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1.  Genotypic and Phenotypic Profiles of Escherichia coli Isolates Belonging to Clinical Sequence Type 131 (ST131), Clinical Non-ST131, and Fecal Non-ST131 Lineages from India 
Antimicrobial Agents and Chemotherapy  2014;58(12):7240-7249.
In view of the epidemiological success of CTX-M-15-producing lineages of Escherichia coli and particularly of sequence type 131 (ST131), it is of significant interest to explore its prevalence in countries such as India and to determine if antibiotic resistance, virulence, metabolic potential, and/or the genetic architecture of the ST131 isolates differ from those of non-ST131 isolates. A collection of 126 E. coli isolates comprising 43 ST131 E. coli, 40 non-ST131 E. coli, and 43 fecal E. coli isolates collected from a tertiary care hospital in India was analyzed. These isolates were subjected to enterobacterial repetitive intergenic consensus (ERIC)-based fingerprinting, O typing, phylogenetic grouping, antibiotic sensitivity testing, and virulence and antimicrobial resistance gene (VAG) detection. Representative isolates from this collection were also analyzed by multilocus sequence typing (MLST), conjugation, metabolic profiling, biofilm production assay, and zebra fish lethality assay. All of the 43 ST131 E. coli isolates were exclusively associated with phylogenetic group B2 (100%), while most of the clinical non-ST131 and stool non-ST131 E. coli isolates were affiliated with the B2 (38%) and A (58%) phylogenetic groups, respectively. Significantly greater proportions of ST131 isolates (58%) than non-ST131 isolates (clinical and stool E. coli isolates, 5% each) were technically identified to be extraintestinal pathogenic E. coli (ExPEC). The clinical ST131, clinical non-ST131, and stool non-ST131 E. coli isolates exhibited high rates of multidrug resistance (95%, 91%, and 91%, respectively), extended-spectrum-β-lactamase (ESBL) production (86%, 83%, and 91%, respectively), and metallo-β-lactamase (MBL) production (28%, 33%, and 0%, respectively). CTX-M-15 was strongly linked with ESBL production in ST131 isolates (93%), whereas CTX-M-15 plus TEM were present in clinical and stool non-ST131 E. coli isolates. Using MLST, we confirmed the presence of two NDM-1-positive ST131 E. coli isolates. The aggregate bioscores (metabolite utilization) for ST131, clinical non-ST131, and stool non-ST131 E. coli isolates were 53%, 52%, and 49%, respectively. The ST131 isolates were moderate biofilm producers and were more highly virulent in zebra fish than non-ST131 isolates. According to ERIC-based fingerprinting, the ST131 strains were more genetically similar, and this was subsequently followed by the genetic similarity of clinical non-ST131 and stool non-ST131 E. coli strains. In conclusion, our data provide novel insights into aspects of the fitness advantage of E. coli lineage ST131 and suggest that a number of factors are likely involved in the worldwide dissemination of and infections due to ST131 E. coli isolates.
PMCID: PMC4249578  PMID: 25246402
2.  Tuberculosis Biliary Stricture Simulating as Cholangiocarcinoma 
PMCID: PMC4413116  PMID: 25954666
Biliary stricture; Obstructive jaundice; Polymerase chain ction (PCR); Tuberculosis
3.  Metastatic Amelanotic Melanoma with Occult Primary Masquerading as Sarcoma 
Malignant melanoma with occult primary is extremely rare. It is found that survival is almost same or even better than the melanomas with known primary site. Surgeons should have a high index of suspicion when a patient presents like sarcoma which bleeds profusely when planning for excision. Here, is an unusual case of young adult which presented initially with granulomatous lymphandenitis in axilla with primary suspicion of tuberculosis, later turning out to be sarcoma on FNAC and MRI. On immunochemistry (IHC), the final diagnosis of amelanotic melanoma was made and further workup did not show up any primary site of origin.
PMCID: PMC4378792  PMID: 25859510
FNAC; Granulomatous lymphadenitis; Sarcoma
4.  Genome dynamics and evolution of Salmonella Typhi strains from the typhoid-endemic zones 
Scientific Reports  2014;4:7457.
Typhoid fever poses significant burden on healthcare systems in Southeast Asia and other endemic countries. Several epidemiological and genomic studies have attributed pseudogenisation to be the major driving force for the evolution of Salmonella Typhi although its real potential remains elusive. In the present study, we analyzed genomes of S. Typhi from different parts of Southeast Asia and Oceania, comprising of isolates from outbreak, sporadic and carrier cases. The genomes showed high genetic relatedness with limited opportunity for gene acquisition as evident from pan-genome structure. Given that pseudogenisation is an active process in S. Typhi, we further investigated core and pan-genome profiles of functional and pseudogenes separately. We observed a decline in core functional gene content and a significant increase in accessory pseudogene content. Upon functional classification, genes encoding metabolic functions formed a major constituent of pseudogenes as well as core functional gene clusters with SNPs. Further, an in-depth analysis of accessory pseudogene content revealed the existence of heterogeneous complements of functional and pseudogenes among the strains. In addition, these polymorphic genes were also enriched in metabolism related functions. Thus, the study highlights the existence of heterogeneous strains in a population with varying metabolic potential and that S. Typhi possibly resorts to metabolic fine tuning for its adaptation.
PMCID: PMC4264004  PMID: 25504040
5.  Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution 
Nucleic Acids Research  2014;43(1):324-335.
The discordant prevalence of Helicobacter pylori and its related diseases, for a long time, fostered certain enigmatic situations observed in the countries of the southern world. Variation in H. pylori infection rates and disease outcomes among different populations in multi-ethnic Malaysia provides a unique opportunity to understand dynamics of host–pathogen interaction and genome evolution. In this study, we extensively analyzed and compared genomes of 27 Malaysian H. pylori isolates and identified three major phylogeographic lineages: hspEastAsia, hpEurope and hpSouthIndia. The analysis of the virulence genes within the core genome, however, revealed a comparable pathogenic potential of the strains. In addition, we identified four genes limited to strains of East-Asian lineage. Our analyses identified a few strain-specific genes encoding restriction modification systems and outlined 311 core genes possibly under differential evolutionary constraints, among the strains representing different ethnic groups. The cagA and vacA genes also showed variations in accordance with the host genetic background of the strains. Moreover, restriction modification genes were found to be significantly enriched in East-Asian strains. An understanding of these variations in the genome content would provide significant insights into various adaptive and host modulation strategies harnessed by H. pylori to effectively persist in a host-specific manner.
PMCID: PMC4288169  PMID: 25452339
6.  Genomes of Two Clinical Isolates of Mycobacterium tuberculosis from Odisha, India 
Genome Announcements  2014;2(2):e00199-14.
We report whole-genome sequences of two clinical isolates of Mycobacterium tuberculosis isolated from patients in Odisha, India. The sequence analysis revealed that these isolates are of an ancestral type and might represent some of the “pristine” isolates in India that have not admixed with other lineages.
PMCID: PMC3961728  PMID: 24652981
7.  Genome anatomy of the gastrointestinal pathogen, Vibrio parahaemolyticus of crustacean origin 
Gut Pathogens  2013;5:37.
Vibrio parahaemolyticus, an important human pathogen, is associated with gastroenteritis and transmitted through partially cooked seafood. It has become a major concern in the production and trade of marine food products. The prevalence of potentially virulent and pathogenic V. parahaemolyticus in raw seafood is of public health significance. Here we describe the genome sequence of a V. parahaemolyticus isolate of crustacean origin which was cultured from prawns in 2008 in Selangor, Malaysia (isolate PCV08-7). The next generation sequencing and analysis revealed that the genome of isolate PCV08-7 has closest similarity to that of V. parahaemolyticus RIMD2210633. However, there are certain unique features of the PCV08-7 genome such as the absence of TDH-related hemolysin (TRH), and the presence of HU-alpha insertion. The genome of isolate PCV08-7 encodes a thermostable direct hemolysin (TDH), an important virulence factor that classifies PCV08-7 isolate to be a serovariant of O3:K6 strain. Apart from these, we observed that there is certain pattern of genetic rearrangements that makes V. parahaemolyticus PCV08-7 a non-pandemic clone. We present detailed genome statistics and important genetic features of this bacterium and discuss how its survival, adaptation and virulence in marine and terrestrial hosts can be understood through the genomic blueprint and that the availability of genome sequence entailing this important Malaysian isolate would likely enhance our understanding of the epidemiology, evolution and transmission of foodborne Vibrios in Malaysia and elsewhere.
PMCID: PMC4029742  PMID: 24330647
Vibrio parahaemolyticus; Genomics; Malaysia; Seafood; Comparative genomics
8.  Multiresistant Uropathogenic Escherichia coli from a Region in India Where Urinary Tract Infections Are Endemic: Genotypic and Phenotypic Characteristics of Sequence Type 131 Isolates of the CTX-M-15 Extended-Spectrum-β-Lactamase-Producing Lineage 
Antimicrobial Agents and Chemotherapy  2012;56(12):6358-6365.
Escherichia coli sequence type 131 (O25b:H4), associated with the CTX-M-15 extended-spectrum beta-lactamases (ESBLs) and linked predominantly to the community-onset antimicrobial-resistant infections, has globally emerged as a public health concern. However, scant attention is given to the understanding of the molecular epidemiology of these strains in high-burden countries such as India. Of the 100 clinical E. coli isolates obtained by us from a setting where urinary tract infections are endemic, 16 ST131 E. coli isolates were identified by multilocus sequence typing (MLST). Further, genotyping and phenotyping methods were employed to characterize their virulence and drug resistance patterns. All the 16 ST131 isolates harbored the CTX-M-15 gene, and half of them also carried TEM-1; 11 of these were positive for blaOXA groups 1 and 12 for aac(6′)-Ib-cr. At least 12 isolates were refractory to four non-beta-lactam antibiotics: ciprofloxacin, gentamicin, sulfamethoxazole-trimethoprim, and tetracycline. Nine isolates carried the class 1 integron. Plasmid analysis indicated a large pool of up to six plasmids per strain with a mean of approximately three plasmids. Conjugation and PCR-based replicon typing (PBRT) revealed that the spread of resistance was associated with the FIA incompatibility group of plasmids. Pulsed-field gel electrophoresis (PFGE) and genotyping of the virulence genes showed a low level of diversity among these strains. The association of ESBL-encoding plasmid with virulence was demonstrated in transconjugants by serum assay. None of the 16 ST131 ESBL-producing E. coli strains were known to synthesize carbapenemase enzymes. In conclusion, our study reports a snapshot of the highly virulent/multiresistant clone ST131 of uropathogenic E. coli from India. This study suggests that the ST131 genotypes from this region are clonally evolved and are strongly associated with the CTX-M-15 enzyme, carry a high antibiotic resistance background, and have emerged as an important cause of community-acquired urinary tract infections.
PMCID: PMC3497203  PMID: 23045357
9.  Genome Sequence and Comparative Pathogenomics Analysis of a Salmonella enterica Serovar Typhi Strain Associated with a Typhoid Carrier in Malaysia 
Journal of Bacteriology  2012;194(21):5970-5971.
Salmonella enterica serovar Typhi is a human pathogen that causes typhoid fever predominantly in developing countries. In this article, we describe the whole genome sequence of the S. Typhi strain CR0044 isolated from a typhoid fever carrier in Kelantan, Malaysia. These data will further enhance the understanding of its host persistence and adaptive mechanism.
PMCID: PMC3486090  PMID: 23045488
10.  Next-Generation Sequencing and De Novo Assembly, Genome Organization, and Comparative Genomic Analyses of the Genomes of Two Helicobacter pylori Isolates from Duodenal Ulcer Patients in India 
Journal of Bacteriology  2012;194(21):5963-5964.
The prevalence of different H. pylori genotypes in various geographical regions indicates region-specific adaptations during the course of evolution. Complete genomes of H. pylori from countries with high infection burdens, such as India, have not yet been described. Herein we present genome sequences of two H. pylori strains, NAB47 and NAD1, from India. In this report, we briefly mention the sequencing and finishing approaches, genome assembly with downstream statistics, and important features of the two draft genomes, including their phylogenetic status. We believe that these genome sequences and the comparative genomics emanating thereupon will help us to clearly understand the ancestry and biology of the Indian H. pylori genotypes, and this will be helpful in solving the so-called Indian enigma, by which high infection rates do not corroborate the minuscule number of serious outcomes observed, including gastric cancer.
PMCID: PMC3486096  PMID: 23045484
11.  Insights from the Genome Sequence of a Salmonella enterica Serovar Typhi Strain Associated with a Sporadic Case of Typhoid Fever in Malaysia 
Journal of Bacteriology  2012;194(18):5124-5125.
Salmonella enterica serovar Typhi is the causative agent of typhoid fever, which causes nearly 21.7 million illnesses and 217,000 deaths globally. Herein, we describe the whole-genome sequence of the Salmonella Typhi strain ST0208, isolated from a sporadic case of typhoid fever in Kuala Lumpur, Malaysia. The whole-genome sequence and comparative genomics allow an in-depth understanding of the genetic diversity, and its link to pathogenicity and evolutionary dynamics, of this highly clonal pathogen that is endemic to Malaysia.
PMCID: PMC3430317  PMID: 22933756
12.  Whole-Genome Sequences and Comparative Genomics of Salmonella enterica Serovar Typhi Isolates from Patients with Fatal and Nonfatal Typhoid Fever in Papua New Guinea 
Journal of Bacteriology  2012;194(18):5122-5123.
Many of the developing countries of the Southeast Asian region are significantly affected by endemic typhoid fever, possibly as a result of marginal living standards. It is an important public health problem in countries such as Papua New Guinea, which is geographically close to some of the foci of endemicity in Asia. The severity of the disease varies in different regions, and this may be attributable to genetic diversity among the native strains. Genome sequence data on strains from different countries are needed to clearly understand their genetic makeup and virulence potential. We describe the genomes of two Salmonella Typhi isolates from patients with fatal and nonfatal cases of typhoid fever in Papua New Guinea. We discuss in brief the underlying sequencing methodology, assembly, genome statistics, and important features of the two draft genomes, which form an essential step in our functional molecular infection epidemiology program centering on typhoid fever. The comparative genomics of these and other isolates would enable us to identify genetic rearrangements and mechanisms responsible for endemicity and the differential severity of pathogenic salmonellae in Papua New Guinea and elsewhere.
PMCID: PMC3430347  PMID: 22933755
13.  Genetic Fine Structure of a Salmonella enterica Serovar Typhi Strain Associated with the 2005 Outbreak of Typhoid Fever in Kelantan, Malaysia 
Journal of Bacteriology  2012;194(13):3565-3566.
Among enteric pathogens, Salmonella enterica serovar Typhi is responsible for the largest number of food-borne outbreaks and fatalities. The ability of the pathogen to cause systemic infection for extended durations leads to a high cost of disease control. Chronic carriers play important roles in the evolution of Salmonella Typhi; therefore, identification and in-depth characterization of isolates from clinical cases and carriers, especially those from zones of endemicity where the pathogen has not been extensively studied, are necessary. Here, we describe the genome sequence of the highly virulent Salmonella Typhi strain BL196/05 isolated during the outbreak of typhoid in Kelantan, Malaysia, in 2005. The whole-genome sequence and comparative genomics of this strain should enable us to understand the virulence mechanisms and evolutionary dynamics of this pathogen in Malaysia and elsewhere.
PMCID: PMC3434757  PMID: 22689247
14.  Genome sequencing and analysis of Salmonella enterica serovar Typhi strain CR0063 representing a carrier individual during an outbreak of typhoid fever in Kelantan, Malaysia 
Gut Pathogens  2012;4:20.
Salmonella Typhi is a human restricted pathogen with a significant number of individuals as asymptomatic carriers of the bacterium. Salmonella infection can be effectively controlled if a reliable method for identification of these carriers is developed. In this context, the availability of whole genomes of carrier strains through high- throughput sequencing and further downstream analysis by comparative genomics approaches is very promising. Herein we describe the genome sequence of a Salmonella Typhi isolate representing an asymptomatic carrier individual during a prolonged outbreak of typhoid fever in Kelantan, Malaysia. Putative genomic coordinates relevant in pathogenesis and persistence of this carrier strain are identified and discussed.
PMCID: PMC3528463  PMID: 23234298
15.  In vitro cytokine profiles and viability of different human cells treated with whole cell lysate of Mycobacterium avium subsp. paratuberculosis 
Gut Pathogens  2012;4:10.
Mycobacterium avium subsp. paratuberculosis (MAP) is a zoonotic pathogen, a very slow growing bacterium which is difficult to isolate and passage in conventional laboratory culture. Although its association with Johne’s disease or paratuberculosis of cattle is well established, it has been only putatively linked to Crohn’s disease in humans. Further, MAP has been recently suggested to be a trigger for other autoimmune diseases such as type-1 diabetes mellitus (T1DM). Recently, some studies have indicated that exposure to MAP is associated with elevated levels of antibodies against MAP lysate although the exact mechanism and significance of the same remains unclear. Further, the cytokine profiles relevant in MAP associated diseases of humans and their exact role in the pathophysiology are not clearly known. We performed in vitro cytokine analyses after exposing different cultured human cells to the whole cell lysate of MAP and found that MAP lysate induces secretion of cytokines IL-1β, IL-6, IL-8, IL-10 and TNF-α by human peripheral blood mononuclear cells (PBMCs). Also, it induces secretion of IL-8 by cultured human stomach adenocarcinoma cells (AGS) and PANC-1(human pancreatic carcinoma cell line) cells. We also found that MAP lysate induced cytotoxicity in PANC-1cells. Collectively, these results provide a much needed base-line data set of cytokines broadly signifying a MAP induced cellular response by human cells.
PMCID: PMC3495707  PMID: 23006537
16.  Massive gene acquisitions in Mycobacterium indicus pranii provide a perspective on mycobacterial evolution 
Nucleic Acids Research  2012;40(21):10832-10850.
Understanding the evolutionary and genomic mechanisms responsible for turning the soil-derived saprophytic mycobacteria into lethal intracellular pathogens is a critical step towards the development of strategies for the control of mycobacterial diseases. In this context, Mycobacterium indicus pranii (MIP) is of specific interest because of its unique immunological and evolutionary significance. Evolutionarily, it is the progenitor of opportunistic pathogens belonging to M. avium complex and is endowed with features that place it between saprophytic and pathogenic species. Herein, we have sequenced the complete MIP genome to understand its unique life style, basis of immunomodulation and habitat diversification in mycobacteria. As a case of massive gene acquisitions, 50.5% of MIP open reading frames (ORFs) are laterally acquired. We show, for the first time for Mycobacterium, that MIP genome has mosaic architecture. These gene acquisitions have led to the enrichment of selected gene families critical to MIP physiology. Comparative genomic analysis indicates a higher antigenic potential of MIP imparting it a unique ability for immunomodulation. Besides, it also suggests an important role of genomic fluidity in habitat diversification within mycobacteria and provides a unique view of evolutionary divergence and putative bottlenecks that might have eventually led to intracellular survival and pathogenic attributes in mycobacteria.
PMCID: PMC3505973  PMID: 22965120
17.  Mycobacterium tuberculosis DosR Regulon Gene Rv0079 Encodes a Putative, ‘Dormancy Associated Translation Inhibitor (DATIN)’ 
PLoS ONE  2012;7(6):e38709.
Mycobacterium tuberculosis is a major human pathogen that has evolved survival mechanisms to persist in an immune-competent host under a dormant condition. The regulation of M. tuberculosis metabolism during latent infection is not clearly known. The dormancy survival regulon (DosR regulon) is chiefly responsible for encoding dormancy related functions of M. tuberculosis. We describe functional characterization of an important gene of DosR regulon, Rv0079, which appears to be involved in the regulation of translation through the interaction of its product with bacterial ribosomal subunits. The protein encoded by Rv0079, possibly, has an inhibitory role with respect to protein synthesis, as revealed by our experiments. We performed computational modelling and docking simulation studies involving the protein encoded by Rv0079 followed by in vitro translation and growth curve analysis experiments, involving recombinant E. coli and Bacille Calmette Guérin (BCG) strains that overexpressed Rv0079. Our observations concerning the interaction of the protein with the ribosomes are supportive of its role in regulation/inhibition of translation. We propose that the protein encoded by locus Rv0079 is a ‘dormancy associated translation inhibitor’ or DATIN.
PMCID: PMC3374827  PMID: 22719925
19.  Genome of a Novel Isolate of Paracoccus denitrificans Capable of Degrading N,N-Dimethylformamide 
Journal of Bacteriology  2011;193(19):5598-5599.
The bacterial genus Paracoccus is comprised of metabolically versatile organisms having diverse degradative capabilities and potential industrial and environmental applications for bioremediation in particular. We report a de novo-assembled sequence and annotation of the genome of a novel isolate of Paracoccus denitrificans originally sourced from coal mine tailings in India. The isolate was capable of utilizing N,N-dimethylformamide (DMF) as a source of carbon and nitrogen and therefore holds potential for bioremediation and mineralization of industrial pollutants. The genome sequence and biological circuitry revealed thereupon will be invaluable in understanding the metabolic capabilities, functioning, and evolution of this important bacterial organism.
PMCID: PMC3187436  PMID: 21914898
20.  Genome of Multidrug-Resistant Uropathogenic Escherichia coli Strain NA114 from India ▿ 
Journal of Bacteriology  2011;193(16):4272-4273.
Uropathogenic Escherichia coli (UPEC) causes serious infections in people at risk and has a significant environmental prevalence due to contamination by human and animal excreta. In developing countries, UPEC assumes importance in certain dwellings because of poor community/personal hygiene and exposure to contaminated water or soil. We report the complete genome sequence of E. coli strain NA114 from India, a UPEC strain with a multidrug resistance phenotype and the capacity to produce extended-spectrum beta-lactamase. The genome sequence and comparative genomics emanating from it will be significant in under-standing the genetic makeup of diverse UPEC strains and in boosting the development of new diagnostics/vaccines.
PMCID: PMC3147708  PMID: 21685291
21.  Modern and Ancestral Genotypes of Mycobacterium tuberculosis from Andhra Pradesh, India 
PLoS ONE  2011;6(11):e27584.
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.
PMCID: PMC3219672  PMID: 22114678
22.  Comparison of Two Multilocus Sequence Based Genotyping Schemes for Leptospira Species 
Several sequence based genotyping schemes have been developed for Leptospira spp. The objective of this study was to genotype a collection of clinical and reference isolates using the two most commonly used schemes and compare and contrast the results.
Methods and Findings
A total of 48 isolates consisting of L. interrogans (n = 40) and L. kirschneri (n = 8) were typed by the 7 locus MLST scheme described by Thaipadungpanit et al., and the 6 locus genotyping scheme described by Ahmed et al., (termed 7L and 6L, respectively). Two L. interrogans isolates were not typed using 6L because of a deletion of three nucleotides in lipL32. The remaining 46 isolates were resolved into 21 sequence types (STs) by 7L, and 30 genotypes by 6L. Overall nucleotide diversity (based on concatenated sequence) was 3.6% and 2.3% for 7L and 6L, respectively. The D value (discriminatory ability) of 7L and 6L were comparable, i.e. 92.0 (95% CI 87.5–96.5) vs. 93.5 (95% CI 88.6–98.4). The dN/dS ratios calculated for each locus indicated that none were under positive selection. Neighbor joining trees were reconstructed based on the concatenated sequences for each scheme. Both trees showed two distinct groups corresponding to L. interrogans and L. kirschneri, and both identified two clones containing 10 and 7 clinical isolates, respectively. There were six instances in which 6L split single STs as defined by 7L into closely related clusters. We noted two discrepancies between the trees in which the genetic relatedness between two pairs of strains were more closely related by 7L than by 6L.
This genetic analysis indicates that the two schemes are comparable. We discuss their practical advantages and disadvantages.
Author Summary
Two independent multilocus sequence based genotyping schemes (denoted here as 7L and 6L for schemes with 7 and 6 loci, respectively) are in use for Leptospira spp., which has led to uncertainty as to which should be adopted by the scientific community. The purpose of this study was to apply the two schemes to a single collection of pathogenic Leptospira, evaluate their performance, and describe the practical advantages and disadvantages of each scheme. We used a variety of phylogenetic approaches to compare the output data and found that the two schemes gave very similar results. 7L has the advantage that it is a conventional multi-locus sequencing typing (MLST) scheme based on housekeeping genes and is supported by a publically accessible database by which genotypes can be readily assigned as known or new sequence types by any investigator, but is currently only applicable to L. interrogans and L. kirschneri. Conversely, 6L can be applied to all pathogenic Leptospira spp., but is not a conventional MLST scheme by design and is not available online. 6L sequences from 271 strains have been released into the public domain, and phylogenetic analysis of new sequences using this scheme requires their download and offline analysis.
PMCID: PMC3210738  PMID: 22087342
23.  Antibodies Recognizing Mycobacterium avium paratuberculosis Epitopes Cross-React with the Beta-Cell Antigen ZnT8 in Sardinian Type 1 Diabetic Patients 
PLoS ONE  2011;6(10):e26931.
The environmental factors at play in the pathogenesis of type 1 diabetes (T1D) remain enigmatic. Mycobacterium avium subspecies paratuberculosis (MAP) is transmitted from dairy herds to humans through food contamination. MAP causes an asymptomatic infection that is highly prevalent in Sardinian T1D patients compared with type 2 diabetes (T2D) and healthy controls. Moreover, MAP elicits humoral responses against several mycobacterial proteins. We asked whether antibodies (Abs) against one of these proteins, namely MAP3865c, which displays a sequence homology with the β-cell protein zinc transporter 8 (ZnT8) could be cross-reactive with ZnT8 epitopes. To this end, Ab responses against MAP3865c were analyzed in Sardinian T1D, T2D and healthy subjects using an enzymatic immunoassay. Abs against MAP3865c recognized two immunodominant transmembrane epitopes in 52–65% of T1D patients, but only in 5–7% of T2D and 3–5% of healthy controls. There was a linear correlation between titers of anti-MAP3865c and anti-ZnT8 Abs targeting these two homologous epitopes, and pre-incubation of sera with ZnT8 epitope peptides blocked binding to the corresponding MAP3865c peptides. These results demonstrate that Abs recognizing MAP3865c epitopes cross-react with ZnT8, possibly underlying a molecular mimicry mechanism, which may precipitate T1D in MAP-infected individuals.
PMCID: PMC3203182  PMID: 22046415
24.  Concurrent Proinflammatory and Apoptotic Activity of a Helicobacter pylori Protein (HP986) Points to Its Role in Chronic Persistence 
PLoS ONE  2011;6(7):e22530.
Helicobacter pylori induces cytokine mediated changes in gastroduodenal pathophysiology, wherein, the activated macrophages at the sub-mucosal space play a central role in mounting innate immune response against the antigens. The bacterium gains niche through persistent inflammation and local immune-suppression causing peptic ulcer disease or chronic gastritis; the latter being a significant risk factor for the development of gastric adenocarcinoma. What favors persistence of H. pylori in the gastric niches is not clearly understood. We report detailed characterization of a functionally unknown gene (HP986), which was detected in patient isolates associated with peptic ulcer and gastric carcinoma. Expression and purification of recombinant HP986 (rHP986) revealed a novel, ∼29 kDa protein in biologically active form which associates with significant levels of humoral immune responses in diseased individuals (p<0.001). Also, it induced significant levels of TNF-α and Interleukin-8 in cultured human macrophages concurrent to the translocation of nuclear transcription factor-κB (NF-κB). Further, the rHP986 induced apoptosis of cultured macrophages through a Fas mediated pathway. Dissection of the underlying signaling mechanism revealed that rHP986 induces both TNFR1 and Fas expression to lead to apoptosis. We further demonstrated interaction of HP986 with TNFR1 through computational and experimental approaches. Independent proinflammatory and apoptotic responses triggered by rHP986 as shown in this study point to its role, possibly as a survival strategy to gain niche through inflammation and to counter the activated macrophages to avoid clearance.
PMCID: PMC3137634  PMID: 21789261
25.  Genomes of Two Chronological Isolates (Helicobacter pylori 2017 and 2018) of the West African Helicobacter pylori Strain 908 Obtained from a Single Patient▿ 
Journal of Bacteriology  2011;193(13):3385-3386.
The diverse clinical outcomes of colonization by Helicobacter pylori reflect the need to understand the genomic rearrangements enabling the bacterium to adapt to host niches and exhibit varied colonization/virulence potential. We describe the genome sequences of the two serial isolates, H. pylori 2017 and 2018 (the chronological subclones of H. pylori 908), cultured in 2003 from the antrum and corpus, respectively, of an African patient who suffered from recrudescent duodenal ulcer disease. When compared with the genome of the parent strain, 908 (isolated from the antrum of the same patient in 1994), the genome sequences revealed genomic alterations relevant to virulence optimization or host-specific adaptation.
PMCID: PMC3133283  PMID: 21515762

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