Immunological consequences of strain variation within the Mycobacterium tuberculosis complex

In 2015, there were an estimated 10.4 million new cases of tuberculosis (TB) globally, making it one of the leading causes of death due to an infectious disease. TB is caused by members of the Mycobacterium tuberculosis complex (MTBC), with human disease resulting from infection by M. tuberculosis sensu stricto and M. africanum. Recent progress in genotyping techniques, in particular the increasing availability of whole genome sequence data, has revealed previously under appreciated levels of genetic diversity within the MTBC. Several studies have shown that this genetic diversity may translate into differences in TB transmission, clinical manifestations of disease, and host immune responses. This suggests the existence of MTBC genotype‐dependent host–pathogen interactions which may influence the outcome of infection and progression of disease. In this review, we highlight the studies demonstrating differences in innate and adaptive immunological outcomes consequent on MTBC genetic diversity, and discuss how these differences in immune response might influence the development of TB vaccines, diagnostics and new therapies.     

Single Nucleotide Polymorphism in Ag85 Genes of Mycobacterium Tuberculosis Complex: Analysis of 178 Clinical Isolates from China and 13 BCG strains

Host immune pressure and associated immune evasion of pathogenic bacteria are key features of host-pathogen co-evolution. Human T-cell epitopes of Mycobacterium tuberculosis (M. tuberculosis) were evolutionarily hyperconserved and thus it was deduced that M. tuberculosis lacks antigenic variation and immune evasion. However, in our previous studies, proteins MPT64, PstS1, Rv0309 and Rv2945c all harbored higher numbers of amino acid substitutions in their T cell epitopes, which suggests their roles in ongoing immune evasion. Here, we used the same set of 180 clinical M. tuberculosis complex (MTBC) isolates from China, amplified the genes encoding Ag85 complex, and compared the sequences. The results showed that Ag85 were hyperconserved in T/B cell epitopes and the genes were more likely to be under purifying selection. The divergence of host immune selection on different proteins may result from different function of the proteins. In addition, A312G of Ag85A and T418C of Ag85B may represent special mutations in BCG strains, which may be used to differentiate M.bovis and BCG strains from MTB strains. Also, C714A in Ag85B seems to be a valuable phylogenetic marker for Beijing strains.     

Consequences of genomic diversity in Mycobacterium tuberculosis

The causative agent of human tuberculosis, Mycobacterium tuberculosis complex (MTBC), comprises seven phylogenetically distinct lineages associated with different geographical regions. Here we review the latest findings on the nature and amount of genomic diversity within and between MTBC lineages. We then review recent evidence for the effect of this genomic diversity on mycobacterial phenotypes measured experimentally and in clinical settings. We conclude that overall, the most geographically widespread Lineage 2 (includes Beijing) and Lineage 4 (also known as Euro-American) are more virulent than other lineages that are more geographically restricted. This increased virulence is associated with delayed or reduced pro-inflammatory host immune responses, greater severity of disease, and enhanced transmission. Future work should focus on the interaction between MTBC and human genetic diversity, as well as on the environmental factors that modulate these interactions.     

Novel DNA Chip Based on a Modified DigiTag2 Assay for High-Throughput Species Identification and Genotyping of Mycobacterium tuberculosis Complex Isolates

A multipurpose high-throughput genotyping tool for the assessment of recent epidemiological data and evolutional pattern in Mycobacterium tuberculosis complex (MTBC) clinical isolates was developed in this study. To facilitate processing, 51 highly informative single nucleotide polymorphisms (SNPs) were selected for discriminating the clinically most relevant MTBC species and genotyping M. tuberculosis into its principle genetic groups (PGGs) and SNP cluster groups (SCGs). Because of the high flexibility of the DigiTag2 assay, the identical protocol and DNA array containing the identical set of probes were applied to the highly GC-rich mycobacterial genome. The specific primers with multiplex amplification and hybridization conditions based on the DigiTag2 principle were optimized and evaluated with 14 MTBC reference strains, 4 nontuberculous mycobacteria (NTM) isolates, and 322 characterized M. tuberculosis clinical isolates. The DNA chip that was developed revealed a 99.85% call rate, a 100% conversion rate, and 99.75% reproducibility. For the accuracy rate, 98.94% of positive calls were consistent with previous molecular characterizations. Our cost-effective technology was capable of simultaneously identifying the MTBC species and the genotypes of 96 M. tuberculosis clinical isolates within 6 h using only simple instruments, such as a thermal cycler, a hybridization oven, and a DNA chip scanner, and less technician skill was required than for other techniques. We demonstrate this approach''s potential as a simple, flexible, and rapid tool for providing clearer information regarding circulating MTBC isolates.     

Variation of the Mycobacterium tuberculosis PE_PGRS 33 gene among clinical isolates

PE_PGRS33, one of about 60 PE_PGRS genes in the Mycobacterium tuberculosis genome, encodes a surface-expressed protein that may be involved in the antigenic variation of M. tuberculosis strains and evasion of the host immune system. While genetic differences between the PE_PGRS33 genes of H37Rv and CDC1551 have been noted, genetic variation in this gene among clinical isolates has not been evaluated. In order to gain a better understanding of the genetic basis for the role of PE_PGRS in antigenic variation and evasion of the host immune system, we investigated the genetic diversity of the PE_PGRS33 gene among 123 clinical M. tuberculosis isolates from a population-based study, using PCR and DNA sequencing. The 123 isolates belonged to principal genetic groups 1, 2, and 3 and had IS6110 copy numbers ranging from 1 to 22. Eighty-four (68.3%) of the 123 isolates were found to have at least one sequence variation in the PE_PGRS33 gene, relative to that of H37Rv. Twenty-five different sequence variations were observed and included three insertions (ranging from 9 to 87 bp), nine deletions (ranging from 1 to 273 bp), one insertion-and-deletion event, and 12 single-nucleotide polymorphisms (six synonymous and six nonsynonymous). Analysis of the relationships among the different PE_PGRS33 gene sequence variations suggests that polymorphisms in the gene are shifting along evolutionary lineages. The observed genetic diversity of the PE_PGRS33 gene supports its role in antigenic variation and can serve as a basis for future investigations of the function of the PE_PGRS33 gene among clinical isolates.     

Development of a Novel PCR Restriction Analysis of the hsp65 Gene as a Rapid Method To Screen for the Mycobacterium tuberculosis Complex and Nontuberculous Mycobacteria in High-Burden Countries

The limitations of conventional methods of identification of Mycobacterium tuberculosis have led to the development of several nucleic acid amplification techniques which have the advantage of being rapid, sensitive, and specific. However, their expense or the need for technical expertise makes it difficult to use them in regions in which tuberculosis is endemic. A novel PCR restriction analysis (PRA) of the hsp65 gene was therefore developed for rapid screening of clinical isolates to identify Mycobacterium spp. The restriction enzymes NruI and BamHI were selected to obtain a limited number of restriction patterns to further differentiate between Mycobacterium tuberculosis complex (MTBC) and nontuberculous mycobacteria (NTM). Three hundred ten isolates from clinical specimens and 24 reference strains were tested. The assay correctly identified 295 of the 310 culture isolates as MTBC, while the remaining 15 isolates were identified as NTM. Of the isolates tested, 135 MTBC strains and all 15 NTM were also confirmed by PRA using Sau96I and CfoI. Thirty-eight randomly selected MTBC strains and all 15 NTM were further confirmed by sequencing. The NruI/BamHI PRA was simple, as it did not require any elaborate analyses. It was cost-effective, rapid, highly sensitive, and specific and did not require technical expertise. The assay can, therefore, be used as a simple screening test not only to detect Mycobacterium spp. but also to differentiate MTBC from NTM in peripheral laboratories with minimal availability of funds.     

The intraspecies diversity of C. albicans triggers qualitatively and temporally distinct host responses that determine the balance between commensalism and pathogenicity

The host immune status is critical for preventing opportunistic infections with Candida albicans. Whether the natural fungal diversity that exists between C. albicans isolates also influences disease development remains unclear. Here, we used an experimental model of oral infection to probe the host response to diverse C. albicans isolates in vivo and found dramatic differences in their ability to persist in the oral mucosa, which inversely correlated with the degree and kinetics of immune activation in the host. Strikingly, the requirement of interleukin (IL)-17 signaling for fungal control was conserved between isolates, including isolates with delayed induction of IL-17. This underscores the relevance of IL-17 immunity in mucosal defense against C. albicans. In contrast, the accumulation of neutrophils and induction of inflammation in the infected tissue was strictly strain dependent. The dichotomy of the inflammatory neutrophil response was linked to the capacity of fungal strains to cause cellular damage and release of alarmins from the epithelium. The epithelium thus translates differences in the fungus into qualitatively distinct host responses. Altogether, this study provides a comprehensive understanding of the antifungal response in the oral mucosa and demonstrates the relevance of evaluating intraspecies differences for the outcome of fungal–host interactions in vivo.     

Prevalence and phylogenetic history of the TcpC virulence determinant in Escherichia coli

Extraintestinal pathogenic Escherichia coli (ExPECs) possess an armament of virulence factors to colonize and infect the host, such as adhesins, toxins, capsules and iron-uptake systems. Recently, we could identify a novel virulence factor of ExPECs that interferes with the innate immune response of the host by interrupting the NF-κB signaling pathway. This protein named TcpC shows considerable homology to motifs of the Tir domain of Toll-like receptors. Here we demonstrate that the tcpC gene is widely distributed among clinical ExPEC isolates with almost half of the E. coli strains from patients suffering pyelonephritis shown to be tcpC positive as compared to only 8% in commensal isolates. However, this gene is only present in phylogenetic group B2 strains. Interestingly, the tcpC gene is strongly associated with presence of the high-pathogenicity island (HPI). The phylogenetic history of the tcpC gene, in the E. coli reference collection (ECOR) and other well-defined E. coli strains, compared to the phylogenetic histories of the HPI and the strains, showed that the tcpC gene (i) is scattered among various B2 subgroups with specific O-types, (ii) has a phylogeny incongruent with the strain phylogeny, but (iii) congruent with the HPI phylogenetic history. This, together with the strong conservation of the tcpC gene, indicates a very recent introduction of this virulence factor into E. coli by horizontal gene transfer which occurred “en bloc” with the HPI at one major hot spot of recombination in the E. coli genome. The present data provide evidence for a strong impact of homologous recombination events in the spread of the TcpC virulence trait among E. coli.     

Performance of lipid fingerprint by routine matrix-assisted laser desorption/ionization time of flight for the diagnosis of Mycobacterium tuberculosis complex species

ObjectivesRapid detection of bacterial pathogens at species and sub-species levels is crucial for appropriate treatment, infection control, and public health management. Currently, one of the challenges in clinical microbiology is the discrimination of mycobacterial sub-species within the M. tuberculosis complex (MTBC). Our objective was to evaluate the ability of a biosafe mycobacterial lipid-based approach to identify MTBC cultures and sub-species.MethodsA blinded study was conducted using 90 mycobacterial clinical isolate strains comprising MTBC strains sub-cultured in Middlebrook 7H11 medium supplemented with 10% oleic-acid, dextrose, catalase growth supplement and incubated for up to 6 weeks at 37°C and using the following seven reference strains (M. tuberculosis H37Rv, M canettii, M. africanum, M. pinnipedii, M. caprae, M. bovis, and M. bovis BCG) grown under the same conditions, to set the reference lipid database and test it against the 90 MTBC clinical isolates. Cultured mycobacteria were heat-inactivated and loaded onto the matrix-assisted laser desorption/ionization target followed by the addition of the matrix. Acquisition of the data was performed using the positive ion mode.ResultsBased on the identification of clear and defined lipid signatures from the seven reference strains, the method that we developed was fast (<10 minutes) and produced interpretable profiles for all but four isolates, caused by poor ionization giving an n = 86 with interpretable spectra. The sensitivity and specificity of the matrix-assisted laser desorption/ionization time of flight were 94.4 (95% CI, 86.4–98.5) and 94.4 (95% CI, 72.7–99.9), respectively.ConclusionsMycobacterial lipid profiling provides a means of rapid, safe, and accurate discrimination of species within the MTBC.     

Induction of Rapid Cell Death by an Environmental Isolate of Legionella pneumophila in Mouse Macrophages

Legionella pneumophila, the etiological agent for Legionnaires'' disease, is ubiquitous in the aqueous environment, where it replicates as an intracellular parasite of free-living protozoa. Our understanding of L. pneumophila pathogenicity is obtained mostly from study of derivatives of several clinical isolates, which employ almost identical virulent determinants to exploit host functions. To determine whether environmental L. pneumophila isolates interact similarly with the model host systems, we analyzed intracellular replication of several recently isolated such strains and found that these strains cannot productively grow in bone marrow-derived macrophages of A/J mice, which are permissive for all examined laboratory strains. By focusing on one strain called LPE509, we found that its deficiency in intracellular replication in primary A/J macrophages is not caused by the lack of important pathogenic determinants because this strain replicates proficiently in two protozoan hosts and the human macrophage U937 cell. We also found that in the early phase of infection, the trafficking of this strain in A/J macrophages is similar to that of JR32, a derivative of strain Philadelphia 1. Furthermore, infection of these cells by LPE509 caused extensive cell death in a process that requires the Dot/Icm type IV secretion system. Finally, we showed that the cell death is caused neither by the activation of the NAIP5/NLRC4 inflammasome nor by the recently described caspase 11-dependent pathway. Our results revealed that some environmental L. pneumophila strains are unable to overcome the defense conferred by primary macrophages from mice known to be permissive for laboratory L. pneumophila strains. These results also suggest the existence of a host immune surveillance mechanism differing from those currently known in responding to L. pneumophila infection.     

Sequencing of hsp65 gene for identification of Mycobacterium species isolated from environmental and clinical sources in Rio de Janeiro, Brazil

This study evaluated the biodiversity of 28 clinical and 24 environmental Mycobacterium isolates from Rio de Janeiro, Brazil, by using hsp65 sequences, with the aim of contributing to a better understanding of the genetic diversity and usefulness of this marker. An extensive phylogenetic analysis was performed. The nucleotide diversity was similar between clinical (0.06508) and environmental (0.06221) isolates.     

Differentiation of clinical Mycobacterium tuberculosis complex isolates by their GyrB polymorphism

Purpose: To evaluate the reliability of the gyrB PCR-RFLP technique in differentiating clinical Mycobacterium tuberculosis complex isolates. Materials and Methods: A primer pair MTUB-f and MTUB-r for M. tuberculosis complex (MTBC) was used to differentiate 79 mycobacterial isolates by specific amplification of the 1,020-bp fragment of the gyrB gene (gyrB-PCR1). The MTBC isolates were further differentiated using a set of specific primers MTUB-756-Gf and MTUB-1450-Cr that allowed selective amplification of the gyrB fragment specific for M. tuberculosis (gyrB-PCR2). The DNA polymorphisms in the 1,020-bp gyrB fragment for 7 M. tuberculosis strains confirmed by PCR as well as 2 reference strains; M. tuberculosis H37Rv and M. bovis BCG were analyzed with the restriction enzyme Rsa1. Results: Seventy-seven (97.5%) isolates were positive for gyrB-PCR1 and thus identified as members of M. tuberculosis complex (MTBC) and two (2.6%) isolates were negative and identified as Mycobacteria other than tuberculosis (MOTT). All the M. tuberculosis isolates showed the typical M. tuberculosis specific Rsa1 RFLP patterns (100, 360, 560-bp) while 360 and 480-bp fragments were generated from M. bovis BCG. Conclusion: The gyrB PCR-RFLP using the endonuclease Rsa1 can be used to differentiate M. tuberculosis from M. bovis in clinical isolates.     

Genome Sequencing of Mycobacterium abscessus Isolates from Patients in the United States and Comparisons to Globally Diverse Clinical Strains

Nontuberculous mycobacterial infections caused by Mycobacterium abscessus are responsible for a range of disease manifestations from pulmonary to skin infections and are notoriously difficult to treat, due to innate resistance to many antibiotics. Previous population studies of clinical M. abscessus isolates utilized multilocus sequence typing or pulsed-field gel electrophoresis, but high-resolution examinations of genetic diversity at the whole-genome level have not been well characterized, particularly among clinical isolates derived in the United States. We performed whole-genome sequencing of 11 clinical M. abscessus isolates derived from eight U.S. patients with pulmonary nontuberculous mycobacterial infections, compared them to 30 globally diverse clinical isolates, and investigated intrapatient genomic diversity and evolution. Phylogenomic analyses revealed a cluster of closely related U.S. and Western European M. abscessus subsp. abscessus isolates that are genetically distinct from other European isolates and all Asian isolates. Large-scale variation analyses suggested genome content differences of 0.3 to 8.3%, relative to the reference strain ATCC 19977^T. Longitudinally sampled isolates showed very few single-nucleotide polymorphisms and correlated genomic deletion patterns, suggesting homogeneous infection populations. Our study explores the genomic diversity of clinical M. abscessus strains from multiple continents and provides insight into the genome plasticity of an opportunistic pathogen.     

Clustered,regularly interspaced short palindromic repeat (CRISPR) diversity and virulence factor distribution in avian Escherichia coli

In order to investigate the diverse characteristics of clustered, regularly interspaced short palindromic repeat (CRISPR) arrays and the distribution of virulence factor genes in avian Escherichia coli, 80 E. coli isolates obtained from chickens with avian pathogenic E. coli (APEC) or avian fecal commensal E. coli (AFEC) were identified. Using the multiplex polymerase chain reaction (PCR), five genes were subjected to phylogenetic typing and examined for CRISPR arrays to study genetic relatedness among the strains. The strains were further analyzed for CRISPR loci and virulence factor genes to determine a possible association between their CRISPR elements and their potential virulence. The strains were divided into five phylogenetic groups: A, B1, B2, D and E. It was confirmed that two types of CRISPR arrays, CRISPR1 and CRISPR2, which contain up to 246 distinct spacers, were amplified in most of the strains. Further classification of the isolates was achieved by sorting them into nine CRISPR clusters based on their spacer profiles, which indicates a candidate typing method for E. coli. Several significant differences in invasion-associated gene distribution were found between the APEC isolates and the AFEC isolates. Our results identified the distribution of 11 virulence genes and CRISPR diversity in 80 strains. It was demonstrated that, with the exception of iucD and aslA, there was no sharp demarcation in the gene distribution between the pathogenic (APEC) and commensal (AFEC) strains, while the total number of indicated CRISPR spacers may have a positive correlation with the potential pathogenicity of the E. coli isolates.     

Molecular characterization of Leishamania isolates from China by inter-simple sequence repeat polymerase chain reaction

Leishmania has distinct epidemiological and biological characteristics and causes a variety of clinical symptoms. To understand the genetic diversity and the phylogenetic relationships among Leishmania isolates from China, 29 Leishmania isolates from different geographic origins, vectors, and hosts were analyzed using 21 inter-simple sequence repeat polymerase chain reaction (ISSR-PCR) primers. A total of 864 polymorphic bands were obtained. According to the results of the neighbor-joining phylogenetic tree and principal component analysis, the 29 isolates studied clustered into six groups. Isolates of Leishmania donovani complex from China share the highest similarity with the reference strain of L. donovani (DD8). This study helps to elucidate the genetic relationship among Leishmania isolates from China and similarities between Chinese isolates and World Health Organization reference strains. Furthermore, ISSR-PCR could also be a quick, simple, and reliable method for Leishmania species identification.     

Microsatellite-primed PCR and random primer amplification polymorphic DNA for the identification and epidemiology of dermatophytes

This study demonstrates the capacity of the one-step polymerase chain reaction (PCR) fingerprinting method using the microsatellite primers (GACA)₄ or (GTG)₅ (MSP-PCR) to identify six of the most frequent dermatophyte species causing cutaneous mycosis. PCR with (GACA)₄ was a suitable method to recognise Microsporum canis, Microsporum gypseum, Trichophyton rubrum and Trichophyton interdigitale among 82 Argentinian clinical isolates, producing the most simple and reproducible band profiles. In contrast, the identification of Trichophyton mentagrophytes and Trichophyton tonsurans was achieved using PCR with (GTG)₅. In this way, the sequential application of PCR using (GACA)₄ and (GTG)₅ allowed the successful typification of clinical isolates which had not been determined by mycological standard techniques. In this work, the intraspecies variability among 33 clinical isolates of M. canis was detected using random amplification of polymorphic DNA (RAPD-PCR) with the primers OPI-07 and OPK-20. The genetic variations in the isolates of M. canis were not associated with clinical features of lesions or pet ownership, but a geographical restriction of one genotype was determined with OPK-20, suggesting a clonal diversity related to different ecological niches in certain geographical areas. The results of this work demonstrate that the detection of intraspecies polymorphisms in M. canis by RAPD-PCR may be applied in future molecular epidemiological studies to identify endemic strains, the route of infection in an outbreak or the coexistence of different strains in a single infection.     

Morphological and genetic diversity of symbiotic cyanobacteria from cycads

The morphological and genetic diversity of cyanobacteria associated with cycads was examined using PCR amplification techniques and 16S rRNA gene sequence analysis. Eighteen symbiotic cyanobacteria were isolated from different cycad species. One of the symbiotic isolates was a species of Calothrix, a genus not previously reported to form symbioses with Cycadaceae family, and the remainder were Nostoc spp. Axenic cyanobacterial strains were compared by DNA amplification using PCR with either short arbitrary primers or primers specific for the repetitive sequences. Based on fingerprint patterns and phenograms, it was revealed that cyanobacterial symbionts exhibit important genetic diversity among host plants, both within and between cycad populations. A phylogenetic analysis based on 16S rRNA gene sequence analysis revealed that most of the symbiotic cyanobacterial isolates fell into well‐separated clades. (© 2010 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)     

Evaluation of a Method Using Three Genomic Guided Escherichia coli Markers for Phylogenetic Typing of E. coli Isolates of Various Genetic Backgrounds

Genotyping and characterization of bacterial isolates are essential steps in the identification and control of antibiotic-resistant bacterial infections. Recently, one novel genotyping method using three genomic guided Escherichia coli markers (GIG-EM), dinG, tonB, and dipeptide permease (DPP), was reported. Because GIG-EM has not been fully evaluated using clinical isolates, we assessed this typing method with 72 E. coli collection of reference (ECOR) environmental E. coli reference strains and 63 E. coli isolates of various genetic backgrounds. In this study, we designated 768 bp of dinG, 745 bp of tonB, and 655 bp of DPP target sequences for use in the typing method. Concatenations of the processed marker sequences were used to draw GIG-EM phylogenetic trees. E. coli isolates with identical sequence types as identified by the conventional multilocus sequence typing (MLST) method were localized to the same branch of the GIG-EM phylogenetic tree. Sixteen clinical E. coli isolates were utilized as test isolates without prior characterization by conventional MLST and phylogenetic grouping before GIG-EM typing. Of these, 14 clinical isolates were assigned to a branch including only isolates of a pandemic clone, E. coli B2-ST131-O25b, and these results were confirmed by conventional typing methods. Our results suggested that the GIG-EM typing method and its application to phylogenetic trees might be useful tools for the molecular characterization and determination of the genetic relationships among E. coli isolates.     

Genetic diversity,the virulence gene profile and antimicrobial resistance of clinical mastitis-associated Escherichia coli

Escherichia coli is a common cause of bovine mastitis, particularly around parturition and early lactation when the host is immunosuppressed. Isolates (n = 37) recovered from cases of clinical mastitis in Ireland were characterised with respect to genotypic diversity, phylogenetic group, virulence gene profile and antimicrobial susceptibility. The isolates were genotypically diverse, belonging to 19 different sequence types. However, the majority (86%) belonged to phylogenetic groups A or B1, groups commonly associated with commensal E. coli. The isolates encoded few virulence genes with iss (increased serum survival, 41%), lpfA (long polar fimbriae, 19%) and astA (enteroaggregative heat-stable toxin, 14%) among the most common virulence genes detected. The only virulence gene to differ in frequency between the phylogenetic groups was lpfA, found exclusively in B1. Resistance to at least one antimicrobial was detected in 16% of isolates. Three isolates were multidrug-resistant, with one resistant to seven antibiotics. There was no relationship between antimicrobial resistance and phylogenetic group. These results indicate that many cases of clinical E. coli mastitis in Ireland may be caused by opportunistic commensal organisms lacking specific virulence genes. However, the organisms represent a reservoir of antimicrobial resistance determinants with the potential to disseminate determinants to other organisms.