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July 7, 2019

High metabolic versatility of different toxigenic and non-toxigenic Clostridioides difficile isolates.

Clostridioides difficile (formerly Clostridium difficile) is a major nosocomial pathogen with an increasing number of community-acquired infections causing symptoms from mild diarrhea to life-threatening colitis. The pathogenicity of C. difficile is considered to be mainly associated with the production of genome-encoded toxins A and B. In addition, some strains also encode and express the binary toxin CDT. However; a large number of non-toxigenic C. difficile strains have been isolated from the human gut and the environment. In this study, we characterized the growth behavior, motility and fermentation product formation of 17 different C. difficile isolates comprising five different major genomic clades and five different toxin inventories in relation to the C. difficile model strains 630?erm and R20291. Within 33 determined fermentation products, we identified two yet undescribed products (5-methylhexanoate and 4-(methylthio)-butanoate) of C. difficile. Our data revealed major differences in the fermentation products obtained after growth in a medium containing casamino acids and glucose as carbon and energy source. While the metabolism of branched chain amino acids remained comparable in all isolates, the aromatic amino acid uptake and metabolism and the central carbon metabolism-associated fermentation pathways varied strongly between the isolates. The patterns obtained followed neither the classification of the clades nor the ribotyping patterns nor the toxin distribution. As the toxin formation is strongly connected to the metabolism, our data allow an improved differentiation of C. difficile strains. The observed metabolic flexibility provides the optimal basis for the adaption in the course of infection and to changing conditions in different environments including the human gut. Copyright © 2017 Elsevier GmbH. All rights reserved.

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July 7, 2019

Toolkit for automated and rapid discovery of structural variants.

Structural variations (SV) are broadly defined as genomic alterations that affect > 50 bp of DNA, which are shown to have significant effect on evolution and disease. The advent of high throughput sequencing (HTS) technologies and the ability to perform whole genome sequencing (WGS), makes it feasible to study these variants in depth. However, discovery of all forms of SV using WGS has proven to be challenging as the short reads produced by the predominant HTS platforms (<200bp for current technologies) and the fact that most genomes include large amounts of repeats make it very difficult to unambiguously map and accurately characterize such variants. Furthermore, existing tools for SV discovery are primarily developed for only a few of the SV types, which may have conflicting sequence signatures (i.e. read pairs, read depth, split reads) with other, untargeted SV classes. Here we are introduce a new framework, Tardis, which combines multiple read signatures into a single package to characterize most SV types simultaneously, while preventing such conflicts. Tardis also has a modular structure that makes it easy to extend for the discovery of additional forms of SV. Copyright © 2017. Published by Elsevier Inc.

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July 7, 2019

Prevalence of colistin resistance gene (mcr-1) containing Enterobacteriaceae in feces of patients attending a tertiary care hospital and detection of a mcr-1 containing, colistin susceptible E. coli.

The emergence of the plasmid-mediated mcr colistin resistance gene in the community poses a potential threat for treatment of patients, especially when hospitalized. The aim of this study was to determine the prevalence of all currently known mcr mediated colistin resistance gene in fecal samples of patients attending a tertiary care hospital. From November 2014 until July 2015, fecal samples of patients attending the Leiden University Medical Center were collected and screened for presence of mcr using real-time PCR. Two of 576 patients were positive for mcr-1, resulting in a prevalence of 0.35%, whereas no mcr-2 was found. One of these samples was culture negative, the second sample contained a blaCMY-2 and mcr-1 containing E.coli. This strain belonged to Sequence Type 359 and serotype O177:H21. The mcr-1 containing E.coli was phenotypically susceptible to colistin with a MIC of = 0.25mg/l, due to a 1329bp transposon IS10R inserted into the mcr-1 gene as identified by WGS. This prevalence study shows that mcr-1 is present in low levels patients out of the community attending a hospital. Furthermore the study underlines the importance of phenotypical confirmation of molecular detection of a mcr-1 gene.

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July 7, 2019

Biofilm formation potential of heat-resistant Escherichia coli dairy isolates and the complete genome of multidrug-resistant, heat-resistant strain FAM21845.

We tested the biofilm formation potential of 30 heat-resistant and 6 heat-sensitive Escherichia coli dairy isolates. Production of curli and cellulose, static biofilm formation on polystyrene (PS) and stainless steel surfaces, biofilm formation under dynamic conditions (Bioflux), and initial adhesion rates (IAR) were evaluated. Biofilm formation varied greatly between strains, media, and assays. Our results highlight the importance of the experimental setup in determining biofilm formation under conditions of interest, as correlation between different assays was often not a given. The heat-resistant, multidrug-resistant (MDR) strain FAM21845 showed the strongest biofilm formation on PS and the highest IAR and was the only strain that formed significant biofilms on stainless steel under conditions relevant to the dairy industry, and it was therefore fully sequenced. Its chromosome is 4.9 Mb long, and it harbors a total of five plasmids (147.2, 54.2, 5.8, 2.5, and 1.9 kb). The strain carries a broad range of genes relevant to antimicrobial resistance and biofilm formation, including some on its two large conjugative plasmids, as demonstrated in plate mating assays.IMPORTANCE In biofilms, cells are embedded in an extracellular matrix that protects them from stresses, such as UV radiation, osmotic shock, desiccation, antibiotics, and predation. Biofilm formation is a major bacterial persistence factor of great concern in the clinic and the food industry. Many tested strains formed strong biofilms, and especially strains such as the heat-resistant, MDR strain FAM21845 may pose a serious issue for food production. Strong biofilm formation combined with diverse resistances (some encoded on conjugative plasmids) may allow for increased persistence, coselection, and possible transfer of these resistance factors. Horizontal gene transfer may conceivably occur in the food production setting or the gastrointestinal tract after consumption. Copyright © 2017 Marti et al.

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July 7, 2019

Analysis of complete genome sequence and major surface antigens of Neorickettsia helminthoeca, causative agent of salmon poisoning disease.

Neorickettsia helminthoeca, a type species of the genus Neorickettsia, is an endosymbiont of digenetic trematodes of veterinary importance. Upon ingestion of salmonid fish parasitized with infected trematodes, canids develop salmon poisoning disease (SPD), an acute febrile illness that is particularly severe and often fatal in dogs without adequate treatment. We determined and analysed the complete genome sequence of N. helminthoeca: a single small circular chromosome of 884 232 bp encoding 774 potential proteins. N. helminthoeca is unable to synthesize lipopolysaccharides and most amino acids, but is capable of synthesizing vitamins, cofactors, nucleotides and bacterioferritin. N. helminthoeca is, however, distinct from majority of the family Anaplasmataceae to which it belongs, as it encodes nearly all enzymes required for peptidoglycan biosynthesis, suggesting its structural hardiness and inflammatory potential. Using sera from dogs that were experimentally infected by feeding with parasitized fish or naturally infected in southern California, Western blot analysis revealed that among five predicted N. helminthoeca outer membrane proteins, P51 and strain-variable surface antigen were uniformly recognized. Our finding will help understanding pathogenesis, prevalence of N. helminthoeca infection among trematodes, canids and potentially other animals in nature to develop effective SPD diagnostic and preventive measures. Recent progresses in large-scale genome sequencing have been uncovering broad distribution of Neorickettsia spp., the comparative genomics will facilitate understanding of biology and the natural history of these elusive environmental bacteria.© 2017 The Authors. Microbial Biotechnology published by John Wiley & Sons Ltd and Society for Applied Microbiology.

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July 7, 2019

Complete genome sequences of 12 isolates of Listeria monocytogenes belonging to serotypes 1/2a, 1/2b, and 4b obtained from food products and food-processing environments in Canada.

Listeria monocytogenes is the etiological agent for an often fatal foodborne illness known as listeriosis. Here, we present the complete genome sequences of 12 L. monocytogenes isolates representing the three most common serotypes of this pathogen (1/2a, 1/2b, and 4b), collected in Canada from different food products and environmental sources.© Crown copyright 2017.

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July 7, 2019

Whole genome and core genome multilocus sequence typing and single nucleotide polymorphism analyses of Listeria monocytogenes associated with an outbreak linked to cheese, United States, 2013.

Epidemiological findings of a listeriosis outbreak in 2013 implicated Hispanic-style cheese produced by Company A, and pulsed-field gel electrophoresis (PFGE) and whole genome sequencing (WGS) were performed on clinical isolates and representative isolates collected from Company A cheese and environmental samples during the investigation. The results strengthened the evidence for cheese as the vehicle. Surveillance sampling and WGS three months later revealed that the equipment purchased by Company B from Company A yielded an environmental isolate highly similar to all outbreak isolates. The whole genome and core genome multilocus sequence typing and single nucleotide polymorphism (SNP) analyses were compared to demonstrate the maximum discriminatory power obtained by using multiple analyses, which were needed to differentiate outbreak-associated isolates from a PFGE-indistinguishable isolate collected in a non-implicated food source in 2012. This unrelated isolate differed from the outbreak isolates by only 7 to 14 SNPs, and as a result, minimum spanning tree by the whole genome analyses and certain variant calling approach and phylogenetic algorithm for core genome-based analyses could not provide the differentiation between unrelated isolates. Our data also suggest that SNP/allele counts should always be combined with WGS clustering generated by phylogenetically meaningful algorithms on sufficient number of isolates, and SNP/allele threshold alone is not sufficient evidence to delineate an outbreak. The putative prophages were conserved across all the outbreak isolates. All outbreak isolates belonged to clonal complex 5 and serotype 1/2b, had an identical inlA sequence, which did not have premature stop codons.IMPORTANCE In this outbreak, multiple analytical approaches were used for maximum discriminatory power. A PFGE-matched, epidemiologically unrelated isolate had high genetic similarity to the outbreak-associated isolates, with as few as only 7 SNP differences. Therefore, the SNP/allele threshold should not be used as the only evidence to define the scope of an outbreak. It is critical that the SNP/allele counts be complemented by WGS clustering generated by phylogenetically meaningful algorithms to distinguish outbreak-associated isolates from epidemiologically unrelated isolates. Careful selection of a variant calling approach and phylogenetic algorithm is critical for core genome-based analyses. The whole genome-based analyses were able to construct the highly resolved phylogeny needed to support the findings of the outbreak investigation. Ultimately, epidemiologic evidence and multiple WGS analyses should be combined to increase the confidence in outbreak investigations. Copyright © 2017 Chen et al.

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July 7, 2019

Complete genome sequences of five representative Staphylococcus aureus ST398 strains from five major sequence heterogeneity groups of a diverse isolate collection.

Staphylococcus aureus sequence type 398 (ST398) is a rapidly emerging livestock-associated strain causing zoonotic disease in humans. The course of pathogen evolution remains unclear, prompting whole-genome comparative studies in attempts to elucidate this issue. We present the full, annotated genomes of five newly isolated representative ST398 strains from five major sequence heterogeneity groups of our diverse isolate collection. Copyright © 2017 McClure and Zhang.

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July 7, 2019

Genetic analysis of Neisseria meningitidis sequence type 7 serogroup X originating from serogroup A.

Neisseria meningitidis causes meningococcal disease, often resulting in fulminant meningitis, sepsis, and death. Vaccination programs have been developed to prevent infection of this pathogen, but serogroup replacement is a problem. Capsular switching has been an important survival mechanism for N. meningitidis, allowing the organism to evolve in the present vaccine era. However, related mechanisms have not been completely elucidated. Genetic analysis of capsular switching between diverse serogroups would help further our understanding of this pathogen. In this study, we analyzed the genetic characteristics of the sequence type 7 (ST-7) serogroup X strain that was predicted to arise from ST-7 serogroup A at the genomic level. By comparing the genomic structures and sequences, ST-7 serogroup X was closest to ST-7 serogroup A, whereas eight probable recombination regions, including the capsular gene locus, were identified. This indicated that serogroup X originated from serogroup A by recombination leading to capsular switching. The recombination involved approximately 8,540 bp from the end of the ctrC gene to the middle of the galE gene. There were more recombination regions and strain-specific single-nucleotide polymorphisms in serogroup X than in serogroup A genomes. However, no specific gene was found for each serogroup except those in the capsule gene locus. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Complete genome sequence of Mycoplasma bovis strain 08M.

Mycoplasma bovis is a major bacterial pathogen that can cause respiratory disease, mastitis, and arthritis in cattle. We report here the complete and annotated genome sequence of M. bovis strain 08M, isolated from a calf lung with pneumonia in China. Copyright © 2017 Chen et al.

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July 7, 2019

Antibiotic resistance markers in Burkholderia pseudomallei strain Bp1651 identified by genome sequence analysis.

Burkholderia pseudomallei Bp1651 is resistant to several classes of antibiotics that are usually effective for treatment of melioidosis, including tetracyclines, sulfonamides, and ß-lactams such as penicillins (amoxicillin-clavulanic acid), cephalosporins (ceftazidime), and carbapenems (imipenem and meropenem). We sequenced, assembled, and annotated the Bp1651 genome and analyzed the sequence using comparative genomic analyses with susceptible strains, keyword searches of the annotation, publicly available antimicrobial resistance prediction tools, and published reports. More than 100 genes in the Bp1651 sequence were identified as potentially contributing to antimicrobial resistance. Most notably, we identified three previously uncharacterized point mutations in penA, which codes for a class A ß-lactamase and was previously implicated in resistance to ß-lactam antibiotics. The mutations result in amino acid changes T147A, D240G, and V261I. When individually introduced into select agent-excluded B. pseudomallei strain Bp82, D240G was found to contribute to ceftazidime resistance and T147A contributed to amoxicillin-clavulanic acid and imipenem resistance. This study provides the first evidence that mutations in penA may alter susceptibility to carbapenems in B. pseudomallei Another mutation of interest was a point mutation affecting the dihydrofolate reductase gene folA, which likely explains the trimethoprim resistance of this strain. Bp1651 was susceptible to aminoglycosides likely because of a frameshift in the amrB gene, the transporter subunit of the AmrAB-OprA efflux pump. These findings expand the role of penA to include resistance to carbapenems and may assist in the development of molecular diagnostics that predict antimicrobial resistance and provide guidance for treatment of melioidosis. Copyright © 2017 American Society for Microbiology.

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July 7, 2019

Genome sequences for Streptomyces spp. isolated from disease-suppressive soils and long-term ecological research sites.

We report here the high-quality genome sequences of three Streptomyces spp. isolated as part of a long-term study of microbial soil ecology. Streptomyces sp. strain GS93-23 was isolated from naturally disease-suppressive soil (DSS) in Grand Rapids, MN, and Streptomyces sp. strains S3-4 and 3211-3 were isolated from experimental plots in the Cedar Creek Ecosystem Science Reserve (CCESR). Copyright © 2017 Heinsch et al.

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July 7, 2019

Transcriptome Remodeling of Acinetobacter baumannii during Infection and Treatment.

Acinetobacter baumannii is an increasingly common multidrug-resistant pathogen in health care settings. Although the genetic basis of antibiotic resistance mechanisms has been extensively studied, much less is known about how genetic variation contributes to other aspects of successful infections. Genetic changes that occur during host infection and treatment have the potential to remodel gene expression patterns related to resistance and pathogenesis. Longitudinal sets of multidrug-resistant A. baumannii isolates from eight patients were analyzed by RNA sequencing (RNA-seq) to identify differentially expressed genes and link them to genetic changes contributing to transcriptional variation at both within-patient and population levels. The number of differentially expressed genes among isolates from the same patient ranged from 26 (patient 588) to 145 (patient 475). Multiple patients had isolates with differential gene expression patterns related to mutations in the pmrAB and adeRS two-component regulatory system genes, as well as significant differences in genes related to antibiotic resistance, iron acquisition, amino acid metabolism, and surface-associated proteins. Population level analysis revealed 39 genetic regions with clade-specific differentially expressed genes, for which 19, 8, and 3 of these could be explained by insertion sequence mobilization, recombination-driven sequence variation, and intergenic mutations, respectively. Multiple types of mutations that arise during infection can significantly remodel the expression of genes that are known to be important in pathogenesis. IMPORTANCE Health care-associated multidrug-resistant Acinetobacter baumannii can cause persistent infections in patients, but bacterial cells must overcome host defenses and antibiotic therapies to do so. Genetic variation arises during host infection, and new mutations are often enriched in genes encoding transcriptional regulators, iron acquisition systems, and surface-associated structures. In this study, genetic variation was shown to result in transcriptome remodeling at the level of individual patients and across phylogenetic groups. Differentially expressed genes include those related to capsule modification, iron acquisition, type I pili, and antibiotic resistance. Population level transcriptional variation reflects genome dynamics over longer evolutionary time periods, and convergent transcriptional changes support the adaptive significance of these regions. Transcriptional changes can be attributed to multiple types of genomic change, but insertion sequence mobilization had a predominant effect. The transcriptional effects of mutations that arise during infection highlight the rapid adaptation of A. baumannii during host exposure. Copyright © 2017 Wright et al.

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July 7, 2019

Zinc resistance within swine associated methicillin resistant staphylococcus aureus (MRSA) Isolates in the USA is associated with MLST lineage.

Zinc resistance in livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) sequence type (ST) 398 is primarily mediated by the czrC gene co-located with the mecA gene, encoding methicillin resistance, within the type V SCCmec element. Because czrC and mecA are located within the same mobile genetic element, it has been suggested that the use of in feed zinc as an antidiarrheal agent has the potential to contribute to the emergence and spread of MRSA in swine through increased selection pressure to maintain the SCCmec element in isolates obtained from pigs. In this study we report the prevalence of the czrC gene and phenotypic zinc resistance in US swine associated LA-MRSA ST5 isolates, MRSA ST5 isolates from humans with no swine contact, and US swine associated LA-MRSA ST398 isolates. We demonstrate that the prevalence of zinc resistance in US swine associated LA-MRSA ST5 isolates was significantly lower than the prevalence of zinc resistance in MRSA ST5 isolates from humans with no swine contact, swine associated LA-MRSA ST398 isolates, and previous reports describing zinc resistance in other LA-MRSA ST398 isolates. Collectively our data suggest that selection pressure associated with zinc supplementation in feed is unlikely to have played a significant role in the emergence of LA-MRSA ST5 in the US swine population. Additionally, our data indicate that zinc resistance is associated with MLST lineage suggesting a potential link between genetic lineage and carriage of resistance determinants.Importance Our data suggest that coselection thought to be associated with the use of zinc in feed as an antimicrobial agent is not playing a role in the emergence of livestock-associated methicillin resistant Staphylococcus aureus (LA-MRSA) ST5 in the US swine population. Additionally, our data indicate that zinc resistance is more associated with multi locus sequence type (MLST) lineage suggesting a potential link between genetic lineage and carriage of resistance markers. This information is important to public health professionals, veterinarians, producers, and consumers. Copyright © 2017 American Society for Microbiology.

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