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September 22, 2019

Genomic analysis of consecutive Acinetobacter baumannii strains from a single patient.

Acinetobacter baumannii is one of the most important nosocomial pathogens, and thus it is required to investigate how it disseminate in hospitals and infect patients. We performed whole genome sequencing for 24 A. baumannii strains isolated successively from the blood of a single patient to evaluate whether repeated infections were due to re-infection or relapse infection and to investigate within-host evolution. The whole genome of the first strain, BL1, was sequenced de novo using the PacBio RSII system. BL2-BL24, were sequenced with an Illumina Hiseq4000 and mapped to the genome sequences of BL1. We identified 42 single-nucleotide variations among the strains. The SNVs differentiated the strains into three groups, BL1, BL2-BL16, and BL17-BL24, indicating that the patient suffered from re-infections or co-infections by similar, but different strains. The results also showed that A. baumannii strains in each group were rather stable at the genomic level. Our study emphasizes the importance of intensive infection control.

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September 22, 2019

Genomic surveillance of Enterococcus faecium reveals limited sharing of strains and resistance genes between livestock and humans in the United Kingdom.

Vancomycin-resistant Enterococcus faecium (VREfm) is a major cause of nosocomial infection and is categorized as high priority by the World Health Organization global priority list of antibiotic-resistant bacteria. In the past, livestock have been proposed as a putative reservoir for drug-resistant E. faecium strains that infect humans, and isolates of the same lineage have been found in both reservoirs. We undertook cross-sectional surveys to isolate E. faecium (including VREfm) from livestock farms, retail meat, and wastewater treatment plants in the United Kingdom. More than 600 isolates from these sources were sequenced, and their relatedness and antibiotic resistance genes were compared with genomes of almost 800 E. faecium isolates from patients with bloodstream infection in the United Kingdom and Ireland. E. faecium was isolated from 28/29 farms; none of these isolates were VREfm, suggesting a decrease in VREfm prevalence since the last UK livestock survey in 2003. However, VREfm was isolated from 1% to 2% of retail meat products and was ubiquitous in wastewater treatment plants. Phylogenetic comparison demonstrated that the majority of human and livestock-related isolates were genetically distinct, although pig isolates from three farms were more genetically related to human isolates from 2001 to 2004 (minimum of 50?single-nucleotide polymorphisms [SNPs]). Analysis of accessory (variable) genes added further evidence for distinct niche adaptation. An analysis of acquired antibiotic resistance genes and their variants revealed limited sharing between humans and livestock. Our findings indicate that the majority of E. faecium strains infecting patients are largely distinct from those from livestock in this setting, with limited sharing of strains and resistance genes.IMPORTANCE The rise in rates of human infection caused by vancomycin-resistant Enterococcus faecium (VREfm) strains between 1988 to the 2000s in Europe was suggested to be associated with acquisition from livestock. As a result, the European Union banned the use of the glycopeptide drug avoparcin as a growth promoter in livestock feed. While some studies reported a decrease in VREfm in livestock, others reported no reduction. Here, we report the first livestock VREfm prevalence survey in the UK since 2003 and the first large-scale study using whole-genome sequencing to investigate the relationship between E. faecium strains in livestock and humans. We found a low prevalence of VREfm in retail meat and limited evidence for recent sharing of strains between livestock and humans with bloodstream infection. There was evidence for limited sharing of genes encoding antibiotic resistance between these reservoirs, a finding which requires further research. Copyright © 2018 Gouliouris et al.

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September 22, 2019

Spread of the florfenicol resistance floR gene among clinical Klebsiella pneumoniae isolates in China.

Florfenicol is a derivative of chloramphenicol that is used only for the treatment of animal diseases. A key resistance gene for florfenicol, floR, can spread among bacteria of the same and different species or genera through horizontal gene transfer. To analyze the potential transmission of resistance genes between animal and human pathogens, we investigated floR in Klebsiella pneumoniae isolates from patient samples. floR in human pathogens may originate from animal pathogens and would reflect the risk to human health of using antimicrobial agents in animals.PCR was used to identify floR-positive strains. The floR genes were cloned, and the minimum inhibitory concentrations (MICs) were determined to assess the relative resistance levels of the genes and strains. Sequencing and comparative genomics methods were used to analyze floR gene-related sequence structure as well as the molecular mechanism of resistance dissemination.Of the strains evaluated, 20.42% (67/328) were resistant to florfenicol, and 86.96% (20/23) of the floR-positive strains demonstrated high resistance to florfenicol with MICs =512 µg/mL. Conjugation experiments showed that transferrable plasmids carried the floR gene in three isolates. Sequencing analysis of a plasmid approximately 125 kb in size (pKP18-125) indicated that the floR gene was flanked by multiple copies of mobile genetic elements. Comparative genomics analysis of a 9-kb transposon-like fragment of pKP18-125 showed that an approximately 2-kb sequence encoding lysR-floR-virD2 was conserved in the majority (79.01%, 83/105) of floR sequences collected from NCBI nucleotide database. Interestingly, the most similar sequence was a 7-kb fragment of plasmid pEC012 from an Escherichia coli strain isolated from a chicken.Identified on a transferable plasmid in the human pathogen K. pneumoniae, the floR gene may be disseminated through horizontal gene transfer from animal pathogens. Studies on the molecular mechanism of resistance gene dissemination in different bacterial species of animal origin could provide useful information for preventing or controlling the spread of resistance between animal and human pathogens.

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September 22, 2019

Impacts of horizontal gene transfer on the compact genome of the clavulanic acid-producing Streptomyces strain F613-1.

Mobile genetic elements involved in mediating horizontal transfer events contribute to bacterial evolution, and bacterial genomic plasticity and instability result in variation in functional genetic information in Streptomyces secondary metabolism. In a previous study, we reported the complete genome sequence of the industrial Streptomyces strain F613-1, which produces high yields of clavulanic acid. In this study, we used comparative genomics and bioinformatics to investigate the unique genomic features of this strain. Taken together, comparative genomics were used to systematically investigate secondary metabolism capabilities and indicated that frequent exchange of genetic materials between Streptomyces replicons may shape the remarkable diversities in their secondary metabolite repertoires. Moreover, a 136.9-kb giant region of plasticity (RGP) was found in the F613-1 chromosome, and the chromosome and plasmid pSCL4 are densely packed with an exceptionally large variety of potential secondary metabolic gene clusters, involving several determinants putatively accounting for antibiotic production. In addition, the differences in the architecture and size of plasmid pSCL4 between F613-1 and ATCC 27064 suggest that the pSCL4 plasmid could evolve from pSCL4-like and pSCL2-like extrachromosomal replicons. Furthermore, the genomic analyses revealed that strain F613-1 has developed specific genomic architectures and genetic patterns that are well suited to meet the requirements of industrial innovation processes.

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September 22, 2019

Conjugative transfer of a novel Staphylococcal plasmid encoding the biocide resistance gene, qacA.

Staphylococcus aureus is the leading cause of skin and soft tissue infections (SSTI). Some S. aureus strains harbor plasmids that carry genes that affect resistance to biocides. Among these genes, qacA encodes the QacA Multidrug Efflux Pump that imparts decreased susceptibility to chlorhexidine, a biocide used ubiquitously in healthcare facilities. Furthermore, chlorhexidine has been considered as a S. aureus decolonization strategy in community settings. We previously conducted a chlorhexidine-based SSTI prevention trial among Ft. Benning Army trainees. Analysis of a clinical isolate (C02) from that trial identified a novel qacA-positive plasmid, pC02. Prior characterization of qacA-containing plasmids is limited and conjugative transfer of those plasmids has not been demonstrated. Given the implications of increased biocide resistance, herein we characterized pC02. In silico analysis identified genes typically associated with conjugative plasmids. Moreover, pC02 was efficiently transferred to numerous S. aureus strains and to Staphylococcus epidermidis. We screened additional qacA-positive S. aureus clinical isolates and pC02 was present in 27% of those strains; other unique qacA-harboring plasmids were also identified. Ten strains were subjected to whole genome sequencing. Sequence analysis combined with plasmid screening studies suggest that qacA-containing strains are transmitted among military personnel at Ft. Benning and that strains carrying qacA are associated with SSTIs within this population. The identification of a novel mechanism of qacA conjugative transfer among Staphylococcal strains suggests a possible future increase in the prevalence of antiseptic tolerant bacterial strains, and an increase in the rate of infections in settings where these agents are commonly used.

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September 22, 2019

Acquired interbacterial defense systems protect against interspecies antagonism in the human gut microbiome

The genomes of bacteria derived from the gut microbiota are replete with pathways that mediate contact-dependent interbacterial antagonism. However, the role of direct interactions between co-resident microbes in driving microbiome composition is not well understood. Here we report the widespread occurrence of acquired interbacterial defense (AID) gene clusters in the human gut microbiome. These clusters are found on predicted mobile elements and encode arrays of immunity genes that confer protection against interbacterial toxin-mediated antagonism in vitro and in gnotobiotic mice. We find that Bacteroides ovatus strains containing AID systems that inactivate B. fragilis toxins delivered between cells by the type VI secretion system are enriched in samples lacking detectable B. fragilis. Moreover, these strains display significantly higher abundance in gut metagenomes than strains without AID systems. Finally, we identify a recombinase-associated AID subtype present broadly in Bacteroidales genomes with features suggestive of active gene acquisition. Our data suggest that neutralization of contact-dependent interbacterial antagonism via AID systems plays an important role in shaping human gut microbiome ecology.

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September 22, 2019

Complete Genome Sequence of Massilia oculi sp. nov. CCUG 43427T (=DSM 26321T), the Type Strain of M. oculi, and Comparison with Genome Sequences of Other Massilia Strains.

Massilia oculi sp. nov. of type strain CCUG 43427T is a Gram-negative, rod-shaped, nonspore-forming bacterium, which was recently isolated from the eye of a patient suffering from endophthalmitis and was described as novel species in Massilia genus. In this study, we present the complete genome sequence of this strain by using Pacbio SMRT cell platform and compare this sequence with the genomes of 30 Massilia representative strains. Also, a comprehensive search was conducted for genes and proteins involved in antibiotic resistance and pathogenicity. The genome of CCUG 43427T is 5,844,653 bp with 65.55% GC content. This genome contains four prophages and four genomic islands (GIs). The cobalt/zinc/cadmium transporter locus CzcABCD is included in these GIs. This GI was predicted to play important role in bacterial heavy-metal tolerance. The in silico genome analysis also revealed that this strain contains a lot of antibiotic resistance and pathogenicity related genes. This result suggested that this strain may has evolved a wide arsenal of weapons for pathogenicity and survival. Genome comparison among CCUG 43427T and other 30 Massilia strains revealed that more than 400 genes are unique in CCUG 43427T. Among these, one gene cluster, which was annotated to be important for LOS biosynthesis, catalytic mechanism and the substrate specificity of the enzyme, was predicted to be horizontally transferred by using phylogenies and biased GC content.

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September 22, 2019

Identification of DNA base modifications by means of Pacific Biosciences RS Sequencing technology.

Whole phage genomes can be sequenced readily using one or a combination of next generation sequencing (NGS) technologies. One of the most recently developed NGS platforms, the so-called Single-Molecule Real-Time (SMRT) sequencing approach provided by the PacBio RS platform, is particularly useful in providing complete (i.e., un-gapped) genome sequences, but differs from other technologies in that the platform also allows for downstream analysis to identify nucleotides that have been modified by DNA methylation. Here, we describe the methodological approach for the detection of genomic methylation motifs by means of SMRT sequencing.

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September 22, 2019

Construction of stable fluorescent laboratory control strains for several food safety relevant Enterobacteriaceae.

Using naturally-occurring bacterial strains as positive controls in testing protocols is typically feared due to the risk of cross-contaminating samples. We have developed a collection of strains which express Green Fluorescent Protein (GFP) at high-level, permitting rapid screening of the following species on selective or non-selective plates: Escherichia coli O157:H7, Shigella sonnei, S. flexneri, Salmonella enterica subsp. Enterica serovar Gaminera, S. Mbandaka, S. Tennesse, S. Minnesota, S. Senftenberg and S. Typhimurium. These new strains fluoresce when irradiated with UV light and maintain this phenotype in absence of antibiotic selection. Recombinants were phenotypically equivalent to the parent strain, except for S. Tennessee Sal66 that appeared Lac- on Xylose Lysine Deoxycholate (XLD) agar plates and Lac+ on Mac Conkey and Hektoen Enteric agar plates. Analysis of closed whole genome sequences revealed that Sal66 had lost one lactose operon; slower rates of lactose metabolism may affect lactose fermentation on XLD agar. These fluorescent enteric control strains were challenging to develop and should provide an easy and effective means of identifying cross-contamination. Published by Elsevier Ltd.

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September 22, 2019

Cloning and characterization of short-chain N-acyl homoserine lactone-producing Enterobacter asburiae strain L1 from lettuce leaves.

In gram-negative bacteria, bacterial communication or quorum sensing (QS) is achieved using common signaling molecules known as N-acyl homoserine lactones (AHL). We have previously reported the genome of AHL-producing bacterium, Enterobacter asburiae strain L1. In silico analysis of the strain L1 genome revealed the presence of a pair of luxI/R genes responsible for AHL-type QS, designated as easIR. In this work, the 639 bp luxI homolog, encoding 212 amino acids, have been cloned and overexpressed in Escherichia coli BL21 (DE3)pLysS. The purified protein (~25 kDa) shares high similarity to several members of the LuxI family among different E asburiae strains. Our findings showed that the heterologously expressed EasI protein has activated violacein production by AHL biosensor Chromobacterium violaceum CV026 as the wild-type E. asburiae. The mass spectrometry analysis showed the production of N-butanoyl homoserine lactone and N-hexanoyl homoserine lactone from induced E. coli harboring the recombinant EasI, suggesting that EasI is a functional AHL synthase. E. asburiae strain L1 was also shown to possess biofilm-forming characteristic activity using crystal violet binding assay. This is the first report on cloning and characterization of the luxI homolog from E. asburiae.© 2018 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd.

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September 22, 2019

Complete genome sequence of Leuconostoc citreum EFEL2700, a host strain for transformation of pCB vectors.

Leuconostoc citreum is an important lactic acid bacterium used as a starter culture for producing kimchi, the traditional Korean fermented vegetables. An efficient host strain for plasmid transformation, L. citreum EFEL2700, was isolated from kimchi, and it has been frequently used for genetic engineering of L. citreum. In this study, we report the whole genome sequence of the strain and its genetic characteristics. Genome assembly yielded 5 contigs (1 chromosome and 4 plasmids), and the complete genome contained 1,923,830 base pairs (bp) with a G?+?C content of 39.0%. Average nucleotide identity analysis showed high homology (= 99%) to the reference strain L. citreum KM 20. The smallest plasmid (4.3 kbp) was used as an Escherichia coli shuttle vector (pCB) for heterologous gene expression, and L. citreum EFEL2700 showed the highest transformation efficiency, 6.7?×?104 CFU µg-1 DNA. Genetic analysis of the genome enabled the construction of primary metabolic pathway showing a typical hetero-type lactic acid fermentation. Notably, no core genes for primary metabolism were observed in plasmid 4 and it could be eliminated to create an efficient host for gene transformation. This report will facilitate the understanding and application of L. citreum EFEL2700 as a food-grade microbial cell factory.Copyright © 2018. Published by Elsevier B.V.

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September 22, 2019

Implications of stx loss for clinical diagnostics of Shiga toxin-producing Escherichia coli.

The dynamics related to the loss of stx genes from Shiga toxin-producing Escherichia coli remain unclear. Current diagnostic procedures have shortcomings in the detection and identification of STEC. This is partly owing to the fact that stx genes may be lost during an infection or in the laboratory. The aim of the present study was to provide new insight into in vivo and in vitro stx loss in order to improve diagnostic procedures. Results from the study support the theory that loss of stx is a strain-related phenomenon and not induced by patient factors. It was observed that one strain could lose stx both in vivo and in vitro. Whole genome comparison of stx-positive and stx-negative isolates from the same patient revealed that different genomic rearrangements, such as complete or partial loss of the parent prophage, may be factors in the loss of stx. Of diagnostic interest, it was shown that patients can be co-infected with different E. coli pathotypes. Therefore, identification of eae-positive, but stx-negative isolates should not be interpreted as “Shiga toxin-lost” E. coli without further testing. Growth and recovery of STEC were supported by different selective agar media for different strains, arguing for inclusion of several media in STEC diagnostics.

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September 22, 2019

Paenibacillus seodonensis sp. nov., isolated from a plant of the genus Campanula.

Strain DCT-19T, representing a Gram-stain-positive, rodshaped, aerobic bacterium, was isolated from a native plant belonging to the genus Campanula on Dokdo, the Republic of Korea. Comparative analysis of the 16S rRNA gene sequence showed that this strain was closely related to Paenibacillus amylolyticus NRRL NRS-290T (98.6%, 16S rRNA gene sequence similarity), Paenibacillus tundrae A10bT (98.1%), and Paenibacillus xylanexedens NRRL B-51090T (97.6%). DNADNA hybridization indicated that this strain had relatively low levels of DNA-DNA relatedness with P. amylolyticus NRRL NRS-290T (30.0%), P. xylanexedens NRRL B-51090T (29.0%), and P. tundrae A10bT (24.5%). Additionally, the genomic DNA G + C content of DCT-19T was 44.8%. The isolated strain grew at pH 6.0-8.0 (optimum, pH 7.0), 0-4% (w/v) NaCl (optimum, 0%), and a temperature of 15-45°C (optimum 25-30°C). The sole respiratory quinone in the strain was menaquinone-7, and the predominant fatty acids were C15:0 anteiso, C16:0 iso, and C16:0. In addition, the major polar lipids were diphosphatidylglycerol and phosphatidylethanolamine. Based on its phenotypic properties, genotypic distinctiveness, and chemotaxonomic features, strain DCT-19T is proposed as a novel species in the genus Paenibacillus, for which the name Paenibacillus seodonensis sp. nov. is proposed (=KCTC 43009T =LMG 30888T). The type strain of Paenibacillus seodonensis is DCT-19T.

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September 22, 2019

Staying alive: growth and survival of Bifidobacterium animalis subsp. animalis under in vitro and in vivo conditions.

Members of the Bifidobacterium genus are widely used as probiotics in fermented milk products. Bifidobacterium animalis subsp. animalis CNCM I-4602 grows and survives poorly in reconstituted skimmed milk (RSM). Availing of genome and transcriptome information, this poor growth and survival phenotype in milk was substantially improved by the addition of certain compounds, such as yeast extract, uric acid, glutathione, cysteine, ferrous sulfate, and a combination of magnesium sulfate and manganese sulfate. Carbohydrate utilization of CNCM I-4602 was also investigated, allowing the identification of several carbohydrate utilization gene clusters, and highlighting this strain’s inability to utilize lactose, unlike the type strain of this subspecies, B. animalis subsp. animalis ATCC25527 and the B. animalis subsp. lactis subspecies. In addition, the ability of B. animalis subsp. animalis CNCM I-4602 to colonize a murine model was investigated, which showed that this strain persists in the murine gut for a period of at least 4 weeks. Associated in vivo transcriptome analysis revealed that, among other genes, a gene cluster encoding a predicted type IVb tight adherence (Tad) pilus was upregulated, indicating that this extracellular structure plays a role in the colonization/adaptation of the murine gastrointestinal tract by this strain.

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September 22, 2019

Long-read sequencing technology indicates genome-wide effects of non-B DNA on polymerization speed and error rate.

DNA conformation may deviate from the classical B-form in ~13% of the human genome. Non-B DNA regulates many cellular processes; however, its effects on DNA polymerization speed and accuracy have not been investigated genome-wide. Such an inquiry is critical for understanding neurological diseases and cancer genome instability. Here, we present the first simultaneous examination of DNA polymerization kinetics and errors in the human genome sequenced with Single-Molecule Real-Time (SMRT) technology. We show that polymerization speed differs between non-B and B-DNA: It decelerates at G-quadruplexes and fluctuates periodically at disease-causing tandem repeats. Analyzing polymerization kinetics profiles, we predict and validate experimentally non-B DNA formation for a novel motif. We demonstrate that several non-B motifs affect sequencing errors (e.g., G-quadruplexes increase error rates), and that sequencing errors are positively associated with polymerase slowdown. Finally, we show that highly divergent G4 motifs have pronounced polymerization slowdown and high sequencing error rates, suggesting similar mechanisms for sequencing errors and germline mutations.© 2018 Guiblet et al.; Published by Cold Spring Harbor Laboratory Press.

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