Targeted Computational Approaches for Mining Functional Elements in Metagenomes

Thesis (Ph.D.) - Indiana University, Informatics, 2012Metagenomics enables the genomic study of uncultured microorganisms by directly extracting the genetic material from microbial communities for sequencing. Fueled by the rapid development of Next Generation Sequencing (NGS) technology, metagenomics research has been revolutionizing the field of microbiology, revealing the taxonomic and functional composition of many microbial communities and their impacts on almost every aspect of life on Earth. Analyzing metagenomes (a metagenome is the collection of genomic sequences of an entire microbial community) is challenging: metagenomic sequences are often extremely short and therefore lack genomic contexts needed for annotating functional elements, while whole-metagenome assemblies are often poor because a metagenomic dataset contains reads from many different species. Novel computational approaches are still needed to get the most out of the metagenomes. In this dissertation, I first developed a binning algorithm (AbundanceBin) for clustering metagenomic sequences into groups, each containing sequences from species of similar abundances. AbundanceBin provides accurate estimations of the abundances of the species in a microbial community and their genome sizes. Application of AbundanceBin prior to assembly results in better assemblies of metagenomes--an outcome crucial to downstream analyses of metagenomic datasets. In addition, I designed three targeted computational approaches for assembling and annotating protein coding genes and other functional elements from metagenomic sequences. GeneStitch is an approach for gene assembly by connecting gene fragments scattered in different contigs into longer genes with the guidance of reference genes. I also developed two specialized assembly methods: the targeted-assembly method for assembling CRISPRs (Clustered Regularly Interspersed Short Palindromic Repeats), and the constrained-assembly method for retrieving chromosomal integrons. Applications of these methods to the Human Microbiome Project (HMP) datasets show that human microbiomes are extremely dynamic, reflecting the interactions between community members (including bacteria and viruses)

Analysis of multiply antibiotic-resistant Acinetobacter baumannii belonging to Global Clone 1

Analysis of multiply antibiotic-resistant Acinetobacter baumannii belonging to Global Clone 1. A. baumannii resistant to multiple antibiotics challenges the treatment of infections caused by this organism. Multiply-antibiotic resistant isolates mainly belong to two major global clones GC1 and GC2. The objective of this study was to analyze the antibiotic resistance in Australian GC1s. Twenty six MAR GC1 isolates were recovered from Australian hospitals and examined to determine the genetic basis of their resistance. The results showed the variety of mobile elements, genomic islands, plasmids, transposons and insertion sequences that incorporate antibiotic resistance genes. AbaR islands were found to carry genes conferring resistance to multiple antibiotics. IS26-mediated deletions and homologous recombination between Tn6018 copies in the AbaR islands were shown to be responsible for generating new variants. AbaR0, the ancestor of AbaRs was found and all of the variants seen so far could be derived from it. Carbapenem resistance was rare but three strains carried the oxa23 carbapenem resistance gene in an AbaR4 island, which includes a backbone related to that of AbaR islands. In one isolate, AbaR4 was found where AbaR-type islands are usually found indicating that transposon backbone of AbaR4 can target the same position as AbaR0/AbaR3. The other two strains carried AbaR4 in a conjugative plasmid that can potentially disseminate the oxa23 gene into strains of different types. The small plasmid pRAY* was responsible for introducing the tobramycin resistance gene aadB into GC1s. Hence, this study shows the significance of plasmids incorporating additional determinants in GC1s. The most unexpected finding was horizontal transfer of DNA segments that contain ISAba1-ampC generating resistance to third generation cephalosporins. Overall, Australian GC1 isolates included a diverse collection. However, two outbreak GC1 strains were identified in a single ward of one of the Sydney hospitals. Outbreak strains persisted for a period of time and were then replaced by a GC2 strain

Antimicrobial resistance, plasmid profiles and sequence typing of enterotoxigenic escherichia coli isolates causing colibacillosis in neonatal and weaning piglets of South Africa.

Master of Science in Genietics. University of KwaZulu-Natal, Pietermaritzburg 2016.Abstract available in PDF file

Analysis of multiply antibiotic-resistant Acinetobacter baumannii belonging to Global Clone 1

Analysis of multiply antibiotic-resistant Acinetobacter baumannii belonging to Global Clone 1. A. baumannii resistant to multiple antibiotics challenges the treatment of infections caused by this organism. Multiply-antibiotic resistant isolates mainly belong to two major global clones GC1 and GC2. The objective of this study was to analyze the antibiotic resistance in Australian GC1s. Twenty six MAR GC1 isolates were recovered from Australian hospitals and examined to determine the genetic basis of their resistance. The results showed the variety of mobile elements, genomic islands, plasmids, transposons and insertion sequences that incorporate antibiotic resistance genes. AbaR islands were found to carry genes conferring resistance to multiple antibiotics. IS26-mediated deletions and homologous recombination between Tn6018 copies in the AbaR islands were shown to be responsible for generating new variants. AbaR0, the ancestor of AbaRs was found and all of the variants seen so far could be derived from it. Carbapenem resistance was rare but three strains carried the oxa23 carbapenem resistance gene in an AbaR4 island, which includes a backbone related to that of AbaR islands. In one isolate, AbaR4 was found where AbaR-type islands are usually found indicating that transposon backbone of AbaR4 can target the same position as AbaR0/AbaR3. The other two strains carried AbaR4 in a conjugative plasmid that can potentially disseminate the oxa23 gene into strains of different types. The small plasmid pRAY* was responsible for introducing the tobramycin resistance gene aadB into GC1s. Hence, this study shows the significance of plasmids incorporating additional determinants in GC1s. The most unexpected finding was horizontal transfer of DNA segments that contain ISAba1-ampC generating resistance to third generation cephalosporins. Overall, Australian GC1 isolates included a diverse collection. However, two outbreak GC1 strains were identified in a single ward of one of the Sydney hospitals. Outbreak strains persisted for a period of time and were then replaced by a GC2 strain

A study of clinical strains of Pseudomonas Aeruginosa and the investigation of antibiotic resistance mechanisms in the multidrug resistant strain PA13

Thirteen clinical strains of bacteria from two Irish hospitals were identified as Pseudomonas aeruginosa using classical methods, API 20NE and Biolog GN. Their identification was confirmed by 16S rRNA gene sequencing. The antibiotic resistance profiles of the isolates were determined against forty-one antibiotics belonging to eleven distinct classes. All the isolates were resistant to penicillin G, ampicillin, cephalothin, cloxacillin, oxacillin, amoxicillin, cefotaxime, moxalactam, sulphatriad cotrimoxazole, chloramphenicol and tetracycline. All were sensitive to ceftazidime, piperacillin-tazobactam, cefepime, ceftriaxone, meropenem, aztreonam, amikacin, apramycin, butirosin A, lividomycin and colistin sulphate. One of the isolates, PA13, was resistant to a further fourteen antibiotics and was identified as a multidrug resistant strain. A 2.2 kbp PCR product was amplified from P. aeruginosa PA13. When this product was sequenced it was found to contain four open reading frames. BLASTN analysis identified these as being an integrase gene (ORF1), an aminoglycoside acetyltransferase gene, aac(6’)-Ib (ORF2), an oxacillinase gene (ORF3) and a quaternary ammonium compound resistance gene (ORF4). The presence of the integrase gene and the quaternary ammonium compound gene suggested that the genes were on a Class 1 integron. The acetyltransferase aac(6’)-Ib gene contained the mutant type of the enzyme with a leucine substitution by serine at position 119. Two expression vectors were chosen to investigate the novel oxacillinase gene. One was a commercially available vector, pET-28a (Novagen) and the other was an in-house vector, pPC. The gene was successfully cloned into both vectors. Following induction the desired protein was not expressed in either the soluble or insoluble fractions

Mobile mercury resistance transposons: surveillance and resistance gene cassette variation in wastewater

In a wastewater environment, mercury resistance and other antimicrobial metal resistance genes have been observed despite their lack of use clinically. The hypotheses explored whether the change in populations to a rural wastewater treatment plant affects the abundance of Tn21 and similarly identify potential co–occurrence antimicrobial resistance genes carried within or alongside Tn21 and Tn21-like transposable elements. Finally, Tn21 is known to be carried by a wide range of Gram-negative bacteria, however without being able to cross-link Tn21 to the host it is not possible to identify in large scale samples which organisms may in fact carry the mobile element within an environmental sample. Results showed that large-scale population changes impacted the abundance of Tn21 and the carriage of co-occurrent resistance genes. Wastewater treatment processing was also shown to reduce diversity of Tn21 gene cassette arrays of the class I integron and therefore not remove the presence of antimicrobial resistance genes disseminating into the environment. The studies highlight the need for intervention within the wastewater treatment process

Mobile mercury resistance transposons: surveillance and resistance gene cassette variation in wastewater

In a wastewater environment, mercury resistance and other antimicrobial metal resistance genes have been observed despite their lack of use clinically. The hypotheses explored whether the change in populations to a rural wastewater treatment plant affects the abundance of Tn21 and similarly identify potential co–occurrence antimicrobial resistance genes carried within or alongside Tn21 and Tn21-like transposable elements. Finally, Tn21 is known to be carried by a wide range of Gram-negative bacteria, however without being able to cross-link Tn21 to the host it is not possible to identify in large scale samples which organisms may in fact carry the mobile element within an environmental sample. Results showed that large-scale population changes impacted the abundance of Tn21 and the carriage of co-occurrent resistance genes. Wastewater treatment processing was also shown to reduce diversity of Tn21 gene cassette arrays of the class I integron and therefore not remove the presence of antimicrobial resistance genes disseminating into the environment. The studies highlight the need for intervention within the wastewater treatment process

Quantification Of Antimicrobial Resistance Genes In Urban Agricultural Soil

The increased dissemination of antibiotic resistance genes and their acquisition by clinically relevant microbes in the environmental setting is becoming a global alarming issue. Environmental areas that encompass anthropogenic pressure such as pharmaceutical manufacturing effluents, aquaculture facilities, municipal wastewater systems, chemical industry effluents and animal husbandry facilities are hotspots of ARGs and ARBs. The main objective of the present study was to investigate the prevalence, identification, and quantification of class 1 integron (intI1) and common antibiotic resistance genes (Sul2, TetM, blaTEM) in the urban agricultural soil. Quantitative PCR was implemented to determine the abundance of ARGs in the soil. Standardization of intI1 gene copy number (106 copies gram-1) and ARGs (Sul2, TetM, blaTEM) was performed and the absolute abundance of resistance genes was normalized by bacterial cell. Correlation between intI1 and Sul2 gene with significance level of p \u3c 0.05 was observed. This study suggests that ARGs are common in the environment including urban agricultural soil that receives no animal wastes or wastewater. Mobile genetic elements (MGE) may play an important role in spreading ARGs in the environment

The Antimicrobial Resistance Plasmid Mobilome of Salmonella enterica and Related Enteric Species and Factors that Influence the Transfer Efficiency.

The dynamic distribution of antimicrobial resistance genes in Salmonella enterica is considered a public health risk. S. enterica is one of the most important etiological agents of foodborne illness and has a critical impact on global human health. In S. enterica and related species, mobile genetic elements (MGEs) serve as primary vehicles for the dissemination of antibiotic resistance genes in the bacterial evolution. This dissemination can be impacted by different selective pressures that leads to diverse antibiotic response phenotypes. This project focusses on the dynamics of antimicrobial resistance genes, particularly exploring the transfer efficiency of multidrug resistance plasmids in S. enterica using a combination of in silico and in vitro techniques. Plasmid physiology is part of an essential genetic engineering tool has mediated the dynamic transfer and spread of antimicrobial resistance in Salmonella isolates. Several phenotypic and molecular techniques were selected in these studies to better understand the molecular biology and epidemiology of resistance plasmids. These methods included whole genome sequencing (WGS) analyses, replicon (incompatibility; Inc) typing and conjugation studies of plasmids from S. enterica and related species. Multiple in silico analyses were used to evaluate WGS and plasmid sequencing data to examine the distribution of MGEs, AMR, biocide, disinfectant, and heavy metal resistance genes across the different plasmid types and to develop a plasmid characterization database. The database supported the development of the computational algorithms to process the WGS data from S. enterica isolates and is very useful for identifying the plasmid Inc types and their specific conjugal transfer systems, which are valuable to study the diversity and dissemination of plasmids associated with AMR in S. enterica and other Enterobacteriaceae. In vitro studies explored the impact of different antimicrobial exposures on conjugal transfer potential of plasmids. For some S. enterica strains, exposure to different concentrations of tetracycline or chloramphenicol led to differences in the efficiency of AMR plasmid transfer. These data help to better understand the distribution of resistance genes and provide a useful method for a comprehensive molecular studies of plasmid transfer dynamics. The impact of these findings clarifies the role of the conjugation dynamics in the dissemination of antimicrobial resistance in S. enterica and related species, which can potentially impact the transfer of resistance genes within the gastrointestinal microbiome. Further studies are required to identify the underlying genetic mechanisms for Salmonella resistance plasmid transfer using approaches like RNA-sequencing methods to extend the understanding of the regulation of genetic pathways during conjugal transfer. Overall, the research study expands our knowledge of plasmid transfer dynamic and the provides tools that can be used to better understand AMR gene transfer among Salmonella enterica isolates which can have an impact in clinical and diagnostic laboratories as well as in epidemiological surveillance