Exserohilum rostratum was the major cause of an outbreak of fungal infections linked to injections of contaminated methylprednisolone acetate. Because almost 14,000 persons were exposed to product that was possibly contaminated with multiple fungal pathogens, there was unprecedented need for a rapid throughput diagnostic test that could detect both E. rostratum and other unusual agents of fungal infection. Here we report development of a novel PCR test that allowed for rapid and specific detection of fungal DNA in cerebrospinal fluid (CSF), other body fluids and tissues of infected individuals. The test relied on direct purification of free-circulating fungal DNA from fluids and subsequent PCR amplification and sequencing. Using this method, we detected Exserohilum rostratum DNA in 123 samples from 114 case-patients (28% of 413 case-patients for whom 627 samples were available), and Cladosporium DNA in one sample from one case-patient. PCR with novel Exserohilum-specific ITS-2 region primers detected 25 case-patients with samples that were negative using broad-range ITS primers. Compared to fungal culture, this molecular test was more sensitive: of 139 case-patients with an identical specimen tested by culture and PCR, E. rostratum was recovered in culture from 19 (14%), but detected by PCR in 41 (29%), showing a diagnostic sensitivity of 29% for PCR compared to 14% for culture in this patient group. The ability to rapidly confirm the etiologic role of E. rostratum in these infections provided an important contribution in the public health response to this outbreak.

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 molle , 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.

Two unusual catalase-negative, Gram-stain-positive, Vagococcus-like isolates that were referred to the CDC Streptococcus Laboratory for identification are described. Strain SS1994(T) was isolated from ground beef and strain SS1995(T) was isolated from a human foot wound. Comparative 16S rRNA gene sequence analysis of isolates SS1994(T) and SS1995(T) against Vagococcus type strain sequences supported their inclusion in the genus Vagococcus. Strain SS1994(T) 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 SS1995(T) (98.6 %), Vagococcus carniphilus (98.0 %), Vagococcus acidifermentans (98.0 %) and Vagococcus fluvialis (97.9 %). The 16S rRNA gene sequence of strain SS1995(T) was most similar to V. penaei (99.1 %), followed by SS1994(T) (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 SS1994(T) and SS1995(T) 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 SS1994(T)=(CCUG 70831(T)=LMG 30164(T)) and Vagococcusvulneris SS1995(T)=(CCUG 70832(T)=LMG 30165(T)) are proposed.

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.

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 beta-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.

Three isolates of a previously reported novel catalase-negative, Gram-stain-positive, coccoid, alpha-haemolytic, Streptococcus species that were associated with meningoencephalitis in naive weanling mice were further evaluated to confirm their taxonomic status and to determine additional phenotypic and molecular characteristics. Comparative 16S rRNA gene sequence analysis showed nearly identical intra-species sequence similarity (>/=99.9 %), and revealed the closest phylogenetically related species, Streptococcus acidominimus and Streptococcus cuniculi, with 97.0 and 97.5 % sequence similarity, respectively. The rpoB, sodA and recN genes were identical for the three isolates and were 87.6, 85.7 and 82.5 % similar to S. acidominimus and 89.7, 86.2 and 80.7 % similar to S. cuniculi, respectively. In silico DNA-DNA hybridization analyses of mouse isolate 12-5202(T) against S. acidominimus CCUG 27296(T) and S. cuniculi CCUG 65085(T) produced estimated values of 26.4 and 25.7 % relatedness, and the calculated average nucleotide identity values were 81.9 and 81.7, respectively. These data confirm the taxonomic status of 12-5202(T) as a distinct Streptococcus species, and we formally propose the type strain, Streptococcusazizii 12-5202(T) (=CCUG 69378(T)=DSM 103678(T)). The genome of Streptococcus azizii sp. nov. 12-5202(T) contains 2062 total genes with a size of 2.34 Mbp, and an average G+C content of 42.76 mol%.

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.

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.

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.

An atypically large outbreak of Elizabethkingia anophelis infections occurred in Wisconsin. Here we show that it was caused by a single strain with thirteen characteristic genomic regions. Strikingly, the outbreak isolates show an accelerated evolutionary rate and an atypical mutational spectrum. Six phylogenetic sub-clusters with distinctive temporal and geographic dynamics are revealed, and their last common ancestor existed approximately one year before the first recognized human infection. Unlike other E. anophelis, the outbreak strain had a disrupted DNA repair mutY gene caused by insertion of an integrative and conjugative element. This genomic change probably contributed to the high evolutionary rate of the outbreak strain and may have increased its adaptability, as many mutations in protein-coding genes occurred during the outbreak. This unique discovery of an outbreak caused by a naturally occurring mutator bacterial pathogen provides a dramatic example of the potential impact of pathogen evolutionary dynamics on infectious disease epidemiology.

The inclusion of molecular methods in the characterization of the novel species Enterococcus horridus necessitated the sequencing and assembly of the genomes of the closely related Enterococcus rotai and Enterococcus silesiacus Sequencing using Illumina technology in combination with optical mapping led to the generation of closed genomes for both isolates.

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.

Eight Gram-negative bacteria (B4199T, C6819, C6918, D2441, D3318, E1086, E1148 and E5571) were identified during a retrospective study of unidentified strains from a historical collection held in the Special Bacteriology Reference Laboratory at the Centers for Disease Control and Prevention. The strains were isolated from eight patients: five female, two male and one not specified. No ages were indicated for the patients. The sources were urine (3), leg tissue (2), foot wound, lung tissue and deep liver. The strains originated from seven different states across the United States of America (Colorado, Connecticut (2), Indiana, North Carolina, Oregon and Pennsylvania). The strains grew at 10 to 42 degrees C, were non-motile, alkalitolerant, slightly halophilic, microaerophilic, catalase and oxidase positive. The DNA G + C content was 47.3 to 47.8 mol%. The major cellular fatty acids were tetradecanoic acid (C14:0), hexadecanoic acid (C16:0) and 11-octadecenoic acid (C18:1omega7c). Polar lipids detected were phosphatidylglycerol (PG), phosphatidylethanolamine (PE), diphosphatidylglycerol (DPG) and an unknown phospholipid; the only respiratory quinone detected was the ubiquinone Q-9 (100 %). 16S rRNA gene sequence analysis produced results with 95.6 % similarity to Pseudomonas caeni (DSM 24390T) and 95.2 % similarity to Thiopseudomonas denitrificans (X2T). The results of the biochemical, chemotaxonomic and phylogenetic analyses between the study strains and some related type strains indicate that these strains represent a novel species of a new genus within the family Pseudomonadaceae, for which the name Oblitimonas alkaliphila gen. nov., sp. nov. is proposed. The type strain is B4199T (=DSM 100830T = CCUG 67636T).

Phenotypic, genotypic and antimicrobial characteristics of six phenotypically distinct human clinical isolates that most closely resembled the type strain of Streptococcus halichoeri isolated from a seal are presented. Sequencing of the 16S rRNA, rpoB, sodA and recN genes, comparative whole genome analysis, conventional biochemical and rapid ID 32 STREP identification methods, and antimicrobial susceptibility testing were performed on the human isolates, the type strain of S. halichoeri, and type strains of closely related species. The six human clinical isolates were biochemically indistinguishable from each other and showed 100% 16S rRNA, rpoB, sodA and recN gene sequence similarity. Comparative 16S rRNA gene sequencing analysis revealed 98.6% similarity to S. halichoeri CCUG 48324T, 97.9% to S. canis ATCC 43496T and 97.8% to S. ictaluri ATCC BAA-1300T. A 3530 bp fragment of the rpoB gene was 98.8% similar to S. halichoeriT, 84.6% to S. canisT and 83.8% to S. ictaluriT. S. halichoeriT and the human clinical isolates were susceptible to the antimicrobials tested based on CLSI guidelines for Streptococcus species viridans group with the exception of tetracycline and erythromycin. The human isolates were phenotypically distinct from the type strain isolated from a seal; comparative whole genome sequence analysis confirmed that the human isolates were S. halichoeri. On the basis of these results, a novel subspecies, Streptococcus halichoeri subsp. hominis is proposed for the human isolates and S. halichoeri subsp. halichoeri for the grey seal isolates. The type strain of the novel subspecies is SS1844T = CCUG 67100T = LMG 28801T.

The CDC Special Bacteriology Reference Laboratory (SBRL) collection of human clinical pathogens contains several strains from the genus Devosia, usually found environmentally. We provide here the complete genome of strain H5989, which was isolated from a human cerebrospinal fluid (CSF) specimen and represents a putative novel species in the genus Devosia.

In September 2012, the Centers for Disease Control and Prevention (CDC) initiated an outbreak investigation of fungal infections linked to injection of contaminated methylprednisolone acetate (MPA). Between 2 October 2012 and 14 February 2013, the CDC laboratory received 799 fungal isolates or human specimens, including cerebrospinal fluid (CSF), synovial fluid, and abscess tissue, from 469 case patients in 19 states. A novel broad-range PCR assay and DNA sequencing were used to evaluate these specimens. Although Aspergillus fumigatus was recovered from the index case, Exserohilum rostratum was the primary pathogen in this outbreak and was also confirmed from unopened MPA vials. Exserohilum rostratum was detected or confirmed in 191 specimens or isolates from 150 case patients, primarily from Michigan (n = 67 patients), Tennessee (n = 26), Virginia (n = 20), and Indiana (n = 16). Positive specimens from Michigan were primarily abscess tissues, while positive specimens from Tennessee, Virginia, and Indiana were primarily CSF. E. rostratum antifungal susceptibility MIC50 and MIC90 values were determined for voriconazole (1 and 2 mug/ml, respectively), itraconazole (0.5 and 1 mug/ml), posaconazole (0.5 and 1 mug/ml), isavuconazole (4 and 4 mug/ml), and amphotericin B (0.25 and 0.5 mug/ml). Thirteen other mold species were identified among case patients, and four other fungal genera were isolated from the implicated MPA vials. The clinical significance of these other fungal species remains under investigation. The laboratory response provided significant support to case confirmation, enabled linkage between clinical isolates and injected vials of MPA, and described significant features of the fungal agents involved in this large multistate outbreak.

Ninety-seven animal, human, and dairy Streptococcus porcinus or Streptococcus pseudoporcinus isolates in the CDC Streptococcus strain collection were evaluated based on DNA-DNA reassociation, 16S rRNA and rpoB gene sequencing, conventional biochemical and rapid ID 32 STREP identification methods, and antimicrobial susceptibility testing to determine their taxonomic status, characteristics for species differentiation, antimicrobial susceptibility and relevance of clinical source. Nineteen of the 97 isolates (1 human, 18 swine) were identified as S. porcinus. The remaining 72 human isolates and 6 dairy isolates were identified as S. pseudoporcinus. The use of 16S rRNA or rpoB gene sequencing was required to differentiate S. porcinus from S. pseudoporcinus. The human and dairy S. pseudoporcinus isolates were biochemically distinct from each other as well as distinct by 16S rRNA and rpoB gene sequencing. Therefore, we propose the subspecies denominations S. pseudoporcinus subsp. hominis subsp. nov. for the human isolates and S. pseudoporcinus subsp. lactis subsp. nov. for the dairy isolates. Most strains were susceptible to the antimicrobials tested, with the exception of tetracycline. Two strains of each species were also resistant to clindamycin and erythromycin and carried the erm(A) (S. pseudoporcinus) or the erm(B) (S. porcinus) genes. S. porcinus was identified from a single human isolate recovered from a wound in an abattoir worker. S. pseudoporcinus was primarily isolated from the genitourinary tract of women, but was also associated with blood, placental, and wound infections. Isolates reacting with group B antiserum and demonstrating wide beta hemolysis should be suspected of being S. pseudoporcinus and not S. agalactiae.

Members of the Mycobacterium chelonae-abscessus complex represent Mycobacterium species that cause invasive infections in immunocompetent and immunocompromised hosts. We report the detection of a new pathogen that had been misidentified as M. chelonae with an atypical antimicrobial drug susceptibility profile. The discovery prompted a multicenter investigation of 26 patients. Almost all patients were from the northeastern United States, and most had underlying sinus or pulmonary disease. Infected patients had clinical features similar to those with M. abscessus infections. Taxonomically, the new pathogen shared molecular identity with members of the M. chelonae-abscessus complex. Multilocus DNA target sequencing, DNA-DNA hybridization, and deep multilocus sequencing (43 full-length genes) support a new taxon for these microorganisms. Because most isolates originated in Pennsylvania, we propose the name M. franklinii sp. nov. This investigation underscores the need for accurate identification of Mycobacterium spp. to detect new pathogens implicated in human disease.

In this study, 16 human clinical isolates of Dietzia species previously misidentified as Rhodococcus equi were evaluated using phenotypic methods, including traditional and commercial (API Coryne) biochemical tests, antimicrobial susceptibility testing, and 16S rRNA gene and gyrB gene sequencing. Positive results for both the hydrolysis of adenine and Christie-Atkins-Munch-Petersen (CAMP) reaction allowed for differentiation between the Dietzia isolates and the type strain of Rhodococcus equi; however, traditional and commercial phenotypic profiles could not be used to reliably identify Dietzia species. The analysis of 16S rRNA gene and gyrB gene sequences could discriminate all Dietzia strains from the type strain of R. equi. Most Dietzia species had distinct 16S rRNA gene and gyrB gene sequences; however, the 16S rRNA gene sequences of the type strains of D. schimae and D. cercidiphylli were identical to D. maris and D. natronolimnaea, respectively. Based on comparative sequence analysis, five clinical isolates clustered with D. maris/D. schimae and nine with D. natronolimnaea/D. cercidiphylli. The two remaining isolates were found to be most closely related to the D. cinnamea/D. papillomatosis clade. Even though molecular analyses were not sufficiently discriminative to accurately identify all Dietzia species, the method was able to reliably identify isolates that were previously misidentified by phenotypic methods to the genus level.

Novel nontoxigenic Corynebacterium diphtheriae was isolated from a domestic cat with severe otitis. Contact investigation and carrier study of human and animal contacts yielded 3 additional, identical isolates from cats, although no evidence of zoonotic transmission was identified. Molecular methods distinguished the feline isolates from known C. diphtheriae.

BACKGROUND: A quadrivalent meningococcal conjugate vaccine (MCV4) was licensed in the United States in 2005; no serogroup B vaccine is available. Neisseria meningitidis changes its capsular phenotype through capsular switching, which has implications for vaccines that do not protect against all serogroups. METHODS: Meningococcal isolates from 10 Active Bacterial Core surveillance sites from 2000 through 2005 were analyzed to identify changes occurring after MCV4 licensure. Isolates were characterized by multilocus sequence typing (MLST) and outer membrane protein gene sequencing. Isolates expressing capsular polysaccharide different from that associated with the MLST lineage were considered to demonstrate capsular switching. RESULTS: Among 1160 isolates, the most common genetic lineages were the sequence type (ST)-23, ST-32, ST-11, and ST-41/44 clonal complexes. Of serogroup B and Y isolates, 8 (1.5%) and 3 (0.9%), respectively, demonstrated capsular switching, compared with 36 (12.9%) for serogroup C ([Formula: see text]); most serogroup C switches were from virulent serogroup B and/or serogroup Y lineages. CONCLUSIONS: A limited number of genetic lineages caused the majority of invasive meningococcal infections. A substantial proportion of isolates had evidence of capsular switching. The high prevalence of capsular switching requires surveillance to detect changes in the meningococcal population structure that may affect the effectiveness of meningococcal vaccines.

The proportion of meningococcal disease in the US, South Africa, and Israel caused by Neisseria meningitidis serogroup Y (NmY) was greater than the worldwide average during the period 1999-2002. Genotypic characterization of 300 NmY isolates by MLST, 16S rRNA gene sequencing and PorA variable region typing was conducted to determine the relationships of the isolates from these 3 countries. 70 different genotypes were found. Two groups of ST-23 clonal complex isolates accounted for 88% of the US isolates, 12% of the South African isolates, and 96% of the isolates from Israel. The single common clone (ST-23/16S-19/P1.5-2,10-1) represented 57%, 5% and 35% of the NmY isolates from the US, South Africa and Israel. The predominant clone in South Africa (ST-175/16S-21/P1.5-1,2-2) and 11 other closely related clones made up 77% of the South African study isolates and were not found among the isolates from the US or Israel. ST-175 was the predicted founder of the ST-175 clonal complex, and isolates of ST-175 and related STs have been described previously in other African countries. Continued active surveillance and genetic characterization of NmY isolates causing disease in the US, South Africa, and Israel will provide valuable data for local and global epidemiology, allow monitoring for any expansion of existing clonal complexes and detection of the emergence of new virulent clones in the population.