invasive meningococcal disease (iMD) due to serogroup Y Neisseria meningitidis emerged in europe during the 2000s. Draft genomes of serogroup Y isolates in Sweden revealed that although the population structure of these isolates was similar to other serogroup Y isolates internationally, a distinct strain (YI) and more specifically a sublineage (1) of this strain was responsible for the increase of serogroup Y iMD in Sweden. We performed single molecule real-time (SMRt) sequencing on eight serogroup Y isolates from different sublineages to unravel the genetic and epigenetic factors delineating them, in order to understand the serogroup Y emergence. extensive comparisons between the serogroup Y sublineages of all coding sequences, complex genomic regions, intergenic regions, and methylation motifs revealed small point mutations in genes mainly encoding hypothetical and metabolic proteins, and non-synonymous variants in genes involved in adhesion, iron acquisition, and endotoxin production. the methylation motif cAcnnnnntAc was only found in isolates of sublineage 2. Only seven genes were putatively differentially expressed, and another two genes encoding hypothetical proteins were only present in sublineage 2. These data suggest that the serogroup Y IMD increase in Sweden was most probably due to small changes in genes important for colonization and transmission. The Gram-negative encapsulated bacterium Neisseria meningitidis is a common commensal found exclusively in the human nasopharyngeal mucosa. It is the leading cause of epidemic meningitis and sepsis 1. Invasive meningococcal disease (IMD) is mainly caused by meningococci expressing specific capsular groups (i.e. serogroups) and belonging to particular hyperinvasive lineages 2,3 , which have a changing global distribution over time. An increase in IMD due to serogroup Y occurred in the United States in the 1990s, and from the end of the 2000s this was also the case in Europe 4,5. This serogroup was the most prevalent cause of IMD in Sweden between 2010 and 2015, representing 53% of all IMD in 2015 6. Characterization by multilocus sequence typing (MLST) and sequencing of the antigens FetA, FHbp, PenA, PorA, and PorB, revealed that three serogroup Y strain types were responsible for IMD in Sweden, in particular those with the genotype Y: P1.5-2, 10-1: F4-1: ST-23 clonal complex 23 (cc23) along with PorB allele 3-36, FHbp allele 25, and PenA allele 22, referred to as strain YI 7. Illumina whole genome sequencing (WGS) of 185 serogroup Y genomes from Sweden showed that the majority of those causing IMD clustered with strain YI, belonging to the WGS lineage 23.1 8. Analysis of genes core to the meningococcus (cgMLST) revealed that this cluster, although antigenically identical, contained an average of 100 core loci with allelic differences, delineating it into sublineages 1 and 2 8. Analysis on a limited selection of 177 loci hypothesized to play a role in meningococcal virulence showed that 10 of these loci differed between the two sublineages. Because 213 core...
Background: Urine is amongst the most frequent specimens submitted to clinical microbiology laboratories and many are negative for uropathogens. Same day reporting on urines negative for bacteria and WBC could impact inappropriate antimicrobial use. As well, same day reporting on the presence of bacteria/WBC may influence decisions regarding empiric antimicrobial therapy. We evaluated the UF1000 flow cytometry on urines submitted for culture and susceptibility testing. Methods: Urines submitted to Clinical Microbiology Royal University Hospital, Saskatoon, SK, Canada were used. Raw and boric acid preserved urines were included in the evaluation from inpatients and outpatients. Approximately 1 ml of urine was placed in a separate tube for flow cytometry performed twice on each specimen. All urines were inoculated to tryptic soy agar containing 5% sheep red blood cells and MacConkey agar and incubated in O 2 for 18-24 hours. Culture results were correlated with bacterial/ WBC counts from flow cytometry readings. Results: A total of 827 urines were analyzed (393 preserved, 434 raw). For raw urines, 142 (32.7%) had ≤10 bacterial cells/ul, 4 (0.9%) had ≥25 WBC/ul by FC; 239 (55.1%) had ≤50 bacterial cells/ul, 18 (4%) had ≥25 WBC/ul. Of the 239 urines with ≤50 bacteria/µl, 200 (83.7%) were 'no growth' or 'mixed' by culture, 0 (100%) urines had >100 million bacteria/L. Similar results were seen for preserved urines: 139 (35.4%) had ≤10 bacteria/µl, 7 (1%) had ≥25 WBC/µl, 195 (49.6%) had ≤50 bacteria/µl, 17 (4.3%) had ≥25 WBC/µl. Of the 195 preserved urines with ≤50 bacteria/µl, 167 (85.6%) were 'no growth' or 'mixed' by culture, 0 (100%) urine had >100 million bacteria/L. Conclusion: Screening of urine using flow cytometry has the potential to identify up to 50% of urines that do not require culture and can be same day reported as negative. A cut off of ≤50 bacteria/µl correlated with insignificant colony counts from urine culture; a higher cut off would further reduce the number of urines for culture. Our laboratory processes approximately 80,000 urines per year and with the possibility of 50% same day reported as negative influences workload, work flow and likely positively impacts antimicrobial stewardship programs. We are currently implementing this technology.OS 1-2 Using MALDI-TOF and selected gene (16S rRNA gene, gyrB gene) sequencing to practical and accurate identify clinical important viridans group streptococci Background: Clinical significant Viridans Group Streptococci (VGS) identification is important but problematic in particular for the Streptococcus mitis group. MALDI-TOF MS is very practical for clinical identification but for some problematic species, selected gene sequencing is necessary in order to make the identification more reliable. Few studies have investigated the performance of MALDI-TOF MS system in VGS identification. Methods: Using 16S rRNA gene or gyrB gene sequencing as a gold standard, the performance of two MALDI-TOF MS instruments in the identification of 181 VGS clinical isolates was...
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