Corynebacterium godavarianum Jani et al. 2018 and Corynebacterium hadale Wei et al. 2018 are both later heterotypic synonyms of Corynebacterium gottingense Atasayar et al. 2017, proposal of an emended description of Corynebacterium gottingense Atasayar et al. 2017.

Seven strains of an unidentifiable Corynebacterium species recovered from blood cultures, urine or cerebrospinal fluid over 26 years, closest to but differentiated from Corynebacterium imitans by 16S rRNA gene and partial rpoB gene sequencing, were studied. In November 2017, Atasayar et al. described a blood culture isolate as Corynebacterium gottingense sp. nov., which had >99 % similarity by 16S rRNA gene sequencing to the Canadian strains. In January 2018, Jani et al. described Corynebacterium godavarianum sp. nov., recovered from the Godavari River, India, which also had >99 % similarity by 16S/rpoB sequencing to the Canadian strains and C. gottingense. In May 2018, Wei et al. described Corynebacterium hadale recovered from hadopelagic water; this too had >99 % similarity by 16S rRNA gene sequencing to C. gottingense, C. godavarianum and the Canadian strains. C. gottingense DSM 103494T and C. godavarianum LMG 29598T were acquired and whole genome sequencing was performed (not previously done). Results were compared with genomes from C. hadale (GenBank accession NQMQ01) and the Canadian isolates. We found that these ten genomes formed a single taxon when compared using digital DNA-DNAhybridization, average nucleotide identity using blastn and average amino acid identity criteria but exhibited some subtle biochemical and chemotaxonomic differences. Heuristically, we propose that C. godavarianum and C. hadale are later heterotypic synonyms of, and the Canadian isolates are identifiable as, C. gottingense. We provide an emended description of Corynebacterium gottingense Atasayar et al. 2017; genomes ranged from 2.48 to 2.69 Mb (C. gottingense DSM 103494T, 2.62 Mb) with G+C content of 65.1-65.6 mol% (WGS), recovered from clinical and environmental sites.

Emendation of the Genus Auritidibacter Yassin et al. 2011 and Auritidibacter ignavus Yassin et al. 2011 based on features observed from Canadian and Swiss clinical isolates and whole-genome sequencing analysis.

Auritidibacter ignavus is a Gram-stain-positive bacillus derived from otorrhea. Four strains derived from ear discharges in Canada and Switzerland, with features consistent with but distinguishable from Auritidibacter ignavus IMMIB L-1656T (accession number FN554542) by 16S rRNA gene sequencing (97.5 % similarity), were thought to represent a novel species of the genus Auritidibacter. Auritidibacter ignavus DSM 45359T (=IMMIB L-1656T) was acquired to compare with Canadian and Swiss strains by whole-genome sequencing (WGS). Unexpectedly, those isolates were observed to be consistent with A. ignavus DSM 45359T by WGS (ANIb scores >98 %), MALDI-TOF (Bruker), cellular fatty acid analysis and biochemically (some differences were observed). A nearly full 16S rRNA gene sequence could not be readily prepared from A. ignavus DSM 45359T, even after multiple attempts. A 16S rRNA gene chimeric consensus sequence created from the genome assembly of A. ignavus DSM 45359T had only 97.5 % similarity to that of A. ignavus IMMIB L-1656T, implying that 16S rRNA sequence accession number FN554542 could not be replicated. We concluded that our isolates of members of the genus Auritidibacter were consistent with A. ignavus DSM 45359T, did not represent a novel species, and that the sequence corresponding to FN554542 was not reproducible. By WGS, A. ignavus DSM 45359T had genome of 2.53×106 bp with a DNA G+C content of 59.34%, while genomes of Canadian and Swiss isolates ranged from 2.47 to 2.59×106 bp with DNA G+C contents of 59.3-59.52 %. A. ignavus NML 100628 (=NCTC 14178=LMG 30897) did not demonstrate a rodcoccus cycle. Emendation of Auritidibacter ignavus was proposed based on these results.

Cutaneous diphtheria in the urban poor population of Vancouver, British Columbia, Canada: a 10-year review.

Between 1998 and 2007, records from 33 patients with cutaneous diphtheria from Vancouver's inner city were reviewed. Cases were associated with injection drug use and poverty. Coinfections with Staphylococcus aureus, Streptococcus pyogenes, and Arcanobacterium haemolyticum occurred. Corynebacterium diphtheriae is endemic in Vancouver's urban core, with strains of multilocus sequence type (MLST) 76 predominating.

Legionella pneumophila monoclonal antibody subgroups and DNA sequence types isolated in Canada between 1981 and 2009: Laboratory Component of National Surveillance.

Legionella pneumophila (Lp) is a significant cause of nosocomial, community-acquired, and travel-associated pneumonia in industrialized regions. Legionellosis has been a nationally notifiable disease in Canada since 1986, with an average of 75 cases reported annually; however, only the most severe, and often fatal, cases are reported or investigated. Here, epidemiological relationships, types, and distribution of Lp referrals to the Canadian national reference center were studied. Lp strains from different years, sources, and geographic locations were subtyped using a sequence-based typing (SBT) scheme and by the 'Joly' and/or 'Dresden' monoclonal antibody panels. Included were 128 epidemiologically unrelated clinical and 86 unrelated environmental strains. Sixty-four (index of diversity [IOD] = 0.964) and 45 (IOD = 0.888) sequence types (STs) were observed among clinical and environmental sources, respectively. Serogroup (sg) 1 was represented by 60.2% (77/128) and 52.3% (45/86) of clinical and environmental strains, respectively, and 63.6% (49/77) and 15.6% (7/45) of those were mAb2-positive, respectively. Serogroup 1, ST1 accounted for 14.1% (18/128) and 30.2% (26/86) of unrelated clinical and environmental isolates, respectively. This database will serve as a basis for Canadian epidemiological surveillance efforts and is linked to global surveillance initiatives curated by the European Working Group for Legionella Infections (EWGLI) network.

Increase in detection of Corynebacterium diphtheriae in Canada: 2006-2019.

BACKGROUND: Increasingly, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) has been used to provide rapid, inexpensive and precise identification of bacteria, including Corynebacterium species. Only three Corynebacterium species are able to produce diphtheria toxin (DT), and strains recovered may be either toxin-producing or non-toxin-producing. It appears the more precise bacterial identification provided by MALDI-TOF systems has led to an increase in requests submitted to the National Microbiology Laboratory (NML) for toxin testing. OBJECTIVE: To describe the number of isolates identified as C. diphtheriae, C. ulcerans and C. pseudotuberculosis, submitted to the NML between January 2006 and July 30, 2019, including their geographic area, source, and whether they produce DT. METHODS: Referrals to the NML of human or animal isolates that were identified as any of those three Corynebacterium species were studied with respect to province, source and toxigenicity. Species identification was confirmed and then specimens were tested by polymerase chain reaction for the presence of tox genes and, if positive, for expression of DT by the modified Elek method. Analysis was descriptive. RESULTS: Over the study period, 639 isolates were identified as C. diphtheriae, 22 isolates as C. ulcerans; no isolates were identified as C. pseudotuberculosis. There was an increase in C. diphtheriae referrals for DT testing: from eight per year in 2006 to an average of 15 per month in 2019, or a 1,200% increase over the 13.6-year period. The referrals were primarily from western Canada (n=609/639; 95%). Most (638/639, 99%) were human isolates and most were obtained from cutaneous sites. Of those isolates, 87/639 (13.6%) were found to be toxigenic and 552/639 (86.4%) non-toxigenic. Among C. ulcerans referrals, 17/22 (77%) were from humans and five (23%) were from animals, with 10/22 (45%) being toxigenic. CONCLUSION: There has been a marked increase in referrals to the NML for DT testing of Corynebacterium species. This could be due to the enhanced ability to identify these bacteria using MALDI-TOF systems. Ongoing monitoring will help to assess whether the increase is due solely to increased precision of diagnosis or whether these are emerging cutaneous pathogens.

Complete Genome Assembly of an Auritidibacter ignavus Isolate Obtained from an Ear Infection in Switzerland and a Comparison to Global Isolates.

We present the whole-genome sequence of an isolate of Auritidibacter ignavus, associated with ear infections. This complete assembly was compared to genomes of four global isolates, which revealed a high diversity within the species.

West Nile virus activity in the United States, 2001.

West Nile virus (WNV) first appeared in the naive environment of the Western Hemisphere in 1999 in New York. Genetic analysis determined that the virus was introduced into the United States from the Mediterranean Basin. This review discusses the spread of the virus in 2001 from the initial focus in Queens, New York, to widespread activity in the eastern and midwestern United States. It concentrates on viral ecology, epizootiology, pathology, prediction, and prevention. Research questions to further our understanding of the transmission cycle of WNV are discussed, including host-preference studies, molecular confirmation of implicated mosquito vectors, and survival of WNV in the temperate environment of the United States. Comparisons are drawn with two other arboviruses enzootic in the United States, eastern equine encephalitis, and St. Louis encephalitis viruses. Although not recently introduced, these two viruses also demonstrated increased activity in the United States in 2001.

A complex neutralization domain of bluetongue virus serotype 17 defines a virulence-associated marker.

A panel of seven monoclonal antibodies (MAb) was used to characterize a virulence-associated marker on bluetongue virus serotype 17 (BLU-17). These MAbs poorly neutralize virulent BLU-17 isolates, but effectively neutralize avirulent isolates (2). The MAbs immunoprecipitated VP2, an outer capsid protein, of both virulent and avirulent BLU-17 isolates despite their failure to neutralize the virulent isolates. The molecular mass (M(r)) of VP2 was calculated from the mobility in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The M(r) of VP2 was estimated as 100,000 Da for the virulent isolates and 97,500 Da for the avirulent isolates. The seven MAbs were tested in a competitive enzyme-linked immunosorbent assay (ELISA) and found to bind at least three overlapping epitopes. In addition, neutralization-resistant variants were selected for five different MAbs. The Variants were tested in virus neutralization assays against the panel of seven MAbs, and three major neutralization patterns were observed, again suggesting at least three distinct epitopes. Minor differences within each neutralization pattern were also observed. The results from the binding and neutralization studies suggested that the seven MAbs define a complex neutralization domain on VP2, comprising at least three overlapping epitopes.

West Nile virus infection in birds and mammals.

West Nile virus (WNV) was found throughout New York State in year 2000. The epicenter was located in New York City with a high level of activity in the immediately surrounding counties, including Rockland, Westchester, Nassau, and Suffolk. During 2000, WNV testing was performed by the Wadsworth Center on 3,687 dead birds, representing 153 species, 46 families, and 18 orders. There were 1,203 WNV-positive birds, representing 63 species, 30 families and 14 orders. The percentage of WNV-positive birds was 33% for all birds tested throughout the state, with no significant difference in infection rates in migratory versus resident birds, although significantly more resident birds were submitted for testing. The highest apparent mortality for the entire season was observed in American crows in Staten Island, a location that also showed the highest minimal infection rate in Culex pipiens complex mosquitoes. Studies examining tissue tropism of WNV in corvids and noncorvids from the epicenter and from remote locations indicated that the kidney was the most consistently infected tissue in birds, regardless of level of infection. The brain was the next most consistently positive tissue. The differences in infection among the tissues were most apparent when low levels of virus were present. Experimental mouse inoculation demonstrated a classical flavivirus infection pattern.

Clostridium lavalense sp. nov., a glycopeptide-resistant species isolated from human faeces.

Two vancomycin-resistant, strictly anaerobic, Gram-positive, rod-shaped, spore-forming organisms (strains CCRI-9842(T) and CCRI-9929) isolated from human faecal specimens in Québec, Canada, and Australia were characterized using phenotypic, biochemical and molecular taxonomic methods. Pairwise analysis of the 16S rRNA gene sequences showed that both strains were closely related to each other genetically (displaying 99.2 % sequence similarity) and represented a previously unknown subline within the Clostridium coccoides rRNA group of organisms (rRNA cluster XIVa of the genus Clostridium). Strains CCRI-9842(T) and CCRI-9929 used carbohydrates as fermentable substrates, producing acetic acid as the major product of glucose metabolism. The novel strains were most closely related to Clostridium asparagiforme, Clostridium bolteae and Clostridium clostridioforme, but morphological, biochemical and phylogenetic studies demonstrated that they represent a previously unidentified species of the genus Clostridium. This was confirmed by the unique cellular fatty acid composition of strains CCRI-9842(T) and CCRI-9929. Therefore, on the basis of data from the polyphasic taxonomic analysis, it is proposed that strains CCRI-9842(T) and CCRI-9929 represent a novel species of the genus Clostridium, for which the name Clostridium lavalense sp. nov. is proposed. The type strain is CCRI-9842(T) (=CCUG 54291(T)=JCM 14986(T)=NML 03-A-015(T)).

Ruminococcus gauvreauii sp. nov., a glycopeptide-resistant species isolated from a human faecal specimen.

A novel strictly anaerobic, vancomycin-resistant, Gram-positive coccus (strain CCRI-16,110(T)) was isolated from a human faecal specimen. This strain was characterized using morphological, biochemical and molecular taxonomic methods. The organism was unable to hydrolyse aesculin and failed to produce acid from cellobiose, d-lactose and alpha-raffinose. Acetic acid was the sole product of glucose fermentation by the organism. On the basis of 16S rRNA and tuf gene sequence comparison, strain CCRI-16,110(T) was most closely related to species of the genus Ruminococcus and formed a hitherto unknown sublineage within the Clostridium coccoides rRNA cluster of organisms (cluster XIVa). Based on phenotypic and phylogenetic evidence, a novel species, Ruminococcus gauvreauii sp. nov., is proposed. The type strain is CCRI-16,110(T) (=NML 060141(T) =CCUG 54,292(T) =JCM 14987(T)).

West Nile virus in the western hemisphere.

West Nile virus first appeared in the western hemisphere in 1999 in New York. Genetic analysis determined that the virus was introduced from the Mediterranean Basin. This review discusses the establishment of West Nile virus in the naïve environment of the northeastern USA, its ecology, epizootiology, pathology, prevention and prediction, as well as laboratory studies that have been conducted to elucidate the transmission cycle.

Cellular fatty acid composition as an adjunct to the identification of asporogenous, aerobic gram-positive rods.

Cellular fatty acid (CFA) compositions of 561 asporogenous, aerobic gram-positive rods were analyzed by gas-liquid chromatography as an adjunct to their identification when grown on blood agar at 35 degrees C. The organisms could be divided into two groups. In the first group (branched-chain type), which included coryneform CDC groups A-3, A-4, and A-5; some strains of B-1 and B-3; "Corynebacterium aquaticum"; Brevibacterium liquefaciens; Rothia dentocariosa; and Listeria spp., the rods had sizable quantities of antiesopentadecanoic (Ca15:0) and anteisoheptadecanoic (Ca17:0) acids. Other species with these types of CFA included B. acetylicum, which contained large amounts of isotridecanoic (Ci13:0) and anteisotridecanoic (Ca13:0) acids. CFAs useful for distinguishing among Jonesia denitrificans, Oerskovia spp., some strains of CDC groups B-1 and B-3, Kurthia spp., and Propionibacterium avidum were hexadecanoic (C 16:0) acid, isopentadecanoic (Ci15:0) acid, and Ca15:0). The second group (straight-chained type), which included Actinomyces pyogenes; Arcanobacterium haemolyticum; C. bovis; C. cystitidis; C. diphtheriae; C. flavescens, "C. gentalium"; C. jeikeium; C. kutscheri; C. matruchotii; C .minutissimum; C. mycetoides; C. pilosum; C. pseudodiphtheriticum; "C. pseudogenitalium"; C. pseudotuberculosis; C. renale; CDC groups 1, 2, ANF-1, D-2, E, F-1, F-2, G-1, G-2, and I-2; C. striatum; "C. tuberculostearicum"; C. ulcerans; C. vitarumen; C. xerosis; and Erysipelothrix rhusiopathiae, was typified by significant quantities of hexadecanoic (C16:0) and oleic acids (C18:cis9), with differences in the amounts of linoleic acid (C18:2), stearic acid (C18:0), an unnamed peak (equivalent chain length, 14.966), and small quantities of other known saturated and unsaturated fatty acids. CFA composition of these organisms was sufficiently discriminatory to assist in classification but could not be used as the sole means of identification.

Mutations in the E2 glycoprotein of Venezuelan equine encephalitis virus confer heparan sulfate interaction, low morbidity, and rapid clearance from blood of mice.

The arbovirus, Venezuelan equine encephalitis virus (VEE), causes disease in humans and equines during periodic outbreaks. A murine model, which closely mimics the encephalitic form of the disease, was used to study mechanisms of attenuation. Molecularly cloned VEE viruses were used: a virulent, epizootic, parental virus and eight site-specific glycoprotein mutants derived from the parental virus. Four of these mutants were selected in vitro for rapid binding and penetration, resulting in positive charge changes in the E2 glycoprotein from glutamic acid or threonine to lysine (N. L. Davis, N. Powell, G. F. Greenwald, L. V. Willis, B. J. Johnson, J. F. Smith, and R. E. Johnston, Virology 183, 20-31, 1991). Tissue culture adaptation also selected for the ability to bind heparan sulfate as evidenced by inhibition of plaque formation by heparin, decreased infectivity for CHO cells deficient for heparan sulfate, and tight binding to heparin-agarose beads. In contrast, the parental virus and three other mutants did not use heparan sulfate as a receptor. All eight mutants were partially or completely attenuated with respect to mortality in adult mice after a subcutaneous inoculation, and the five mutants that interacted with heparan sulfate in vitro had low morbidity (0-50%). These same five mutants were cleared rapidly from the blood after an intravenous inoculation. In contrast, the parental virus and the other three mutants were cleared very slowly. In summary, the five VEE viruses that contain tissue-culture-selected mutations interacted with cell surface heparan sulfate, and this interaction correlated with low morbidity and rapid clearance from the blood. We propose that one mechanism of attenuation is rapid viral clearance in vivo due to binding of the virus to ubiquitous heparan sulfate.

Characteristics of rare or recently described corynebacterium species recovered from human clinical material in Canada.

Nineteen new Corynebacterium species or taxa described since 1995 have been associated with human disease. We report the characteristics of 72 strains identified as or most closely resembling 14 of these newer, medically relevant Corynebacterium species or taxa, as well as describe in brief an isolate of Corynebacterium bovis, a rare pathogen for humans. The bacteria studied in this report were nearly all derived from human clinical specimens and were identified by a polyphasic approach. Most were characterized by nearly full 16S rRNA gene sequence analysis. Some isolates were recovered from previously unreported sources and exhibited unusual phenotypes or represented the first isolates found outside Europe. Products of fermentation, with emphasis on the presence or absence of propionic acid, were also studied in order to provide an additional characteristic with which to differentiate among phenotypically similar species.

Clinical microbiology of coryneform bacteria.

Coryneform bacteria are aerobically growing, asporogenous, non-partially-acid-fast, gram-positive rods of irregular morphology. Within the last few years, there has been a massive increase in the number of publications related to all aspects of their clinical microbiology. Clinical microbiologists are often confronted with making identifications within this heterogeneous group as well as with considerations of the clinical significance of such isolates. This review provides comprehensive information on the identification of coryneform bacteria and outlines recent changes in taxonomy. The following genera are covered: Corynebacterium, Turicella, Arthrobacter, Brevibacterium, Dermabacter. Propionibacterium, Rothia, Exiguobacterium, Oerskovia, Cellulomonas, Sanguibacter, Microbacterium, Aureobacterium, "Corynebacterium aquaticum," Arcanobacterium, and Actinomyces. Case reports claiming disease associations of coryneform bacteria are critically reviewed. Minimal microbiological requirements for publications on disease associations of coryneform bacteria are proposed.

Most Corynebacterium xerosis strains identified in the routine clinical laboratory correspond to Corynebacterium amycolatum.

A comprehensive study was performed on 25 bacterial clinical isolates originally identified as Corynebacterium xerosis. Three reference strains of C. xerosis were also included in the study. On the basis of a variety of phenotypic characteristics tested, all strains could be divided into two separate clusters: reference strains ATCC 373 (the type strain of C. xerosis) and ATCC 7711 showed yellow-pigmented, dry, rough colonies, fermented 5-keto-gluconate, exhibited strong leucine arylamidase and alpha-glucosidase activities, produced lactate as the major end product of glucose metabolism, were susceptible to most of the 19 antimicrobial agents tested, and showed an inhibition zone around disks containing the vibriocidal compound O/129. In contrast, the remaining 26 strains including reference strain NCTC 7243 as well as all clinical isolates formed white-grayish, dry, slightly rough colonies, did not ferment 5-keto-gluconate, exhibited only weak leucine arylamidase and no alpha-glucosidase activity, produced large amounts of propionic acid as the end product of glucose metabolism, and were resistant to most antimicrobial agents tested, including O/129. Chemotaxonomic (cellular fatty acids, mycolic acids, and G+C content) and molecular genetic (16S rRNA gene sequence) investigations revealed that the strains of the second cluster unambiguously belonged to the species C. amycolatum. Our data suggest that most strains reported in the literature as C. xerosis are probably misidentified and correspond to C. amycolatum.

Sequence and cognitive analyses of two virulence-associated markers of bluetongue virus serotype 17.

Genome segments 2 and 3 were completely sequenced for one virulent and one avirulent bluetongue serotype 17 (BLU-17). These two segments were previously shown to exhibit virulence-associated markers. The marker on segment 2 was characterized as a change in the neutralization domain on its protein product, VP2. The nucleotide sequences for segments 2 were 94.5% identical, and their predicted proteins differed by 34 amino acids or 3.7%. Three clusters of variability were identified which may be involved with viral neutralization. These variable regions were compared to mutations for published monoclonal antibody-resistant variants of BLU. The marker on segment 3 was characterized as a mobility shift in polyacrylamide gel electrophoresis (PAGE). The nucleotide sequences were 95.0% identical, and their predicted proteins differed by four amino acids or 0.4%. These amino acid changes were relatively conserved; therefore, they are not likely responsible for virulence. The segment 3 sequences were compared to published sequences, and evidence was found to suggest that the virulent isolate had naturally reassorted between a BLU-17 and BLU-10 isolate.