June 1, 2021  |  

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens.

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic states. Therefore, sequencing methods which provide complete, de novo genome assemblies and epigenomes are necessary to fully characterize infectious disease agents in an unbiased, hypothesis-free manner. Hybrid assembly methods have been described that combine long sequence reads from SMRT DNA Sequencing with short reads (SMRT CCS (circular consensus) or second-generation reads), wherein the short reads are used to error-correct the long reads which are then used for assembly. We have developed a new paradigm for microbial de novo assemblies in which SMRT sequencing reads from a single long insert library are used exclusively to close the genome through a hierarchical genome assembly process, thereby obviating the need for a second sample preparation, sequencing run, and data set. We have applied this method to achieve closed de novo genomes with accuracies exceeding QV50 (>99.999%) for numerous disease outbreak samples, including E. coli, Salmonella, Campylobacter, Listeria, Neisseria, and H. pylori. The kinetic information from the same SMRT Sequencing reads is utilized to determine epigenomes. Approximately 70% of all methyltransferase specificities we have determined to date represent previously unknown bacterial epigenetic signatures. With relatively short sequencing run times and automated analysis pipelines, it is possible to go from an unknown DNA sample to its complete de novo genome and epigenome in about a day.


June 1, 2021  |  

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non-pathogenic to pathogenic states. Therefore, sequencing methods which provide complete, de novo genome assemblies and epigenomes are necessary to fully characterize infectious disease agents in an unbiased, hypothesis-free manner. Hybrid assembly methods have been described that combine long sequence reads from SMRT DNA sequencing with short, high-accuracy reads (SMRT (circular consensus sequencing) CCS or second-generation reads) to generate long, highly accurate reads that are then used for assembly. We have developed a new paradigm for microbial de novo assemblies in which long SMRT sequencing reads (average readlengths >5,000 bases) are used exclusively to close the genome through a hierarchical genome assembly process, thereby obviating the need for a second sample preparation, sequencing run and data set. We have applied this method to achieve closed de novo genomes with accuracies exceeding QV50 (>99.999%) to numerous disease outbreak samples, including E. coli, Salmonella, Campylobacter, Listeria, Neisseria, and H. pylori. The kinetic information from the same SMRT sequencing reads is utilized to determine epigenomes. Approximately 70% of all methyltransferase specificities we have determined to date represent previously unknown bacterial epigenetic signatures. The process has been automated and requires less than 1 day from an unknown DNA sample to its complete de novo genome and epigenome.


June 1, 2021  |  

New discoveries from closing Salmonella genomes using Pacific Biosciences continuous long reads.

The newer hierarchical genome assembly process (HGAP) performs de novo assembly using data from a single PacBio long insert library. To assess the benefits of this method, DNA from several Salmonella enterica serovars was isolated from a pure culture. Genome sequencing was performed using Pacific Biosciences RS sequencing technology. The HGAP process enabled us to close sixteen Salmonella subsp. enterica genomes and their associated mobile elements: The ten serotypes include: Salmonella enterica subsp. enterica serovar Enteritidis (S. Enteritidis) S. Bareilly, S. Heidelberg, S. Cubana, S. Javiana and S. Typhimurium, S. Newport, S. Montevideo, S. Agona, and S. Tennessee. In addition, we were able to detect novel methyltransferases (MTases) by using the Pacific Biosciences kinetic score distributions showing that each serovar appears to have a novel methylation pattern. For example while all Salmonella serovars examined so far have methylase specific activity for 5’-GATC-3’/3’-CTAG-5’ and 5’-CAGAG-3’/3’-GTCTC-5’ (underlined base indicates a modification), S. Heidelberg is uniquely specific for 5’-ACCANCC-3’/3’-TGGTNGG-5’, while S. Typhimurium has uniquely methylase specific for 5′-GATCAG-3’/3′- CTAGTC-5′ sites, for the samples examined so far. We believe that this may be due to the unique environments and phages that these serotypes have been exposed to. Furthermore, our analysis identified and closed a variety of plasmids such as mobilization plasmids, antimicrobial resistance plasmids and IncX plasmids carrying a Type IV secretion system (T4SS). The VirB/D4 T4SS apparatus is important in that it assists with rapid dissemination of antibiotic resistance and virulence determinants. Presently, only limited information exists regarding the genotypic characterization of drug resistance in S. Heidelberg isolates derived from various host species. Here, we characterize two S. Heidelberg outbreak isolates from two different outbreaks. Both isolates contain the IncX plasmid of approximately 35 kb, and carried the genes virB1, virB2, virB3/4, virB5, virB6, virB7, virB8, virB9, virB10, virB11, virD2, and virD4, that are associated with the T4SS. In addition, the outbreak isolate associated with ground turkey carries a 4,473 bp mobilization plasmid and an incompatibility group (Inc) I1 antimicrobial resistance plasmid encoding resistance to gentamicin (aacC2), beta-lactam (bl2b_tem), streptomycin (aadAI) and tetracycline (tetA, tetR) while the outbreak isolate associated with chicken breast carries the IncI1 plasmid encoding resistance to gentamicin (aacC2), streptomycin (aadAI) and sulfisoxazole (sul1). Using this new technology we explored the genetic elements present in resistant pathogens which will achieve a better understanding of the evolution of Salmonella.


April 21, 2020  |  

Dual Role of gnaA in Antibiotic Resistance and Virulence in Acinetobacter baumannii.

Acinetobacter baumannii is an important Gram-negative pathogen in hospital-related infections. However, treatment options for A. baumannii infections have become limited due to multidrug resistance. Bacterial virulence is often associated with capsule genes found in the K locus, many of which are essential for biosynthesis of the bacterial envelope. However, the roles of other genes in the K locus remain largely unknown. From an in vitro evolution experiment, we obtained an isolate of the virulent and multidrug-resistant A. baumannii strain MDR-ZJ06, called MDR-ZJ06M, which has an insertion by the ISAba16 transposon in gnaA (encoding UDP-N-acetylglucosamine C-6 dehydrogenase), a gene found in the K locus. The isolate showed an increased resistance toward tigecycline, whereas the MIC decreased in the case of carbapenems, cephalosporins, colistin, and minocycline. By using knockout and complementation experiments, we demonstrated that gnaA is important for the synthesis of lipooligosaccharide and capsular polysaccharide and that disruption of the gene affects the morphology, drug susceptibility, and virulence of the pathogen.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Genomic Analysis of Emerging Florfenicol-Resistant Campylobacter coli Isolated from the Cecal Contents of Cattle in the United States.

Genomic analyses were performed on florfenicol-resistant (FFNr) Campylobacter coli isolates recovered from cattle, and the cfr(C) gene-associated multidrug resistance (MDR) plasmid was characterized. Sixteen FFNrC. coli isolates recovered between 2013 and 2018 from beef cattle were sequenced using MiSeq. Genomes and plasmids were found to be closed for three of the isolates using the PacBio system. Single nucleotide polymorphisms (SNPs) across the genome and the structures of MDR plasmids were investigated. Conjugation experiments were performed to determine the transferability of cfr(C)-associated MDR plasmids. The spectrum of resistance encoded by the cfr(C) gene was further investigated by agar dilution antimicrobial susceptibility testing. All 16 FFNr isolates were MDR and exhibited coresistance to ciprofloxacin, nalidixic acid, clindamycin, and tetracycline. All isolates shared the same resistance genotype, carrying aph (3′)-III, hph, ?aadE (truncated), blaOXA-61, cfr(C), and tet(O) genes plus a mutation of GyrA (T86I). The cfr(C), aph (3′)-III, hph, ?aadE, and tet(O) genes were colocated on transferable MDR plasmids ranging in size from 48 to 50?kb. These plasmids showed high sequence homology with the pTet plasmid and carried several Campylobacter virulence genes, including virB2, virB4, virB5, VirB6, virB7, virB8, virb9, virB10, virB11, and virD4 The cfr(C) gene conferred resistance to florfenicol (8 to 32?µg/ml), clindamycin (512 to 1,024?µg/ml), linezolid (128 to 512?µg/ml), and tiamulin (1,024?µg/ml). Phylogenetic analysis showed SNP differences ranging from 11 to 2,248 SNPs among the 16 isolates. The results showed that the cfr(C) gene located in the conjugative pTet MDR/virulence plasmid is present in diverse strains, where it confers high levels of resistance to several antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. This report highlights the power of genomic antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.IMPORTANCECampylobacter is a leading cause of foodborne diarrheal illness worldwide, with more than one million cases each year in the United States alone. The global emergence of antimicrobial resistance in this pathogen has become a growing public health concern. Florfenicol-resistant (FFNr) Campylobacter has been very rare in the United States. In this study, we employed whole-genome sequencing to characterize 16 multidrug-resistant Campylobacter coli isolates recovered from cattle in the United States. A gene [cfr(C)] was found to be responsible for resistance not only to florfenicol but also to several other antimicrobials, including linezolid, a critical drug for treating infections by Gram-positive bacteria in humans. The results showed that cfr(C) is located in a conjugative pTet MDR/virulence plasmid. This report highlights the power of antimicrobial resistance surveillance to uncover the intricacies of transmissible coresistance and provides information that is needed for accurate risk assessment and mitigation strategies.


April 21, 2020  |  

Retrospective whole-genome sequencing analysis distinguished PFGE and drug-resistance-matched retail meat and clinical Salmonella isolates.

Non-typhoidal Salmonella is a leading cause of outbreak and sporadic-associated foodborne illnesses in the United States. These infections have been associated with a range of foods, including retail meats. Traditionally, pulsed-field gel electrophoresis (PFGE) and antibiotic susceptibility testing (AST) have been used to facilitate public health investigations of Salmonella infections. However, whole-genome sequencing (WGS) has emerged as an alternative tool that can be routinely implemented. To assess its potential in enhancing integrated surveillance in Pennsylvania, USA, WGS was used to directly compare the genetic characteristics of 7 retail meat and 43 clinical historic Salmonella isolates, subdivided into 3 subsets based on PFGE and AST results, to retrospectively resolve their genetic relatedness and identify antimicrobial resistance (AMR) determinants. Single nucleotide polymorphism (SNP) analyses revealed that the retail meat isolates within S. Heidelberg, S. Typhimurium var. O5- subset 1 and S. Typhimurium var. O5- subset 2 were separated from each primary PFGE pattern-matched clinical isolate by 6-12, 41-96 and 21-81 SNPs, respectively. Fifteen resistance genes were identified across all isolates, including fosA7, a gene only recently found in a limited number of Salmonella and a =95?%?phenotype to genotype correlation was observed for all tested antimicrobials. Moreover, AMR was primarily plasmid-mediated in S. Heidelberg and S. Typhimurium var. O5- subset 2, whereas AMR was chromosomally carried in S. Typhimurium var. O5- subset 1. Similar plasmids were identified in both the retail meat and clinical isolates. Collectively, these data highlight the utility of WGS in retrospective analyses and enhancing integrated surveillance for Salmonella from multiple sources.


April 21, 2020  |  

Biomimetic hydroxyapatite nanocrystals are an active carrier for Salmonella bacteriophages.

The use of bacteriophages represents a valid alternative to conventional antimicrobial treatments, overcoming the widespread bacterial antibiotic resistance phenomenon. In this work, we evaluated whether biomimetic hydroxyapatite (HA) nanocrystals are able to enhance some properties of bacteriophages. The final goal of this study was to demonstrate that biomimetic HA nanocrystals can be used for bacteriophage delivery in the context of bacterial infections, and contribute – at the same time – to enhance some of the biological properties of the same bacteriophages such as stability, preservation, antimicrobial activity, and so on.Phage isolation and characterization were carried out by using Mitomycin C and following double-layer agar technique. The biomimetic HA water suspension was synthesized in order to obtain nanocrystals with plate-like morphology and nanometric dimensions. The interaction of phages with the HA was investigated by dynamic light scattering and Zeta potential analyses. The cytotoxicity and intracellular killing activities of the phage-HA complex were evaluated in human hepatocellular carcinoma HepG2 cells. The bacterial inhibition capacity of the complex was assessed on chicken minced meat samples infected with Salmonella Rissen.Our data highlighted that the biomimetic HA nanocrystal-bacteriophage complex was more stable and more effective than phages alone in all tested experimental conditions.Our results evidenced the important contribution of biomimetic HA nanocrystals: they act as an excellent carrier for bacteriophage delivery and enhance its biological characteristics. This study confirmed the significant role of the mineral HA when it is complexed with biological entities like bacteriophages, as it has been shown for molecules such as lactoferrin.


April 21, 2020  |  

Deciphering bacterial epigenomes using modern sequencing technologies.

Prokaryotic DNA contains three types of methylation: N6-methyladenine, N4-methylcytosine and 5-methylcytosine. The lack of tools to analyse the frequency and distribution of methylated residues in bacterial genomes has prevented a full understanding of their functions. Now, advances in DNA sequencing technology, including single-molecule, real-time sequencing and nanopore-based sequencing, have provided new opportunities for systematic detection of all three forms of methylated DNA at a genome-wide scale and offer unprecedented opportunities for achieving a more complete understanding of bacterial epigenomes. Indeed, as the number of mapped bacterial methylomes approaches 2,000, increasing evidence supports roles for methylation in regulation of gene expression, virulence and pathogen-host interactions.


April 21, 2020  |  

Dysbiosis and Variation in Predicted Functions of the Granulation Tissue Microbiome in HPV Positive and Negative Severe Chronic Periodontitis.

Retrospective analysis has already shown correlation between severe Chronic Periodontitis (CP) cases with human papiloma virus (HPV). Hence, we aimed to explore deep-seated infected granulation tissue removed during periodontal flap surgery procedures for residential bacterial species between HPV+ and HVP- CP cases, which may serve as good predisposition marker for oral cancer. All CP-granulation samples showed the prominence of Firmicutes, Proteobacteria, and Bacteroidetes phyla with an abundance of gram negative anaerobes, except Streptococcus. In Beta diversity nonmetric multidimensional scaling plot, the random distribution of species was observed between HPV+ and HPV- CP granulation-samples. However, an abundance of Capnocytophaga ochracea was observed in HPV+ CP samples (p<0.05), while Porphyromonas endodontalis, Macellibacteroides fermentas, Treponema phagedenis, and Campylobacter rectus species were highly abundant in HPV- CP samples (p<0.05). The differential species richness leads altered functions related to mismatch-repair and nucleotide excision-repair and cytoskeleton-proteins. Hence, differential abundance of gram negative bacterial species between HPV+ and HPV- granulation-samples under anaerobic conditions may release virulence factors which may alter pathways favouring carcinogenesis. Hence, these species may serve as good predisposition marker for oral-cancer.


April 21, 2020  |  

Development of CRISPR-Cas systems for genome editing and beyond

The development of clustered regularly interspaced short-palindromic repeat (CRISPR)-Cas systems for genome editing has transformed the way life science research is conducted and holds enormous potential for the treatment of disease as well as for many aspects of biotech- nology. Here, I provide a personal perspective on the development of CRISPR-Cas9 for genome editing within the broader context of the field and discuss our work to discover novel Cas effectors and develop them into additional molecular tools. The initial demonstra- tion of Cas9-mediated genome editing launched the development of many other technologies, enabled new lines of biological inquiry, and motivated a deeper examination of natural CRISPR-Cas systems, including the discovery of new types of CRISPR-Cas systems. These new discoveries in turn spurred further technological developments. I review these exciting discoveries and technologies as well as provide an overview of the broad array of applications of these technologies in basic research and in the improvement of human health. It is clear that we are only just beginning to unravel the potential within microbial diversity, and it is quite likely that we will continue to discover other exciting phenomena, some of which it may be possible to repurpose as molecular technologies. The transformation of mysterious natural phenomena to powerful tools, however, takes a collective effort to discover, characterize, and engineer them, and it has been a privilege to join the numerous researchers who have contributed to this transformation of CRISPR-Cas systems.


April 21, 2020  |  

Urinary tract colonization is enhanced by a plasmid that regulates uropathogenic Acinetobacter baumannii chromosomal genes.

Multidrug resistant (MDR) Acinetobacter baumannii poses a growing threat to global health. Research on Acinetobacter pathogenesis has primarily focused on pneumonia and bloodstream infections, even though one in five A. baumannii strains are isolated from urinary sites. In this study, we highlight the role of A. baumannii as a uropathogen. We develop the first A. baumannii catheter-associated urinary tract infection (CAUTI) murine model using UPAB1, a recent MDR urinary isolate. UPAB1 carries the plasmid pAB5, a member of the family of large conjugative plasmids that represses the type VI secretion system (T6SS) in multiple Acinetobacter strains. pAB5 confers niche specificity, as its carriage improves UPAB1 survival in a CAUTI model and decreases virulence in a pneumonia model. Comparative proteomic and transcriptomic analyses show that pAB5 regulates the expression of multiple chromosomally-encoded virulence factors besides T6SS. Our results demonstrate that plasmids can impact bacterial infections by controlling the expression of chromosomal genes.


April 21, 2020  |  

Arcobacter cryaerophilus Isolated From New Zealand Mussels Harbor a Putative Virulence Plasmid.

A wide range of Arcobacter species have been described from shellfish in various countries but their presence has not been investigated in Australasia, in which shellfish are a popular delicacy. Since several arcobacters are considered to be emerging pathogens, we undertook a small study to evaluate their presence in several different shellfish, including greenshell mussels, oysters, and abalone (paua) in New Zealand. Arcobacter cryaerophilus, a species associated with human gastroenteritis, was the only species isolated, from greenshell mussels. Whole-genome sequencing revealed a range of genomic traits in these strains that were known or associated virulence factors. Furthermore, we describe the first putative virulence plasmid in Arcobacter, containing lytic, immunoavoidance, adhesion, antibiotic resistance, and gene transfer traits, among others. Complete genome sequence determination using a combination of long- and short-read genome sequencing strategies, was needed to identify the plasmid, clearly identifying its benefits. The potential for plasmids to disseminate virulence traits among Arcobacter and other species warrants further consideration by researchers interested in the risks to public health from these organisms.


April 21, 2020  |  

Survival Mechanisms of Campylobacter hepaticus Identified by Genomic Analysis and Comparative Transcriptomic Analysis of in vivo and in vitro Derived Bacteria.

Chickens infected with Campylobacter jejuni or Campylobacter coli are largely asymptomatic, however, infection with the closely related species, Campylobacter hepaticus, can result in Spotty Liver Disease (SLD). C. hepaticus has been detected in the liver, bile, small intestine and caecum of SLD affected chickens. The survival and colonization mechanisms that C. hepaticus uses to colonize chickens remain unknown. In this study, we compared the genome sequences of 14 newly sequenced Australian isolates of C. hepaticus, isolates from outbreaks in the United Kingdom, and reference strains of C. jejuni and C. coli, with the aim of identifying virulence genes associated with SLD. We also carried out global comparative transcriptomic analysis between C. hepaticus recovered from the bile of SLD infected chickens and C. hepaticus grown in vitro. This revealed how the bacteria adapt to proliferate in the challenging host environment in which they are found. Additionally, biochemical experiments confirmed some in silico metabolic predictions. We found that, unlike other Campylobacter sp., C. hepaticus encodes glucose and polyhydroxybutyrate metabolism pathways. This study demonstrated the metabolic plasticity of C. hepaticus, which may contribute to survival in the competitive, nutrient and energy-limited environment of the chicken. Transcriptomic analysis indicated that gene clusters associated with glucose utilization, stress response, hydrogen metabolism, and sialic acid modification may play an important role in the pathogenicity of C. hepaticus. An understanding of the survival and virulence mechanisms that C. hepaticus uses will help to direct the development of effective intervention methods to protect birds from the debilitating effects of SLD.


April 21, 2020  |  

Comparative Genomic Analyses Reveal Core-Genome-Wide Genes Under Positive Selection and Major Regulatory Hubs in Outlier Strains of Pseudomonas aeruginosa.

Genomic information for outlier strains of Pseudomonas aeruginosa is exiguous when compared with classical strains. We sequenced and constructed the complete genome of an environmental strain CR1 of P. aeruginosa and performed the comparative genomic analysis. It clustered with the outlier group, hence we scaled up the analyses to understand the differences in environmental and clinical outlier strains. We identified eight new regions of genomic plasticity and a plasmid pCR1 with a VirB/D4 complex followed by trimeric auto-transporter that can induce virulence phenotype in the genome of strain CR1. Virulence genotype analysis revealed that strain CR1 lacked hemolytic phospholipase C and D, three genes for LPS biosynthesis and had reduced antibiotic resistance genes when compared with clinical strains. Genes belonging to proteases, bacterial exporters and DNA stabilization were found to be under strong positive selection, thus facilitating pathogenicity and survival of the outliers. The outliers had the complete operon for the production of vibrioferrin, a siderophore present in plant growth promoting bacteria. The competence to acquire multidrug resistance and new virulence factors makes these strains a potential threat. However, we identified major regulatory hubs that can be used as drug targets against both the classical and outlier groups.


April 21, 2020  |  

Resource Concentration Modulates the Fate of Dissimilated Nitrogen in a Dual-Pathway Actinobacterium.

Respiratory ammonification and denitrification are two evolutionarily unrelated dissimilatory nitrogen (N) processes central to the global N cycle, the activity of which is thought to be controlled by carbon (C) to nitrate (NO3-) ratio. Here we find that Intrasporangium calvum C5, a novel dual-pathway denitrifier/respiratory ammonifier, disproportionately utilizes ammonification rather than denitrification when grown under low C concentrations, even at low C:NO3- ratios. This finding is in conflict with the paradigm that high C:NO3- ratios promote ammonification and low C:NO3- ratios promote denitrification. We find that the protein atomic composition for denitrification modules (NirK) are significantly cost minimized for C and N compared to ammonification modules (NrfA), indicating that limitation for C and N is a major evolutionary selective pressure imprinted in the architecture of these proteins. The evolutionary precedent for these findings suggests ecological importance for microbial activity as evidenced by higher growth rates when I. calvum grows predominantly using its ammonification pathway and by assimilating its end-product (ammonium) for growth under ammonium-free conditions. Genomic analysis of I. calvum further reveals a versatile ecophysiology to cope with nutrient stress and redox conditions. Metabolite and transcriptional profiles during growth indicate that enzyme modules, NrfAH and NirK, are not constitutively expressed but rather induced by nitrite production via NarG. Mechanistically, our results suggest that pathway selection is driven by intracellular redox potential (redox poise), which may be lowered when resource concentrations are low, thereby decreasing catalytic activity of upstream electron transport steps (i.e., the bc1 complex) needed for denitrification enzymes. Our work advances our understanding of the biogeochemical flexibility of N-cycling organisms, pathway evolution, and ecological food-webs.


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