Screening and Genomic Characterization of Filamentous Hemagglutinin-Deficient Bordetella pertussis.

Despite high vaccine coverage, pertussis cases in the United States have increased over the last decade. Growing evidence suggests that disease resurgence results, in part, from genetic divergence of circulating strain populations away from vaccine references. The United States employs acellular vaccines exclusively, and current Bordetella pertussis isolates are predominantly deficient in at least one immunogen, pertactin (Prn). First detected in the United States retrospectively in a 1994 isolate, the rapid spread of Prn deficiency is likely vaccine driven, raising concerns about whether other acellular vaccine immunogens experience similar pressures, as further antigenic changes could potentially threaten vaccine efficacy. We developed an electrochemiluminescent antibody capture assay to monitor the production of the acellular vaccine immunogen filamentous hemagglutinin (Fha). Screening 722 U.S. surveillance isolates collected from 2010 to 2016 identified two that were both Prn and Fha deficient. Three additional Fha-deficient laboratory strains were also identified from a historic collection of 65 isolates dating back to 1935. Whole-genome sequencing of deficient isolates revealed putative, underlying genetic changes. Only four isolates harbored mutations to known genes involved in Fha production, highlighting the complexity of its regulation. The chromosomes of two Fha-deficient isolates included unexpected structural variation that did not appear to influence Fha production. Furthermore, insertion sequence disruption of fhaB was also detected in a previously identified pertussis toxin-deficient isolate that still produced normal levels of Fha. These results demonstrate the genetic potential for additional vaccine immunogen deficiency and underscore the importance of continued surveillance of circulating B. pertussis evolution in response to vaccine pressure.

Enterococcus crotali sp. nov., isolated from faecal material of a timber rattlesnake.

A facultatively anaerobic, Gram-stain-positive bacterium, designated ETRF1T, was found in faecal material of a timber rattlesnake (Crotalus horridus). Based on a comparative 16S rRNA gene sequence analysis, the isolate was assigned to the genus Enterococcus. The 16S rRNA gene sequence of strain ETRF1T showed >97 % similarity to that of the type strains of Enterococcus rotai, E. caccae, E. silesiacus, E haemoperoxidus, E. ureasiticus, E. moraviensis, E. plantarum, E. quebecensis, E. ureilyticus, E. termitis, E. rivorum and E. faecalis. The organism could be distinguished from these 12 phylogenetically related enterococci using conventional biochemical testing, the Rapid ID32 Strep system, comparative pheS and rpoA gene sequence analysis, and comparative whole genome sequence analysis. The estimated in silico DNA-DNA hybridization values were <70 %, and average nucleotide identity values were <96 %, when compared to these 12 species, further validating that ETRF1T represents a unique species within the genus Enterococcus. On the basis of these analyses, strain ETRF1T (=CCUG 65857T=LMG 28312T) is proposed as the type strain of a novel species, Enterococcus crotali sp. nov.

Molecular detection of airborne Coccidioides in Tucson, Arizona.

Environmental surveillance of the soil-dwelling fungus Coccidioides is essential for the prevention of Valley fever, a disease primarily caused by inhalation of the arthroconidia. Methods for collecting and detecting Coccidioides in soil samples are currently in use by several laboratories; however, a method utilizing current air sampling technologies has not been formally demonstrated for the capture of airborne arthroconidia. In this study, we collected air/dust samples at two sites (Site A and Site B) in the endemic region of Tucson, Arizona, and tested a variety of air samplers and membrane matrices. We then employed a single-tube nested qPCR assay for molecular detection. At both sites, numerous soil samples (n = 10 at Site A and n = 24 at Site B) were collected and Coccidioides was detected in two samples (20%) at Site A and in eight samples (33%) at Site B. Of the 25 air/dust samples collected at both sites using five different air sampling methods, we detected Coccidioides in three samples from site B. All three samples were collected using a high-volume sampler with glass-fiber filters. In this report, we describe these methods and propose the use of these air sampling and molecular detection strategies for environmental surveillance of Coccidioides.

Conserved Patterns of Symmetric Inversion in the Genome Evolution of Bordetella Respiratory Pathogens.

Whooping cough (pertussis), primarily caused by Bordetella pertussis, has resurged in the United States, and circulating strains exhibit considerable chromosome structural fluidity in the form of rearrangement and deletion. The genus Bordetella includes additional pathogenic species infecting various animals, some even causing pertussis-like respiratory disease in humans; however, investigation of their genome evolution has been limited. We studied chromosome structure in complete genome sequences from 167 Bordetella species isolates, as well as 469 B. pertussis isolates, to gain a generalized understanding of rearrangement patterns among these related pathogens. Observed changes in gene order primarily resulted from large inversions and were only detected in species with genomes harboring multicopy insertion sequence (IS) elements, most notably B. holmesii and B. parapertussis While genomes of B. pertussis contain >240 copies of IS481, IS elements appear less numerous in other species and yield less chromosome structural diversity through rearrangement. These data were further used to predict all possible rearrangements between IS element copies present in Bordetella genomes, revealing that only a subset is observed among circulating strains. Therefore, while it appears that rearrangement occurs less frequently in other species than in B. pertussis, these clinically relevant respiratory pathogens likely experience similar mutation of gene order. The resulting chromosome structural fluidity presents both challenges and opportunity for the study of Bordetella respiratory pathogens.IMPORTANCE Bordetella pertussis is the primary agent of whooping cough (pertussis). The Bordetella genus includes additional pathogens of animals and humans, including some that cause pertussis-like respiratory illness. The chromosome of B. pertussis has previously been shown to exhibit considerable structural rearrangement, but insufficient data have prevented comparable investigation in related species. In this study, we analyze chromosome structure variation in several Bordetella species to gain a generalized understanding of rearrangement patterns in this genus. Just as in B. pertussis, we observed inversions in other species that likely result from common mutational processes. We used these data to further predict additional, unobserved inversions, suggesting that specific genome structures may be preferred in each species.

Identification of five major and two minor genotypes of varicella-zoster virus strains: a practical two-amplicon approach used to genotype clinical isolates in Australia and New Zealand.

Whole genome phylogenetic analysis in this study resolved a total of five major genotypes among the 22 varicella-zoster virus (VZV) strains or isolates for which complete genomic sequences are available. Consistent with earlier publications we have designated these genotypes European 1 (E1), European 2 (E2), Japanese (J), mosaic 1 (M1), and mosaic 2 (M2). Single nucleotide polymorphism (SNP) analysis performed in a whole-genome alignment revealed that VZV isolates of all five genotypes can be accurately genotyped using SNPs from two amplicons: open reading frame 22 (ORF22) and either ORF21 or ORF50. This modified approach identifies all of the genotypes observed using any of the published genotyping protocols. Of 165 clinical varicella and zoster isolates from Australia and New Zealand typed using this approach, 67 of 127 eastern Australian isolates were E1, 30 were E2, 16 were J, 10 were M1, and 4 were M2; 25 of 38 New Zealand isolates were E1, 8 were E2, and 5 were M1. VZV strain diversity in eastern Australia is thus broader than has been described for any other region, including Europe, Africa, and North America. J strains were far more prevalent than previously observed in countries other than Japan. Two-amplicon typing was in complete accord with genotypes derived using SNP in multiple ORFs (ORFs 1, 21, 22, 38, 50, 54, and 62). Two additional minor genotypes, M3 and M4, could also be resolved using two-amplicon typing.

Genome Sequence of the Amphotericin B-Resistant Candida duobushaemulonii Strain B09383.

Candida duobushaemulonii is a drug-resistant yeast that can cause invasive candidiasis. Here, we report the first genome sequence of C. duobushaemulonii, isolate B09383, generated using PacBio sequencing technology. The estimated genome size was 12.5 Mb with a GC content of 46.84%.

PacBio Genome Sequences of Escherichia coli Serotype O157:H7, Diffusely Adherent E. coli, and Salmonella enterica Strains, All Carrying Plasmids with an mcr-1 Resistance Gene.

We report here Illumina-corrected PacBio whole-genome sequences of an Escherichia coli serotype O157:H7 strain (2017C-4109), an E. coli serotype O[undetermined]:H2 strain (2017C-4173W12), and a Salmonella enterica subsp. enterica serovar Enteritidis strain (2017K-0021), all of which carried the mcr-1 resistance gene on an IncI2 or IncX4 plasmid. We also determined that pMCR-1-CTSe is identical to a previously published plasmid, pMCR-1-CT.

Genomic insights into multidrug-resistance, mating and virulence in Candida auris and related emerging species.

Candida auris is an emergent multidrug-resistant fungal pathogen causing increasing reports of outbreaks. While distantly related to C. albicans and C. glabrata, C. auris is closely related to rarely observed and often multidrug-resistant species from the C. haemulonii clade. Here, we analyze near complete genome assemblies for the four C. auris clades and three related species, and map intra- and inter-species rearrangements across the seven chromosomes. Using RNA-Seq-guided gene predictions, we find that most mating and meiosis genes are conserved and that clades contain either the MTLa or MTLα mating loci. Comparing the genomes of these emerging species to those of other Candida species identifies genes linked to drug resistance and virulence, including expanded families of transporters and lipases, as well as mutations and copy number variants in ERG11. Gene expression analysis identifies transporters and metabolic regulators specific to C. auris and those conserved with related species which may contribute to differences in drug response in this emerging fungal clade.

High-Quality Whole-Genome Sequences for 77 Shiga Toxin-Producing Escherichia coli Strains Generated with PacBio Sequencing.

Shiga toxin-producing Escherichia coli (STEC) is an enteric foodborne pathogen that can cause mild to severe illness. Here, we report the availability of high-quality whole-genome sequences for 77 STEC strains generated using the PacBio sequencing platform.

High-Quality Complete and Draft Genome Sequences for Three Escherichia spp. and Three Shigella spp. Generated with Pacific Biosciences and Illumina Sequencing and Optical Mapping.

Escherichia spp., including E. albertii and E. coli, Shigella dysenteriae, and S. flexneri are causative agents of foodborne disease. We report here reference-level whole-genome sequences of E. albertii (2014C-4356), E. coli (2011C-4315 and 2012C-4431), S. dysenteriae (BU53M1), and S. flexneri (94-3007 and 71-2783).

Genome Sequence of a Multidrug-Resistant Candida haemulonii Isolate from a Patient with Chronic Leg Ulcers in Israel.

Candida haemulonii is an emerging multidrug-resistant yeast that can cause invasive candidiasis. Here, we report the first genome sequence of C. haemulonii (isolate B11899) generated using PacBio sequencing technology. The estimated genome size was 13.3 Mb, with a GC content of 45.19%.

High-Quality Whole-Genome Sequences for 59 Historical Shigella Strains Generated with PacBio Sequencing.

Shigella spp. are enteric pathogens that cause shigellosis. We report here the high-quality whole-genome sequences of 59 historical Shigella strains that represent the four species and a variety of serotypes.

Twelve Complete Reference Genomes of Clinical Isolates in the Capnocytophaga Genus.

We report here 1 near-complete genome sequence and 12 complete genome sequences for clinical Capnocytophaga isolates. Total read coverages ranged from 211× to 737×, and genome sizes ranged from 2.41 Mb to 3.10 Mb. These genomes will enable a more comprehensive taxonomic evaluation of the Capnocytophaga genus.

Complete Genome Sequences of Bordetella pertussis Isolates with Novel Pertactin-Deficient Deletions.

Clinical isolates of the respiratory pathogen Bordetella pertussis in the United States have become predominantly deficient for the acellular vaccine immunogen pertactin through various independent mutations. Here, we report the complete genome sequences for four B. pertussis isolates that harbor novel deletions responsible for pertactin deficiency.

High-Quality Genome Sequence of an Escherichia coli O157 Strain Carrying an mcr-1 Resistance Gene Isolated from a Patient in the United States.

Enterobacteriaceae carrying plasmid-mediated colistin resistance have been found around the world. We report here the high-quality whole-genome sequence of an Escherichia coli O157:H48 isolate (2016C-3936C1) from Connecticut that carried the mcr-1 resistance gene on an IncX4-type plasmid.

Complete Circularized Genome Sequences of Four Strains of Elizabethkingia anophelis, Including Two Novel Strains Isolated from Wild-Caught Anopheles sinensis.

We provide complete circularized genome sequences of two mosquito-derived Elizabethkingia anophelis strains with draft sequences currently in the public domain (R26 and Ag1), and two novel E. anophelis strains derived from a different mosquito species, Anopheles sinensis (AR4-6 and AR6-8). The genetic similarity of all four mosquito-derived strains is remarkable.

New mosaic subgenotype of varicella-zoster virus in the USA: VZV detection and genotyping by oligonucleotide-microarray.

A rapid and sensitive microarray-based method was used to distinguish the three major circulating genotypes of varicella-zoster virus (VZV). The method analyzes five variable positions located in a 447-nucleotide variable region 1 of open reading frame 22 (ORF 22r1); these single nucleotide polymorphisms (SNP) display in stably occurring patterns specific to each of the VZV genotypes established in previously published studies. Pairs of short oligonucleotide probes (oligoprobes) with sequences corresponding to all of the observed SNP were used to detect specific sequences. Fluorescently labeled ssRNA samples for hybridization with a chip were prepared by in vitro T7 polymerase driven transcription of the amplicons of ORF 22r1, followed by chemical labeling with Cy5 into RNA sample. Ratios between fluorescent hybridization signals from each pair of oligoprobes were used to assess the sequence at each SNP. We evaluated six reference VZV strains and 130 VZV clinical specimens to validate the method. The microarray method accurately identified strains isolated in the US in 2001-2002, representing all major genotypes as determined using more extensive sequence analysis, correctly assigning strains to genotypes E (81.5%), J (3%) and M (15.5%). In addition, a new M variant (M3) was identified.

Complete Genome Sequences for Three Chromosomes of the Burkholderia stabilis Type Strain (ATCC BAA-67).

We report here the complete annotated genome sequence of the Burkholderia stabilis type strain ATCC BAA-67. There were three circular chromosomes with a combined size of 8,527,947 bp and G+C composition of 66.4%. These characteristics closely resemble the genomes of other sequenced members of the Burkholderia cepacia complex.

Three Genome Sequences of Legionella pneumophila subsp. pascullei Associated with Colonization of a Health Care Facility.

Here, we report the complete genome sequences of three Legionella pneumophila subsp. pascullei strains (including both serogroup 1 and 5 strains) that were found in the same health care facility in 1982 and 2012.

Complete Genome Sequences of Four Strains from the 2015-2016 Elizabethkingia anophelis Outbreak.

The complete circularized genome sequences of selected specimens from the largest known Elizabethkingia anophelis outbreak to date are described here. Genomic rearrangements observed among the outbreak strains are discussed.