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July 7, 2019

Population structure and acquisition of the vanB resistance determinant in German clinical isolates of Enterococcus faecium ST192.

In the context of the global action plan to reduce the dissemination of antibiotic resistances it is of utmost importance to understand the population structure of resistant endemic bacterial lineages and to elucidate how bacteria acquire certain resistance determinants. Vancomycin resistant enterococci represent one such example of a prominent nosocomial pathogen on which nation-wide population analyses on prevalent lineages are scarce and data on how the bacteria acquire resistance, especially of the vanB genotype, are still under debate. With respect to Germany, an increased prevalence of VRE was noted in recent years. Here, invasive infections caused by sequence type ST192 VRE are often associated with the vanB-type resistance determinant. Hence, we analyzed 49 vanB-positive and vanB-negative E. faecium isolates by means of whole genome sequencing. Our studies revealed a distinct population structure and that spread of the Tn1549-vanB-type resistance involves exchange of large chromosomal fragments between vanB-positive and vanB-negative enterococci rather than independent acquisition events. In vitro filter-mating experiments support the hypothesis and suggest the presence of certain target sequences as a limiting factor for dissemination of the vanB element. Thus, the present study provides a better understanding of how enterococci emerge into successful multidrug-resistant nosocomial pathogens.

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July 7, 2019

Complete genome sequence of Turicibacter sp. strain H121, isolated from the feces of a contaminated germ-free mouse.

Turicibacterbacteria are commonly detected in the gastrointestinal tracts and feces of humans and animals, but their phylogeny, ecological role, and pathogenic potential remain unclear. We present here the first complete genome sequence ofTuricibactersp. strain H121, which was isolated from the feces of a mouse line contaminated following germ-free derivation. Copyright © 2016 Auchtung et al.

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July 7, 2019

First report of cfr-encoding plasmids in the pandemic sequence type (ST) 22 methicillin-resistant Staphylococcus aureus Staphylococcal cassette chromosome mec type-IV clone.

Linezolid is often the drug of last resort for serious methicillin-resistant Staphylococcus aureus (MRSA) infections. Linezolid resistance is mediated by mutations in 23S rRNA and genes for ribosomal proteins, cfr encoding phenicol, lincosamide, oxazolidinone, pleuromutilin and streptogramin A (PhLOPSA) resistance, its homolgue cfr(B) or optrA conferring oxazolidinone and phenicol resistance. Linezolid resistance is rare in S. aureus, and cfr even rarer. This study investigated the clonality and linezolid resistance mechanisms of two MRSA isolates from patients in separate Irish hospitals. Isolates were subjected to cfr PCR, PhLOPSA susceptibility testing, 23S rRNA PCR and sequencing, DNA microarray profiling, spa typing, pulsed-field gel electrophoresis (PFGE), plasmid curing and conjugative transfer. Whole-genome sequencing was used for single nucleotide variant (SNV) analysis, multilocus-sequence typing, L-protein mutation identification, cfr-plasmid sequence analysis and optrA and cfr(B) detection. Isolates M12/0145 and M13/0401 exhibited linezolid MICs of 64 and 16 mg/liter, respectively, and harbored identical 23S rRNA and L22 mutations, but M12/0145 exhibited the mutation in 2/6 23S rRNA alleles compared to 1/5 in M13/0401. Both isolates were ST22-MRSA-IV/t032, harbored cfr, exhibited the PhLOPSA phenotype and lacked optrA and cfr(B). They differed by five PFGE bands and 603 SNVs. Isolate M12/0145 harbored cfr and fexA on a 41-kb conjugative pSCFS3-type plasmid, whereas M13/0401 harbored cfr and lsa(B) on a novel 27-kb plasmid. This is the first report of cfr in the pandemic ST22-MRSA-IV clone. Different cfr plasmids and mutations associated with linezolid resistance in genotypically distinct ST22-MRSA-IV isolates highlights that prudent management of linezolid use is essential. Copyright © 2016 Shore et al.

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July 7, 2019

Regulation of genetic flux between bacteria by restriction-modification systems.

Restriction-modification (R-M) systems are often regarded as bacteria’s innate immune systems, protecting cells from infection by mobile genetic elements (MGEs). Their diversification has been recently associated with the emergence of particularly virulent lineages. However, we have previously found more R-M systems in genomes carrying more MGEs. Furthermore, it has been suggested that R-M systems might favor genetic transfer by producing recombinogenic double-stranded DNA ends. To test whether R-M systems favor or disfavor genetic exchanges, we analyzed their frequency with respect to the inferred events of homologous recombination and horizontal gene transfer within 79 bacterial species. Genetic exchanges were more frequent in bacteria with larger genomes and in those encoding more R-M systems. We created a recognition target motif predictor for Type II R-M systems that identifies genomes encoding systems with similar restriction sites. We found more genetic exchanges between these genomes, independently of their evolutionary distance. Our results reconcile previous studies by showing that R-M systems are more abundant in promiscuous species, wherein they establish preferential paths of genetic exchange within and between lineages with cognate R-M systems. Because the repertoire and/or specificity of R-M systems in bacterial lineages vary quickly, the preferential fluxes of genetic transfer within species are expected to constantly change, producing time-dependent networks of gene transfer.

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July 7, 2019

Antibiotic resistance mechanisms of Myroides sp.

Bacteria of the genus Myroides (Myroides spp.) are rare opportunistic pathogens. Myroides sp. infections have been reported mainly in China. Myroides sp. is highly resistant to most available antibiotics, but the resistance mechanisms are not fully elucidated. Current strain identification methods based on biochemical traits are unable to identify strains accurately at the species level. While 16S ribosomal RNA (rRNA) gene sequencing can accurately achieve this, it fails to give information on the status and mechanisms of antibiotic resistance, because the 16S rRNA sequence contains no information on resistance genes, resistance islands or enzymes. We hypothesized that obtaining the whole genome sequence of Myroides sp., using next generation sequencing methods, would help to clarify the mechanisms of pathogenesis and antibiotic resistance, and guide antibiotic selection to treat Myroides sp. infections. As Myroides sp. can survive in hospitals and the environment, there is a risk of nosocomial infections and pandemics. For better management of Myroides sp. infections, it is imperative to apply next generation sequencing technologies to clarify the antibiotic resistance mechanisms in these bacteria.

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July 7, 2019

Near-Complete Genome Sequence of Clostridium paradoxum Strain JW-YL-7.

Clostridium paradoxum strain JW-YL-7 is a moderately thermophilic anaerobic alkaliphile isolated from the municipal sewage treatment plant in Athens, GA. We report the near-complete genome sequence of C. paradoxum strain JW-YL-7 obtained by using PacBio DNA sequencing and Pilon for sequence assembly refinement with Illumina data. Copyright © 2016 Lancaster et al.

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July 7, 2019

The absence of a mature cell wall sacculus in stable Listeria monocytogenes L-form cells is independent of peptidoglycan synthesis.

L-forms are cell wall-deficient variants of otherwise walled bacteria that maintain the ability to survive and proliferate in absence of the surrounding peptidoglycan sacculus. While transient or unstable L-forms can revert to the walled state and may still rely on residual peptidoglycan synthesis for multiplication, stable L-forms cannot revert to the walled form and are believed to propagate in the complete absence of peptidoglycan. L-forms are increasingly studied as a fundamental biological model system for cell wall synthesis. Here, we show that a stable L-form of the intracellular pathogen Listeria monocytogenes features a surprisingly intact peptidoglycan synthesis pathway including glycosyl transfer, in spite of the accumulation of multiple mutations during prolonged passage in the cell wall-deficient state. Microscopic and biochemical analysis revealed the presence of peptidoglycan precursors and functional glycosyl transferases, resulting in the formation of peptidoglycan polymers but without the synthesis of a mature cell wall sacculus. In conclusion, we found that stable, non-reverting L-forms, which do not require active PG synthesis for proliferation, may still continue to produce aberrant peptidoglycan.

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July 7, 2019

Emergence of host-adapted Salmonella Enteritidis through rapid evolution in an immunocompromised host.

Host adaptation is a key factor contributing to the emergence of new bacterial, viral and parasitic pathogens. Many pathogens are considered promiscuous because they cause disease across a range of host species, while others are host-adapted, infecting particular hosts(1). Host adaptation can potentially progress to host restriction where the pathogen is strictly limited to a single host species and is frequently associated with more severe symptoms. Host-adapted and host-restricted bacterial clades evolve from within a broader host-promiscuous species and sometimes target different niches within their specialist hosts, such as adapting from a mucosal to a systemic lifestyle. Genome degradation, marked by gene inactivation and deletion, is a key feature of host adaptation, although the triggers initiating genome degradation are not well understood. Here, we show that a chronic systemic non-typhoidal Salmonella infection in an immunocompromised human patient resulted in genome degradation targeting genes that are expendable for a systemic lifestyle. We present a genome-based investigation of a recurrent blood-borne Salmonella enterica serotype Enteritidis (S. Enteritidis) infection covering 15 years in an interleukin (IL)-12 ß-1 receptor-deficient individual that developed into an asymptomatic chronic infection. The infecting S. Enteritidis harbored a mutation in the mismatch repair gene mutS that accelerated the genomic mutation rate. Phylogenetic analysis and phenotyping of multiple patient isolates provides evidence for a remarkable level of within-host evolution that parallels genome changes present in successful host-restricted bacterial pathogens but never before observed on this timescale. Our analysis identifies common pathways of host adaptation and demonstrates the role that immunocompromised individuals can play in this process.

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July 7, 2019

The challenges of implementing next generation sequencing across a large healthcare system, and the molecular epidemiology and antibiotic susceptibilities of carbapenemase-producing bacteria in the healthcare system of the U.S. Department of Defense.

We sought to: 1) provide an overview of the genomic epidemiology of an extensive collection of carbapenemase-producing bacteria (CPB) collected in the U.S. Department of Defense health system; 2) increase awareness of the public availability of the sequences, isolates, and customized antimicrobial resistance database of that system; and 3) illustrate challenges and offer mitigations for implementing next generation sequencing (NGS) across large health systems.Prospective surveillance and system-wide implementation of NGS.288-hospital healthcare network.All phenotypically carbapenem resistant bacteria underwent CarbaNP® testing and PCR, followed by NGS. Commercial (Newbler and Geneious), on-line (ResFinder), and open-source software (Btrim, FLASh, Bowtie2, an Samtools) were used for assembly, SNP detection and clustering. Laboratory capacity, throughput, and response time were assessed. From 2009 through 2015, 27,000 multidrug-resistant Gram-negative isolates were submitted. 225 contained carbapenemase-encoding genes (most commonly blaKPC, blaNDM, and blaOXA23). These were found in 15 species from 146 inpatients in 19 facilities. Genetically related CPB were found in more than one hospital. Other clusters or outbreaks were not clonal and involved genetically related plasmids, while some involved several unrelated plasmids. Relatedness depended on the clustering algorithm used. Transmission patterns of plasmids and other mobile genetic elements could not be determined without ultra-long read, single-molecule real-time sequencing. 80% of carbapenem-resistant phenotypes retained susceptibility to aminoglycosides, and 70% retained susceptibility to fluoroquinolones. However, among the CPB-confirmed genotypes, fewer than 25% retained susceptibility to aminoglycosides or fluoroquinolones.Although NGS is increasingly acclaimed to revolutionize clinical practice, resource-constrained environments, large or geographically dispersed healthcare networks, and military or government-funded public health laboratories are likely to encounter constraints and challenges as they implement NGS across their health systems. These include lack of standardized definitions and quality control metrics, limitations of short-read sequencing, insufficient bandwidth, and the current limited availability of very expensive and scarcely available sequencing platforms. Possible solutions and mitigations are also proposed.

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July 7, 2019

Complete genome sequence of Enterococcus faecium ATCC 700221.

We report the complete genome sequence of a vancomycin-resistant isolate of Enterococcus faecium derived from human feces. The genome comprises one chromosome of 2.9 Mb and three plasmids. The strain harbors a plasmid-borne vanA-type vancomycin resistance locus and is a member of multilocus sequencing type (MLST) cluster ST-17. Copyright © 2016 McKenney et al.

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July 7, 2019

Accelerated dysbiosis of gut microbiota during aggravation of DSS-induced colitis by a butyrate-producing bacterium.

Butyrate-producing bacteria (BPB) are potential probiotic candidates for inflammatory bowel diseases as they are often depleted in the diseased gut microbiota. However, here we found that augmentation of a human-derived butyrate-producing strain, Anaerostipes hadrus BPB5, significantly aggravated colitis in dextran sulphate sodium (DSS)-treated mice while exerted no detrimental effect in healthy mice. We explored how the interaction between BPB5 and gut microbiota may contribute to this differential impact on the hosts. Butyrate production and severity of colitis were assessed in both healthy and DSS-treated mice, and gut microbiota structural changes were analysed using high-throughput sequencing. BPB5-inoculated healthy mice showed no signs of colitis, but increased butyrate content in the gut. In DSS-treated mice, BPB5 augmentation did not increase butyrate content, but induced significantly more severe disease activity index and much higher mortality. BPB5 didn’t induce significant changes of gut microbiota in healthy hosts, but expedited the structural shifts 3 days earlier toward the disease phase in BPB5-augmented than DSS-treated animals. The differential response of gut microbiota in healthy and DSS-treated mice to the same potentially beneficial bacterium with drastically different health consequences suggest that animals with dysbiotic gut microbiota should also be employed for the safety assessment of probiotic candidates.

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July 7, 2019

Complete genomes of Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, two phylogenetically distinct probiotics.

Several spore-forming strains of Bacillus are marketed as probiotics due to their ability to survive harsh gastrointestinal conditions and confer health benefits to the host. We report the complete genomes of two commercially available probiotics, Bacillus coagulans S-lac and Bacillus subtilis TO-A JPC, and compare them with the genomes of other Bacillus and Lactobacillus. The taxonomic position of both organisms was established with a maximum-likelihood tree based on twenty six housekeeping proteins. Analysis of all probiotic strains of Bacillus and Lactobacillus reveal that the essential sporulation proteins are conserved in all Bacillus probiotic strains while they are absent in Lactobacillus spp. We identified various antibiotic resistance, stress-related, and adhesion-related domains in these organisms, which likely provide support in exerting probiotic action by enabling adhesion to host epithelial cells and survival during antibiotic treatment and harsh conditions.

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July 7, 2019

Characterization of the first cultured representative of Verrucomicrobia subdivision 5 indicates the proposal of a novel phylum.

The recently isolated strain L21-Fru-AB(T) represents moderately halophilic, obligately anaerobic and saccharolytic bacteria that thrive in the suboxic transition zones of hypersaline microbial mats. Phylogenetic analyses based on 16S rRNA genes, RpoB proteins and gene content indicated that strain L21-Fru-AB(T) represents a novel species and genus affiliated with a distinct phylum-level lineage originally designated Verrucomicrobia subdivision 5. A survey of environmental 16S rRNA gene sequences revealed that members of this newly recognized phylum are wide-spread and ecologically important in various anoxic environments ranging from hypersaline sediments to wastewater and the intestine of animals. Characteristic phenotypic traits of the novel strain included the formation of extracellular polymeric substances, a Gram-negative cell wall containing peptidoglycan and the absence of odd-numbered cellular fatty acids. Unusual metabolic features deduced from analysis of the genome sequence were the production of sucrose as osmoprotectant, an atypical glycolytic pathway lacking pyruvate kinase and the synthesis of isoprenoids via mevalonate. On the basis of the analyses of phenotypic, genomic and environmental data, it is proposed that strain L21-Fru-AB(T) and related bacteria are specifically adapted to the utilization of sulfated glycopolymers produced in microbial mats or biofilms.

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July 7, 2019

A roadmap for gene system development in Clostridium.

Clostridium species are both heroes and villains. Some cause serious human and animal diseases, those present in the gut microbiota generally contribute to health and wellbeing, while others represent useful industrial chassis for the production of chemicals and fuels. To understand, counter or exploit, there is a fundamental requirement for effective systems that may be used for directed or random genome modifications. We have formulated a simple roadmap whereby the necessary gene systems maybe developed and deployed. At its heart is the use of ‘pseudo-suicide’ vectors and the creation of a pyrE mutant (a uracil auxotroph), initially aided by ClosTron technology, but ultimately made using a special form of allelic exchange termed ACE (Allele-Coupled Exchange). All mutants, regardless of the mutagen employed, are made in this host. This is because through the use of ACE vectors, mutants can be rapidly complemented concomitant with correction of the pyrE allele and restoration of uracil prototrophy. This avoids the phenotypic effects frequently observed with high copy number plasmids and dispenses with the need to add antibiotic to ensure plasmid retention. Once available, the pyrE host may be used to stably insert all manner of application specific modules. Examples include, a sigma factor to allow deployment of a mariner transposon, hydrolases involved in biomass deconstruction and therapeutic genes in cancer delivery vehicles. To date, provided DNA transfer is obtained, we have not encountered any clostridial species where this technology cannot be applied. These include, Clostridium difficile, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium botulinum, Clostridium perfringens, Clostridium sporogenes, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium autoethanogenum and even Geobacillus thermoglucosidasius. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

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July 7, 2019

Co-utilization of glucose and xylose by evolved Thermus thermophilus LC113 strain elucidated by (13)C metabolic flux analysis and whole genome sequencing.

We evolved Thermus thermophilus to efficiently co-utilize glucose and xylose, the two most abundant sugars in lignocellulosic biomass, at high temperatures without carbon catabolite repression. To generate the strain, T. thermophilus HB8 was first evolved on glucose to improve its growth characteristics, followed by evolution on xylose. The resulting strain, T. thermophilus LC113, was characterized in growth studies, by whole genome sequencing, and (13)C-metabolic flux analysis ((13)C-MFA) with [1,6-(13)C]glucose, [5-(13)C]xylose, and [1,6-(13)C]glucose+[5-(13)C]xylose as isotopic tracers. Compared to the starting strain, the evolved strain had an increased growth rate (~2-fold), increased biomass yield, increased tolerance to high temperatures up to 90°C, and gained the ability to grow on xylose in minimal medium. At the optimal growth temperature of 81°C, the maximum growth rate on glucose and xylose was 0.44 and 0.46h(-1), respectively. In medium containing glucose and xylose the strain efficiently co-utilized the two sugars. (13)C-MFA results provided insights into the metabolism of T. thermophilus LC113 that allows efficient co-utilization of glucose and xylose. Specifically, (13)C-MFA revealed that metabolic fluxes in the upper part of metabolism adjust flexibly to sugar availability, while fluxes in the lower part of metabolism remain relatively constant. Whole genome sequence analysis revealed two large structural changes that can help explain the physiology of the evolved strain: a duplication of a chromosome region that contains many sugar transporters, and a 5x multiplication of a region on the pVV8 plasmid that contains xylose isomerase and xylulokinase genes, the first two enzymes of xylose catabolism. Taken together, (13)C-MFA and genome sequence analysis provided complementary insights into the physiology of the evolved strain. Copyright © 2016 International Metabolic Engineering Society. Published by Elsevier Inc. All rights reserved.

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