Benchmarking of two bioinformatic workflows for the analysis of whole-genome sequenced Staphylococcus aureus collected from patients with suspected sepsis.

BACKGROUND: The rapidly growing area of sequencing technologies, and more specifically bacterial whole-genome sequencing, could offer applications in clinical microbiology, including species identification of bacteria, prediction of genetic antibiotic susceptibility and virulence genes simultaneously. To accomplish the aforementioned points, the commercial cloud-based platform, 1928 platform (1928 Diagnostics, Gothenburg, Sweden) was benchmarked against an in-house developed bioinformatic pipeline as well as to reference methods in the clinical laboratory. METHODS: Whole-genome sequencing data retrieved from 264 Staphylococcus aureus isolates using the Illumina HiSeq X next-generation sequencing technology was used. The S. aureus isolates were collected during a prospective observational study of community-onset severe sepsis and septic shock in adults at Skaraborg Hospital, in the western region of Sweden. The collected isolates were characterized according to accredited laboratory methods i.e., species identification by MALDI-TOF MS analysis and phenotypic antibiotic susceptibility testing (AST) by following the EUCAST guidelines. Concordance between laboratory methods and bioinformatic tools, as well as concordance between the bioinformatic tools was assessed by calculating the percent of agreement. RESULTS: There was an overall high agreement between predicted genotypic AST and phenotypic AST results, 98.0% (989/1006, 95% CI 97.3-99.0). Nevertheless, the 1928 platform delivered predicted genotypic AST results with lower very major error rates but somewhat higher major error rates compared to the in-house pipeline. There were differences in processing times i.e., minutes versus hours, where the 1928 platform delivered the results faster. Furthermore, the bioinformatic workflows showed overall 99.4% (1267/1275, 95% CI 98.7-99.7) agreement in genetic prediction of the virulence gene characteristics and overall 97.9% (231/236, 95% CI 95.0-99.2%) agreement in predicting the sequence types (ST) of the S. aureus isolates. CONCLUSIONS: Altogether, the benchmarking disclosed that both bioinformatic workflows are able to deliver results with high accuracy aiding diagnostics of severe infections caused by S. aureus. It also illustrates the need of international agreement on quality control and metrics to facilitate standardization of analytical approaches for whole-genome sequencing based predictions.

Sequence-based genotyping of extra-intestinal pathogenic Escherichia coli isolates from patients with suspected community-onset sepsis, Sweden.

Extra-intestinal pathogenic Escherichia coli (ExPEC) strains are responsible for a large number of human infections globally. The management of infections caused by ExPEC has been complicated by the emergence of antimicrobial resistance, most importantly the increasing recognition of isolates producing extended-spectrum ß-lactamases (ESBL). Herein, we used whole-genome sequencing (WGS) on ExPEC isolates for a comprehensive genotypic characterization. Twenty-one ExPEC isolates, nine with and 12 without ESBL-production, from 16 patients with suspected sepsis were sequenced on an Illumina MiSeq platform. Analysis of WGS data was performed with widely used bioinformatics software and tools for genotypic characterization of the isolates. A higher number of plasmids, virulence and resistance genes were observed in the ESBL-producing isolates than the non-ESBL-producing, although not statistically significant due to the low sample size. All nine ESBL-producing ExPEC isolates presented with at least one bla gene, as did three of the 12 without ESBL-production. Multi-locus sequence typing analysis revealed a diversity of sequence types whereas phylogroup A prevailed among isolates both with and without ESBL-production. In conclusion, this limited study shows that analysis of WGS data can be used for genotypic characterization of ExPEC isolates to obtain in-depth information of clinical relevance.

MicroRNA amplification and detection technologies: opportunities and challenges for point of care diagnostics.

The volume of point of care (POC) testing continues to grow steadily due to the increased availability of easy-to-use devices, thus making it possible to deliver less costly care closer to the patient site in a shorter time relative to the central laboratory services. A novel class of molecules called microRNAs have recently gained attention in healthcare management for their potential as biomarkers for human diseases. The increasing interest of miRNAs in clinical practice has led to an unmet need for assays that can rapidly and accurately measure miRNAs at the POC. However, the most widely used methods for analyzing miRNAs, including Northern blot-based platforms, in situ hybridization, reverse transcription qPCR, microarray, and next-generation sequencing, are still far from being used as ideal POC diagnostic tools, due to considerable time, expertize required for sample preparation, and in terms of miniaturizations making them suitable platforms for centralized labs. In this review, we highlight various existing and upcoming technologies for miRNA amplification and detection with a particular emphasis on the POC testing industries. The review summarizes different miRNA targets and signals amplification-based assays, from conventional methods to alternative technologies, such as isothermal amplification, paper-based, oligonucleotide-templated reaction, nanobead-based, electrochemical signaling- based, and microfluidic chip-based strategies. Based on critical analysis of these technologies, the possibilities and feasibilities for further development of POC testing for miRNA diagnostics are addressed and discussed.

Clinical evaluation of commercial nucleic acid amplification tests in patients with suspected sepsis.

BACKGROUND: Sepsis is a serious medical condition requiring timely administered, appropriate antibiotic therapy. Blood culture is regarded as the gold standard for aetiological diagnosis of sepsis, but it suffers from low sensitivity and long turnaround time. Thus, nucleic acid amplification tests (NAATs) have emerged to shorten the time to identification of causative microbes. The aim of the present study was to evaluate the clinical utility in everyday practice in the emergency department of two commercial NAATs in patients suspected with sepsis. METHODS: During a six-week period, blood samples were collected consecutively from all adult patients admitted to the general emergency department for suspicion of a community-onset sepsis and treated with intravenous antibiotics. Along with conventional blood cultures, multiplex PCR (Magicplex™) was performed on whole blood specimens whereas portions from blood culture bottles were used for analysis by microarray-based assay (Prove-it™). The aetiological significance of identified organisms was determined by two infectious disease physicians based on clinical presentation and expected pathogenicity. RESULTS: Among 382 episodes of suspected sepsis, clinically relevant microbes were detected by blood culture in 42 episodes (11%), by multiplex PCR in 37 episodes (9.7%), and by microarray in 32 episodes (8.4%). Although moderate agreement with blood culture (kappa 0.50), the multiplex PCR added diagnostic value by timely detection of 15 clinically relevant findings in blood culture-negative specimens. Results of the microarray corresponded very well to those of blood culture (kappa 0.90), but were available just marginally prior to blood culture results. CONCLUSIONS: The use of NAATs on whole blood specimens in adjunct to current culture-based methods provides a clinical add-on value by allowing for detection of organisms missed by blood culture. However, the aetiological significance of findings detected by NAATs should be interpreted with caution as the high analytical sensitivity may add findings that do not necessarily corroborate with the clinical diagnosis.

Genotypic Characterization of Clinical Klebsiella spp. Isolates Collected From Patients With Suspected Community-Onset Sepsis, Sweden.

Klebsiella is a genus of Gram-negative bacteria known to be opportunistic pathogens that may cause a variety of infections in humans. Highly drug-resistant Klebsiella species, especially K. pneumoniae, have emerged rapidly and are becoming a major concern in clinical management. Although K. pneumoniae is considered the most important pathogen within the genus, the true clinical significance of the other species is likely underrecognized due to the inability of conventional microbiological methods to distinguish between the species leading to high rates of misidentification. Bacterial whole-genome sequencing (WGS) enables precise species identification and characterization that other technologies do not allow. Herein, we have characterized the diversity and traits of Klebsiella spp. in community-onset infections by WGS of clinical isolates (n = 105) collected during a prospective sepsis study in Sweden. The sequencing revealed that 32 of the 82 isolates (39.0%) initially identified as K. pneumoniae with routine microbiological methods based on cultures followed by matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) had been misidentified. Of these, 23 were identified as Klebsiella variicola and nine as other members of the K. pneumoniae complex. Comparisons of the number of resistance genes showed that significantly fewer resistance genes were detected in Klebsiella oxytoca compared to K. pneumoniae and K. variicola (both values of p < 0.001). Moreover, a high proportion of the isolates within the K. pneumoniae complex were predicted to be genotypically multidrug-resistant (MDR; 79/84, 94.0%) in contrast to K. oxytoca (3/16, 18.8%) and Klebsiella michiganensis (0/4, 0.0%). All isolates predicted as genotypically MDR were found to harbor the combination of ß-lactam, fosfomycin, and quinolone resistance markers. Multi-locus sequence typing (MLST) revealed a high diversity of sequence types among the Klebsiella spp. with ST14 (10.0%) and ST5429 (10.0%) as the most prevalent ones for K. pneumoniae, ST146 for K. variicola (12.0%), and ST176 for K. oxytoca (25.0%). In conclusion, the results from this study highlight the importance of using high-resolution genotypic methods for identification and characterization of clinical Klebsiella spp. isolates. Our findings indicate that infections caused by other members of the K. pneumoniae complex than K. pneumoniae are a more common clinical problem than previously described, mainly due to high rates of misidentifications.

Evaluation of QuickFISH and maldi Sepsityper for identification of bacteria in bloodstream infection.

BACKGROUND: Early detection of bacteria and their antibiotic susceptibility patterns are critical to guide therapeutic decision-making for optimal care of septic patients. The current gold standard, blood culturing followed by subculture on agar plates for subsequent identification, is too slow leading to excessive use of broad-spectrum antibiotic with harmful consequences for the patient and, in the long run, the public health. The aim of the present study was to assess the performance of two commercial assays, QuickFISH® (OpGen) and Maldi Sepsityper™ (Bruker Daltonics) for early and accurate identification of microorganisms directly from positive blood cultures. MATERIALS AND METHODS: During two substudies of positive blood cultures, the two commercial assays were assessed against the routine method used at the clinical microbiology laboratory, Unilabs AB, at Skaraborg Hospital, Sweden. RESULTS: The Maldi Sepsityper™ assay enabled earlier microorganism identification. Using the cut-off for definite species identification according to the reference method (>2.0), sufficiently accurate species identification was achieved, but only among Gram-negative bacteria. The QuickFISH® assay was time-saving and showed high concordance with the reference method, 94.8% (95% CI 88.4-98.3), when the causative agent was covered by the QuickFISH® assay. CONCLUSIONS: The use of the commercial assays may shorten the time to identification of causative agents in bloodstream infections and can be a good complement to the current clinical routine diagnostics. Nevertheless, the performance of the commercial assays is considerably affected by the characteristics of the causative agents.

Diagnostic accuracy of procalcitonin, neutrophil-lymphocyte count ratio, C-reactive protein, and lactate in patients with suspected bacterial sepsis.

BACKGROUND: Early recognition is a key factor to achieve improved outcomes for septic patients. Combinations of biomarkers, as opposed to single ones, may improve timely diagnosis and survival. We investigated the performance characteristics of sepsis biomarkers, alone and in combination, for diagnosis of verified bacterial sepsis using Sepsis-2 and Sepsis-3 criteria, respectively. METHODS: Procalcitonin (PCT), neutrophil-lymphocyte count ratio (NLCR), C-reactive protein (CRP), and lactate were determined in a total of 1,572 episodes of adult patients admitted to the emergency department on suspicion of sepsis. All sampling were performed prior to antibiotic administration. Discriminant analysis was used to construct two composite biomarkers consisting of linear combinations of the investigated biomarkers, one including three selected biomarkers (i.e., NLCR, CRP, and lactate), and another including all four (i.e., PCT, NLCR, CRP, and lactate). The diagnostic performances of the composite biomarkers as well as the individual biomarkers were compared using the area under the receiver operating characteristic curve (AUC). RESULTS: For diagnosis of bacterial sepsis based on Sepsis-3 criteria, the AUC for PCT (0.68; 95% CI 0.65-0.71) was comparable to the AUCs for the both composite biomarkers. Using the Sepsis-2 criteria for bacterial sepsis diagnosis, the AUC for the NLCR (0.68; 95% CI 0.65-0.71) but not for the other single biomarkers, was equal to the AUCs for the both composite biomarkers. For diagnosis of severe bacterial sepsis or septic shock based on the Sepsis-2 criteria, the AUCs for both composite biomarkers were significantly greater than those of the single biomarkers (0.85; 95% CI 0.82-0.88 for the composite three-biomarker, and 0.86; 95% CI 0.83-0.89 for the composite four-biomarker). CONCLUSIONS: Combinations of biomarkers can improve the diagnosis of verified bacterial sepsis in the most critically ill patients, but in less severe septic conditions either the NLCR or PCT alone exhibit equivalent performance.

The Escherichia coli BarA-UvrY two-component system is a virulence determinant in the urinary tract.

BACKGROUND: The Salmonella enterica BarA-SirA, the Erwinia carotovora ExpS-ExpA, the Vibrio cholerae BarA-VarA and the Pseudomonas spp GacS-GacA all belong to the same orthologous family of two-component systems as the Escherichia coli BarA-UvrY. In the first four species it has been demonstrated that disruption of this two-component system leads to a clear reduction in virulence of the bacteria. Our aim was to determine if the Escherichia coli BarA-UvrY two-component system is connected with virulence using a monkey cystitis model. RESULTS: Cystitis was generated in Macaque fascularis monkeys by infecting the bladder with a 1:1 mixture of the uropathogenic Escherichia coli isolate DS17 and a derivative where the uvrY gene had been disrupted with a kanamycin resistance gene. Urine was collected through bladder punctuation at subsequent time intervals and the relative amount of uvrY mutant was determined. This showed that inactivation of the UvrY response regulator leads to a reduced fitness. In similar competitions in culture flasks with Luria Broth (LB) the uvrY mutant rather had a higher fitness than the wild type. When the competitions were done in flasks with human urine the uvrY mutant initially had a lower fitness. This was followed by a fluctuation in the level of mutant in the long-term culture, with a pattern that was specific for the individual urines that were tested. Addition of LB to the different urine competition cultures however clearly led to a consistently higher fitness of the uvrY mutant. CONCLUSION: This paper demonstrates that the BarA-UvrY two-component system is a determinant for virulence in a monkey cystitis model. The observed competition profiles strengthen our previous hypothesis that disruption of the BarA-UvrY two-component system impairs the ability of the bacteria to switch between different carbon sources. The urine in the bladder contains several different carbon sources and its composition changes over time. Inability to efficiently switch between the carbon sources may thus provide an explanation to the reduced fitness of the uvrY mutant in the cystitis model.

Toward quantifying the thymic dysfunctional state in mouse models of inflammatory bowel disease.

Inflammatory bowel disease is characterized by a number of immunological alterations, not the least in the T-cell compartment. Numerous animal models of colitis have revealed aberrant thymocyte dynamics associated with skewed thymocyte development. The recent advancements in quantitative methods have proposed critical kinetic alterations in the thymocyte development during the progression of colitis. This review focuses on the aberrant thymocyte dynamics in Gαi2-deficient mice as this mouse model provides most quantitative data of the thymocyte development associated with colitis. Herein, we discuss several dynamic changes during the progression of colitis and propose a hypothesis for the underlying causes for the skewed proportions of the thymocyte populations seen in the Gαi2-deficient mice and in other mouse models of colitis.

Genetic and functional characterization of the Escherichia coli BarA-UvrY two-component system: point mutations in the HAMP linker of the BarA sensor give a dominant-negative phenotype.

The BarA-UvrY two-component system family is strongly associated with virulence but is poorly understood at the molecular level. During our attempts to complement a barA deletion mutant, we consistently generated various mutated BarA proteins. We reasoned that characterization of the mutants would help us to better understand the signal transduction mechanism in tripartite sensors. This was aided by the demonstrated ability to activate the UvrY regulator with acetyl phosphate independently of the BarA sensor. Many of the mutated BarA proteins had poor complementation activity but could counteract the activity of the wild-type sensor in a dominant-negative fashion. These proteins carried point mutations in or near the recently identified HAMP linker, previously implicated in signal transduction between the periplasm and cytoplasm. This created sensor proteins with an impaired kinase activity and a net dephosphorylating activity. Using further site-directed mutagenesis of a HAMP linker-mutated protein, we could demonstrate that the phosphoaccepting aspartate 718 and histidine 861 are crucial for the dephosphorylating activity. Additional analysis of the HAMP linker-mutated BarA sensors demonstrated that a dephosphorylating activity can operate via phosphotransfer within a tripartite sensor dimer in vivo. This also means that a tripartite sensor can be arranged as a dimer even in the dephosphorylating mode.

The Escherichia coli BarA-UvrY two-component system is needed for efficient switching between glycolytic and gluconeogenic carbon sources.

The Escherichia coli BarA and UvrY proteins were recently demonstrated to constitute a novel two-component system, although its function has remained largely elusive. Here we show that mutations in the sensor kinase gene, barA, or the response regulator gene, uvrY, in uropathogenic E. coli drastically affect survival in long-term competition cultures. Using media with gluconeogenic carbon sources, the mutants have a clear growth advantage when competing with the wild type, but using media with carbon sources feeding into the glycolysis leads to a clear growth advantage for the wild type. Results from competitions with mutants in the carbon storage regulation system, CsrA/B, known to be a master switch between glycolysis and gluconeogenesis, led us to propose that the BarA-UvrY two-component system controls the Csr system. Taking these results together, we propose the BarA-UvrY two-component system is crucial for efficient adaptation between different metabolic pathways, an essential function for adaptation to a new environment.

Regulatory circuitry of the CsrA/CsrB and BarA/UvrY systems of Escherichia coli.

The global regulator CsrA (carbon storage regulator) is an RNA binding protein that coordinates central carbon metabolism, activates flagellum biosynthesis and motility, and represses biofilm formation in Escherichia coli. CsrA activity is antagonized by the untranslated RNA CsrB, to which it binds and forms a globular ribonucleoprotein complex. CsrA indirectly activates csrB transcription, in an apparent autoregulatory mechanism. In the present study, we elucidate the intermediate regulatory circuitry of this system. Mutations affecting the BarA/UvrY two-component signal transduction system decreased csrB transcription but did not affect csrA'-'lacZ expression. The uvrY defect was severalfold more severe than that of barA. Both csrA and uvrY were required for optimal barA expression. The latter observation suggests an autoregulatory loop for UvrY. Ectopic expression of uvrY suppressed the csrB-lacZ expression defects caused by uvrY, csrA, or barA mutations; csrA suppressed csrA or barA defects; and barA complemented only the barA mutation. Purified UvrY protein stimulated csrB-lacZ expression approximately sixfold in S-30 transcription-translation reactions, revealing a direct effect of UvrY on csrB transcription. Disruption of sdiA, which encodes a LuxR homologue, decreased the expression of uvrY'-'lacZ and csrB-lacZ fusions but did not affect csrA'-'lacZ. The BarA/UvrY system activated biofilm formation. Ectopic expression of uvrY stimulated biofilm formation by a csrB-null mutant, indicative of a CsrB-independent role for UvrY in biofilm development. Collectively, these results demonstrate that uvrY resides downstream from csrA in a signaling pathway for csrB and that CsrA stimulates UvrY-dependent activation of csrB expression by BarA-dependent and -independent mechanisms.