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  |  

Microbial diversity in the tick Argas japonicus (Acari: Argasidae) with a focus on Rickettsia pathogens.

The soft tick Argas japonicus mainly infests birds and can cause human dermatitis; however, no pathogen has been identified from this tick species in China. In the present study, the microbiota in A. japonicus collected from an epidemic community was explored, and some putative Rickettsia pathogens were further characterized. The results obtained indicated that bacteria in A. japonicus were mainly ascribed to the phyla Proteobacteria, Firmicutes and Actinobacteria. At the genus level, the male A. japonicus harboured more diverse bacteria than the females and nymphs. The bacteria Alcaligenes, Pseudomonas, Rickettsia and Staphylococcus were common in nymphs and adults. The abundance of bacteria belonging to the Rickettsia genus in females and males was 7.27% and 10.42%, respectively. Furthermore, the 16S rRNA gene of Rickettsia was amplified and sequenced, and phylogenetic analysis revealed that 13 sequences were clustered with the spotted fever group rickettsiae (Rickettsia heilongjiangensis and Rickettsia japonica) and three were clustered with Rickettsia limoniae, which suggested that the characterized Rickettsia in A. japonicus were novel putative pathogens and also that the residents were at considerable risk for infection by tick-borne pathogens. © 2019 The Royal Entomological Society.


April 21, 2020  |  

Morphological and genomic characterisation of the hybrid schistosome infecting humans in Europe reveals a complex admixture between Schistosoma haematobium and Schistosoma bovis parasites

Schistosomes cause schistosomiasis, the worldtextquoterights second most important parasitic disease after malaria. A peculiar feature of schistosomes is their ability to produce viable and fertile hybrids. Originally only present in the tropics, schistosomiasis is now also endemic in Europe. Based on two genetic markers the European species had been identified as a hybrid between the ruminant-infective Schistosoma bovis and the human-infective Schistosoma haematobium.Here we describe for the first time the genomic composition of the European schistosome hybrid (77% of S. haematobium and 23% of S. bovis origins), its morphometric parameters and its compatibility with the European vector snail and intermediate host Compatibility is a key parameter for the parasites life cycle progression. We also show that egg morphology (a classical diagnostic parameter) does not allow for differential diagnosis while genetic tests do so. Additionally, we performed genome assembly improvement and annotation of S. bovis, the parental species for which no satisfactory genome assembly was available.For the first time since the discovery of hybrid schistosomes, these results reveal at the whole genomic level a complex admixture of parental genomes highlighting (i) the high permeability of schistosomes to other speciestextquoteright alleles, and (ii) the importance of hybrid formation for pushing species boundaries not only conceptionally but also geographically.


April 21, 2020  |  

Complete Genome Sequence of Leptospira kmetyi LS 001/16, Isolated from a Soil Sample Associated with a Leptospirosis Patient in Kelantan, Malaysia.

The Gram-negative pathogenic spirochetal bacteria Leptospira spp. cause leptospirosis in humans and livestock animals. Leptospira kmetyi strain LS 001/16 was isolated from a soil sample associated with a leptospirosis patient in Kelantan, which is among the states in Malaysia with a high reported number of disease cases. Here, we report the complete genome sequence of Leptospira kmetyi strain LS 001/16. Copyright © 2019 Yusof et al.


April 21, 2020  |  

Detection of VIM-1-Producing Enterobacter cloacae and Salmonella enterica Serovars Infantis and Goldcoast at a Breeding Pig Farm in Germany in 2017 and Their Molecular Relationship to Former VIM-1-Producing S. Infantis Isolates in German Livestock Production.

In 2011, VIM-1-producing Salmonella enterica serovar Infantis and Escherichia coli were isolated for the first time in four German livestock farms. In 2015/2016, highly related isolates were identified in German pig production. This raised the issue of potential reservoirs for these isolates, the relation of their mobile genetic elements, and potential links between the different affected farms/facilities. In a piglet-producing farm suspicious for being linked to some blaVIM-1 findings in Germany, fecal and environmental samples were examined for the presence of carbapenemase-producing Enterobacteriaceae and Salmonella spp. Newly discovered isolates were subjected to Illumina whole-genome sequencing (WGS) and S1 pulsed-field gel electrophoresis (PFGE) hybridization experiments. WGS data of these isolates were compared with those for the previously isolated VIM-1-producing Salmonella Infantis isolates from pigs and poultry. Among 103 samples, one Salmonella Goldcoast isolate, one Salmonella Infantis isolate, and one Enterobacter cloacae isolate carrying the blaVIM-1 gene were detected. Comparative WGS analysis revealed that the blaVIM-1 gene was part of a particular Tn21-like transposable element in all isolates. It was located on IncHI2 (ST1) plasmids of ~290 to 300?kb with a backbone highly similar (98 to 100%) to that of reference pSE15-SA01028. SNP analysis revealed a close relationship of all VIM-1-positive S Infantis isolates described since 2011. The findings of this study demonstrate that the occurrence of the blaVIM-1 gene in German livestock is restricted neither to a certain bacterial species nor to a certain Salmonella serovar but is linked to a particular Tn21-like transposable element located on transferable pSE15-SA01028-like IncHI2 (ST1) plasmids, being present in all of the investigated isolates from 2011 to 2017.IMPORTANCE Carbapenems are considered one of few remaining treatment options against multidrug-resistant Gram-negative pathogens in human clinical settings. The occurrence of carbapenemase-producing Enterobacteriaceae in livestock and food is a major public health concern. Particularly the occurrence of VIM-1-producing Salmonella Infantis in livestock farms is worrisome, as this zoonotic pathogen is one of the main causes for human salmonellosis in Europe. Investigations on the epidemiology of those carbapenemase-producing isolates and associated mobile genetic elements through an in-depth molecular characterization are indispensable to understand the transmission of carbapenemase-producing Enterobacteriaceae along the food chain and between different populations to develop strategies to prevent their further spread.Copyright © 2019 Roschanski et al.


April 21, 2020  |  

Comparative Transcriptomic Profiling of Yersinia enterocolitica O:3 and O:8 Reveals Major Expression Differences of Fitness- and Virulence-Relevant Genes Indicating Ecological Separation.

Yersinia enterocolitica is a zoonotic pathogen and an important cause of bacterial gastrointestinal infections in humans. Large-scale population genomic analyses revealed genetic and phenotypic diversity of this bacterial species, but little is known about the differences in the transcriptome organization, small RNA (sRNA) repertoire, and transcriptional output. Here, we present the first comparative high-resolution transcriptome analysis of Y. enterocolitica strains representing highly pathogenic phylogroup 2 (serotype O:8) and moderately pathogenic phylogroup 3 (serotype O:3) grown under four infection-relevant conditions. Our transcriptome sequencing (RNA-seq) approach revealed 1,299 and 1,076 transcriptional start sites and identified strain-specific sRNAs that could contribute to differential regulation among the phylogroups. Comparative transcriptomics further uncovered major gene expression differences, in particular, in the temperature-responsive regulon. Multiple virulence-relevant genes are differentially regulated between the two strains, supporting an ecological separation of phylogroups with certain niche-adapted properties. Strong upregulation of the ystA enterotoxin gene in combination with constitutive high expression of cell invasion factor InvA further showed that the toxicity of recent outbreak O:3 strains has increased. Overall, our report provides new insights into the specific transcriptome organization of phylogroups 2 and 3 and reveals gene expression differences contributing to the substantial phenotypic differences that exist between the lineages. IMPORTANCE Yersinia enterocolitica is a major diarrheal pathogen and is associated with a large range of gut-associated diseases. Members of this species have evolved into different phylogroups with genotypic variations. We performed the first characterization of the Y. enterocolitica transcriptional landscape and tracked the consequences of the genomic variations between two different pathogenic phylogroups by comparing their RNA repertoire, promoter usage, and expression profiles under four different virulence-relevant conditions. Our analysis revealed major differences in the transcriptional outputs of the closely related strains, pointing to an ecological separation in which one is more adapted to an environmental lifestyle and the other to a mostly mammal-associated lifestyle. Moreover, a variety of pathoadaptive alterations, including alterations in acid resistance genes, colonization factors, and toxins, were identified which affect virulence and host specificity. This illustrates that comparative transcriptomics is an excellent approach to discover differences in the functional output from closely related genomes affecting niche adaptation and virulence, which cannot be directly inferred from DNA sequences.


April 21, 2020  |  

Whole-genome sequence of the oriental lung fluke Paragonimus westermani.

Foodborne infections caused by lung flukes of the genus Paragonimus are a significant and widespread public health problem in tropical areas. Approximately 50 Paragonimus species have been reported to infect animals and humans, but Paragonimus westermani is responsible for the bulk of human disease. Despite their medical and economic importance, no genome sequence for any Paragonimus species is available.We sequenced and assembled the genome of P. westermani, which is among the largest of the known pathogen genomes with an estimated size of 1.1 Gb. A 922.8 Mb genome assembly was generated from Illumina and Pacific Biosciences (PacBio) sequence data, covering 84% of the estimated genome size. The genome has a high proportion (45%) of repeat-derived DNA, particularly of the long interspersed element and long terminal repeat subtypes, and the expansion of these elements may explain some of the large size. We predicted 12,852 protein coding genes, showing a high level of conservation with related trematode species. The majority of proteins (80%) had homologs in the human liver fluke Opisthorchis viverrini, with an average sequence identity of 64.1%. Assembly of the P. westermani mitochondrial genome from long PacBio reads resulted in a single high-quality circularized 20.6 kb contig. The contig harbored a 6.9 kb region of non-coding repetitive DNA comprised of three distinct repeat units. Our results suggest that the region is highly polymorphic in P. westermani, possibly even within single worm isolates.The generated assembly represents the first Paragonimus genome sequence and will facilitate future molecular studies of this important, but neglected, parasite group.


April 21, 2020  |  

The bacteriocin from the prophylactic candidate Streptococcus suis 90-1330 is widely distributed across S. suis isolates and appears encoded in an integrative and conjugative element.

The Gram-positive a-hemolytic Streptococcus suis is a major pathogen in the swine industry and an emerging zoonotic agent that can cause several systemic issues in both pigs and humans. A total of 35 S. suis serotypes (SS) have been identified and genotyped into > 700 sequence types (ST) by multilocus sequence typing (MLST). Eurasian ST1 isolates are the most virulent of all S. suis SS2 strains while North American ST25 and ST28 strains display moderate to low/no virulence phenotypes, respectively. Notably, S. suis 90-1330 is an avirulent Canadian SS2-ST28 isolate producing a lantibiotic bacteriocin with potential prophylactic applications. To investigate the suitability of this strain for such purposes, we sequenced its complete genome using the Illumina and PacBio platforms. The S. suis 90-1330 bacteriocin was found encoded in a locus cargoed in what appears to be an integrative and conjugative element (ICE). This bacteriocin locus was also found to be widely distributed across several streptococcal species and in a few Staphylococcus aureus strains. Because the locus also confers protection from the bacteriocin, the potential prophylactic benefits of using this strain may prove limited due to the spread of the resistance to its effects. Furthermore, the S. suis 90-1330 genome was found to code for genes involved in blood survival, suggesting that strain may not be a benign as previously thought.


April 21, 2020  |  

Transmission of ESBL-producing Escherichia coli between broilers and humans on broiler farms.

ESBL and AmpC ß-lactamases are an increasing concern for public health. Studies suggest that ESBL/pAmpC-producing Escherichia coli and their plasmids carrying antibiotic resistance genes can spread from broilers to humans working or living on broiler farms. These studies used traditional typing methods, which may not have provided sufficient resolution to reliably assess the relatedness of these isolates.Eleven suspected transmission events among broilers and humans living/working on eight broiler farms were investigated using whole-genome short-read (Illumina) and long-read sequencing (PacBio). Core genome MLST (cgMLST) was performed to investigate the occurrence of strain transmission. Horizontal plasmid and gene transfer were analysed using BLAST.Of eight suspected strain transmission events, six were confirmed. The isolate pairs had identical ESBL/AmpC genes and fewer than eight allelic differences according to the cgMLST, and five had an almost identical plasmid composition. On one of the farms, cgMLST revealed that the isolate pairs belonging to ST10 from a broiler and a household member of the farmer had 475 different alleles, but that the plasmids were identical, indicating horizontal transfer of mobile elements rather than strain transfer. Of three suspected horizontal plasmid transmission events, one was confirmed. In addition, gene transfer between plasmids was found.The present study confirms transmission of strains as well as horizontal plasmid and gene transfer between broilers and farmers and household members on the same farm. WGS is an important tool to confirm suspected zoonotic strain and resistance gene transmission. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.


April 21, 2020  |  

A prophage and two ICESa2603-family integrative and conjugative elements (ICEs) carrying optrA in Streptococcus suis.

To investigate the presence and transfer of the oxazolidinone/phenicol resistance gene optrA and identify the genetic elements involved in the horizontal transfer of the optrA gene in Streptococcus suis.A total of 237 S. suis isolates were screened for the presence of the optrA gene by PCR. Whole-genome DNA of three optrA-positive strains was completely sequenced using the Illumina MiSeq and Pacbio RSII platforms. MICs were determined by broth microdilution. Transferability of the optrA gene in S. suis was investigated by conjugation. The presence of circular intermediates was examined by inverse PCR.The optrA gene was present in 11.8% (28/237) of the S. suis strains. In three strains, the optrA gene was flanked by two copies of IS1216 elements in the same orientation, located either on a prophage or on ICESa2603-family integrative and conjugative elements (ICEs), including one tandem ICE. In one isolate, the optrA-carrying ICE transferred with a frequency of 2.1?×?10-8. After the transfer, the transconjugant displayed elevated MICs of the respective antimicrobial agents. Inverse PCRs revealed that circular intermediates of different sizes were formed in the three optrA-carrying strains, containing one copy of the IS1216E element and the optrA gene alone or in combination with other resistance genes.A prophage and two ICESa2603-family ICEs (including one tandem ICE) associated with the optrA gene were identified in S. suis. The association of the optrA gene with the IS1216E elements and its location on either a prophage or ICEs will aid its horizontal transfer. © The Author(s) 2019. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy. All rights reserved. For permissions, please email: journals.permissions@oup.com.


April 21, 2020  |  

A systematic review of the Trypanosoma cruzi genetic heterogeneity, host immune response and genetic factors as plausible drivers of chronic chagasic cardiomyopathy.

Chagas disease is a complex tropical pathology caused by the kinetoplastid Trypanosoma cruzi. This parasite displays massive genetic diversity and has been classified by international consensus in at least six Discrete Typing Units (DTUs) that are broadly distributed in the American continent. The main clinical manifestation of the disease is the chronic chagasic cardiomyopathy (CCC) that is lethal in the infected individuals. However, one intriguing feature is that only 30-40% of the infected individuals will develop CCC. Some authors have suggested that the immune response, host genetic factors, virulence factors and even the massive genetic heterogeneity of T. cruzi are responsible of this clinical pattern. To date, no conclusive data support the reason why a few percentages of the infected individuals will develop CCC. Therefore, we decided to conduct a systematic review analysing the host genetic factors, immune response, cytokine production, virulence factors and the plausible association of the parasite DTUs and CCC. The epidemiological and clinical implications are herein discussed.


April 21, 2020  |  

Multidrug-Resistant Bovine Salmonellosis Predisposing for Severe Human Clostridial Myonecrosis.

BACKGROUND The overuse of antibiotics in animals promotes the development of multidrug-resistance predisposing for severe polymicrobial human infections. CASE REPORT We describe a case of spontaneous clostridial myonecrosis due to ulcerative colonic infection with multidrug-resistant Salmonella enterica subsp. enterica, serotype 4,[5],12: i: -. Serotyping of the colonic Salmonella isolate in the index case and the bovine farm outbreak isolates from where the patient worked indicated they were both serotype I 4,[5],12: i: -, which is linked with a multitude of large reported disease outbreaks. Further analysis revealed that they are highly genetically related and antibiotic susceptibility testing indicated that they are phenotypically identical. CONCLUSIONS Enteritis due to human acquisition of multidrug-resistant Salmonella from cattle led to the invasion and dissemination of Clostridium septicum resulting in devastating myonecrotic disease. This highlights the ramifications of co-existence and evolution of pathogenic bacteria in animals and humans and lends support to reducing the use of antibiotics in animals.


April 21, 2020  |  

Genomic analysis of three Clostridioides difficile isolates from urban water sources.

We investigated inflow of a wastewater treatment plant and sediment of an urban lake for the presence of Clostridioides difficile by cultivation and PCR. Among seven colonies we sequenced the complete genomes of three: two non-toxigenic isolates from wastewater and one toxigenic isolate from the urban lake. For all obtained isolates, a close genomic relationship with human-derived isolates was observed.Copyright © 2019 Elsevier Ltd. All rights reserved.


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