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September 22, 2019

Comparative genomics of Czech vaccine strains of Bordetella pertussis.

Bordetella pertussis is a strictly human pathogen causing the respiratory infectious disease called whooping cough or pertussis. B. pertussis adaptation to acellular pertussis vaccine pressure has been repeatedly highlighted, but recent data indicate that adaptation of circulating strains started already in the era of the whole cell pertussis vaccine (wP) use. We sequenced the genomes of five B. pertussis wP vaccine strains isolated in the former Czechoslovakia in the pre-wP (1954-1957) and early wP (1958-1965) eras, when only limited population travel into and out of the country was possible. Four isolates exhibit a similar genome organization and form a distinct phylogenetic cluster with a geographic signature. The fifth strain is rather distinct, both in genome organization and SNP-based phylogeny. Surprisingly, despite isolation of this strain before 1966, its closest sequenced relative appears to be a recent isolate from the US. On the genome content level, the five vaccine strains contained both new and already described regions of difference. One of the new regions contains duplicated genes potentially associated with transport across the membrane. The prevalence of this region in recent isolates indicates that its spread might be associated with selective advantage leading to increased strain fitness.

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September 22, 2019

Convergent evolution of complex genomic rearrangements in two fungal meiotic drive elements.

Meiotic drive is widespread in nature. The conflict it generates is expected to be an important motor for evolutionary change and innovation. In this study, we investigated the genomic consequences of two large multi-gene meiotic drive elements, Sk-2 and Sk-3, found in the filamentous ascomycete Neurospora intermedia. Using long-read sequencing, we generated the first complete and well-annotated genome assemblies of large, highly diverged, non-recombining regions associated with meiotic drive elements. Phylogenetic analysis shows that, even though Sk-2 and Sk-3 are located in the same chromosomal region, they do not form sister clades, suggesting independent origins or at least a long evolutionary separation. We conclude that they have in a convergent manner accumulated similar patterns of tandem inversions and dense repeat clusters, presumably in response to similar needs to create linkage between genes causing drive and resistance.

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September 22, 2019

Whole genome sequencing and microsatellite analysis of the Plasmodium falciparum E5 NF54 strain show that the var, rifin and stevor gene families follow Mendelian inheritance.

Plasmodium falciparum exhibits a high degree of inter-isolate genetic diversity in its variant surface antigen (VSA) families: P. falciparum erythrocyte membrane protein 1, repetitive interspersed family (RIFIN) and subtelomeric variable open reading frame (STEVOR). The role of recombination for the generation of this diversity is a subject of ongoing research. Here the genome of E5, a sibling of the 3D7 genome strain is presented. Short and long read whole genome sequencing (WGS) techniques (Ilumina, Pacific Bioscience) and a set of 84 microsatellites (MS) were employed to characterize the 3D7 and non-3D7 parts of the E5 genome. This is the first time that VSA genes in sibling parasites were analysed with long read sequencing technology.Of the 5733 E5 genes only 278 genes, mostly var and rifin/stevor genes, had no orthologues in the 3D7 genome. WGS and MS analysis revealed that chromosomal crossovers occurred at a rate of 0-3 per chromosome. var, stevor and rifin genes were inherited within the respective non-3D7 or 3D7 chromosomal context. 54 of the 84 MS PCR fragments correctly identified the respective MS as 3D7- or non-3D7 and this correlated with var and rifin/stevor gene inheritance in the adjacent chromosomal regions. E5 had 61 var and 189 rifin/stevor genes. One large non-chromosomal recombination event resulted in a new var gene on chromosome 14. The remainder of the E5 3D7-type subtelomeric and central regions were identical to 3D7.The data show that the rifin/stevor and var gene families represent the most diverse compartments of the P. falciparum genome but that the majority of var genes are inherited without alterations within their respective parental chromosomal context. Furthermore, MS genotyping with 54 MS can successfully distinguish between two sibling progeny of a natural P. falciparum cross and thus can be used to investigate identity by descent in field isolates.

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September 22, 2019

Genomic analysis of the Phalaenopsis pathogen Dickeya sp. PA1, representing the emerging species Dickeya fangzhongdai.

Dickeya sp. strain PA1 is the causal agent of bacterial soft rot in Phalaenopsis, an important indoor orchid in China. PA1 and a few other strains were grouped into a novel species, Dickeya fangzhongdai, and only the orchid-associated strains have been shown to cause soft rot symptoms.We constructed the complete PA1 genome sequence and used comparative genomics to explore the differences in genomic features between D. fangzhongdai and other Dickeya species.PA1 has a 4,979,223-bp circular genome with 4269 predicted protein-coding genes. D. fangzhongdai was phylogenetically similar to Dickeya solani and Dickeya dadantii. The type I to type VI secretion systems (T1SS-T6SS), except for the stt-type T2SS, were identified in D. fangzhongdai. The three phylogenetically similar species varied significantly in terms of their T5SSs and T6SSs, as did the different D. fangzhongdai strains. Genomic island (GI) prediction and synteny analysis (compared to D. fangzhongdai strains) of PA1 also indicated the presence of T5SSs and T6SSs in strain-specific regions. Two typical CRISPR arrays were identified in D. fangzhongdai and in most other Dickeya species, except for D. solani. CRISPR-1 was present in all of these Dickeya species, while the presence of CRISPR-2 varied due to species differentiation. A large polyketide/nonribosomal peptide (PK/NRP) cluster, similar to the zeamine biosynthetic gene cluster in Dickeya zeae rice strains, was discovered in D. fangzhongdai and D. solani. The D. fangzhongdai and D. solani strains might recently have acquired this gene cluster by horizontal gene transfer (HGT).Orchid-associated strains are the typical members of D. fangzhongdai. Genomic analysis of PA1 suggested that this strain presents the genomic characteristics of this novel species. Considering the absence of the stt-type T2SS, the presence of CRISPR loci and the zeamine biosynthetic gene cluster, D. fangzhongdai is likely a transitional form between D. dadantii and D. solani. This is supported by the later acquisition of the zeamine cluster and the loss of CRISPR arrays by D. solani. Comparisons of phylogenetic positions and virulence determinants could be helpful for the effective quarantine and control of this emerging species.

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September 22, 2019

Coculture of marine Streptomyces sp. with Bacillus sp. produces a newpiperazic acid-bearing cyclic peptide.

Microbial culture conditions in the laboratory, which conventionally involve the cultivation of one strain in one culture vessel, are vastly different from natural microbial environments. Even though perfectly mimicking natural microbial interactions is virtually impossible, the cocultivation of multiple microbial strains is a reasonable strategy to induce the production of secondary metabolites, which enables the discovery of new bioactive natural products. Our coculture of marine Streptomyces and Bacillus strains isolated together from an intertidal mudflat led to discover a new metabolite, dentigerumycin E (1). Dentigerumycin E was determined to be a new cyclic hexapeptide incorporating three piperazic acids, N-OH-Thr, N-OH-Gly, ß-OH-Leu, and a pyran-bearing polyketide acyl chain mainly by analysis of its NMR and MS spectroscopic data. The putative PKS-NRPS biosynthetic gene cluster for dentigerumycin E was found in the Streptomyces strain, providing clear evidence that this cyclic peptide is produced by the Streptomyces strain. The absolute configuration of dentigerumycin E was established based on the advanced Marfey’s method, ROESY NMR correlations, and analysis of the amino acid sequence of the ketoreductase domain in the biosynthetic gene cluster. In biological evaluation of dentigerumycin E (1) and its chemical derivatives [2-N,16-N-deoxydenteigerumycin E (2) and dentigerumycin methyl ester (3)], only dentigerumycin E exhibited antiproliferative and antimetastatic activities against human cancer cells, indicating that N-OH and carboxylic acid functional groups are essential for the biological activity.

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September 22, 2019

Conversion of methionine to cysteine in Lactobacillus paracasei depends on the highly mobile cysK-ctl-cysE gene cluster.

Milk and dairy products are rich in nutrients and are therefore habitats for various microbiomes. However, the composition of nutrients can be quite diverse, in particular among the sulfur containing amino acids. In milk, methionine is present in a 25-fold higher abundance than cysteine. Interestingly, a fraction of strains of the species L. paracasei – a flavor-enhancing adjunct culture species – can grow in medium with methionine as the sole sulfur source. In this study, we focus on genomic and evolutionary aspects of sulfur dependence in L. paracasei strains. From 24 selected L. paracasei strains, 16 strains can grow in medium with methionine as sole sulfur source. We sequenced these strains to perform gene-trait matching. We found that one gene cluster – consisting of a cysteine synthase, a cystathionine lyase, and a serine acetyltransferase – is present in all strains that grow in medium with methionine as sole sulfur source. In contrast, strains that depend on other sulfur sources do not have this gene cluster. We expanded the study and searched for this gene cluster in other species and detected it in the genomes of many bacteria species used in the food production. The comparison to these species showed that two different versions of the gene cluster exist in L. paracasei which were likely gained in two distinct events of horizontal gene transfer. Additionally, the comparison of 62 L. paracasei genomes and the two versions of the gene cluster revealed that this gene cluster is mobile within the species.

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September 22, 2019

Complete genomic analysis of a kingdom crossing Klebsiella variicola isolate.

Bacterial isolate X39 was isolated from a community-acquired pneumonia patient in Beijing, China. A phylogenetic tree based on rpoB genes and average nucleotide identity data confirmed that isolate X39 belonged to Klebsiella variicola. The genome of K. variicola X39 contained one circular chromosome and nine plasmids. Comparative genomic analyses with other K. variicola isolates revealed that K. variicola X39 contained the most unique genes. Of these unique genes, many were prophages and transposases. Many virulence factors were shared between K. variicola X39 and Klebsiella pneumoniae F1. The pathogenicity of K. variicola X39 was compared with that of K. pneumoniae F1 in an abdominal infection model. The results indicated that K. variicola X39 was less virulent than typical clinical K. pneumoniae F1. The genome of K. variicola X39 also contained some genes involved in plant colonization, nitrogen fixation, and defense against oxidative stress. GFP-labeled K. variicola X39 could colonize maize as an endophytic bacterium. We concluded that K. variicola X39 was a kingdom-crossing strain.

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September 22, 2019

Type II restriction modification system in Ureaplasma parvum OMC-P162 strain.

Ureaplasma parvum serovar 3 strain, OMC-P162, was isolated from the human placenta of a preterm delivery at 26 weeks’ gestation. In this study, we sequenced the complete genome of OMC-P162 and compared it with other serovar 3 strains isolated from patients with different clinical conditions. Ten unique genes in OMC-P162, five of which encoded for hypothetical proteins, were identified. Of these, genes UPV_229 and UPV_230 formed an operon whose open reading frames were predicted to code for a DNA methyltransferase and a hypothetical protein, respectively. DNA modification analysis of the OMC-P162 genome identified N4-methylcytosine (m4C) and N6-methyladenine (m6A), but not 5-methylocytosine (m5C). UPV230 recombinant protein displayed endonuclease activity and recognized the CATG sequence, resulting in a blunt cut between A and T. This restriction enzyme activity was identical to that of the cultivated OMC-P162 strain, suggesting that this restriction enzyme was naturally expressed in OMC-P162. We designated this enzyme as UpaP162. Treatment of pT7Blue plasmid with recombinant protein UPV229 completely blocked UpaP162 restriction enzyme activity. These results suggest that the UPV_229 and UPV_230 genes act as a type II restriction-modification system in Ureaplasma OMC-P162.

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September 22, 2019

Discovery of mcr-1-mediated colistin resistance in a highly virulent Escherichia coli lineage.

Resistance to last-line polymyxins mediated by the plasmid-borne mobile colistin resistance gene (mcr-1) represents a new threat to global human health. Here we present the complete genome sequence of an mcr-1-positive multidrug-resistant Escherichia coli strain (MS8345). We show that MS8345 belongs to serotype O2:K1:H4, has a large 241,164-bp IncHI2 plasmid that carries 15 other antibiotic resistance genes (including the extended-spectrum ß-lactamase blaCTX-M-1) and 3 putative multidrug efflux systems, and contains 14 chromosomally encoded antibiotic resistance genes. MS8345 also carries a large ColV-like virulence plasmid that has been associated with E. coli bacteremia. Whole-genome phylogeny revealed that MS8345 clusters within a discrete clade in the sequence type 95 (ST95) lineage, and MS8345 is very closely related to the highly virulent O45:K1:H4 clone associated with neonatal meningitis. Overall, the acquisition of a plasmid carrying resistance to colistin and multiple other antibiotics in this virulent E. coli lineage is concerning and might herald an era where the empirical treatment of ST95 infections becomes increasingly more difficult.IMPORTANCEEscherichia coli ST95 is a globally disseminated clone frequently associated with bloodstream infections and neonatal meningitis. However, the ST95 lineage is defined by low levels of drug resistance amongst clinical isolates, which normally provides for uncomplicated treatment options. Here, we provide the first detailed genomic analysis of an E. coli ST95 isolate that has both high virulence potential and resistance to multiple antibiotics. Using the genome, we predicted its virulence and antibiotic resistance mechanisms, which include resistance to last-line antibiotics mediated by the plasmid-borne mcr-1 gene. Finding an ST95 isolate resistant to nearly all antibiotics that also has a high virulence potential is of major clinical importance and underscores the need to monitor new and emerging trends in antibiotic resistance development in this important global lineage. Copyright © 2018 Forde et al.

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September 22, 2019

Distribution of the pco gene cluster and associated genetic determinants among swine Escherichia coli from a controlled feeding trial.

Copper is used as an alternative to antibiotics for growth promotion and disease prevention. However, bacteria developed tolerance mechanisms for elevated copper concentrations, including those encoded by the pco operon in Gram-negative bacteria. Using cohorts of weaned piglets, this study showed that the supplementation of feed with copper concentrations as used in the field did not result in a significant short-term increase in the proportion of pco-positive fecal Escherichia coli. The pco and sil (silver resistance) operons were found concurrently in all screened isolates, and whole-genome sequencing showed that they were distributed among a diversity of unrelated E. coli strains. The presence of pco/sil in E. coli was not associated with elevated copper minimal inhibitory concentrations (MICs) under a variety of conditions. As found in previous studies, the pco/sil operons were part of a Tn7-like structure found both on the chromosome or on plasmids in the E. coli strains investigated. Transfer of a pco/sil IncHI2 plasmid from E. coli to Salmonellaenterica resulted in elevated copper MICs in the latter. Escherichia coli may represent a reservoir of pco/sil genes transferable to other organisms such as S. enterica, for which it may represent an advantage in the presence of copper. This, in turn, has the potential for co-selection of resistance to antibiotics.

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September 22, 2019

Comparative genome analysis and evaluation of probiotic characteristics of Lactobacillus plantarum strain JDFM LP11.

In the current study, the probiotic potential of approximately 250 strains of lactic acid bacteria (LAB) isolated from piglet fecal samples were investigated; among them Lactobacillus plantarum strain JDFM LP11, which possesses significant probiotic potential, with enhanced acid/bile tolerance, attachment to porcine intestinal epithelial cells (IPEC-J2), and antimicrobial activity. The genetic characteristics of strain JDFM LP11 were explored by performing whole genome sequencing (WGS) using a PacBio system. The circular draft genome have a total length of 3,206,883 bp and a total of 3,021 coding sequences were identified. Phylogenetically, three genes, possibly related to survival and metabolic activity in the porcine host, were identified. These genes encode p60, lichenan permease IIC component, and protein TsgA, which are a putative endopeptidase, a component of the phosphotransferase system (PTS), and a major facilitator in the gut environment, respectively. Our findings suggest that understanding the functional and genetic characteristics of L. plantarum strain JDFM LP11, with its candidate genes for gut health, could provide new opportunities and insights into applications in the animal food and feed additive industries.

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September 22, 2019

Repeat elements organise 3D genome structure and mediate transcription in the filamentous fungus Epichloë festucae.

Structural features of genomes, including the three-dimensional arrangement of DNA in the nucleus, are increasingly seen as key contributors to the regulation of gene expression. However, studies on how genome structure and nuclear organisation influence transcription have so far been limited to a handful of model species. This narrow focus limits our ability to draw general conclusions about the ways in which three-dimensional structures are encoded, and to integrate information from three-dimensional data to address a broader gamut of biological questions. Here, we generate a complete and gapless genome sequence for the filamentous fungus, Epichloë festucae. We use Hi-C data to examine the three-dimensional organisation of the genome, and RNA-seq data to investigate how Epichloë genome structure contributes to the suite of transcriptional changes needed to maintain symbiotic relationships with the grass host. Our results reveal a genome in which very repeat-rich blocks of DNA with discrete boundaries are interspersed by gene-rich sequences that are almost repeat-free. In contrast to other species reported to date, the three-dimensional structure of the genome is anchored by these repeat blocks, which act to isolate transcription in neighbouring gene-rich regions. Genes that are differentially expressed in planta are enriched near the boundaries of these repeat-rich blocks, suggesting that their three-dimensional orientation partly encodes and regulates the symbiotic relationship formed by this organism.

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September 22, 2019

Therapeutic potential of a new jumbo phage that infects Vibrio coralliilyticus, a widespread coral pathogen.

Biological control using bacteriophages is a promising approach for mitigating the devastating effects of coral diseases. Several phages that infect Vibrio coralliilyticus, a widespread coral pathogen, have been isolated, suggesting that this bacterium is permissive to viral infection and is, therefore, a suitable candidate for treatment by phage therapy. In this study, we combined functional and genomic approaches to evaluate the therapeutic potential of BONAISHI, a novel V. coralliilyticus phage, which was isolated from the coral reef in Van Phong Bay (Vietnam). BONAISHI appears to be strictly lytic for several pathogenic strains of V. coralliilyticus and remains infectious over a broad range of environmental conditions. This candidate has an unusually large dsDNA genome (303 kb), with no genes that encode known toxins or implicated in lysogeny control. We identified several proteins involved in host lysis, which may offer an interesting alternative to the use of whole bacteriophages for controlling V. coralliilyticus. A preliminary therapy test showed that adding BONAISHI to an infected culture of Symbiodinium sp. cells reduced the impact of V. coralliilyticus on Symbiodinium sp. photosynthetic activity. This study showed that BONAISHI is able to mitigate V. coralliilyticus infections, making it a good candidate for phage therapy for coral disease.

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September 22, 2019

Update on Tetracycline Susceptibility of Pediococcus acidilactici Based on Strains Isolated from Swiss Cheese and Whey.

Bacterial strains used as starter cultures in the production of fermented foods may act as reservoirs for antibiotic resistance (AbR) genes. To avoid the introduction of such genes into the food chain, the presence of acquired AbR in bacterial strains added to food must be tested. Standard protocols and microbiological cut-off values have been defined to provide practitioners with a basis for evaluating whether their bacterial isolates harbor an acquired resistance to a given antibiotic. Here, we tested the AbR of 24 strains of Pediococcus acidilactici by using the standard protocol and microbiological cut-off values recommended by the European Food Safety Authority. Phenotypic data were complemented by searching for known AbR genes using an in silico analysis of whole genomes. The majority (54.2%) of the strains were able to grow at a tetracycline concentration above the defined cut-off, even though only one strain carried a known tetracycline resistance gene, tetM. The same strain also carried the AbR gene of an erythromycin resistance methylase, ermA, and displayed resistance toward clindamycin and erythromycin. Our results bolster the scarce data on the sensitivity of P. acidilactici to tetracycline and suggest that the microbiological cut-off recommended by the European Food Safety Authority for this antibiotic should be amended.

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September 22, 2019

Antimicrobial resistance profile of mcr-1 positive clinical isolates of Escherichia coli in China From 2013 to 2016.

Multidrug-resistant (MDR) Escherichia coli poses a great challenge for public health in recent decades. Polymyxins have been reconsidered as a valuable therapeutic option for the treatment of infections caused by MDR E. coli. A plasmid-encoded colistin resistance gene mcr-1 encoding phosphoethanolamine transferase has been recently described in Enterobacteriaceae. In this study, a total of 123 E. coli isolates obtained from patients with diarrheal diseases in China were used for the genetic analysis of colistin resistance in clinical isolates. Antimicrobial resistance profile of polymyxin B (PB) and 11 commonly used antimicrobial agents were determined. Among the 123 E. coli isolates, 9 isolates (7.3%) were resistant to PB and PCR screening showed that seven (5.7%) isolates carried the mcr-1 gene. A hybrid sequencing analysis using single-molecule, real-time (SMRT) sequencing and Illumina sequencing was then performed to resolve the genomes of the seven mcr-1 positive isolates. These seven isolates harbored multiple plasmids and are MDR, with six isolates carrying one mcr-1 positive plasmid and one isolate (14EC033) carrying two mcr-1 positive plasmids. These eight mcr-1 positive plasmids belonged to the IncX4, IncI2, and IncP1 types. In addition, the mcr-1 gene was the solo antibiotic resistance gene identified in the mcr-1 positive plasmids, while the rest of the antibiotic resistance genes were mostly clustered into one or two plasmids. Interestingly, one mcr-1 positive isolate (14EC047) was susceptible to PB, and we showed that the activity of MCR-1-mediated colistin resistance was not phenotypically expressed in 14EC047 host strain. Furthermore, three isolates exhibited resistance to PB but did not carry previously reported mcr-related genes. Multilocus sequence typing (MLST) showed that these mcr-1 positive E. coli isolates belonged to five different STs, and three isolates belonged to ST301 which carried multiple virulence factors related to diarrhea. Additionally, the mcr-1 positive isolates were all susceptible to imipenem (IMP), suggesting that IMP could be used to treat infection caused by mcr-1 positive E. coli isolates. Collectively, this study showed a high occurrence of mcr-1 positive plasmids in patients with diarrheal diseases of Guangzhou in China and the abolishment of the MCR-1 mediated colistin resistance in one E. coli isolate.

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