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Asset Tag: Microbiology

April 21, 2020

Plasmid-encoded tet(X) genes that confer high-level tigecycline resistance in Escherichia coli.

Tigecycline is one of the last-resort antibiotics to treat complicated infections caused by both multidrug-resistant Gram-negative and Gram-positive bacteria1. Tigecycline resistance has sporadically occurred in recent years, primarily due to chromosome-encoding mechanisms, such as overexpression of efflux pumps and ribosome protection2,3. Here, we report the emergence of the plasmid-mediated mobile tigecycline resistance mechanism Tet(X4) in Escherichia coli isolates from China, which is capable of degrading all tetracyclines, including tigecycline and the US FDA newly approved eravacycline. The tet(X4)-harbouring IncQ1 plasmid is highly transferable, and can be successfully mobilized and stabilized in recipient clinical and laboratory strains of Enterobacteriaceae bacteria. It is noteworthy that tet(X4)-positive E.?coli strains, including isolates co-harbouring mcr-1, have been widely detected in pigs, chickens, soil and dust samples in China. In vivo murine models demonstrated that the presence of Tet(X4) led to tigecycline treatment failure. Consequently, the emergence of plasmid-mediated Tet(X4) challenges the clinical efficacy of the entire family of tetracycline antibiotics. Importantly, our study raises concern that the plasmid-mediated tigecycline resistance may further spread into various ecological niches and into clinical high-risk pathogens. Collective efforts are in urgent need to preserve the potency of these essential antibiotics.

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April 21, 2020

Antibiotic susceptibility of plant-derived lactic acid bacteria conferring health benefits to human.

Lactic acid bacteria (LAB) confer health benefits to human when administered orally. We have recently isolated several species of LAB strains from plant sources, such as fruits, vegetables, flowers, and medicinal plants. Since antibiotics used to treat bacterial infection diseases induce the emergence of drug-resistant bacteria in intestinal microflora, it is important to evaluate the susceptibility of LAB strains to antibiotics to ensure the safety and security of processed foods. The aim of the present study is to determine the minimum inhibitory concentration (MIC) of antibiotics against several plant-derived LAB strains. When aminoglycoside antibiotics, such as streptomycin (SM), kanamycin (KM), and gentamicin (GM), were evaluated using LAB susceptibility test medium (LSM), the MIC was higher than when using Mueller-Hinton (MH) medium. Etest, which is an antibiotic susceptibility assay method consisting of a predefined gradient of antibiotic concentrations on a plastic strip, is used to determine the MIC of antibiotics world-wide. In the present study, we demonstrated that Etest was particularly valuable while testing LAB strains. We also show that the low susceptibility of the plant-derived LAB strains against each antibiotic tested is due to intrinsic resistance and not acquired resistance. This finding is based on the whole-genome sequence information reflecting the horizontal spread of the drug-resistance genes in the LAB strains.

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April 21, 2020

A putative microcin amplifies Shiga toxin 2a production of Escherichia coli O157: H7

Escherichia coli O157:H7 is a foodborne pathogen, implicated in various multi-state outbreaks. It encodes Shiga toxin on a prophage, and Shiga toxin production is linked to phage induction. An E. coli strain, designated 0.1229, was identified that amplified Stx2a production when co-cultured with E. coli O157:H7 strain PA2. Growth of PA2 in 0.1229 cell-free supernatants had a similar effect, even when supernatants were heated to 100°C for 10 min, but not after treatment with Proteinase K. The secreted molecule was shown to use TolC for export and the TonB system for import. The genes sufficient for production of this molecule were localized to a 5.2 kb region of a 12.8 kb plasmid. This region was annotated, identifying hypothetical proteins, a predicted ABC transporter, and a cupin superfamily protein. These genes were identified and shown to be functional in two other E. coli strains, and bioinformatic analyses identified related gene clusters in similar and distinct bacterial species. These data collectively suggest E. coli 0.1229 and other E. coli produce a microcin that induces the SOS response in target bacteria. Besides adding to the limited number of microcins known to be produced by E. coli, this study provides an additional mechanism by which stx2a expression is increased in response to the gut microflora.

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April 21, 2020

Genetically diverse uropathogenic Escherichia coli adopt a common transcriptional program in patients with urinary tract infections

Uropathogenic Escherichia coli (UPEC) is the major causative agent of uncomplicated urinary tract infections (UTIs). A common virulence genotype of UPEC strains responsible for UTIs is yet to be defined, due to the large variation of virulence factors observed in UPEC strains. We hypothesized that studying UPEC functional responses in patients might reveal universal UPEC features that enable pathogenesis. Here we identify a transcriptional program shared by genetically diverse UPEC strains isolated from 14 patients during uncomplicated UTIs. Strikingly, this in vivo gene expression program is marked by upregulation of translational machinery, providing a mechanism for the rapid growth within the host. Our analysis indicates that switching to a more specialized catabolism and scavenging lifestyle in the host allows for the increased translational output. Our study identifies a common transcriptional program underlying UTIs and illuminates the molecular underpinnings that likely facilitate the fast growth rate of UPEC in infected patients.

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April 21, 2020

A megaplasmid family responsible for dissemination of multidrug resistance in Pseudomonas

Multidrug resistance (MDR) represents a global threat to health. Although plasmids can play an important role in the dissemination of MDR, they have not been commonly linked to the emergence of antimicrobial resistance in the pathogen Pseudomonas aeruginosa. We used whole genome sequencing to characterize a collection of P. aeruginosa clinical isolates from a hospital in Thailand. Using long-read sequence data we obtained complete sequences of two closely related megaplasmids (>420 kb) carrying large arrays of antibiotic resistance genes located in discrete, complex and dynamic resistance regions, and revealing evidence of extensive duplication and recombination events. A comprehensive pangenomic and phylogenomic analysis indicated that 1) these large plasmids comprise a family present in different members of the Pseudomonas genus and associated with multiple sources (geographical, clinical or environmental); 2) the megaplasmids encode diverse niche-adaptive accessory traits, including multidrug resistance; 3) the pangenome of the megaplasmid family is highly flexible and diverse, comprising a substantial core genome (average of 48% of plasmid genes), but with individual members carrying large numbers of unique genes. The history of the megaplasmid family, inferred from our analysis of the available database, suggests that members carrying multiple resistance genes date back to at least the 1970s.

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April 21, 2020

Integrative functional genomics decodes herpes simplex virus 1

Since the genome of herpes simplex virus 1 (HSV-1) was first sequenced more than 30 years ago, its predicted 80 genes have been intensively studied. Here, we unravel the complete viral transcriptome and translatome during lytic infection with base-pair resolution by computational integration of multi-omics data. We identified a total of 201 viral transcripts and 284 open reading frames (ORFs) including all known and 46 novel large ORFs. Multiple transcript isoforms expressed from individual gene loci explain translation of the vast majority of novel viral ORFs as well as N-terminal extensions (NTEs) and truncations thereof. We show that key viral regulators and structural proteins possess NTEs, which initiate from non-canonical start codons and govern subcellular protein localization and packaging. We validated a novel non-canonical large spliced ORF in the ICP0 locus and identified a 93 aa ORF overlapping ICP34.5 that is thus also deleted in the FDA-approved oncolytic virus Imlygic. Finally, we extend the current nomenclature to include all novel viral gene products. Taken together, this work provides a valuable resource for future functional studies, vaccine design and oncolytic therapies.

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April 21, 2020

Long metabarcoding of the eukaryotic rDNA operon to phylogenetically and taxonomically resolve environmental diversity

High-throughput environmental DNA metabarcoding has revolutionized the analysis of microbial diversity, but this approach is generally restricted to amplicon sizes below 500 base pairs. These short regions contain limited phylogenetic signal, which makes it impractical to use environmental DNA in full phylogenetic inferences. However, new long-read sequencing technologies such as the Pacific Biosciences platform may provide sufficiently large sequence lengths to overcome the poor phylogenetic resolution of short amplicons. To test this idea, we amplified soil DNA and used PacBio Circular Consensus Sequencing (CCS) to obtain a ~4500 bp region of the eukaryotic rDNA operon spanning most of the small (18S) and large subunit (28S) ribosomal RNA genes. The CCS reads were first treated with a novel curation workflow that generated 650 high-quality OTUs containing the physically linked 18S and 28S regions of the long amplicons. In order to assign taxonomy to these OTUs, we developed a phylogeny-aware approach based on the 18S region that showed greater accuracy and sensitivity than similarity-based and phylogenetic placement-based methods using shorter reads. The taxonomically-annotated OTUs were then combined with available 18S and 28S reference sequences to infer a well-resolved phylogeny spanning all major groups of eukaryotes, allowing to accurately derive the evolutionary origin of environmental diversity. A total of 1019 sequences were included, of which a majority (58%) corresponded to the new long environmental CCS reads. Comparisons to the 18S-only region of our amplicons revealed that the combined 18S-28S genes globally increased the phylogenetic resolution, recovering specific groupings otherwise missing. The long-reads also allowed to directly investigate the relationships among environmental sequences themselves, which represents a key advantage over the placement of short reads on a reference phylogeny. Altogether, our results show that long amplicons can be treated in a full phylogenetic framework to provide greater taxonomic resolution and a robust evolutionary perspective to environmental DNA.

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April 21, 2020

Convergent evolution of linked mating-type loci in basidiomycetes: an ancient fusion event that has stood the test of time

Sexual development is a key evolutionary innovation of eukaryotes. In many species, mating involves interaction between compatible mating partners that can undergo cell and nuclear fusion and subsequent steps of development including meiosis. Mating compatibility in fungi is governed by mating type determinants, which are localized at mating type (MAT) loci. In basidiomycetes, the ancestral state is hypothesized to be tetrapolar (bifactorial), with two genetically unlinked MAT loci containing homeodomain transcription factor genes (HD locus) and pheromone and pheromone receptor genes (P/R locus), respectively. Alleles at both loci must differ between mating partners for completion of sexual development. However, there are also basidiomycete species with bipolar (unifactorial) mating systems, which can arise through genomic linkage of the HD and P/R loci. In the order Tremellales, which is comprised of mostly yeast-like species, bipolarity is found only in the human pathogenic Cryptococcus species. Here, we describe the analysis of MAT loci from the Trichosporonales, a sister order to the Tremellales. We analyzed genome sequences from 29 strains that belong to 24 species, including two new genome sequences generated in this study. Interestingly, in all of the species analyzed, the MAT loci are fused and a single HD gene is present in each mating type. This is similar to the organization in the pathogenic Cryptococci, which also have linked MAT loci and carry only one HD gene per MAT locus instead of the usual two HD genes found in the vast majority of basidiomycetes. However, the HD and P/R allele combinations in the Trichosporonales are different from those in the pathogenic Cryptococcus species. The differences in allele combinations compared to the bipolar Cryptococci as well as the existence of tetrapolar Tremellales sister species suggest that fusion of the HD and P/R loci and differential loss of one of the two HD genes per MAT allele occurred independently in the Trichosporonales and pathogenic Cryptococci. This finding supports the hypothesis of convergent evolution at the molecular level towards fused mating-type regions in fungi, similar to previous findings in other fungal groups. Unlike the fused MAT loci in several other basidiomycete lineages though, the gene content and gene order within the fused MAT loci are highly conserved in the Trichosporonales, and there is no apparent suppression of recombination extending from the MAT loci to adjacent chromosomal regions, suggesting different mechanisms for the evolution of physically linked MAT loci in these groups.

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April 21, 2020

Antibiotic production is organized by a division of labour in Streptomyces

One of the hallmark behaviors of social groups is division of labour, where different group members become specialized to carry out complementary tasks. By dividing labour, cooperative groups of individuals increase their efficiency, thereby raising group fitness even if these specialized behaviors reduce the fitness of individual group members. Here we provide evidence that antibiotic production in colonies of the multicellular bacterium Streptomyces coelicolor is coordinated by a division of labour. We show that S. coelicolor colonies are genetically heterogeneous due to massive amplifications and deletions to the chromosome. Cells with gross chromosomal changes produce an increased diversity of secondary metabolites and secrete significantly more antibiotics; however, these changes come at the cost of dramatically reduced individual fitness, providing direct evidence for a trade-off between secondary metabolite production and fitness. Finally, we show that colonies containing mixtures of mutant strains and their parents produce significantly more antibiotics, while colony-wide spore production remains unchanged. Our work demonstrates that by generating mutants that are specialized to hyper-produce antibiotics, streptomycetes reduce the colony-wide fitness costs of secreted secondary metabolites while maximizing the yield and diversity of these products.

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April 21, 2020

Neighbor predation linked to natural competence fosters the transfer of large genomic regions in Vibrio cholerae.

Natural competence for transformation is a primary mode of horizontal gene transfer. Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. However, the prevalence of non-degraded DNA with sufficient coding capacity is not well understood. In this context, we previously showed that naturally competent Vibrio cholerae use their type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors. Here, we explored the conditions of the DNA released through T6SS-mediated killing versus passive cell lysis and the extent of the transfers that occur due to these conditions. We show that competent V. cholerae acquire DNA fragments with a length exceeding 150 kbp in a T6SS-dependent manner. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters the exchange of genetic material with sufficient coding capacity to significantly accelerate bacterial evolution. © 2019, Matthey et al.

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April 21, 2020

Identification and characterization of OmpT-like proteases in uropathogenic Escherichia coli clinical isolates

Bacterial colonization of the urogenital tract is limited by innate defenses, including the production of antimicrobial peptides (AMPs). Uropathogenic Escherichia coli (UPEC) resist AMP-killing to cause a range of urinary tract infections (UTIs) including asymptomatic bacteriuria, cystitis, pyelonephritis, and sepsis. UPEC strains have high genomic diversity and encode numerous virulence factors that differentiate them from non-UTI causing strains, including ompT. As OmpT homologues cleave and inactivate AMPs, we hypothesized that high OmpT protease activity-levels contribute to UPEC colonization during symptomatic UTIs. Therefore, we measured OmpT activity in 58 UPEC clinical isolates. While heterogeneous OmpT activities were observed, OmpT activity was significantly greater in UPEC strains isolated from patients with symptomatic infections. Unexpectedly, UPEC strains exhibiting the greatest protease activities harboured an additional ompT-like gene called arlC (ompTp). The presence of two OmpT-like proteases in some UPEC isolates led us to compare the substrate specificities of OmpT-like proteases found in E. coli. While all three cleaved AMPs, cleavage efficiency varied on the basis of AMP size and secondary structure. Our findings suggest the presence ArlC and OmpT in the same UPEC isolate may confer a fitness advantage by expanding the range of target substrates.

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April 21, 2020

Complete genome sequence and evolution analysis of Psychrobacter sp. YP14 from Gammaridea Gastrointestinal Microbiota of Yap Trench

Psychrobacter sp. YP14, a moderately psychrophilic bacterium belonging to the class Gammaproteobacteria, was isolated from Gammaridea Gastrointestinal Microbiota of Yap Trench. The strain has one circular chromosome of 2,895,311 bp with a 44.66% GC content, consisting of 2333 protein-coding genes, 53 tRNA genes and 9 rRNA genes. Four plasmids were completely assembled and their sizes were 13,712 bp, 19711 bp, 36270 bp, 8194 bp, respectively. In particular, a putative open reading frame (ORF) for dienelactone hydrolase (DLH) related to degradation of chlorinated aromatic hydrocarbons. To get an better understanding of the evolution of Psychrobacter sp. YP14 in this genus, six Psychrobacter strains (G, PRwf-1, DAB_AL43B, AntiMn-1,P11G5, P2G3), with publicly available complete genome, were selected and comparative genomics analysis were performed among them. The closest phylogenetic relationship was identified between strains G and K5 based on 16s gene and ANI (average nucleotide identity) values. Analysis of the pan-genome structure found that YP14 has fewer COG clusters associated with transposons and prophage which indicates fewer sequence rearrangements compared with PRwf-1. Besides, stress response-related genes of strain YP14 demonstrates that it has less strategies to cope with extreme environment, which is consistent with its intestinal habitat. The difference of metabolism and strategies coped with stress response of YP14 are more conducive to the study of microbial survival and metabolic mechanisms in deep sea environment.

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April 21, 2020

Multiple Long-read Sequencing Survey of Herpes Simplex Virus Lytic Transcriptome

Long-read sequencing (LRS) has become increasingly important in RNA research due to its strength in resolving complex transcriptomic architectures. In this regard, currently two LRS platforms have demonstrated adequate performance: the Single Molecule Real-Time Sequencing by Pacific Biosciences (PacBio) and the nanopore sequencing by Oxford Nanopore Technologies (ONT). Even though these techniques produce lower coverage and are more error prone than short-read sequencing, they continue to be more successful in identifying transcript isoforms including polycistronic and multi-spliced RNA molecules, as well as transcript overlaps. Recent reports have successfully applied LRS for the investigation of the transcriptome of viruses belonging to various families. These studies have substantially increased the number of previously known viral RNA molecules. In this work, we used the Sequel and MinION technique from PacBio and ONT, respectively, to characterize the lytic transcriptome of the herpes simplex virus type 1 (HSV-1). In most samples, we analyzed the poly(A) fraction of the transcriptome, but we also performed random oligonucleotide-based sequencing. Besides cDNA sequencing, we also carried out native RNA sequencing. Our investigations identified more than 160 previously undetected transcripts, including coding and non-coding RNAs, multi-splice transcripts, as well as polycistronic and complex transcripts. Furthermore, we determined previously unsubstantiated transcriptional start sites, polyadenylation sites, and splice sites. A large number of novel transcriptional overlaps were also detected. Random-primed sequencing revealed that each convergent gene pair produces non-polyadenylated read-through RNAs overlapping the partner genes. Furthermore, we identified novel replication-associated transcripts overlapping the HSV-1 replication origins, and novel LAT variants with very long 5’ regions, which are co-terminal with the LAT-0.7kb transcript. Overall, our results demonstrated that the HSV-1 transcripts form an extremely complex pattern of overlaps, and that entire viral genome is transcriptionally active. In most viral genes, if not in all, both DNA strands are expressed.

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April 21, 2020

Evidence of extensive intraspecific noncoding reshuffling in a 169-kb mitochondrial genome of a basidiomycetous fungus

Comparative genomics of fungal mitochondrial genomes (mitogenomes) have revealed a remarkable pattern of rearrangement between and within major phyla owing to horizontal gene transfer (HGT) and recombination. The role of recombination was exemplified at a finer evolutionary time scale in basidiomycetes group of fungi as they display a diversity of mitochondrial DNA (mtDNA) inheritance patterns. Here, we assembled mitogenomes of six species from the Hymenochaetales order of basidiomycetes and examined 59 mitogenomes from two genetic lineages of Pyrrhoderma noxium. Gene order is largely colinear while intergene regions are major determinants of mitogenome size variation. Substantial sequence divergence was found in shared introns consistent with high HGT frequency observed in yeasts, but we also identified a rare case where an intron was retained in five species since speciation. In contrast to the hyperdiversity observed in nuclear genomes of P. noxium, mitogenomes’ intraspecific polymorphisms at protein coding sequences are extremely low. Phylogeny based on introns revealed turnover as well as exchange of introns between two lineages. Strikingly, some strains harbor a mosaic origin of introns from both lineages. Analysis of intergenic sequence indicated substantial differences between and within lineages, and an expansion may be ongoing as a result of exchange between distal intergenes. These findings suggest that the evolution in mtDNAs is usually lineage specific but chimeric mitotypes are frequently observed, thus capturing the possible evolutionary processes shaping mitogenomes in a basidiomycete. The large mitogenome sizes reported in various basidiomycetes appear to be a result of interspecific reshuffling of intergenes.

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April 21, 2020

Complete genome sequence and annotation of the laboratory reference strain Shigella flexneri serovar 5a M90T and genome-wide transcription start site determination

Background Shigella is a Gram-negative facultative intracellular bacterium that causes bacillary dysentery in humans. Shigella invades cells of the colonic mucosa owing to its virulence plasmid-encoded Type 3 Secretion System (T3SS), and multiplies in the target cell cytosol. Although the laboratory reference strain S. flexneri serotype 5a M90T has been extensively used to understand the molecular mechanisms of pathogenesis, its complete genome sequence is not available, thereby greatly limiting studies employing high-throughput sequencing and systems biology approaches. Results We have sequenced, assembled, annotated and manually curated the full genome of S. flexneri 5a M90T. This yielded two complete circular contigs, the chromosome and the virulence plasmid (pWR100). To obtain the genome sequence, we have employed long-read PacBio DNA sequencing followed by polishing with Illumina RNA-seq data. This provides a new pipeline to prepare gapless, highly accurate genome sequences. Furthermore, we have performed genome-wide analysis of transcriptional start sites and determined the length of 5’ untranslated regions (5’-UTRs) at typical culture conditions for the inoculum of in vitro infection experiments. We identified 6,723 primary TSS (pTSS) and 7,328 secondary TSS (sTSS). The S. flexneri 5a M90T annotated genome sequence and the transcriptional start sites are integrated into RegulonDB (http://regulondb.ccg.unam.mx) and RSAT (http://embnet.ccg.unam.mx/rsat/) to use its analysis tools in S. flexneri 5a M90T genome. Conclusions We provide the first complete genome for S. flexneri serotype 5a, specifically the laboratory reference strain M90T. Our work opens the possibility of employing S. flexneri M90T in high-quality systems biology studies such as transcriptomic and differential expression analyses or in genome evolution studies. Moreover, the catalogue of TSS that we report here can be used in molecular pathogenesis studies as a resource to know which genes are transcribed before infection of host cells. The genome sequence, together with the analysis of transcriptional start sites, is also a valuable tool for precise genetic manipulation of S. flexneri 5a M90T. The hybrid pipeline that we report here combining genome sequencing with long-reads technology and polishing with RNAseq data defines a powerful strategy for genome assembly, polishing and annotation in any type of organism.

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