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.

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.

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.

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)).

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.

West Nile virus infection in birds and mosquitoes, New York State, 2000.

West Nile (WN) virus was found throughout New York State in 2000, with the epicenter in New York City and surrounding counties. We tested 3,403 dead birds and 9,954 mosquito pools for WN virus during the transmission season. Sixty-three avian species, representing 30 families and 14 orders, tested positive for WN virus. The highest proportion of dead birds that tested positive for WN virus was in American Crows in the epicenter (67% positive, n=907). Eight mosquito species, representing four genera, were positive for WN virus. The minimum infection rate per 1,000 mosquitoes (MIR) was highest for Culex pipiens in the epicenter: 3.53 for the entire season and 7.49 for the peak week of August 13. Staten Island had the highest MIR (11.42 for Cx. pipiens), which was associated with the highest proportion of dead American Crows that tested positive for WN virus (92%, n=48) and the highest number of human cases (n=10).

High-throughput detection of West Nile virus RNA.

The recent outbreaks of West Nile virus (WNV) in the northeastern United States and other regions of the world have made it essential to develop an efficient protocol for surveillance of WNV. In the present report, we describe a high-throughput procedure that combines automated RNA extraction, amplification, and detection of WNV RNA. The procedure analyzed 96 samples in approximately 4.5 h. A robotic system, the ABI Prism 6700 Automated Nucleic Acid workstation, extracted RNA and set up reactions for real-time reverse transcription (RT)-PCR in a 96-well format. The robot extracted RNA with a recovery as efficient as that of a commercial RNA extraction kit. A real-time RT-PCR assay was used to detect and quantitate WNV RNA. Using in vitro transcribed RNA, we estimated the detection limit of the real-time RT-PCR to be approximately 40 copies of RNA. A standard RT-PCR assay was optimized to a sensitivity similar to that of the real-time RT-PCR. The standard assay can be reliably used to test a small number of samples or to confirm previous test results. Using internal primers in a nested RT-PCR, we increased the sensitivity by approximately 10-fold compared to that of the standard RT-PCR. The results of the study demonstrated for the first time that the use of an automated system for the purpose of large-scale viral RNA surveillance dramatically increased the speed and efficiency of sample throughput for diagnosis.

Diversity within reference strains of Corynebacterium matruchotii includes Corynebacterium durum and a novel organism.

Corynebacterium matruchotii has been the subject of numerous dental pathogenesis studies. The purpose of the present study was to resolve concerns about diversity within the reference strains of C. matruchotii through analysis of seven strains procured from the American Type Culture Collection (ATCC). Analysis of whole-cell fatty acid profiles with the library generation software of Microbial ID Inc. revealed that three types of organisms have been deposited in the ATCC as C. matruchotii. These three groups of organisms were also distinguishable by DNA-DNA dot blot hybridization, by sequences of two hypervariable regions of the 16S rRNA gene, and by the pyrrolidonyl arylamidase test. These studies indicate that two C. matruchotii reference strains, ATCC 33449 and ATCC 33822, are members of the recently proposed species, Corynebacterium durum. The colonial morphology and biochemical reactions of the C. durum strains are more diverse than originally reported. Strain ATCC 43833 is unique and represents a novel species. In addition to the type strain, ATCC 14266, true members of the species C. matruchotii include ATCC strains 14265, 33806, and 43832 plus two reference strains, L2 and Richardson 13, which comprise the vast majority of strains used in dental pathogenesis research with this species.

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.

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.

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.

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.

Differentiation of clinical Helicobacter pullorum isolates from related Helicobacter and Campylobacter species.

BACKGROUND: Helicobacter pullorum, first detected in the liver and intestinal contents of poultry, was defined as a new species in 1994. This organism has since been isolated from humans with gastroenteritis. Phenotypic as well as genotypic methods have been used to identify H. pullorum associated with cases of human disease. MATERIALS AND METHODS: Clinical isolates were submitted for identification to the National Laboratory for Enteric Pathogens by Provincial Public Health Laboratories within Canada. Phenotypic characterization was conducted using a variety of growth and biochemical tests including oxidase, catalase, indoxyl acetate, H2S production in triple sugar iron (TSI) agar, antimicrobial susceptibility testing, and fatty acid analysis. Genotypic identification was performed using a polymerase chain reaction-restriction fragment-length polymorphism (PCR-RFLP) analysis of a 1-kb fragment of the Helicobacter 16S rRNA gene. RESULTS: During the last 7 years (1993-1999) a total of 11 isolates of H. pullorum were detected from patients with gastroenteritis for inclusion in this study. Typically, these isolates were oxidase and catalase positive, produced optimal growth at 42 degrees C, and produced H2S in TSI. Of these 11 isolates, 1 showed DNase activity, while another did not produce H2S in TSI, and only 2 showed tolerance to 1% bile. Antimicrobial susceptibility assays indicated that 6 of the 11 strains were resistant to nalidixic acid. The fatty acid profiles of the isolates were similar to each other and provided a distinguishing profile from the other related species. Genetically identical and distinct species-specific restriction fragment-length polymorphism (RFLP) patterns were produced using the restriction enzymes Bsr I and Dde I. CONCLUSION: Phenotypic and genotypic procedures were used to identify H. pullorum. Interspecies phenotypic variability was apparent and supported the use of a polyphasic approach for identification. Similarities to the more prominent human pathogens Campylobacter coli and C. lari were also noted. The use of a combination of phenotypic and, in particular, genotypic markers for H. pullorum should prove valuable both for epidemiological investigations and for the diagnosis of disease related to this emerging human pathogen.

Infection of neonatal mice with sindbis virus results in a systemic inflammatory response syndrome.

Laboratory strains of viruses may contain cell culture-adaptive mutations which result in significant quantitative and qualitative alterations in pathogenesis compared to natural virus isolates. This report suggests that this is the case with Sindbis virus strain AR339. A cDNA clone comprising a consensus sequence of Sindbis virus strain AR339 has been constructed (W. B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). This clone (pTR339) regenerates a sequence predicted to be very close to that of the original AR339 isolate by eliminating several cell culture-adaptive mutations present in individual laboratory strains of the virus (K. L. McKnight et al., J. Virol. 70:1981-1989, 1996). It thus provides a unique reagent for study of the pathogenesis of Sindbis virus strain AR339 in mice. Neonatal mouse pathogenesis of virus (TR339) generated from the pTR339 clone was compared with that of virus from a cDNA clone of the cell culture-passaged laboratory AR339 strain, TRSB, and virus from a clone of a more highly cell culture-adapted strain, HR(sp) (Toto 50). The sequence of TRSB differs from the consensus at three coding positions, while Toto 50 differs at eight codons and one nucleotide in the 5' nontranslated region. Both cell culture-adapted strains contain mutations associated with heparan sulfate (HS)-dependent attachment to cells (W.B. Klimstra, K. D. Ryman, and R. E. Johnston, J. Virol. 72:7357-7366, 1998). TR339 caused 100% mortality with an average survival time (AST) of 1.7 +/- 0.25 days. While TRSB also caused 100% mortality, the AST was extended to 2.9 +/- 0.52 days. The more extensively cell culture-adapted virus Toto 50 caused only 30% mortality with an AST extended to 11.0 +/- 4.8 days. TRSB and TR339 induced high serum levels of alpha/beta interferon, gamma interferon, tumor necrosis factor alpha, interleukin-6, and corticosterone and induced pathology reminiscent of lipopolysaccharide-induced endotoxic shock, a type of systemic inflammatory response syndrome. However, the reduced intensity of this response in TRSB-infected mice correlated with the increased AST. Toto 50 failed to induce the shock-like cytokine cascade. In situ hybridization studies indicated that TR339 and TRSB replicated in identical tissues, but the TRSB signal was less widespread at early times postinfection. While Toto 50 also replicated in similar tissues, the extent of replication was severely restricted and mice developed lesions characteristic of encephalitis. A single mutation in TRSB at E2 position 1 (Arg) conferred HS-dependent attachment to cells and was associated with reduced cytokine induction and extended AST in vivo.

Corynebacterium sundsvallense sp. nov., from human clinical specimens.

Three strains of a previously undescribed catalase-positive non-lipophilic coryneform bacterium isolated from human clinical specimens were characterized by phenotypic and molecular taxonomic methods. Morphologically the unknown bacterium consisted of pleomorphic rods, some of which displayed bulges/knobs at their ends. All three strains were similar in that they produced acid from fructose, glucose, maltose and sucrose and were urease-positive. Chemotaxonomic investigations revealed the presence of meso-diaminopimelic acid and short-chain mycolic acids consistent with the genus Corynebacterium sensu stricto. Comparative 16S rRNA gene sequencing showed that the three strains are genealogically highly related and constitute a new subline within the genus Corynebacterium, displaying > 3% sequence divergence with recognized species. The unknown bacterium was distinguished from currently validly published Corynebacterium species by phenotypic tests, including electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from clinical specimens be classified as Corynebacterium sundsvallense sp. nov. The type strain is CCUG 36622T.