Sabiha Shaik

Comparative genomic analysis of Helicobacter pylori from Malaysia identifies three distinct lineages suggestive of differential evolution

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.

Molecular Epidemiology and Genome Dynamics of New Delhi Metallo-β-Lactamase-Producing Extraintestinal Pathogenic Escherichia coli Strains from India

ABSTRACT The global dissemination and increasing incidence of carbapenem-resistant, Gram-negative organisms have resulted in acute public health concerns. Here, we present a retrospective multicenter study on molecular characterization of metallo-β-lactamase (MBL)-producing clinical Escherichia coli isolates recovered from extraintestinal infections in two hospitals in Pune, India. We screened a large sample size of 510 E. coli isolates for MBL production wherein we profiled their molecular determinants, antimicrobial resistance phenotypes, functional virulence properties, genomic features, and transmission dynamics. Approximately 8% of these isolates were MBL producers, the majority of which were of the NDM-1 (69%) type, followed by NDM-5 (19%), NDM-4 (5.5%), and NDM-7 (5.5%). MBL producers were resistant to all antibiotics tested except for colistin, fosfomycin, and chloramphenicol, which were effective to various extents. Plasmids were found to be an effective means of dissemination of NDM genes and other resistance traits. All MBL producers adhered to and invaded bladder epithelial (T24) cells and demonstrated significant serum resistance. Genomic analysis of MBL-producing E. coli isolates revealed higher resistance but a moderate virulence gene repertoire. A subset of NDM-1-positive E. coli isolates was identified as dominant sequence type 101 (ST101) while two strains belonging to ST167 and ST405 harbored NDM-5. A majority of MBL-producing E. coli strains revealed unique genotypes, suggesting that they were clonally unrelated. Overall, the coexistence of virulence and carbapenem resistance in clinical E. coli isolates is of serious concern. Moreover, the emergence of NDM-1 among the globally dominant E. coli ST101 isolates warrants stringent surveillance and control measures.

Genomic and Functional Portrait of a Highly Virulent, CTX-M-15-Producing H30-Rx Subclone of Escherichia coli Sequence Type 131

ABSTRACT Escherichia coli sequence type 131 (ST131) is a pandemic clone associated with multidrug-resistant, extraintestinal infections, attributable to the presence of the CTX-M-15 extended-spectrum β-lactamase gene and mutations entailing fluoroquinolone resistance. Studies on subclones within E. coli ST131 are critically required for targeting and implementation of successful control efforts. Our study comprehensively analyzed the genomic and functional attributes of the H30-Rx subclonal strains NA097 and NA114, belonging to the ST131 lineage. We carried out whole-genome sequencing, comparative analysis, phenotypic virulence assays, and profiling of the antibacterial responses of THP1 cells infected with these subclones. Phylogenomic analysis suggested that the strains were clonal in nature and confined entirely to a single clade. Comparative genomic analysis revealed that the virulence and resistance repertoires were comparable among the H30-Rx ST131 strains except for the commensal ST131 strain SE15. Similarly, seven phage-specific regions were found to be strongly associated with the H30-Rx strains but were largely absent in the genome of SE15. Phenotypic analysis confirmed the virulence and resistance similarities between the two strains. However, NA097 was found to be more robust than NA114 in terms of virulence gene carriage (dra operon), invasion ability (P < 0.05), and antimicrobial resistance (streptomycin resistance). RT2 gene expression profiling revealed generic upregulation of key proinflammatory responses in THP1 cells, irrespective of ST131 lineage status. In conclusion, our study provides comprehensive, genome-inferred insights into the biology and immunological properties of ST131 strains and suggests clonal diversification of genomic and phenotypic features within the H30-Rx subclone of E. coli ST131.

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

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.

Whole-Genome Sequences and Comparative Genomics of Salmonella enterica Serovar Typhi Isolates from Patients with Fatal and Nonfatal Typhoid Fever in Papua New Guinea

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.

Contig-Layout-Authenticator (CLA): A Combinatorial Approach to Ordering and Scaffolding of Bacterial Contigs for Comparative Genomics and Molecular Epidemiology

A wide variety of genome sequencing platforms have emerged in the recent past. High-throughput platforms like Illumina and 454 are essentially adaptations of the shotgun approach generating millions of fragmented single or paired sequencing reads. To reconstruct whole genomes, the reads have to be assembled into contigs, which often require further downstream processing. The contigs can be directly ordered according to a reference, scaffolded based on paired read information, or assembled using a combination of the two approaches. While the reference-based approach appears to mask strain-specific information, scaffolding based on paired-end information suffers when repetitive elements longer than the size of the sequencing reads are present in the genome. Sequencing technologies that produce long reads can solve the problems associated with repetitive elements but are not necessarily easily available to researchers. The most common high-throughput technology currently used is the Illumina short read platform. To improve upon the shortcomings associated with the construction of draft genomes with Illumina paired-end sequencing, we developed Contig-Layout-Authenticator (CLA). The CLA pipeline can scaffold reference-sorted contigs based on paired reads, resulting in better assembled genomes. Moreover, CLA also hints at probable misassemblies and contaminations, for the users to cross-check before constructing the consensus draft. The CLA pipeline was designed and trained extensively on various bacterial genome datasets for the ordering and scaffolding of large repetitive contigs. The tool has been validated and compared favorably with other widely-used scaffolding and ordering tools using both simulated and real sequence datasets. CLA is a user friendly tool that requires a single command line input to generate ordered scaffolds.

Genome dynamics and molecular infection epidemiology of multi-drug resistant Helicobacter pullorum isolates obtained from broiler and country chickens in India

ABSTRACT Some life-threatening, foodborne, and zoonotic infections are transmitted through poultry birds. Inappropriate and indiscriminate use of antimicrobials in the livestock industry has led to an increased prevalence of multidrug-resistant bacteria with epidemic potential. Here, we present a functional molecular epidemiological analysis entailing the phenotypic and whole-genome sequence-based characterization of 11 H. pullorum isolates from broiler and free-range chickens sampled from retail wet markets in Hyderabad City, India. Antimicrobial susceptibility tests revealed all of the isolates to be resistant to multiple antibiotic classes such as fluoroquinolones, cephalosporins, sulfonamides, and macrolides. The isolates were also found to be extended-spectrum β-lactamase producers and were even resistant to clavulanic acid. Whole-genome sequencing and comparative genomic analysis of these isolates revealed the presence of five or six well-characterized antimicrobial resistance genes, including those encoding a resistance-nodulation-division efflux pump(s). Phylogenetic analysis combined with pan-genome analysis revealed a remarkable degree of genetic diversity among the isolates from free-range chickens; in contrast, a high degree of genetic similarity was observed among broiler chicken isolates. Comparative genomic analysis of all publicly available H. pullorum genomes, including our isolates (n = 16), together with the genomes of 17 other Helicobacter species, revealed a high number (8,560) of H. pullorum-specific protein-encoding genes, with an average of 535 such genes per isolate. In silico virulence screening identified 182 important virulence genes and also revealed high strain-specific gene content in isolates from free-range chickens (average, 34) compared to broiler chicken isolates. A significant prevalence of prophages (ranging from 1 to 9) and a significant presence of genomic islands (0 to 4) were observed in free-range and broiler chicken isolates. Taken together, these observations provide significant baseline data for functional molecular infection epidemiology of nonpyloric Helicobacter species such as H. pullorum by unraveling their evolution in chickens and their possible zoonotic transmission to humans. IMPORTANCE Globally, the poultry industry is expanding with an ever-growing consumer base for chicken meat. Given this, food-associated transmission of multidrug-resistant bacteria represents an important health care issue. Our study involves a critical baseline approach directed at genome sequence-based epidemiology and transmission dynamics of H. pullorum, a poultry pathogen having established zoonotic potential. We believe our studies would facilitate the development of surveillance systems that ensure the safety of food for humans and guide public health policies related to the use of antibiotics in animal feed in countries such as India. We sequenced 11 new genomes of H. pullorum as a part of this study. These genomes would provide much value in addition to the ongoing comparative genomic studies of helicobacters.

Genome anatomy of the gastrointestinal pathogen, Vibrio parahaemolyticus of crustacean origin

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.

Genome dynamics and evolution of Salmonella Typhi strains from the typhoid-endemic zones

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.

Genome sequencing and analysis of Salmonella enterica serovar Typhi strain CR0063 representing a carrier individual during an outbreak of typhoid fever in Kelantan, Malaysia

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.