Comparative Whole-Genome Analysis of Clinical Isolates Reveals Characteristic Architecture of Mycobacterium tuberculosis Pangenome

The tubercle complex consists of closely related mycobacterium species which appear to be variants of a single species. Comparative genome analysis of different strains could provide useful clues and insights into the genetic diversity of the species. We integrated genome assemblies of 96 strains from Mycobacterium tuberculosis complex (MTBC), which included 8 Indian clinical isolates sequenced and assembled in this study, to understand its pangenome architecture. We predicted genes for all the 96 strains and clustered their respective CDSs into homologous gene clusters (HGCs) to reveal a hard-core, soft-core and accessory genome component of MTBC. The hard-core (HGCs shared amongst 100% of the strains) was comprised of 2,066 gene clusters whereas the soft-core (HGCs shared amongst at least 95% of the strains) comprised of 3,374 gene clusters. The change in the core and accessory genome components when observed as a function of their size revealed that MTBC has an open pangenome. We identified 74 HGCs that were absent from reference strains H37Rv and H37Ra but were present in most of clinical isolates. We report PCR validation on 9 candidate genes depicting 7 genes completely absent from H37Rv and H37Ra whereas 2 genes shared partial homology with them accounting to probable insertion and deletion events. The pangenome approach is a promising tool for studying strain specific genetic differences occurring within species. We also suggest that since selecting appropriate target genes for typing purposes requires the expected target gene be present in all isolates being typed, therefore estimating the core-component of the species becomes a subject of prime importance.     

Epidemiology of Clinical Isolates of Mycobacterium tuberculosis at Ibadan, Nigeria

Despite the huge burden of tuberculosis (TB) in Nigeria, case detection rate of infectious cases still remain low, thus constituting obstacle to eradication of the disease in the community. We carried out a 15 month (1st January 2008 to 30th March 2009) retrospective review of epidemiology of clinical isolates of M. tuberculosis isolated at TB regional reference laboratory at the department of Medical Microbiology and Parasitology, University College Hospital, Ibadan, Nigeria. Fifty isolates were recovered from 720 specimens during the period of study with a recovery rate of 6.9%. Sixty- two (8.6%) of the specimens were contaminated. Thirty eight (76.0%) isolates were from the specimens of male subjects and 12 (24.0%) from female subjects giving a male to female ratio of 3.2: 1.0 Majority (62.0%) of the isolates were from subjects aged 20 years and above with an isolation rate of 7.3% while only two clinical isolates (4.0%) were recovered from specimens from children. A high yield of 20.8% was recovered from specimen collected from Hausa ethnic group who predominantly domiciled in a particular part of the metropolis. In terms of socio-economic status, clinical isolates recovered from specimens from unskilled workers (76.0%) was more than thrice from that obtained from the professionals (24.0%). Seven (14.0%) of the total isolates were recovered from extra-pulmonary lesions while the majority 43 (86.0%) were for pulmonary TB. The isolation rate from children and extra-pulmonary sites are low. This suggests a need to pay more attention to diagnosis of childhood and extra-pulmonary TB in Ibadan, Nigeria.Keywords: M. tuberculosis, Isolates, Epidemiology, Ibadan.     

A note on wildlife poisoning cases from Kerala,South India

Wildlife poisoning is an important conservation threat for endangered species in India. There are no publications in the scientific literature that identify the specific poisons or chemicals involved in wildlife poisoning cases from the state of Kerala. In this report, all cases of wildlife mortality recorded between 2011 and 2013 at the office of the Assistant Forest Veterinary Officer, Periyar Tiger Reserve in Kerala were reviewed and cases where poisoning was considered as a differential diagnosis were identified. Specific poisons or chemicals were identified in three cases, while in a fourth, poisoning was determined to have occurred based on physical traces of the poison in gut contents. The poisons identified include carbofuran (a carbamate pesticide) in a bonnet macaque (Macaca radiata), warfarin (a rodenticide) in a mortality event involving four wild boars (Sus scrofa), endosulfan (an organochlorine pesticide) toxicity in a gaur (Bos gaurus) and imidacloprid (a neonicotinoid pesticide) toxicity in a wild adult Asian elephant (Elephas maximus). This communication thus reports for the first time on the specific chemical compounds identified in wildlife poisoning cases from Kerala state and argues for greater regulation of the sale and use of such toxic compounds in India.     

IUCD acceptors in Trivandrum,Kerala, South India

Abstract

Previous tests of the economic theory of fertility using multivariate regression techniques have generally analyzed only cross‐sectional data. Some students of fertility, notably Janowitz (1973a,b), have argued that such studies must also be conducted with change data. This paper presents tests of the economic theory of fertility with both cross‐sectional and change data for the 48 contiguous states. The cross‐sectional analyses examined the effects of male and female income on the marital fertility of 20–24 year olds for 1950, 1960, and 1970 while controlling for the effects of education, urbanization, race, religion, and population density. Controlling for the effects of the same noneconomic variables, a second set of multivariate regression analyses examined the effects of changes in male and female income on changes in the level of marital fertility of 20–24 year olds during the 1950–60 and 1960–70 periods. Both the analyses with cross‐sectional data and change scores provided support for the economic theory.     

Efflux Pump Gene Expression in Multidrug-Resistant Mycobacterium tuberculosis Clinical Isolates

Isoniazid (INH) and rifampicin (RIF) are the two most effective drugs in tuberculosis therapy. Understanding the molecular mechanisms of resistance to these two drugs is essential to quickly diagnose multidrug-resistant (MDR) tuberculosis and extensive drug-resistant tuberculosis. Nine clinical Mycobacterium tuberculosis isolates resistant to only INH and RIF and 10 clinical pan-sensitive isolates were included to evaluate the expression of 20 putative drug efflux pump genes and sequence mutations in rpoB (RIF), katG (INH), the inhA promoter (INH), and oxyR-ahpC (INH). Nine and three MDR isolates were induced to overexpress efflux pump genes by INH and RIF, respectively. Eight and two efflux pump genes were induced to overexpress by INH and RIF in MDR isolates, respectively. drrA, drrB, efpA, jefA (Rv2459), mmr, Rv0849, Rv1634, and Rv1250 were overexpressed under INH or RIF stress. Most efflux pump genes were overexpressed under INH stress in a MDR isolates that carried the wild-type katG, inhA, and oxyR-ahpC associated with INH resistance than in those that carried mutations. The expression levels of 11 genes (efpA, Rv0849, Rv1250, P55 (Rv1410c), Rv1634, Rv2994, stp, Rv2459, pstB, drrA, and drrB) without drug inducement were significantly higher (P < 0.05) in nine MDR isolates than in 10 pan-sensitive isolates. In conclusion, efflux pumps may play an important role in INH acquired resistance in MDR M. tuberculosis, especially in those strains having no mutations in genes associated with INH resistance; basal expression levels of some efflux pump genes are higher in MDR isolates than in pan-sensitive isolates and the basal expressional differences may be helpful to diagnose and treat resistant tuberculosis.     

Draft Genome Sequences of Two Neisseria meningitidis Serogroup C Clinical Isolates

Neisseria meningitidis is a human-specific pathogen known for its capability to cause sepsis and meningitis. Here we report the availability of 2 draft genome sequences obtained from patients infected during the same epidemic outbreak. Both bacterial isolates belong to serogroup C, but their genome sequences show local and remarkable differences compared with each other or with the reference genome of strain FAM18.Neisseria meningitidis is found as a commensal organism of the human nasopharynx in 8 to 25% of the adult population (9), but sporadically, it is able to cross the mucosa and reach the bloodstream, causing severe septicemia and meningitis. Even though the reasons triggering these pathogenic outbreaks are not well understood, several factors related either to the host or the bacterium have been proposed 3, 8).So far, complete genome sequences for N. meningitidis serogroups A (strain Z2491 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AL157959","term_id":"30407145","term_text":"AL157959"}}AL157959]) (4), B (strain MC58 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AE002098","term_id":"66731897","term_text":"AE002098"}}AE002098]) (10), and C (strains FAM18, 8013, and 053442 [GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM421808","term_id":"120865607","term_text":"AM421808"}}AM421808, {"type":"entrez-nucleotide","attrs":{"text":"FM999788","term_id":"261391559","term_text":"FM999788"}}FM999788, and {"type":"entrez-nucleotide","attrs":{"text":"CP000381","term_id":"161594571","term_text":"CP000381"}}CP000381, respectively]) (1, 5, 6) have been reported, together with the unencapsulated strain α14 (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AM889136","term_id":"254667570","term_text":"AM889136"}}AM889136) (7). Here we announce the availability of 2 draft genome sequences for N. meningitidis serogroup C, strains K1207 and S0108, isolated from the same epidemic cluster which occurred in the Veneto region in northern Italy during the 2007-2008 winter (2).The genomes were sequenced using 454 pyrosequencing (Roche), combining shotgun and 30-kb paired-end strategies, according to the manufacturer''s recommendations. The coverage was nearly 27×, and assemblies were performed with Newbler. We obtained 223 and 226 contigs for the 2 genomes, which were finally mapped in 17 and 16 scaffolds, respectively. From both samples, we also isolated a 7-kb plasmid, whose sequence was nearly identical to that of pJS-B, already available in GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"NC_004758","term_id":"30250494","term_text":"NC_004758"}}NC_004758).The first analysis was performed by comparing sequences of the two isolates with the most similar complete genome available, strain FAM18. This analysis showed that the genome lengths were almost identical (about 2.2 Mb) and GC contents were comparable (51.91% in both isolates versus 51.62% of strain FAM18). Then, to identify potential differences in coding sequence content, the contigs obtained for both isolates were aligned with those for strain FAM18 using MEGABLAST (11) and LASTZ tools, which showed that in the genomes of the two N. meningitidis isolates, several genes were missing or nonfunctional because of the presence of insertions or deletions. For example, a couple of FAM18 outer membrane proteins (NMC0214 and NMC0215) were completely missing in both genomes, due to a 3-kb deletion, and no homologues were present in other genomic regions.Sequences that did not map on the genome of strain FAM18 were investigated by performing a BLAST analysis on a nonredundant database. Interestingly, besides genes or partial genes belonging to the other completely sequenced N. meningitidis serogroup C strain 053442, the genomes of our isolates contained coding sequences from N. meningitidis serogroups A and B, from other Neisseria species, such as N. gonorrhoeae, N. cinerea, and N. mucosa, and even from other bacterial species, such as cobyrinic acid ac-diamide synthase from Shewanella baltica, attesting once more to the great capability of horizontal gene transfer, which is peculiar to this microorganism.A detailed report of our two isolates will be included in a future publication, with the results of a full comparative analysis between the genomes.     

Draft Genome Sequences of Two Campylobacter jejuni Clinical Isolates,NW and D2600

The Campylobacter jejuni human clinical isolates NW and D2600 colonized C57BL/6 interleukin-10-deficient (IL-10^−/−) mice without inducing a robust inflammatory response (J. A. Bell et al., BMC Microbiol. 9:57, 2009). We announce draft genome sequences of NW and D2600 to facilitate comparisons with strains that induce gastrointestinal inflammation in this mouse model.     

Nucleotide Polymorphism Associated with Ethambutol Resistance in Clinical Isolates of Mycobacterium tuberculosis

Ethambutol (EMB) is a first-line drug used for antitubercular therapy in combination with other drugs as recommended by World Health Organization DOTS/DOTS-Plus regimens. EMB is also effective in the treatment of opportunistic mycobacterial infections in patients with human immunodeficiency virus. The emb locus has been considered as a drug target for EMB, and substitutions of codon 306 in Mycobacterium tuberculosis gene embB have been shown to be the most frequent and predictive mutations for EMB resistance. The aim of the present study was to detect embB and embC gene mutations in EMB-resistant clinical isolates. A total of 23 isolates of M. tuberculosis from patients with pulmonary tuberculosis were included in the study. Drug sensitivity was tested by proportion method and E-test. All 23 isolates were EMB resistant. Primers to amplify the embB and embC gene were designed, and polymerase chain reaction products were subjected for sequence analysis. H37Rv standard laboratory strain was used as control. Nucleotide sequencing showed that 16 strains had a mutation in the embB gene. The most common mutation observed in the embB gene was at codon 306, followed by mutations at codons 299 and 378 in 4 and 2 isolates, respectively. Novel mutations have been reported at codons 239, 240, 247, 282, 311, 368, 397, 446, 469, and 471. Sequence analysis of the embC gene showed mutation in 8 isolates at codon 270. Novel mutations in embC have been reported at codons 251 and 254. The most common nucleotide polymorphism in our isolates was at codons 306 and 299 in the embB gene and at codon 270 in the embC gene. A mutation at codon 306 was usually associated with high-level ethambutol resistance.     

Modern and ancestral genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India

Traditionally, the distribution of the Mycobacterium tuberculosis genotypes in India has been characterized by widespread prevalence of ancestral lineages (TbD1+ strains and variants) in the south and the modern forms (TbD1(-) CAS and variants) predominating in the north of India. The pattern was, however, not clearly known in the south-central region such as Hyderabad and the rest of the state of Andhra Pradesh where the prevalence of both tuberculosis (TB) and human immunodeficiency virus (HIV) infection is one of the highest in the country; this area has been the hotspot of TB vaccine trials. Spoligotyping of 101 clinical isolates obtained from Hyderabad and rural Andhra Pradesh confirmed the occurrence of major genogroups such as the ancestral (or the TbD1+ type or the East African Indian (EAI) type), the Central Asian (CAS) or Delhi type and the Beijing lineage in Andhra Pradesh. Sixty five different spoligotype patterns were observed for the isolates included in this study; these were further analyzed based on specific genetic signatures/mutations. It was found that the major genogroups, CAS and "ancestral," were almost equally prevalent in our collection but followed a north-south compartmentalization as was also reported previously. However, we observed a significant presence of MANU lineage in south Andhra Pradesh, which was earlier reported to be overwhelmingly present in Mumbai. This study portrays genotypic diversity of M. tuberculosis from the Indian state of Andhra Pradesh and provides a much needed snapshot of the strain diversity that will be helpful in devising effective TB control programs in this part of the world.     

Clinical Significance of Mycobacterium kansasii Isolates from Respiratory Specimens

The clinical significance of Mycobacterium kansasii respiratory isolates is uncertain. The aims of this study were to determine the clinical relevance of M. kansasii isolates and to identify the clinical features and outcomes of M. kansasii lung disease. We reviewed the medical records of 104 patients from whom at least one respiratory M. kansasii isolate was obtained from January 2003 to July 2014 at Samsung Medical Center, South Korea. Of these 104 patients, 54 (52%) met the diagnostic criteria for nontuberculous mycobacterial lung disease; among them, 41 (76%) patients received antibiotic treatment for a median time of 15.0 months (interquartile range [IQR], 7.0–18.0 months). The remaining 13 (24%) without overt disease progression were observed for a median period of 24.0 months (IQR, 5.0–34.5 months). Patients with M. kansasii lung disease exhibited various radiographic findings of lung disease, including the fibrocavitary form (n = 24, 44%), the nodular bronchiectatic form (n = 17, 32%), and an unclassifiable form (n = 13, 24%). The fibrocavitary form was more common in patients who received treatment (n = 23, 56%), while the nodular bronchiectatic form was more common in patients with M. kansasii lung disease who did not receive treatment (n = 9, 70%). None of the patients with a single sputum isolate (n = 18) developed M. kansasii disease over a median follow-up period of 12.0 months (IQR, 4.0–26.5 months). In total, 52% of all patients with M. kansasii respiratory isolates exhibited clinically significant disease. Moreover, patients with M. kansasii lung disease displayed diverse radiographic findings in addition to the fibrocavitary form. The nodular bronchiectatic form was more common in patients with M. kansasii lung disease with an indolent clinical course. Thus, since the clinical significance of a single M. kansasii respiratory isolate is not definite, strict adherence to recommended diagnostic criteria is advised.     

Aerobic reduction of perchlorate by bacteria isolated in Kerala, South India

Possibility of perchlorate reduction by microbes raises hope for an eco-friendly mode of degradation of this toxic rocket fuel. This study reports 3 isolates (A1, A2 and A3) capable of molybdenum-independent degradation of perchlorate under aerobic conditions. The rate of degradation was the highest when perchlorate concentration was 17 mM, and then 3.2 mM, 4.7 mM and 4.1 mM of perchlorate was reduced by isolates A1, A2 and A3 (respectively) after 72 h at 28°C under aerobic conditions. Presence of perchlorate at a concentration higher than 17 mM resulted in some inhibition of perchlorate reduction. 16S ribosomal RNA gene analysis revealed isolate A1 to bePseudomonas stutzeri (Proteobacteria) while isolates A2 ad A3 where found to belong to the genusArthrobacter (Actinobacteria). The study, apart from demonstrating ribotyping as a rapid method of identification of economically important soil microbes, also raised prospects for using artificial consortia for environmental degradation of perchlorate, without apparent domination ofDechloromonas spp. (a group of microbes known for perchlorate remediation in the environment).     

Two new species of freshwater Ostracoda of the genus Parastenocypris Hartmann, 1964 from Kerala,India

Parastenocypris goddeerisi n.sp. and P. achandii n.sp. are described from temporary habitats in Kerala, southern India. The morphology of both males and females is illustrated and the new species are compared to the known representatives of the genus. Stenocypris fernandoi Neale, described from Sri Lanka, is here referred to Parastenocypris.     

Detection of Mutations in pncA in Mycobacterium tuberculosis Clinical Isolates from Nepal in Association with Pyrazinamide Resistance

Without the proper information on pyrazinamide (PZA) susceptibility of Mycobacterium tuberculosis (MTB), PZA is inappropriately recommended for the treatment of both susceptible and multidrug-resistant tuberculosis (MDR-TB) in Nepal. This study aimed to collect information regarding PZA susceptibility in MTB isolates from Nepal by analyzing pncA and its upstream regulatory region (URR). A total of 211 MTB isolates were included in this study. Sequence analysis of pncA and its URR was performed to assess PZA resistance. First-line drug susceptibility testing, spoligotyping, and sequence analysis of rpoB, katG, the inhA regulatory region, gyrA, gyrB, and rrs were performed to assess their association with pncA mutation. Sequencing results reveal that 125 (59.2%) isolates harbored alterations in pncA and its URR. A total of 57 different mutation types (46 reported and 11 novel) were scattered throughout the whole length of the pncA gene. Eighty-seven isolates (41.2%) harbored mutations in pncA, causing PZA resistance in MTB. There was a more significant association of pncA alterations in MDR/pre-extensively drug-resistant (Pre-XDR) TB than in mono-resistant/pan-susceptible TB (p < 0.005). This first report on the increasing level of PZA resistance in DR-TB in Nepal highlights the importance of PZA susceptibility testing before DR-TB treatment.     

Population Structure among Mycobacterium tuberculosis Isolates from Pulmonary Tuberculosis Patients in Colombia

Background

Phylogeographic composition of M. tuberculosis populations reveals associations between lineages and human populations that might have implications for the development of strategies to control the disease. In Latin America, lineage 4 or the Euro-American, is predominant with considerable variations among and within countries. In Colombia, although few studies from specific localities have revealed differences in M. tuberculosis populations, there are still areas of the country where this information is lacking, as is a comparison of Colombian isolates with those from the rest of the world.

Principal Findings

A total of 414 M. tuberculosis isolates from adult pulmonary tuberculosis cases from three Colombian states were studied. Isolates were genotyped using IS6110-restriction fragment length polymorphism (RFLP), spoligotyping, and 24-locus Mycobacterial interspersed repetitive units variable number tandem repeats (MIRU-VNTRs). SIT42 (LAM9) and SIT62 (H1) represented 53.3% of isolates, followed by 8.21% SIT50 (H3), 5.07% SIT53 (T1), and 3.14% SIT727 (H1). Composite spoligotyping and 24-locus MIRU- VNTR minimum spanning tree analysis suggest a recent expansion of SIT42 and SIT62 evolved originally from SIT53 (T1). The proportion of Haarlem sublineage (44.3%) was significantly higher than that in neighboring countries. Associations were found between M. tuberculosis MDR and SIT45 (H1), as well as HIV-positive serology with SIT727 (H1) and SIT53 (T1).

Conclusions

This study showed the population structure of M. tuberculosis in several regions from Colombia with a dominance of the LAM and Haarlem sublineages, particularly in two major urban settings (Medellín and Cali). Dominant spoligotypes were LAM9 (SIT 42) and Haarlem (SIT62). The proportion of the Haarlem sublineage was higher in Colombia compared to that in neighboring countries, suggesting particular conditions of co-evolution with the corresponding human population that favor the success of this sublineage.     

Characterization of the Genetic Diversity of Extensively-Drug Resistant Mycobacterium tuberculosis Clinical Isolates from Pulmonary Tuberculosis Patients in Peru

Background

Peru holds the fourth highest burden of tuberculosis in the Americas. Despite an apparently well-functioning DOTS control program, the prevalence of multidrug resistant tuberculosis (MDR-TB) continues to increase. To worsen this situation, cases of extensively drug resistance tuberculosis (XDR-TB) have been detected. Little information exists about the genetic diversity of drug-susceptible vs. MDR-TB and XDR-TB.

Methods

Cryopreserved samples of XDR strains from 2007 to 2009 (second semester), were identified and collected. Starting from 227 frozen samples, a total of 142 XDR-TB strains of Mycobacterium tuberculosis complex (MTBC; 1 isolate per patient) were retained for this study. Each strain DNA was analyzed by spoligotyping and the 15-loci Mycobacterial Interspersed Repetitive Unit (MIRU-15).

Results

Among the 142 isolates analyzed, only 2 samples (1.41%) could not be matched to any lineage. The most prevalent sublineage was Haarlem (43.66%), followed by T (27.46%), LAM (16.2%), Beijing (9.15%), and X clade (1.41%). Spoligotype analysis identified clustering for 128/142 (90.1%) isolates vs. 49/142 (34.5%) with MIRUs. Of the samples, 90.85% belonged to retreated patients. The drug resistant profile demonstrated that 62.67% showed resistance to injectable drugs capreomycin (CAP) and kanamycin (KAN) vs. 15.5% to CAP alone and 21.8% to KAN alone. The SIT219/T1 and SIT50/H3 were the most prevalent patterns in our study. The spoligoforest analysis showed that SIT53/T1 was at the origin of many of the T lineage strains as well as a big proportion of Haarlem lineage strains (SIT50/H3, followed by SIT47/H1, SIT49/H3, and SIT2375/H1), as opposed to the SIT1/Beijing strains that did not appear to evolve into minor Beijing sublineages among the XDR-TB strains.

Conclusion

In contrast with other Latin-American countries where LAM sublineage is the most predominant, we found the Haarlem to be the most common followed by T sublineage among the XDR-TB strains.     

中国耐多药结核分枝杆菌临床分离株rpoB基因突变特点

为阐明中国耐多药结核分枝杆菌临床分离株rpoB基因的突变特征,对86株结核分枝杆菌临床分离株rpoB基因两个区域,包括81个碱基利福平抗药性决定区(rifampin resistance determining region,RRDR)和V176F区进行序列测定。其中72株耐多药分离株中的65株rpoB基因的RRDR区存在22种不同类型突变、21种点突变和一个插入突变。最常见的突变部位分别位于密码子531(41％)、526(40％)和516(4％),10％耐药分离株未检测到突变。鉴定了RRDR内6个新的等位基因,以及RRDR外部区域5个新的突变。所有分离菌株V176均无突变。     

Multilocus Sequence Typing of Clinical Isolates of Cryptococcus from India

Mycopathologia - Cryptococcosis is a life-threatening infection caused by Cryptococcus neoformans and C. gattii species complex. In the present study, to understand the molecular epidemiology of...