Division of the genus Chryseobacterium: Observation of discontinuities in amino acid identity values, a possible consequence of major extinction events, guides transfer of nine species to the genus Epilithonimonas, eleven species to the genus Kaistella, and three species to the genus Halpernia gen. nov., with description of Kaistella daneshvariae sp. nov. and Epilithonimonas vandammei sp. nov. derived from clinical specimens.

The genus Chryseobacterium in the family Weeksellaceae is known to be polyphyletic. Amino acid identity (AAI) values were calculated from whole-genome sequences of species of the genus Chryseobacterium, and their distribution was found to be multi-modal. These naturally-occurring non-continuities were leveraged to standardise genus assignment of these species. We speculate that this multi-modal distribution is a consequence of loss of biodiversity during major extinction events, leading to the concept that a bacterial genus corresponds to a set of species that diversified since the Permian extinction. Transfer of nine species (Chryseobacterium arachidiradicis, Chryseobacterium bovis, Chryseobacterium caeni, Chryseobacterium hispanicum, Chryseobacterium hominis, Chryseobacterium hungaricum,, Chryseobacterium pallidum and Chryseobacterium zeae) to the genus Epilithonimonas and eleven (Chryseobacterium anthropi, Chryseobacterium antarcticum, Chryseobacterium carnis, Chryseobacterium chaponense, Chryseobacterium haifense, Chryseobacterium jeonii, Chryseobacterium montanum, Chryseobacterium palustre, Chryseobacterium solincola, Chryseobacterium treverense and Chryseobacterium yonginense) to the genus Kaistella is proposed. Two novel species are described: Kaistella daneshvariae sp. nov. and Epilithonimonas vandammei sp. nov. Evidence is presented to support the assignment of Planobacterium taklimakanense to a genus apart from Chryseobacterium, to which Planobacterium salipaludis comb nov. also belongs. The novel genus Halpernia is proposed, to contain the type species Halpernia frigidisoli comb. nov., along with Halpernia humi comb. nov., and Halpernia marina comb. nov.

A Real-Time Multiplex PCR Assay for Detection of Elizabethkingia Species and Differentiation between Elizabethkingia anophelis and E. meningoseptica.

Nosocomial infections of Elizabethkingia species can have fatal outcomes if not identified and treated properly. The current diagnostic tools available require culture and isolation, which can extend the reporting time and delay treatment. Using comparative genomics, we developed an efficient multiplex real-time PCR for the simultaneous detection of all known species of Elizabethkingia, as well as differentiating the two most commonly reported species, Elizabethkingia anophelis and Elizabethkingia meningoseptica.

Vagococcus bubulae sp. nov., isolated from ground beef, and Vagococcus vulneris sp. nov., isolated from a human foot wound.

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994T was isolated from ground beef and strain SS1995T was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994T and SS1995T against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994T showed high sequence similarity (>97.0 %) to the two most recently proposed species, Vagococcus martis (99.2 %) and Vagococcus teuberi (99.0 %) followed by Vagococcus penaei (98.8 %), strain SS1995T (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995T was most similar to V. penaei (99.1 %), followed by SS1994T (98.6 %), V. martis (98.4 %), V. teuberi (98.1 %), V. acidifermentans (97.8 %), and both V. carniphilus and V. fluvialis (97.5 %). A polyphasic taxonomic study using conventional biochemical and the rapid ID 32 STREP system, MALDI-TOF MS, cell fatty acid analysis, pairwise sequence comparisons of the 16S rRNA, rpoA, rpoB, pheS and groL genes, and comparative core and whole genome sequence analyses revealed that strains SS1994T and SS1995T were two novel Vagococcus species. The novel taxonomic status of the two isolates was confirmed with core genome phylogeny, average nucleotide identity <84 % and in silico DNA-DNA hybridization <28 % to any other Vagococcus species. The names Vagococcusbubulae SS1994T=(CCUG 70831T=LMG 30164T) and Vagococcusvulneris SS1995T=(CCUG 70832T=LMG 30165T) are proposed.

Molecular Evidence of Sexual Transmission of Ebola Virus.

A suspected case of sexual transmission from a male survivor of Ebola virus disease (EVD) to his female partner (the patient in this report) occurred in Liberia in March 2015. Ebola virus (EBOV) genomes assembled from blood samples from the patient and a semen sample from the survivor were consistent with direct transmission. The genomes shared three substitutions that were absent from all other Western African EBOV sequences and that were distinct from the last documented transmission chain in Liberia before this case. Combined with epidemiologic data, the genomic analysis provides evidence of sexual transmission of EBOV and evidence of the persistence of infective EBOV in semen for 179 days or more after the onset of EVD. (Funded by the Defense Threat Reduction Agency and others.).

Revisiting the taxonomy of the genus Elizabethkingia using whole-genome sequencing, optical mapping, and MALDI-TOF, along with proposal of three novel Elizabethkingia species: Elizabethkingia bruuniana sp. nov., Elizabethkingia ursingii sp. nov., and Elizabethkingia occulta sp. nov.

The genus Elizabethkingia is genetically heterogeneous, and the phenotypic similarities between recognized species pose challenges in correct identification of clinically derived isolates. In addition to the type species Elizabethkingia meningoseptica, and more recently proposed Elizabethkingia miricola, Elizabethkingia anophelis and Elizabethkingia endophytica, four genomospecies have long been recognized. By comparing historic DNA-DNA hybridization results with whole genome sequences, optical maps, and MALDI-TOF mass spectra on a large and diverse set of strains, we propose a comprehensive taxonomic revision of this genus. Genomospecies 1 and 2 contain the type strains E. anophelis and E. miricola, respectively. Genomospecies 3 and 4 are herein proposed as novel species named as Elizabethkingia bruuniana sp. nov. (type strain, G0146T = DSM 2975T = CCUG 69503T = CIP 111191T) and Elizabethkingia ursingii sp. nov. (type strain, G4122T = DSM 2974T = CCUG 69496T = CIP 111192T), respectively. Finally, the new species Elizabethkingia occulta sp. nov. (type strain G4070T = DSM 2976T = CCUG 69505T = CIP 111193T), is proposed.

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.

Bacterial composition in a metropolitan drinking water distribution system utilizing different source waters.

We investigated the bacterial composition of water samples from two service areas within a drinking water distribution system (DWDS), each associated with a different primary source of water (groundwater, GW; surface water, SW) and different treatment process. Community analysis based on 16S rRNA gene clone libraries indicated that Actinobacteria (Mycobacterium spp.) and α-Proteobacteria represented nearly 43 and 38% of the total sequences, respectively. Sequences closely related to Legionella, Pseudomonas, and Vibrio spp. were also identified. In spite of the high number of sequences (71%) shared in both areas, multivariable analysis revealed significant differences between the GW and SW areas. While the dominant phylotypes where not significantly contributing in the ordination of samples, the populations associated with the core of phylotypes (1-10% in each sample) significantly contributed to the differences between both service areas. Diversity indices indicate that the microbial community inhabiting the SW area is more diverse and contains more distantly related species coexisting with local assemblages as compared with the GW area. The bacterial community structure of SW and GW service areas were dissimilar, suggesting that their respective source water and/or water quality parameters shaped by the treatment processes may contribute to the differences in community structure observed.

A Method Detection Limit for Bacillus anthracis Spores in Water Using an Automated Waterborne Pathogen Concentrator.

The method detection limit (MDL, 99% chance of detecting a positive result in a single replicate), as per the United States Code of Federal Regulations, was determined for a protocol using an ultrafiltration based automated waterborne pathogen concentration device. Bacillus anthracis Sterne strain spores were seeded at low levels into 100 L reagent water samples. Suspect colonies were confirmed through morphological, chemical, and genetic tests. Samples of 100 L (n=14) of reagent water were seeded with five B. anthracis CFUs each. To confirm the estimated detection limit, a second set (n=19) of 100 L reagent water samples were seeded at a higher level (7 CFUs). The second estimate of the MDL could not be pooled with the first, due to significant difference in variance. A third trial (n=7) seeded with 10 CFUs produced an estimate of the MDL that could be pooled with the higher previous estimate. Another trial consisting of eight 100 L samples of tap water were seeded with approximately 7 CFUs. Recovery in these samples was not significantly different from the pooled MDL. Theoretically a concentration of 4.6 spores/100 L would be required for detection 95% of the time, based on a Poisson distribution. The calculated pooled MDL, based on experimental data was approximately 6 B. anthracis CFU/100 L (95% confidence interval 4.8 to 8.4). Detection at this level was achieved in municipal water samples.

Using the BDFX40 Automated Continuous Blood Culture System to Isolate and Recover Streptobacillus moniliformis in the Presence of 0.05% SPS: A 55-Year, 56-Strain Retrospective Study.

Rat bite fever and Haverhill fever are often difficult to diagnose in a clinical setting. This difficulty results in part from clinicians and laboratory professionals not being able to reliably recover the causative agent Streptobacillus moniliformis using culture-based methods. After utilizing an automated continuous-monitoring blood culture bottle system, we showed that the organism can be reliably cultured when a blood volume inoculum of 10 mL is used. Further, we showed that when the above recommendation is followed, sodium polyanethole sulfonate (up to a concentration of 0.05% w/v) in commercially purchased blood culture bottle formulations seems to be inactivated, allowing for the growth and detection of S. moniliformis. Herein, we offer data and methods used to overcome these clinical limitations. This is a comprehensive study of the historical collection of S. moniliformis isolates maintained by our facility and believed to be the largest of its kind to date.

A real-time multiplex PCR assay for detection of the causative agents of rat bite fever, Streptobacillus moniliformis and zoonoticStreptobacillus species.

Rat bite fever (RBF) caused by Streptobacillus moniliformis has been described as a diagnostic challenge. While it has a favorable prognosis with treatment, timely diagnosis is hindered by the lack of culture-free identification methods. Here we present a multiplex real-time PCR assay that detects the zoonotic Streptobacillus spp. as well as differentiate the primary causative agent of RBF, Streptobacillus moniliformis. The performance of this assay was evaluated using mock clinical specimens for blood, serum, and urine. Analytical sensitivity was determined to be 3-4 genome equivalents (GE)/µl for the zoonotic Streptobacillus spp. target, and 1-2 GE/µl for the S. moniliformis specific target. The assay correctly detected only the intended targets with no cross-reactivity identified. The pathogen was detected in all spiked matrices and not detected in the negative non-spiked specimens. This rapid diagnostic assay may permit quicker diagnosis of RBF patients.

Complete Genome Sequence of Rhodococcus sp. Strain W8901, a Human Clinical Specimen, Assembled Using MiSeq and MinION Sequence Data.

Rhodococcus sp. strain W8901 is a Gram-positive, aerobic, mycolic acid-containing coccobacillus obtained from a patient with acute lymphocytic leukemia. Here, we report on the complete, circular genome sequence obtained using Illumina MiSeq and Oxford Nanopore Technologies MinION reads in order to better resolve the phylogeny of a rare pathogen.

Complete and Circularized Genome Assemblies of the Kroppenstedtia eburnea Genus Type Strain and the Kroppenstedtia pulmonis Species Type Strain with MiSeq and MinION Sequence Data.

Kroppenstedtia eburnea DSM 45196T and Kroppenstedtia pulmonis W9323T are aerobic, Gram-positive, filamentous, chemoorganotrophic thermoactinomycetes. Here, we report on the complete and circular genome assemblies generated using Illumina MiSeq and Oxford Nanopore Technologies MinION reads. Putative gene clusters predicted to be involved in the production of secondary metabolites were also identified.

Complete Genome Sequence of Nocardia farcinica W6977T Obtained by Combining Illumina and PacBio Reads.

The complete genome sequence of the Nocardia farcinica type strain was obtained by combining Illumina HiSeq and PacBio reads, producing a single 6.29-Mb chromosome and 2 circular plasmids. Bioinformatic analysis identified 5,991 coding sequences, including putative genes for virulence, microbial resistance, transposons, and biosynthesis gene clusters.

Draft Genome Sequence of Kroppenstedtia sanguinis X0209T, a Clinical Isolate Recovered from Human Blood.

Kroppenstedtia sanguinis X0209T, a thermoactinomycete, was isolated from the blood of a patient in Sweden. We report on the draft genome sequence obtained with an Illumina MiSeq instrument. The assembled genome totaled 3.73 Mb and encoded 3,583 proteins. Putative genes for virulence, transposons, and biosynthetic gene clusters have been identified.

Enhancing laboratory capacity during Ebola virus disease (EVD) heightened surveillance in Liberia: lessons learned and recommendations.

INTRODUCTION: Following a declaration by the World Health Organization that Liberia had successfully interrupted Ebola virus transmission on May 9th, 2015; the country entered a period of enhanced surveillance. The number of cases had significantly reduced prior to the declaration, leading to closure of eight out of eleven Ebola testing laboratories. Enhanced surveillance led to an abrupt increase in demand for laboratory services. We report interventions, achievements, lessons learned and recommendations drawn from enhancing laboratory capacity. METHODS: Using archived data, we reported before and after interventions that aimed at increasing laboratory capacity. Laboratory capacity was defined by number of laboratories with Ebola Virus Disease (EVD) testing capacity, number of competent staff, number of specimens tested, specimen backlog, daily and surge testing capacity, and turnaround time. Using Stata 14 (Stata Corporation, College Station, TX, USA), medians and trends were reported for all continuous variables. RESULTS: Between May and December 2015, interventions including recruitment and training of eight staff, establishment of one EVD laboratory facility, implementation of ten Ebola GeneXpert diagnostic platforms, and establishment of working shifts yielded an 8-fold increase in number of specimens tested, a reduction in specimens backlog to zero, and restoration of turn-around time to 24 hours. This enabled a more efficient surveillance system that facilitated timely detection and containment of two EVD clusters observed thereafter. CONCLUSION: Effective enhancement of laboratory services during high demand periods requires a combination of context-specific interventions. Building and ensuring sustainability of local capacity is an integral part of effective surveillance and disease outbreak response efforts.

Genotypic differences between strains of the opportunistic pathogen Corynebacterium bovis isolated from humans, cows, and rodents.

Corynebacterium bovis is an opportunistic bacterial pathogen shown to cause eye and prosthetic joint infections as well as abscesses in humans, mastitis in dairy cattle, and skin disease in laboratory mice and rats. Little is known about the genetic characteristics and genomic diversity of C. bovis because only a single draft genome is available for the species. The overall aim of this study was to sequence and compare the genome of C. bovis isolates obtained from different species, locations, and time points. Whole-genome sequencing was conducted on 20 C. bovis isolates (six human, four bovine, nine mouse and one rat) using the Illumina MiSeq platform and submitted to various comparative analysis tools. Sequencing generated high-quality contigs (over 2.53 Mbp) that were comparable to the only reported assembly using C. bovis DSM 20582T (97.8 ± 0.36% completeness). The number of protein-coding DNA sequences (2,174 ± 12.4) was similar among all isolates. A Corynebacterium genus neighbor-joining tree was created, which revealed Corynebacterium falsenii as the nearest neighbor to C. bovis (95.87% similarity), although the reciprocal comparison shows Corynebacterium jeikeium as closest neighbor to C. falsenii. Interestingly, the average nucleotide identity demonstrated that the C. bovis isolates clustered by host, with human and bovine isolates clustering together, and the mouse and rat isolates forming a separate group. The average number of genomic islands and putative virulence factors were significantly higher (p<0.001) in the mouse and rat isolates as compared to human/bovine isolates. Corynebacterium bovis' pan-genome contained a total of 3,067 genes of which 1,354 represented core genes. The known core genes of all isolates were primarily related to ''metabolism" and ''information storage/processing." However, most genes were classified as ''function unknown" or "unclassified". Surprisingly, no intact prophages were found in any isolate; however, almost all isolates had at least one complete CRISPR-Cas system.

Draft Reference Genome Sequence of Corynebacterium mastitidis 16-1433, Isolated from a Mouse.

We report here a nearly complete draft genome sequence for a Corynebacterium mastitidis isolate from a mouse. The total read coverage is 198×, and the genome size is 2,264,319 bp with a 69.04% GC content. This genome complements the only other genome available for C. mastitidis, which was obtained from a sheep.

Complete Genome Sequence of Streptacidiphilus sp. Strain 15-057A, Obtained from Bronchial Lavage Fluid.

Streptacidiphilus sp. strain 15-057A was isolated from a bronchial lavage sample and represents the only member of the genus not isolated from acidic soils. A single circular chromosome of 7.01 Mb was obtained by combining Illumina and PacBio sequencing data. Bioinformatic analysis detected 63 putative secondary biosynthetic gene clusters and recognized 43 transposons.

The draft genomes of Elizabethkingia anophelis of equine origin are genetically similar to three isolates from human clinical specimens.

We report the isolation and characterization of two Elizabethkingia anophelis strains (OSUVM-1 and OSUVM-2) isolated from sources associated with horses in Oklahoma. Both strains appeared susceptible to fluoroquinolones and demonstrated high MICs to all cell wall active antimicrobials including vancomycin, along with aminoglycosides, fusidic acid, chloramphenicol, and tetracycline. Typical of the Elizabethkingia, both draft genomes contained multiple copies of ß-lactamase genes as well as genes predicted to function in antimicrobial efflux. Phylogenetic analysis of the draft genomes revealed that OSUVM-1 and OSUVM-2 differ by only 6 SNPs and are in a clade with 3 strains of Elizabethkingia anophelis that were responsible for human infections. These findings therefore raise the possibility that Elizabethkingia might have the potential to move between humans and animals in a manner similar to known zoonotic pathogens.

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.