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July 7, 2019  |  

Simultaneous emergence of multidrug-resistant Candida auris on 3 continents confirmed by whole-genome sequencing and epidemiological analyses.

Candida auris, a multidrug-resistant yeast that causes invasive infections, was first described in 2009 in Japan and has since been reported from several countries.To understand the global emergence and epidemiology of C. auris, we obtained isolates from 54 patients with C. auris infection from Pakistan, India, South Africa, and Venezuela during 2012-2015 and the type specimen from Japan. Patient information was available for 41 of the isolates. We conducted antifungal susceptibility testing and whole-genome sequencing (WGS).Available clinical information revealed that 41% of patients had diabetes mellitus, 51% had undergone recent surgery, 73% had a central venous catheter, and 41% were receiving systemic antifungal therapy when C. auris was isolated. The median time from admission to infection was 19 days (interquartile range, 9-36 days), 61% of patients had bloodstream infection, and 59% died. Using stringent break points, 93% of isolates were resistant to fluconazole, 35% to amphotericin B, and 7% to echinocandins; 41% were resistant to 2 antifungal classes and 4% were resistant to 3 classes. WGS demonstrated that isolates were grouped into unique clades by geographic region. Clades were separated by thousands of single-nucleotide polymorphisms, but within each clade isolates were clonal. Different mutations in ERG11 were associated with azole resistance in each geographic clade.C. auris is an emerging healthcare-associated pathogen associated with high mortality. Treatment options are limited, due to antifungal resistance. WGS analysis suggests nearly simultaneous, and recent, independent emergence of different clonal populations on 3 continents. Risk factors and transmission mechanisms need to be elucidated to guide control measures. Published by Oxford University Press for the Infectious Diseases Society of America 2016. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019  |  

Regulation of PI-2b pilus expression in hypervirulent Streptococcus agalactiae ST-17 BM110.

The widely spread Streptococcus agalactiae (also known as Group B Streptococcus, GBS) “hypervirulent” ST17 clone is strongly associated with neonatal meningitis. The PI-2b locus is mainly found in ST17 strains but is also present in a few non ST17 human isolates such as the ST-7 prototype strain A909. Here, we analysed the expression of the PI-2b pilus in the ST17 strain BM110 as compared to the non ST17 A909. Comparative genome analyses revealed the presence of a 43-base pair (bp) hairpin-like structure in the upstream region of PI-2b operon in all 26 ST17 genomes, which was absent in the 8 non-ST17 strains carrying the PI-2b locus. Deletion of this 43-bp sequence in strain BM110 resulted in a 3- to 5-fold increased transcription of PI-2b. Characterization of PI-2b promoter region in A909 and BM110 strains was carried out by RNAseq, primer extension, qRT-PCR and transcriptional fusions with gfp as reporter gene. Our results indicate the presence of a single promoter (Ppi2b) with a transcriptional start site (TSS) mapped 37 bases upstream of the start codon of the first PI-2b gene. The large operon of 16 genes located upstream of PI-2b codes for the group B carbohydrate (also known as antigen B), a major constituent of the bacterial cell wall. We showed that the hairpin sequence located between antigen B and PI-2b operons is a transcriptional terminator. In A909, increased expression of PI-2b probably results from read-through transcription from antigen B operon. In addition, we showed that an extended 5′ promoter region is required for maximal transcription of gfp as a reporter gene in S. agalactiae from Ppi2b promoter. Gene reporter assays performed in Lactococcus lactis strain NZ9000, a related non-pathogenic Gram-positive species, revealed that GBS-specific regulatory factors are required to drive PI-2b transcription. PI-2b expression is up-regulated in the BM110?covR mutant as compared to the parental BM110 strain, but this effect is probably indirect. Collectively, our results indicate that PI-2b expression is regulated in GBS ST17 strains, which may confer a selective advantage in the human host either by reducing host immune responses and/or increasing their dissemination potential.

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July 7, 2019  |  

Population genomic analysis of 1,777 extended-spectrum beta-lactamase-producing Klebsiella pneumoniae isolates, Houston, Texas: unexpected abundance of clonal group 307.

Klebsiella pneumoniae is a major human pathogen responsible for high morbidity and mortality rates. The emergence and spread of strains resistant to multiple antimicrobial agents and documented large nosocomial outbreaks are especially concerning. To develop new therapeutic strategies for K. pneumoniae, it is imperative to understand the population genomic structure of strains causing human infections. To address this knowledge gap, we sequenced the genomes of 1,777 extended-spectrum beta-lactamase-producing K. pneumoniae strains cultured from patients in the 2,000-bed Houston Methodist Hospital system between September 2011 and May 2015, representing a comprehensive, population-based strain sample. Strains of largely uncharacterized clonal group 307 (CG307) caused more infections than those of well-studied epidemic CG258. Strains varied markedly in gene content and had an extensive array of small and very large plasmids, often containing antimicrobial resistance genes. Some patients with multiple strains cultured over time were infected with genetically distinct clones. We identified 15 strains expressing the New Delhi metallo-beta-lactamase 1 (NDM-1) enzyme that confers broad resistance to nearly all beta-lactam antibiotics. Transcriptome sequencing analysis of 10 phylogenetically diverse strains showed that the global transcriptome of each strain was unique and highly variable. Experimental mouse infection provided new information about immunological parameters of host-pathogen interaction. We exploited the large data set to develop whole-genome sequence-based classifiers that accurately predict clinical antimicrobial resistance for 12 of the 16 antibiotics tested. We conclude that analysis of large, comprehensive, population-based strain samples can assist understanding of the molecular diversity of these organisms and contribute to enhanced translational research. IMPORTANCEKlebsiella pneumoniae causes human infections that are increasingly difficult to treat because many strains are resistant to multiple antibiotics. Clonal group 258 (CG258) organisms have caused outbreaks in health care settings worldwide. Using a comprehensive population-based sample of extended-spectrum beta-lactamase (ESBL)-producing K. pneumoniae strains, we show that a relatively uncommon clonal type, CG307, caused the plurality of ESBL-producing K. pneumoniae infections in our patients. We discovered that CG307 strains have been abundant in Houston for many years. As assessed by experimental mouse infection, CG307 strains were as virulent as pandemic CG258 strains. Our results may portend the emergence of an especially successful clonal group of antibiotic-resistant K. pneumoniae. Copyright © 2017 Long et al.

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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  |  

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  |  

Modulation of aneuploidy in Leishmania donovani during adaptation to different in vitro and in vivo environments and its impact on gene expression.

Aneuploidy is usually deleterious in multicellular organisms but appears to be tolerated and potentially beneficial in unicellular organisms, including pathogens. Leishmania, a major protozoan parasite, is emerging as a new model for aneuploidy, since in vitro-cultivated strains are highly aneuploid, with interstrain diversity and intrastrain mosaicism. The alternation of two life stages in different environments (extracellular promastigotes and intracellular amastigotes) offers a unique opportunity to study the impact of environment on aneuploidy and gene expression. We sequenced the whole genomes and transcriptomes of Leishmania donovani strains throughout their adaptation to in vivo conditions mimicking natural vertebrate and invertebrate host environments. The nucleotide sequences were almost unchanged within a strain, in contrast to highly variable aneuploidy. Although high in promastigotes in vitro, aneuploidy dropped significantly in hamster amastigotes, in a progressive and strain-specific manner, accompanied by the emergence of new polysomies. After a passage through a sand fly, smaller yet consistent karyotype changes were detected. Changes in chromosome copy numbers were correlated with the corresponding transcript levels, but additional aneuploidy-independent regulation of gene expression was observed. This affected stage-specific gene expression, downregulation of the entire chromosome 31, and upregulation of gene arrays on chromosomes 5 and 8. Aneuploidy changes in Leishmania are probably adaptive and exploited to modulate the dosage and expression of specific genes; they are well tolerated, but additional mechanisms may exist to regulate the transcript levels of other genes located on aneuploid chromosomes. Our model should allow studies of the impact of aneuploidy on molecular adaptations and cellular fitness.IMPORTANCE Aneuploidy is usually detrimental in multicellular organisms, but in several microorganisms, it can be tolerated and even beneficial. Leishmania-a protozoan parasite that kills more than 30,000 people each year-is emerging as a new model for aneuploidy studies, as unexpectedly high levels of aneuploidy are found in clinical isolates. Leishmania lacks classical regulation of transcription at initiation through promoters, so aneuploidy could represent a major adaptive strategy of this parasite to modulate gene dosage in response to stressful environments. For the first time, we document the dynamics of aneuploidy throughout the life cycle of the parasite, in vitro and in vivo We show its adaptive impact on transcription and its interaction with regulation. Besides offering a new model for aneuploidy studies, we show that further genomic studies should be done directly in clinical samples without parasite isolation and that adequate methods should be developed for this. Copyright © 2017 Dumetz et al.

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July 7, 2019  |  

Evidence for the evolutionary steps leading to mecA-mediated ß-lactam resistance in staphylococci.

The epidemiologically most important mechanism of antibiotic resistance in Staphylococcus aureus is associated with mecA-an acquired gene encoding an extra penicillin-binding protein (PBP2a) with low affinity to virtually all ß-lactams. The introduction of mecA into the S. aureus chromosome has led to the emergence of methicillin-resistant S. aureus (MRSA) pandemics, responsible for high rates of mortality worldwide. Nonetheless, little is known regarding the origin and evolution of mecA. Different mecA homologues have been identified in species belonging to the Staphylococcus sciuri group representing the most primitive staphylococci. In this study we aimed to identify evolutionary steps linking these mecA precursors to the ß-lactam resistance gene mecA and the resistance phenotype. We sequenced genomes of 106 S. sciuri, S. vitulinus and S. fleurettii strains and determined their oxacillin susceptibility profiles. Single-nucleotide polymorphism (SNP) analysis of the core genome was performed to assess the genetic relatedness of the isolates. Phylogenetic analysis of the mecA gene homologues and promoters was achieved through nucleotide/amino acid sequence alignments and mutation rates were estimated using a Bayesian analysis. Furthermore, the predicted structure of mecA homologue-encoded PBPs of oxacillin-susceptible and -resistant strains were compared. We showed for the first time that oxacillin resistance in the S. sciuri group has emerged multiple times and by a variety of different mechanisms. Development of resistance occurred through several steps including structural diversification of the non-binding domain of native PBPs; changes in the promoters of mecA homologues; acquisition of SCCmec and adaptation of the bacterial genetic background. Moreover, our results suggest that it was exposure to ß-lactams in human-created environments that has driven evolution of native PBPs towards a resistance determinant. The evolution of ß-lactam resistance in staphylococci highlights the numerous resources available to bacteria to adapt to the selective pressure of antibiotics.

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July 7, 2019  |  

ALUMINUM RESISTANCE TRANSCRIPTION FACTOR 1 (ART1) contributes to natural variation in aluminum resistance in diverse genetic backgrounds of rice (O. sativa)

Abstract Transcription factors (TFs) regulate the expression of other genes to indirectly mediate stress resistance mechanisms. Therefore, when studying TF-mediated stress resistance, it is important to understand how TFs interact with genes in the genetic background. Here, we fine-mapped the aluminum (Al) resistance QTL Alt12.1 to a 44-kb region containing six genes. Among them is ART1, which encodes a C2H2-type zinc finger TF required for Al resistance in rice. The mapping parents, Al-resistant cv Azucena (tropical japonica) and Al-sensitive cv IR64 (indica), have extensive sequence polymorphism within the ART1 coding region, but similar ART1 expression levels. Using reciprocal near-isogenic lines (NILs) we examined how allele-swapping the Alt12.1 locus would affect plant responses to Al. Analysis of global transcriptional responses to Al stress in roots of the NILs alongside their recurrent parents demonstrated that the presence of the Alt12.1 from Al-resistant Azucena led to greater changes in gene expression in response to Al when compared to the Alt12.1 from IR64 in both genetic backgrounds. The presence of the ART1 allele from the opposite parent affected the expression of several genes not previously implicated in rice Al tolerance. We highlight examples where putatively functional variation in cis-regulatory regions of ART1-regulated genes interacts with ART1 to determine gene expression in response to Al. This ART1–promoter interaction may be associated with transgressive variation for Al resistance in the Azucena × IR64 population. These results illustrate how ART1 interacts with the genetic background to contribute to quantitative phenotypic variation in rice Al resistance.

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July 7, 2019  |  

Tracing origins of the Salmonella Bareilly strain causing a food-borne outbreak in the United States.

Using a novel combination of whole-genome sequencing (WGS) analysis and geographic metadata, we traced the origins of Salmonella Bareilly isolates collected in 2012 during a widespread food-borne outbreak in the United States associated with scraped tuna imported from India.Using next-generation sequencing, we sequenced the complete genome of 100 Salmonella Bareilly isolates obtained from patients who consumed contaminated product, from natural sources, and from unrelated historically and geographically disparate foods. Pathogen genomes were linked to geography by projecting the phylogeny on a virtual globe and produced a transmission network.Phylogenetic analysis of WGS data revealed a common origin for outbreak strains, indicating that patients in Maryland and New York were infected from sources originating at a facility in India.These data represent the first report fully integrating WGS analysis with geographic mapping and a novel use of transmission networks. Results showed that WGS vastly improves our ability to delimit the scope and source of bacterial food-borne contamination events. Furthermore, these findings reinforce the extraordinary utility that WGS brings to global outbreak investigation as a greatly enhanced approach to protecting the human food supply chain as well as public health in general. Published by Oxford University Press for the Infectious Diseases Society of America 2015. This work is written by (a) US Government employee(s) and is in the public domain in the US.

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July 7, 2019  |  

Whole-genome sequencing: opportunities and challenges for public health, food-borne outbreak investigations, and the global food supply.

Food-borne disease is burdensome, af- fecting 1 in 6 persons or an estimated 48 million ill, 128 000 hospitalized, and 3000 deaths in the United States annually. In addition, societal costs from lost lives, lost labor, lost wages, and even lost revenue in the food industry are substan- tial. Globally the burden is even higher, and multinational outbreaks due to the global movement of contaminated foods are being described increasingly. The glo- bal food supply links nations and econo- mies, emphasizing the need to view food safety with an integrated farm-to-fork lens. As predicted, advances in molecular techniques and information management have been transformative for food-borne disease investigation.

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July 7, 2019  |  

High incidence of invasive group A Streptococcus disease caused by strains of uncommon emm types in Thunder Bay, Ontario, Canada.

An outbreak of type emm59 invasive group A Streptococcus (iGAS) disease was declared in 2008 in Thunder Bay District, Northwestern Ontario, two years after a country-wide emm59 epidemic was recognized in Canada. Despite a declining number of emm59 infections since 2010, numerous cases of iGAS disease continue to be reported in the area. We collected clinical information on all iGAS cases recorded in Thunder Bay District from 2008-2013. We also emm typed and sequenced the genomes of all available strains isolated in 2011-2013 from iGAS infections, and from severe cases of soft tissue infections. We used whole-genome data to investigate the population structure of GAS strains of the most frequently isolated emm types. We report increased incidence of iGAS in Thunder Bay compared to the metropolitan area of Toronto/Peel and the province of Ontario. Illicit drug use, alcohol abuse, homelessness and hepatitis C infection were underlying diseases or conditions that might have predisposed patients to iGAS disease. Most cases were caused by clonal strains of “skin” or “generalist” emm types (i.e. emm82, emm87, emm101, emm4, emm83, and emm114), uncommonly seen in other areas of the province. We observed rapid waxing and waning of emm types causing disease and their replacement by other emm types associated with the same tissue tropisms. Thus, iGAS disease in Thunder Bay District predominantly affects a select population of disadvantaged persons and is caused by clonally related strains of a few “skin” and “generalist” emm types less commonly associated with iGAS in other areas of Ontario. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Identification and resolution of microdiversity through metagenomic sequencing of parallel consortia.

To gain a predictive understanding of the interspecies interactions within microbial communities that govern community function, the genomic complement of every member population must be determined. Although metagenomic sequencing has enabled the de novo reconstruction of some microbial genomes from environmental communities, microdiversity confounds current genome reconstruction techniques. To overcome this issue, we performed short-read metagenomic sequencing on parallel consortia, defined as consortia cultivated under the same conditions from the same natural community with overlapping species composition. The differences in species abundance between the two consortia allowed reconstruction of near-complete (at an estimated >85% of gene complement) genome sequences for 17 of the 20 detected member species. Two Halomonas spp. indistinguishable by amplicon analysis were found to be present within the community. In addition, comparison of metagenomic reads against the consensus scaffolds revealed within-species variation for one of the Halomonas populations, one of the Rhodobacteraceae populations, and the Rhizobiales population. Genomic comparison of these representative instances of inter- and intraspecies microdiversity suggests differences in functional potential that may result in the expression of distinct roles in the community. In addition, isolation and complete genome sequence determination of six member species allowed an investigation into the sensitivity and specificity of genome reconstruction processes, demonstrating robustness across a wide range of sequence coverage (9× to 2,700×) within the metagenomic data set. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

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July 7, 2019  |  

Clonal Complex 17 group B Streptococcus strains causing invasive disease in neonates and adults originate from the same genetic pool.

A significant proportion of group B Streptococcus (GBS) neonatal disease, particularly late-onset disease, is associated with strains of serotype III, clonal complex (CC) 17. CC17 strains also cause invasive infections in adults. Little is known about the phylogenetic relationships of isolates recovered from neonatal and adult CC17 invasive infections. We performed whole-genome-based phylogenetic analysis of 93 temporally and geographically matched CC17 strains isolated from both neonatal and adult invasive infections in the metropolitan region of Toronto/Peel, Canada. We also mined the whole-genome data to reveal mobile genetic elements carrying antimicrobial resistance genes. We discovered that CC17 GBS strains causing neonatal and adult invasive disease are interspersed and cluster tightly in a phylogenetic tree, signifying that they are derived from the same genetic pool. We identified limited variation due to recombination in the core CC17 genome. We describe that loss of Pilus Island 1 and acquisition of different mobile genetic elements carrying determinants of antimicrobial resistance contribute to CC17 genetic diversity. Acquisition of some of these mobile genetic elements appears to correlate with clonal expansion of the strains that possess them. Our results provide a genome-wide portrait of the population structure and evolution of a major disease-causing clone of an opportunistic pathogen.

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July 7, 2019  |  

Genomic analyses reveal that partial sequence of an earlier pseudorabies virus in China is originated from a Bartha-vaccine-like strain.

Pseudorabies virus (PRV), the causative agent of Aujeszky?s disease, has gained increased attention in China in recent years as a result of the outbreak of emergent pseudorabies. Several genomic and partial sequences are available for Chinese emergent and European-American strains of PRV, but limited sequence data exist for the earlier Chinese strains. In this study, we determined the complete genomic sequence of one earlier Chinese strain SC and one emergent strain HLJ8. Compared with other known sequences, we demonstrated that PRV strains from distinct geographical regions displayed divergent evolution. Additionally, we report for the first time, a recombination event between PRV strains, and show that strain SC is a recombinant of an endemic Chinese strain and a Bartha-vaccine-like strain. These results contribute to our understanding of PRV evolution. Copyright © 2016 Elsevier Inc. All rights reserved.

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July 7, 2019  |  

Complete closed genome sequences of Salmonella enterica subsp. enterica serotypes Anatum, Montevideo, Typhimurium, and Newport, isolated from beef, cattle, and humans.

Salmonella enterica spp. are a diverse group of bacteria with a wide range of virulence potential. To facilitate genome comparisons across this virulence spectrum, we present eight complete closed genome sequences of four S. enterica serotypes (Anatum, Montevideo, Typhimurium, and Newport), isolated from various cattle samples and from humans. Copyright © 2016 Harhay et al.

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