Structure and variation of CRISPR and CRISPR-flanking regions in deleted-direct repeat region Mycobacterium tuberculosis complex strains.

BACKGROUND: CRISPR and CRISPR-flanking genomic regions are important for molecular epidemiology of Mycobacterium tuberculosis complex (MTBC) strains, and potentially for adaptive immunity to phage and plasmid DNA, and endogenous roles in the bacterium. Genotyping in the Israel National Mycobacterium Reference Center Tel-Aviv of over 1500 MTBC strains from 2008-2013 showed three strains with validated negative 43-spacer spoligotypes, that is, with putatively deleted direct repeat regions (deleted-DR/CRISPR regions). Two isolates of each of three negative spoligotype MTBC (a total of 6 isolates) were subjected to Next Generation Sequencing (NGS). As positive controls, NGS was performed for three intact-DR isolates belonging to T3_Eth, the largest multiple-drug-resistant (MDR)-containing African-origin cluster in Israel. Other controls consisted of NGS reads and complete whole genome sequences from GenBank for 20 intact-DR MTBC and for 1 deleted-DR MTBC strain recognized as CAS by its defining RD deletion. RESULTS: NGS reads from negative spoligotype MTBC mapped to reference H37Rv NC_000962.3 suggested that the DR/CRISPR regions were completely deleted except for retention of the middle IS6110 mobile element. Clonally specific deletion of CRISPR-flanking genes also was observed, including deletion of at least cas2 and cas1 genes. Genomic RD deletions defined lineages corresponding to the major spoligotype families Beijing, EAI, and Haarlem, consistent with 24 loci MIRU-VNTR profiles. Analysis of NGS reads, and analysis of contigs obtained by manual PCR confirmed that all 43 gold standard DR/CRISPR spacers were missing in the deleted-DR genomes. CONCLUSIONS: Although many negative spoligotype strains are recorded as spoligotype-international-type (SIT) 2669 in the SITVIT international database, this is the first time to our knowledge that it has been shown that negative spoligotype strains are found in at least 4 different 24 loci MIRU-VNTR and RD deletion families. We report for the first time negative spoligotype-associated total loss of CRISPR region spacers and repeats, with accompanying clonally specific loss of flanking genes, including at least CRISPR-associated genes cas2 and cas1. Since cas1 deleted E.coli shows increased sensitivity to DNA damage and impaired chromosomal segregation, we discussed the possibility of a similar phenotype in the deleted-DR strains and Beijing family strains as both lack the cas1 gene.

In silico region of difference (RD) analysis of Mycobacterium tuberculosis complex from sequence reads using RD-Analyzer.

BACKGROUND: Whole-genome sequencing is increasingly used in clinical diagnosis of tuberculosis and study of Mycobacterium tuberculosis complex (MTC). MTC consists of several genetically homogenous mycobacteria species which can cause tuberculosis in humans and animals. Regions of difference (RDs) are commonly regarded as gold standard genetic markers for MTC classification. RESULTS: We develop RD-Analyzer, a tool that can accurately infer the species and lineage of MTC isolates from sequence reads based on the presence and absence of a set of 31 RDs. Applied on a publicly available diverse set of 377 sequenced MTC isolates from known major species and lineages, RD-Analyzer achieved an accuracy of 98.14 % (370/377) in species prediction and a concordance of 98.47 % (257/261) in Mycobacterium tuberculosis lineage prediction compared to predictions based on single nucleotide polymorphism markers. By comparing respective sequencing read depths on each genomic position between isolates of different sublineages, we were able to identify the known RD markers in different sublineages of Lineage 4 and provide support for six potential delineating markers having high sensitivities and specificities for sublineage prediction. An extended version of RD-Analyzer was thus developed to allow user-defined RDs for lineage prediction. CONCLUSIONS: RD-Analyzer is a useful and accurate tool for species, lineage and sublineage prediction using known RDs of MTC from sequence reads and is extendable to accepting user-defined RDs for analysis. RD-Analyzer is written in Python and is freely available at https://github.com/xiaeryu/RD-Analyzer .

Whole genome sequencing and comparative genomics of Mycobacterium orygis isolated from different animal hosts to identify specific diagnostic markers.

Introduction: Mycobacterium orygis, a member of MTBC has been identified in higher numbers in the recent years from animals of South Asia. Comparative genomics of this important zoonotic pathogen is not available which can provide data on the molecular difference between other MTBC members. Hence, the present study was carried out to isolate, whole genome sequence M. orygis from different animal species (cattle, buffalo and deer) and to identify molecular marker for the differentiation of M. orygis from other MTBC members. Methods: Isolation and whole genome sequencing of M. orygis was carried out for 9 samples (4 cattle, 4 deer and 1 buffalo) died due to tuberculosis. Comparative genomics employing 53 genomes (44 from database and 9 newly sequenced) was performed to identify SNPs, spoligotype, pangenome structure, and region of difference. Results: M. orygis was isolated from water buffalo and sambar deer which is the first of its kind report worldwide. Comparative pangenomics of all M. orygis strains worldwide (n= 53) showed a closed pangenome structure which is also reported for the first time. Pairwise SNP between TANUVAS_2, TANUVAS_4, TANUVAS_5, TANUVAS_7 and NIRTAH144 was less than 15 indicating that the same M. orygis strain may be the cause for infection. Region of difference prediction showed absence of RD7, RD8, RD9, RD10, RD12, RD301, RD315 in all the M. orygis analyzed. SNPs in virulence gene, PE35 was found to be unique to M. orygis which can be used as marker for identification. Conclusion: The present study is yet another supportive evidence that M. orygis is more prevalent among animals in South Asia and the zoonotic potential of this organism needs to be evaluated.

Genomic differences between sequence types 1 and 104 of Streptococcus suis Serotype 2.

Background: Streptococcus suis is a zoonotic pathogen that can cause invasive infections in humans who are in close contact with infected pigs or contaminated pork-derived products. S. suis serotype 2 sequence type (ST) 1 strains are mostly associated with meningitis, whereas ST104 strains are mostly recovered from sepsis cases in humans. No data are available for comparison of the ST1 and ST104 strains at the genomic level, particularly concerning virulence-associated genes. Thus, genomic comparison of both STs was performed in this study. Methods: An ST1 isolate (ID26154) from the cerebrospinal fluid of a patient with meningitis and an ST104 isolate (ID24525) from the blood of a patient with sepsis were subjected to shotgun pyrosequencing using the 454 GS Junior System. Genomic comparison was conducted between the ST1 isolate and the ST104 isolate using the Artemis Comparison Tool (ACT) to identify the region of differences (RDs) between ST1 and ST104. Results: Fifty-eight RDs were unique to the ST104 genome and were mainly involved in metabolism and cell functional activities, cell wall anchored proteins, bacteriophages and mobile genetic elements, ABC-type transporters, two-component signal transductions, and lantibiotic proteins. Some virulence genes mostly found in ST1 strains were also present in the ST104 genome. Whole-genome comparison is a powerful tool for identifying genomic region differences between different STs of S. suis serotype 2, leading to the identification of the molecular basis of virulence involved in the pathogenesis of the infection.

Identification of region of difference and H37Rv-related deletion in Mycobacterium tuberculosis complex by structural variant detection and genome assembly.

Mycobacterium tuberculosis complex (MTBC), the main cause of TB in humans and animals, is an extreme example of genetic homogeneity, whereas it is still nevertheless separated into various lineages by numerous typing methods, which differ in phenotype, virulence, geographic distribution, and host preference. The large sequence polymorphism (LSP), incorporating region of difference (RD) and H37Rv-related deletion (RvD), is considered to be a powerful means of constructing phylogenetic relationships within MTBC. Although there have been many studies on LSP already, focusing on the distribution of RDs in MTBC and their impact on MTB phenotypes, a crumb of new lineages or sub-lineages have been excluded and RvDs have received less attention. We, therefore, sampled a dataset of 1,495 strains, containing 113 lineages from the laboratory collection, to screen for RDs and RvDs by structural variant detection and genome assembly, and examined the distribution of RvDs in MTBC, including RvD2, RvD5, and cobF region. Consistent with genealogical delineation by single nucleotide polymorphism (SNP), we identified 125 RDs and 5 RvDs at the species, lineage, or sub-lineage levels. The specificities of RDs and RvDs were further investigated in the remaining 10,218 strains, suggesting that most of them were highly specific to distinct phylogenetic groups, could be used as stable genetic markers in genotyping. More importantly, we identified 34 new lineage or evolutionary branch specific RDs and 2 RvDs, also demonstrated the distribution of known RDs and RvDs in MTBC. This study provides novel details about deletion events that have occurred in distinct phylogenetic groups and may help to understand the genealogical differentiation.

Genetic diversity of Mycobacterium tuberculosis clinical isolates in Blantyre, Malawi.

Despite the high burden of tuberculosis (TB) worldwide, specific factors influencing disease transmission remain elusive. Long term epidemiological studies and in vitro experimental models provide evidence of variable relative fitness of Mycobacterium tuberculosis (Mtb) strains but few such studies are available. Large sequence polymorphisms (LSP) are a robust molecular marker and are feasible as an epidemiological investigative tool. Few Mtb molecular epidemiological studies have been reported in Malawi owing to lack of laboratories with molecular tools. We characterized the genetic diversity of Mtb clinical isolates amongst TB patients in Blantyre, Malawi. We genotyped 64 Mtb clinical isolates using LSP-PCR, assigned specific lineages and confirmed 18 of the isolates using SMRT sequencing. The 64 isolates clustered into 4 lineages (L1-L4) with L4 predominating. There were 10/64 (16%) isolates belonging to L1, 6/64 (9%) belonging to L2, 2/64 (3%) belonging to L3 and 46/64 (72%) belonging to L4. Comparison with a previous study done in Karonga revealed concordance in L1 and L4 but discodance in L2 and L3. The phylogenetic tree constructed, comprised of 3/4 lineages present in Blantyre with 3/18 belonging to L1, 3/18 belonging to L2 and 12/18 belonging to L4. Four Mtb lineages were present in Blantyre with L4 predominating. Larger studies are needed to understand the molecular epidemiology of TB in Blantyre in light of increased bi-directional migration with South Africa.

In silico identification of cytotoxic T lymphocyte epitopes encoded by RD5 region of Mycobacterium tuberculosis.

INTRODUCTION: While CD8+ T cells (cytotoxic T lymphocytes, CTLs) play important roles in immunity against Mycobacterium tuberculosis, only a small number of human leukocyte differentiation antigen (HLA) class I-restricted CTL epitopes for TB have been identified. The current study evaluates CTL epitopes of Rv3117 and Rv3120 proteins, two newly found M. tuberculosis region-diffference-5 (RD5)-encoded antigens, and their population coverage. METHODOLOGY: The amino acid sequences of the two proteins were subjected to epitope analysis under HLA-A2, A3 and B7 supertype restriction using NetCTL, SYFPEITHI, BIMAS, NetCTLPan, IEDB, NetMHC and NetMHCPan prediction online servers. RESULTS: Eight RD5-encoded CTL epitopes were identified in the two proteins and the average population coverage of these epitopes was 87.2% among populations worldwide. CONCLUSION: These CTL epitopes that were identified in silico and may have potential use for CD8+ T cell-mediated TB vaccine design.

Genotyping and Resolution of a Case of Osteomyelitis in a 16-Month-Old Boy of Hispanic/African American Ethnicity.

Most cases of osteomyelitis in children are caused by Staphylococcus aureus, although Kingella kingae, various streptococci, and Salmonella species also underlie this condition. Organisms such as Mycobacterium, Histoplasma, and Cryptococcus are much less commonly identified as etiologic agents in osteomyelitis. This case report describes a 16-month-old boy of Hispanic/African American ethnicity who had extensive inflammation of and discharge from his right ankle. Imaging studies supported a diagnosis of osteomyelitis. Acid-fast bacillus (AFB) and routine wound cultures were ordered on the wound discharge. The AFB culture yielded a positive result for Mycobacterium bovis, and molecular diagnostic testing further genotyped the microorganism as Mycobacterium bovis, Bacillus Calmette-Guerin (BCG). Herein, we report a rare case of osteomyelitis that we believe resulted from a BCG vaccine that the patient had received outside the United States.

Identification and characterization of regions of difference between the Salmonella Gallinarum biovar Gallinarum and the Salmonella Gallinarum biovar Pullorum genomes.

Salmonella Gallinarum is the causative agent of fowl typhoid, a severe septicaemic disease that affects birds of all ages, whereas S. Pullorum causes pullorum disease, a systemic disorder affecting primarily young birds. A proportion of birds with pullorum disease become carriers and are thereby able to transmit S. Pullorum vertically. Although these two pathogens cause distinct diseases, they are otherwise phenotypically and genetically similar. Therefore, the small variations that lead to the differences in virulence must have a genetic basis which currently is unknown. In the present study, we compared the genome sequences of S. Gallinarum (strains: SG287/91 and SG9) and S. Pullorum (strains: SP_CDC, SP_RKS, SP_FCAV, SP_S06) and identified 223 regions of difference (RODs), characterized by indels which were detected by using the software Artemis Comparison Tool. Some of the RODs led to pseudogenes frequently formed by frameshifts and premature stop codons in genes primarily involved in virulence and metabolism. We further verified the presence of some conserved RODs by PCR in 26 isolates of S. Gallinarum and 17 of S. Pullorum in order to extrapolate data analyses from genome comparison to field strains. The variations observed in virulence-related genes of S. Gallinarum and S. Pullorum appear not to be sufficient to explain the differences between the distinct biology of infection of fowl typhoid and pullorum disease. Thus, we suggest that the identified pseudogenes affecting metabolism might play a greater role during infection than previously thought.

High level of IFN-γ released from whole blood of human tuberculosis infections following stimulation with Rv2073c of Mycobacterium tuberculosis.

More efficacious and specific biomarkers are urgently needed for better control of tuberculosis (TB), the second leading infectious cause of mortality worldwide. The region of difference 9 (RD9) presents the genome of the causative pathogen Mycobacterium tuberculosis rather than other species of the genus Mycobacterium, which might be promising targets for specific diagnosis, vaccine development and pathogenesis. In this study, two proteins Rv2073c and Rv2074, encoded by the RD9 were expressed and purified from Escherichia coli system. Following stimulation with both proteins, the levels of IFN-γ secreted by T cells from a total of 49 whole blood samples obtained from clinically diagnosed active TB patients, patients with latent TB infections (LTBIs), and healthy donors, were compared with those of the incubation with recombinant fusion protein of CFP21 and MPT64 (rCM). Our results demonstrated that only Rv2073c could induce a higher level of IFN-γ in TB infections than healthy controls and there was a positive correlation between Rv2073c- and rCM-specific IFN-γ levels in TB infections and healthy donors, respectively. These findings indicate that Rv2073c might be a promising antigen for specific diagnostic reagents and vaccine candidates of TB.

A novel peptide interferes with Mycobacterium tuberculosis virulence and survival.

Tuberculosis (TB) is a huge global burden, with new and resistant strains emerging at an alarming rate, necessitating an urgent need for a new class of drug candidates. Here, we report that SL3, a novel 33-amino acid peptide, causes debilitating effects on mycobacterial morphology. Treatment with SL3 drastically inhibits the growth of Mycobacterium tuberculosis in vitro as well as in a pre-clinical mouse model for M.tb infection. Microarray analysis of SL3-expressing strain demonstrates wide-scale transcriptional disruption in M.tb. We therefore believe that SL3 and similar peptides may herald a new approach towards discovering new molecules for TB therapy.

Antimicrobial responses of teleost phagocytes and innate immune evasion strategies of intracellular bacteria.

During infection, macrophage lineage cells eliminate infiltrating pathogens through a battery of antimicrobial responses, where the efficacy of these innate immune responses is pivotal to immunological outcomes. Not surprisingly, many intracellular pathogens have evolved mechanisms to overcome macrophage defenses, using these immune cells as residences and dissemination strategies. With pathogenic infections causing increasing detriments to both aquacultural and wild fish populations, it is imperative to garner greater understanding of fish phagocyte antimicrobial responses and the mechanisms by which aquatic pathogens are able to overcome these teleost macrophage barriers. Insights into the regulation of macrophage immunity of bony fish species will lend to the development of more effective aquacultural prophylaxis as well as broadening our understanding of the evolution of these immune processes. Accordingly, this review focuses on recent advances in the understanding of teleost macrophage antimicrobial responses and the strategies by which intracellular fish pathogens are able to avoid being killed by phagocytes, with a focus on Mycobacterium marinum.

Genetic diversity of Mycobacterium tuberculosis in Mbarara, South Western Uganda.

BACKGROUND: We determined the genetic diversity of mycobacteria isolated from tuberculosis patients in Mbarara Uganda, using region of difference (RD) analysis and spacer oligonucleotide typing (spoligotyping). METHODS: Sputum samples were cultured on Lowenstein Jensen media. The isolates were characterized using RD analysis and spoligotyping. RESULTS: The majority (92.8%) of the patients were new cases, 60% were males and 44% were HIV positive with a mean age of 33.7 years. All the 125 isolates were identified as M.tuberculosis sensu stricto. Most (92.8%) of the isolates were modern strains. Spoligotyping revealed 79 spoligotype patterns, with an overall diversity of 63.2%. Sixty (48%) isolates formed 16 clusters each consisting of 2-15 isolates. Mst (59.2 %) of the isolates were Uganda genotype strains. The major shared spoligotypes in our sample were SIT 135 (T2-Uganda) with 12 isolates and SIT 128 (T2) with 5 isolates. Sixty nine (87%) patterns had not yet been defined in the SpolDB4.0.database. CONCLUSION: The TB epidemic in Mbarara is caused mainly by modern M.tuberculosis strains of the Uganda genotype. The wide diversity of strains may indicate that the majority of the TB cases are reactivation rather than re-infection. However this needs to be ascertained with more discriminative finger printing techniques.