m Rapid development within the field of massive parallel sequencing (MPS) is about to bring this technology within reach for diagnostic microbiology laboratories. We wanted to explore its potential for improving diagnosis and understanding of polymicrobial infections, using bacterial brain abscesses as an example. We conducted a prospective nationwide study on bacterial brain abscesses. Fifty-two surgical samples were included over a 2-year period. The samples were categorized as either spontaneous intracerebral, spontaneous subdural, or postoperative. Bacterial 16S rRNA genes were amplified directly from the specimens and sequenced using Ion Torrent technology, with an average of 500,000 reads per sample. The results were compared to those from culture-and Sanger sequencing-based diagnostics. Compared to culture, MPS allowed for triple the number of bacterial identifications. Aggregatibacter aphrophilus, Fusobacterium nucleatum, and Streptococcus intermedius or combinations of them were found in all spontaneous polymicrobial abscesses. F. nucleatum was systematically detected in samples with anaerobic flora. The increased detection rate for Actinomyces spp. and facultative Gram-negative rods further revealed several species associations. We suggest that A. aphrophilus, F. nucleatum, and S. intermedius are key pathogens for the establishment of spontaneous polymicrobial brain abscesses. In addition, F. nucleatum seems to be important for the development of anaerobic flora. MPS can accurately describe polymicrobial specimens when a sufficient number of reads is used to compensate for unequal species concentrations and principles are defined to discard contaminant bacterial DNA in the subsequent data analysis. This will contribute to our understanding of how different types of polymicrobial infections develop. O ur understanding of polymicrobial infections has been hindered by our limited possibilities for describing them. Recent investigations of bacterial brain abscesses using universal amplification of the bacterial 16S rRNA gene, followed by Sanger sequencing of cloned amplicons, have revealed that only a fraction of the bacteria present are identified by culture (1, 2). Nevertheless, this approach has limitations when it comes to detecting smaller subpopulations in a multispecies community, unless very high numbers of clones are sequenced (3). This is problematic, since the species structure of an abscess may change over time and pathogens important for establishing the infection potentially remain at only low concentrations in the more mature abscesses. Furthermore, the species that are important for maintaining and expanding the abscess might primarily exist close to the abscess wall and do not necessarily dominate in the pus obtained by aspiration. Rapid development within the field of massive parallel sequencing technologies (MPS) is about to provide the diagnostic laboratories with tools that can characterize even the most complex microbial communities. The aim of the present study was to use recent adva...

Objectives To use the nationwide Norwegian surveillance programme on resistant microbes in humans (NORM) to address longitudinal changes in the population structure of Klebsiella pneumoniae isolates from 2001–15, focusing on the emergence and dissemination of ESBL-producing K. pneumoniae in Norway. Methods Among blood (n = 6124) and urinary tract (n = 5496) surveillance isolates from 2001–15, we used Illumina technology to whole genome sequence 201 ESBL-producing isolates from blood (n = 130) and urine (n = 71), and 667 non-ESBL isolates from blood. Complete genomes for four isolates were resolved with Oxford Nanopore sequencing. Results In a highly diverse collection, Klebsiella variicola ssp. variicola caused 24.5% of Klebsiella pneumoniae species complex (KpSC) bacteraemias. ESBL production was limited to K. pneumoniae sensu stricto (98.5%). A diverse ESBL population of 57 clonal groups (CGs) were dominated by MDR CG307 (17%), CG15 (12%), CG70 (6%), CG258 (5%) and CG45 (5%) carrying blaCTX-M-15. Yersiniabactin was significantly more common in ESBL-positive (37.8%) compared with non-ESBL K. pneumoniae sensu stricto isolates (12.7%), indicating convergence of virulence and resistance determinants. Moreover, we found a significantly lower prevalence of yersiniabactin (3.0%, 37.8% and 17.3%), IncFIB (58.7%, 87.9% and 79.4%) and IncFII plasmid replicons (40.5%, 82.8% and 54.2%) in K. variicola ssp. variicola compared with ESBL- and non-ESBL K. pneumoniae sensu stricto isolates, respectively. Conclusions The increase in Norwegian ESBL-producing KpSC during 2010–15 was driven by CG307 and CG15 carrying blaCTX-M-15. K. variicola ssp. variicola was a frequent cause of invasive KpSC infection, but rarely carried ESBLs.

BackgroundBloodstream infections represent serious conditions carrying a high mortality and morbidity rate. Rapid identification of microorganisms and prompt institution of adequate antimicrobial therapy is of utmost importance for a successful outcome. Aiming at the development of a rapid, simplified and efficient protocol, we developed and compared two in-house preparatory methods for the direct identification of bacteria from positive blood culture flasks (BD BACTEC FX system) by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS).Both methods employed saponin and distilled water for erythrocyte lysis. In method A the cellular pellet was overlaid with formic acid on the MALDI TOF target plate for protein extraction, whereas in method B the pellet was exposed to formic acid followed by acetonitrile prior to placing on the target plate.ResultsBest results were obtained by method A. Direct identification was achieved for 81.9 % and 65.8 % (50.3 % and 26.2 % with scores >2.0) of organisms by method A and method B, respectively. Overall concordance with final identification was 100 % to genus and 97.9 % to species level. By applying a lower cut-off score value, the levels of identification obtained by method A and method B increased to 89.3 % and 77.8 % of organisms (81.9 % and 65.8 % identified with scores >1.7), respectively. Using the lowered score criteria, concordance with final results was obtained for 99.3 % of genus and 96.6 % of species identifications.ConclusionThe reliability of results, rapid performance (approximately 25 min) and applicability of in-house method A have contributed to implementation of this robust and cost-effective method in our laboratory.

Introduction. Urinary tract infections are common bacterial infections worldwide. Urine culture is the gold standard method to identify and quantify the presence or absence of bacteria in urine. Flow cytometry, which can differentiate and quantify multiple particles (including bacteria) in the urine, presents an alternative method for rapid screening to rule out bacteriuria. Hypothesis. Adding flow cytometry to identify urine samples without bacteriuria could substantially reduce the number of urine samples that need to be cultured as well as the response time for negative results. However, the level of instrument rinsing between samples could affect sample-to-sample carryover rate, a concept given little attention in previous studies. Aim. We aimed to evaluate urine flow cytometry as a rapid screening method to identify urine samples without significant bacterial growth, including analyses of cross-contamination and sample-to-sample carryover rate. Methodology. We analysed 3919 urine samples by quantitative urine culture and flow cytometry screening (Sysmex UF-5000). Receiver operator characteristic (ROC) curve analyses were used to test method agreement to identify: (a) positive vs. negative culture and (b) mixed vs. pure culture. In addition, we performed carryover and cross-contamination studies. Results. ROC curve analyses identified bacterial count (BACT ml−1) and leucocyte count (WBC µl−1) as possible predictors of bacterial growth in the total material and subpopulations, except pregnant women (n=451). This subgroup was excluded from further analyses, leaving a final 3468 urine samples. Area under the ROC curve was 0.94 (95 % CI 0.93–0.95) and 0.81 (95 % CI 0.79–0.82) for bacterial and leucocyte count, respectively. A bacterial count cut-off of 30 BACT ml−1 resulted in 95.2 % sensitivity and 91.2 % negative predictive value, resulting in approximately 30 % of urine samples that could be reported as negative without culture. Use of high-level rinse modes was necessary to ensure carryover rates <0.05 %. Conclusion. Flow cytometry is a suitable and rapid method to rule out urine samples without significant bacterial growth. Rinses between samples should be adjusted, depending on the cut-off used, to prevent sample-to-sample carryover, whereas cross-contamination can be eliminated by the use of separate urine aliquots for flow cytometry analysis and urine culturing respectively.