics of the Beijing strains in Peru. A total of 200 Beijing family strains were identified from 2140 M. tuberculosis isolates obtained in Lima, Peru, between December 2008 and January 2010. Of them, 198 strains were classified into sublineages, on the basis of 10 sets of single nucleotide polymorphisms (SNPs). They were also subjected to variable number tandem-repeat (VNTR) typing using an international standard set of 15 loci (15-MIRU-VNTR) plus 9 additional loci optimized for Beijing strains. An additional 70 Beijing family strains, isolated between 1999 and 2006 in Lima, were also analyzed in order to make a longitudinal comparison. The Beijing family was the third largest spoligotyping clade in Peru. Its population structure, by SNP typing, was characterized by a high frequency of Sequence Type 10 (ST10), which belongs to a modern subfamily of Beijing strains (178/198, 89.9%). Twelve strains belonged to the ancient subfamily (ST3 [n = 3], ST25 [n = 1], ST19 [n = 8]). Overall, the polymorphic information content for each of the 24 loci values was low. The 24 loci VNTR showed a high clustering rate (80.3%) and a high recent transmission index (RTI_n−1 = 0.707). These strongly suggest the active and on-going transmission of Beijing family strains in the survey area. Notably, 1 VNTR genotype was found to account for 43.9% of the strains. Comparisons with data from East Asia suggested the genotype emerged as a uniquely endemic clone in Peru. A longitudinal comparison revealed the genotype was present in Lima by 1999.     

Organization of the SUC gene family in Saccharomyces.

The SUC gene family of yeast (Saccharomyces) includes six structural genes for invertase (SUC1 through SUC5 and SUC7) found at unlinked chromosomal loci. A given yeast strain does not usually carry SUC+ alleles at all six loci; the natural negative alleles are called suc0 alleles. Cloned SUC2 DNA probes were used to investigate the physical structure of the SUC gene family in laboratory strains, commercial wine strains, and different Saccharomyces species. The active SUC+ genes are homologous. The suc0 allele at the SUC2 locus (suc2(0) in some strains is a silent gene or pseudogene. Other SUC loci carrying suc0 alleles appear to lack SUC DNA sequences. These findings imply that SUC genes have transposed to different chromosomal locations in closely related Saccharomyces strains.     

Integration of heterologous DNA into the genome of Paracoccus denitrificans is mediated by a family of IS1248-related elements and a second type of integrative recombination event.

All members of the IS1248 family residing in the genome of Paracoccus denitrificans have been isolated by using a set of insertion sequence entrapment vectors. The family consists of five closely related members that integrate the entrapment vectors at distinct sites. One of these, IS1248b, was sequenced and, except for a single base change, shown to be identical to the previously isolated IS1248a. Southern analysis of genomic DNA with labeled IS1248 revealed different hybridization patterns for different isolates of P. denitrificans and Thiosphaera pantotropha. No hybridization was observed with DNA from Thiobacillus versutus and more distantly related species. From a comparison of the fingerprints it was shown that one of the members of the IS1248 family found in P. denitrificans DSM413 is absent in strain NCIB8944, although they are catalogued in international strain catalogues as identical strains. Furthermore, strains Pd1222 and Pd1235, both derivatives of P. denitrificans DSM413, were shown to have different patterns of IS1248 hybridizing restriction fragments. In 14 of 18 strains, the entrapment vectors used in this study were incorporated into the genome via IS1248-mediated cointegrate formation. In the other four strains, the entrapment vectors were shown to be integrated through a different mechanism not involving IS1248.     

Isoprenoid pigments in representatives of the family Microbacteriaceae

By using fosmidomycin and mevinolin (inhibitors of the synthesis of isoprenoid pigments), spectrophotometry, and mass spectrometry, the presence of isoprenoid pigments is shown in 71 of the 78 strains under study. All of these strains belong to 11 genera of the family Microbacteriaceae. Yellow, orange, and red pigments are found to have absorption spectra typical of C40-carotenoids. Eight out of the sixteen strains of the genus Microbacterium are able to synthesize neurosporene, a precursor of lycopene and beta-carotene. The biosynthesis of carotenoids in some representatives of the genera Agromyces, Leifsonia, and Microbacterium is induced by light. Inhibition of the biosynthesis of isoprenoid pigments by fosmidomycin suggests that they are synthesized via the nonmevalonate pathway. Twelve strains are found to exhibit both the nonmevalonate and mevalonate pathways of isoprenoid synthesis. These data, together with the difference in the inhibitory concentration of fosmidomycin, can be used for differentiating various taxa within the family Microbacteriaceae.