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High yield extraction of E. coli RNA from human whole blood
Abstract
Purpose:
Studies of bacterial transcriptomics during bloodstream infections are limited to-date because unbiased extraction of bacterial mRNA from whole blood in sufficient quantity and quality has proved challenging. Problems include the high excess of human cells, the presence of PCR inhibitors and the short intrinsic half-life of bacterial mRNA. This study aims to provide a framework for the choice of the most suitable sample preparation method.
Methodology:
Escherichia coli cells were spiked into human whole blood and the bacterial gene expression was stabilized with RNAprotect either immediately or after lysis of the red blood cells with Triton X-100, saponin, ammonium chloride or the commercial MolYsis buffer CM. RNA yield, purity and integrity were assessed by absorbance measurements at 260 and 280 nm, real-time PCR and capillary electrophoresis.
Results:
For low cell numbers, the best mRNA yields were obtained by adding the commercial RNAprotect reagent directly to the sample without prior lyses of the human blood cells. Using this protocol, significant amounts of human RNA were co-purified, however, this had a beneficial impact on the yields of bacterial mRNA. Among the tested lysis agents, Triton X-100 was the most effective and reduced the human RNA background by three to four orders of magnitude.
Conclusion:
For most applications, lysis of the human blood cells is not required. However, co-purified human RNA may interfere with some downstream processes such as RNA sequencing. In this case, blood cell lysis with Triton X-100 is desirable.
... Due to the increasing demand for rapid, culture-independent molecular detection methods, a plethora of multiplex PCR detection systems for E. coli and total coliform bacteria have been developed during the last decade [49,50]. Recently, Krapft et al. revealed a clear correlation between viable cell counts and real-time quantitative PCR (qPCR) data [51]. ...
We established an innovative approach that included direct, viability, and nested PCR for rapid and reliable identification of the fecal indicator organism Escherichia coli (E. coli). Direct PCR enabled successful amplification of the target uidA gene, omitting a prior DNA isolation or purification step. Furthermore, we applied viability PCR (v-PCR) to ensure the detection of only relevant viable bacterial cells. The principle involves the binding of propidium monoazide (PMA), a selective nucleic acid intercalating dye, to accessible DNA of heat killed bacteria cells and, consequently, allows viable and heat killed E. coli cells to be discriminated. To ensure high sensitivity, direct v-PCR was followed by a nested PCR step. The resulting amplicons were analyzed by a rapid 30 min microarray-based DNA hybridization assay for species-specific DNA detection of E. coli. A positive signal was indicated by enzymatically generated silver nanoparticle deposits, which served as robust endpoint signals allowing an immediate visual readout. The presented novel protocol allows the detection of 1 × 101 viable E. coli cells per PCR run.
... Pathogen enrichment from whole blood is frequently combined with the selective lysis of human blood cells in order to release intracellular pathogens that might otherwise remain undetected (Fraunholz and Sinha, 2012) and to deplete factors interfering with PCR (Al-Soud and Radstrom, 2001;Djordjevic et al., 2006;Doring et al., 2008;Garcia et al., 2002). Selective lysis of blood cells can be achieved by addition of hypotonic or chaotropic buffers containing apolar, zwitterionic, or polar detergents, of which only the latter also lyse the nucleus to release human DNA (Brennecke et al., 2017;Caspar et al., 2017;Katholnig et al., 2015;Trung et al., 2016). Bacteria and fungi are able to withstand the osmotic pressure during lysis due to their cell walls and can subsequently be separated from the supernatant containing the lysed blood cells. ...
Blood culture represents the current reference method for the detection of bacteria or fungi in the circulation. To accelerate pathogen identification, molecular diagnostic methods, mainly based on polymerase chain reaction (PCR), have been introduced to ensure early and targeted antibiotic treatment of patients suffering from bloodstream infection. Still, these approaches suffer from a lack of sensitivity and from inhibition of PCR in a number of clinical samples, leading to false negative results. To overcome these limitations, various approaches aiming at the enrichment of pathogens from larger blood volumes prior to the extraction of pathogen DNA, thereby also depleting factors interfering with PCR, have been developed. Here, we provide an overview of current systems for diagnosing bloodstream infection, with a focus on approaches for pre-analytical pathogen enrichment, and highlight emerging applications of pathogen depletion for therapeutic purposes as a potential adjunctive treatment of sepsis patients.
Urinary tract infections (UTIs) are the most prevalent infectious diseases in community and clinical settings. The global emergence of multidrug-resistant (MDR) Escherichia coli causing these UTIs necessitates exploring novel approaches. The use of compounds that attenuate bacterial virulence acting through inhibition of quorum sensing (QS) is gathering momentum nowadays. This research was undertaken to investigate the anti-QS prospects of azithromycin (AZ) against 67 E. coli isolated from urine cultures of patients admitted to Alexandria Main University Hospital. Antibiotic susceptibility testing showed that 94% of these isolates were MDR. Swimming, twitching, and biofilm formation abilities were detected in 60%, 52.5%, and 55.2%, of the isolates, respectively. At its sub-inhibitory concentration, AZ hindered swimming and twitching motilities in 75% and 74.3% of the tested isolates, respectively. It reduced the biofilm formation by percentages ranging from 27% to 73%. Azithromycin downregulated the differential gene expression of luxS, a QS-regulating gene, and the genes encoding motility, thus proving its anti-QS ability. Further, the combinatory activity of AZ with ceftriaxone, ciprofloxacin, colistin, doxycycline, and tigecycline generated 95% of successful combinations. In conclusion, AZ effectively attenuated the QS in urinary E. coli isolates leading to a regression in the production of QS-associated virulence factors and hindrance of biofilm formation, thus delivering an avirulent pathogen. In addition, the synergistic combinations succeeded in circumventing antibiotic resistance of E. coli isolates. Hence, AZ could be regarded as an anti-QS agent that might aid the design of innovative treatment alternatives for the management of UTIs caused by MDR E. coli.
Staphylococcus aureus (S. aureus) as an important pathogen with high stress tolerance poses a serious threat to human health. Ultrasound (US) combined with hydrogen peroxide (H2O2) is considered to be a relatively eco-friendly and efficient approach for pollutant degradation in water, but their synergistic antimicrobial effect has been rarely researched. Therefore, this study investigated the synergetic antimicrobial effects and mechanism of US and H2O2 against S. aureus in water. The results show that US (288 W, 20 kHz) and H2O2 (0.1, 0.25 and 0.5 M) exhibited marked synergistic antibacterial effects against S. aureus, achieving a bacterial reduction ranging from 0.34 to 7.05 log CFU/mL, which was significantly higher than the individual US (0.05–0.48 log reduction CFU/mL) or H2O2 (0.06–4.94 log reduction CFU/mL) treatment. The best synergistic effect was achieved by the combined treatment of US and 0.25 M H2O2 for 6 min (7.05 log reduction CFU/mL). The physiological results show that US combined with H2O2 could disrupt the cell membrane integrity, alter the cell morphology including cell shrinkage, collapse, and fragmentation, decrease the average cell size and cause cell surface oxidation. More importantly, the enhanced generation of hydroxyl radical (OH) was confirmed to play an important role in the synergistic antibacterial effects of US combined with H2O2. These results indicate that the combined treatment of US and H2O2 is a promising technology to reduce S. aureus counts in water.
Fast and reliable detection of infectious pathogens is in many clinical cases decisive for the therapy and prognosis of the patient. Detection methods like PCR are able to meet these requirements. However, to ensure successful amplification and detection, the sample should be purified from inhibiting substances. Since very small sample volumes are used in a PCR run, a previous concentration should also be carried out to achieve a sufficient concentration of the target sequence and thus avoid false negative results. Standard procedures for the preparation of a sample are time-consuming and require many manual steps, a well-equipped microbiological laboratory and well-trained personnel, making them unsuitable for point-of-care-testing (POCT) and resulting in high costs.
This work is focused on the development of lab-on-a-chip (LOC) systems, which are capable of performing all steps of sample preparation. The concentration is based on free-flow electrophoresis (FFE) and allows the preparation of both bacteria and viruses. For the first time it could be shown that the concentration of viruses by FFE is possible, larger volumes can be processed in a few minutes and the electrical conductivity of the sample medium is crucial for a fast and efficient concentration. The extraction was carried out by thermal or thermoelectric lysis and on-chip gel electrophoresis for the purification of DNA and RNA. It was shown that bacterial mRNA can be extracted from undiluted EDTA blood in less than three minutes.
Since the lab-on-a-chip systems can be used to prepare both bacteria and viruses, their process characteristics and technology enable the rapid preparation of complex sample matrices in the least amount of space and automation of the process can be easily implemented, the systems represent a promising approach for POCT and may help to improve a patient's prognosis in the future.
The study was performed to develop a non-culture method to quantify viable loads of Lactobacillus acidophilus NCFM using RNA-based molecular technique. The ‘growth curve’ and ‘cycle threshold curve’ were developed respectively by plate counting and using cycle threshold (C T ) values. ‘Standard curve’ was constructed using cells per milliliter and relative C T values. A maximum viable count (1.5 ± 0.15) × 10 ¹⁰ cells/mL with a minimum C T value 20.18 ± 0.56 was achieved following 18 h of growth. The two parameters were inversely proportional to each other over the exponential growth. The ‘standard curve’ corresponded to equation y = 2E + 28e −2.034x (y = cells/mL, x = C T value; R ² = 0.993), and no sample showed significant difference between ‘plate count’ and relative ‘PCR count’ following the validation process. Industrial adaptation of this method in dairy processing could potentially contribute to a faster enumeration of viable L. acidophilus NCFM compared to plate counting.
Background: Bifidobacterium animalis subsp. lactis HN019 is widely used as a probiotic in various dairy preparations. To ensure optimal health benefits, an adequate number of probiotics should be viable in products, but the culture-based methods require longer periods to account for the strain. Objective: Develop a standard curve to account viable load of B. animalis subsp. lactis HN019 by real-time PCR. Methods: The growth curve was developed according to plate count method, and cycle threshold (CT) curve was prepared using CT values. These two curves were then combined to construct the standard curve. To validate the method, the strain was proliferated in whole milk, and viable loads were enumerated by plate counting and relative PCR counting followed by determining the SEM between the two counts. Results: The growth curve and CT curve, respectively, showed the highest viable load (2.13 × 108 CFU/mL) and lowest CT value (18.29) after 18 h, and during the entire growth phase (0-18 h), viable loads are inversely proportional to the CT values. The standard curve revealed the model y = 2E + 18e-1.233
x
(y = cells/mL, x = CT value; R² = 0.992). In validation, the highest SEM (± 0.70 × 108) was found between the plate count (1.15 × 108 cells/mL) and relative PCR count (1.29 × 108 cells/mL) after cultivating for 14 h. Conclusions: The method could be readily used in dairy industries to quantify viable B. animalis subsp. lactis HN019 by a shorter period. Highlights: Culture-independent enumeration of viable B. animalis subsp. lactis HN019 by real-time PCR.
The human pathogen Mycobacterium tuberculosis has the capacity to escape eradication by professional phagocytes. During infection, M. tuberculosis resists the harsh environment of phagosomes and actively manipulates macrophages and dendritic cells to ensure prolonged intracellular survival. In contrast to other intracellular pathogens, it has remained difficult to capture the transcriptome of mycobacteria during infection due to an unfavorable host-to-pathogen ratio.
We infected the human macrophage-like cell line THP-1 with the attenuated M. tuberculosis surrogate M. bovis Bacillus Calmette–Guérin (M. bovis BCG). Mycobacterial RNA was up to 1000-fold underrepresented in total RNA preparations of infected host cells. We employed microbial enrichment combined with specific ribosomal RNA depletion to simultaneously analyze the transcriptional responses of host and pathogen during infection by dual RNA sequencing. Our results confirm that mycobacterial pathways for cholesterol degradation and iron acquisition are upregulated during infection. In addition, genes involved in the methylcitrate cycle, aspartate metabolism and recycling of mycolic acids were induced. In response to M. bovis BCG infection, host cells upregulated de novo cholesterol biosynthesis presumably to compensate for the loss of this metabolite by bacterial catabolism.
Dual RNA sequencing allows simultaneous capture of the global transcriptome of host and pathogen, during infection. However, mycobacteria remained problematic due to their relatively low number per host cell resulting in an unfavorable bacterium-to-host RNA ratio. Here, we use a strategy that combines enrichment for bacterial transcripts and dual RNA sequencing to provide the most comprehensive transcriptome of intracellular mycobacteria to date. The knowledge acquired into the pathogen and host pathways regulated during infection may contribute to a solid basis for the deployment of novel intervention strategies to tackle infection.
AimsIsolation of Salmonella Typhi from blood culture is the standard diagnostic for confirming typhoid fever but it is unavailable in many developing countries. We previously described a Microwave Accelerated Metal Enhanced Fluorescence (MAMEF)-based assay to detect Salmonella in medium. Attempts to detect Salmonella in blood were unsuccessful, presumably due to the interference of erythrocytes. The objective of this study was to evaluate various blood treatment methods that could be used prior to PCR, real-time PCR or MAMEF to increase sensitivity of detection of Salmonella.Methods and ResultsWe tested ammonium chloride and erythrocyte lysis buffer, water, Lymphocyte Separation Medium, BD Vacutainer® CPT™ Tubes and dextran. Erythrocyte lysis buffer was the best isolation method as it is fast, inexpensive and works with either fresh or stored blood. The sensitivity of PCR- and real-time PCR-detection of Salmonella in spiked blood was improved when whole blood was first lysed using erythrocyte lysis buffer prior to DNA extraction. Removal of erythrocytes and clotting factors also enabled reproducible lysis of Salmonella and fragmentation of DNA, which are necessary for MAMEF sensing.Conclusions
Use of the erythrocyte lysis procedure prior to DNA extraction has enabled improved sensitivity of Salmonella detection by PCR and real-time PCR and has allowed lysis and fragmentation of Salmonella using microwave radiation (for future detection by MAMEF).Significance and Impact of the studyAdaptation of the blood lysis method represents a fundamental breakthrough that improves the sensitivity of DNA-based detection of Salmonella in blood.This article is protected by copyright. All rights reserved.
Sepsis is a severe medical condition characterized by a systemic inflammatory response of the body caused by pathogenic microorganisms in the bloodstream. Blood or plasma is typically used for diagnosis, both containing large amount of human DNA, greatly exceeding the DNA of microbial origin. In order to enrich bacterial DNA, we applied the C0t effect to reduce human DNA background: a model system was set up with human and Escherichia coli (E. coli) DNA to mimic the conditions of bloodstream infections; and this system was adapted to plasma and blood samples from septic patients. As a consequence of the C0t effect, abundant DNA hybridizes faster than rare DNA. Following denaturation and re-hybridization, the amount of abundant DNA can be decreased with the application of double strand specific nucleases, leaving the non-hybridized rare DNA intact. Our experiments show that human DNA concentration can be reduced approximately 100,000-fold without affecting the E. coli DNA concentration in a model system with similarly sized amplicons. With clinical samples, the human DNA background was decreased 100-fold, as bacterial genomes are approximately 1,000-fold smaller compared to the human genome. According to our results, background suppression can be a valuable tool to enrich rare DNA in clinical samples where a high amount of background DNA can be found.
Background
The use of low quality RNA samples in whole-genome gene expression profiling remains controversial. It is unclear if transcript degradation in low quality RNA samples occurs uniformly, in which case the effects of degradation can be corrected via data normalization, or whether different transcripts are degraded at different rates, potentially biasing measurements of expression levels. This concern has rendered the use of low quality RNA samples in whole-genome expression profiling problematic. Yet, low quality samples (for example, samples collected in the course of fieldwork) are at times the sole means of addressing specific questions.
Results
We sought to quantify the impact of variation in RNA quality on estimates of gene expression levels based on RNA-seq data. To do so, we collected expression data from tissue samples that were allowed to decay for varying amounts of time prior to RNA extraction. The RNA samples we collected spanned the entire range of RNA Integrity Number (RIN) values (a metric commonly used to assess RNA quality). We observed widespread effects of RNA quality on measurements of gene expression levels, as well as a slight but significant loss of library complexity in more degraded samples.
Conclusions
While standard normalizations failed to account for the effects of degradation, we found that by explicitly controlling for the effects of RIN using a linear model framework we can correct for the majority of these effects. We conclude that in instances in which RIN and the effect of interest are not associated, this approach can help recover biologically meaningful signals in data from degraded RNA samples.
This study aimed to determine whether the addition of an aminoglycoside to a ß-lactam antibiotic increases the antimicrobial effect during the early phase of Gram-negative severe sepsis/septic shock. A porcine model was selected that considered each animal's individual blood bactericidal capacity. Escherichia coli, susceptible to both antibiotics, was given to healthy pigs intravenously during 3 h. At 2 h, the animals were randomized to a 20-min infusion with either cefuroxime alone (n = 9), a combination of cefuroxime+tobramycin (n = 9), or saline (control, n = 9). Blood samples were collected hourly for cultures and quantitative polymerase chain reaction (PCR). Bacterial growth in the organs after 6 h was chosen as the primary endpoint. A blood sample was obtained at baseline before start of bacterial infusion for ex vivo investigation of the blood bactericidal capacity. At 1 h after the administration of the antibiotics, a second blood sample was taken for ex vivo investigation of the antibiotic-induced blood killing activity. All animals developed severe sepsis/septic shock. Blood cultures and PCR rapidly became negative after completed bacterial infusion. Antibiotic-induced blood killing activity was significantly greater in the combination group than in the cefuroxime group (p<0.001). Growth of bacteria in the spleen was reduced in the two antibiotic groups compared with the controls (p<0.01); no difference was noted between the two antibiotic groups. Bacterial growth in the liver was significantly less in the combination group than in the cefuroxime group (p<0.05). High blood bactericidal capacity at baseline was associated with decreased growth in the blood and spleen (p<0.05). The addition of tobramycin to cefuroxime results in increased antibiotic-induced blood killing activity and less bacteria in the liver than cefuroxime alone. Individual blood bactericidal capacity may have a significant effect on antimicrobial outcome.
For patients suffering from bloodstream infections (BSI) molecular diagnostics from whole blood holds promise to provide fast and adequate treatment. However, this approach is hampered by the need of large blood volumes. Three methods for pathogen DNA isolation from whole blood were compared, i.e. an enzymatic method (MolYsis, 1-5 ml), the novel non-enzymatic procedure (Polaris, 1-5 ml), and a method that does not entail removal of human DNA (Triton-Tris-EDTA EasyMAG, 200 µl). These methods were evaluated by processing blood spiked with 0-1000 CFU/ml of Staphylococcus aureus, Pseudomonas aeruginosa and Candida albicans. Downstream detection was performed with real-time PCR assays. Polaris and MolYsis processing followed by real-time PCRs enabled pathogen detection at clinically relevant concentrations of 1-10 CFU/ml blood. By increasing sample volumes, concurrent lower cycle threshold (Ct) values were obtained at clinically relevant pathogen concentrations, demonstrating the benefit of using larger blood volumes. A 100% detection rate at a concentration of 10 CFU/ml for all tested pathogens was obtained with the Polaris enrichment, whereas comparatively lower detection rates were measured for MolYsis (50-67%) and EasyMAG (58-79%). For the samples with a concentration of 1 CFU/ml Polaris resulted in most optimal detection rates of 70-75% (MolYsis 17-50% and TTE-EasyMAG 20-36%). The Polaris method was more reproducible, less labour intensive, and faster (45 minutes (including Qiagen DNA extraction) vs. 2 hours (MolYsis)). In conclusion, Polaris and MolYsis enrichment followed by DNA isolation and real-time PCR enables reliable and sensitive detection of bacteria and fungi from 5 ml blood. With Polaris results are available within 3 hours, showing potential for improved BSI diagnostics.
The matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been recently introduced in microbiology laboratories for bacterial and fungal identification. This technology could greatly improve the clinical management and guidance for chemotherapy.In this study, we evaluated the performance of two commercial MALDI-TOF MS systems, VITEK MS IVD (bioMerieux) and Microflex LT Biotyper (Bruker Daltonics) on direct bacterial identification on positive blood cultures with the use of the commercial MALDI Sepsityper extraction method. Among the 181 mono-microbial cultures, both systems could generate genus to species level identifications among >90% of specimens [Biotyper (177/181, 97.8%); VITEK MS IVD (167/181; 92.3%)]. Overall, the Biotyper system could generate significantly more accurate identifications than the VITEK MS IVD system (p=0.016 177 vs 167 out of 181 specimens). The Biotyper system was able to identify the minority species among polymicrobial blood cultures.An in-house extraction method was also evaluated towards the Sepsityper on both MALDI-TOF MS systems. The in-house method could generate more correct genus level identifications than the Sepsityper (96.7% vs 93.5%) on the Biotyper system, whereas they exhibited the same performance (88.0% vs 88.0%) on the VITEK MS IVD system.Our study confirmed the practical advantages of MALDI-TOF MS and our in-house extraction method could reduce the reagent cost to US$1 per specimen with shorter turn around-time of 3 hours, which is highly cost-effective for diagnostic microbiology service.
Background
The events leading to sepsis start with an invasive infection of a primary organ of the body followed by an overwhelming systemic response. Intra-abdominal infections are the second most common cause of sepsis. Peritoneal fluid is the primary site of infection in these cases. A microarray-based approach was used to study the temporal changes in cells from the peritoneal cavity of septic mice and to identify potential biomarkers and therapeutic targets for this subset of sepsis patients.
Results
We conducted microarray analysis of the peritoneal cells of mice infected with a non-pathogenic strain of Escherichia coli. Differentially expressed genes were identified at two early (1 h, 2 h) and one late time point (18 h). A multiplexed bead array analysis was used to confirm protein expression for several cytokines which showed differential expression at different time points based on the microarray data. Gene Ontology based hypothesis testing identified a positive bias of differentially expressed genes associated with cellular development and cell death at 2 h and 18 h respectively. Most differentially expressed genes common to all 3 time points had an immune response related function, consistent with the observation that a few bacteria are still present at 18 h.
Conclusions
Transcriptional regulators like PLAGL2, EBF1, TCF7, KLF10 and SBNO2, previously not described in sepsis, are differentially expressed at early and late time points. Expression pattern for key biomarkers in this study is similar to that reported in human sepsis, indicating the suitability of this model for future studies of sepsis, and the observed differences in gene expression suggest species differences or differences in the response of blood leukocytes and peritoneal leukocytes.
Neisseria meningitidis is the major cause of septicemia and meningococcal meningitis. During the course of infection, the bacterium must adapt to
different host environments as a crucial factor for survival and dissemination; in particular, one of the crucial factors
in N. meningitidis pathogenesis is the ability to grow and survive in human blood. We recently showed that N. meningitidis alters the expression of 30% of the open reading frames (ORFs) of the genome during incubation in human whole blood and suggested
the presence of fine regulation at the gene expression level in order to control this step of pathogenesis. In this work,
we used a customized tiling oligonucleotide microarray to define the changes in the whole transcriptional profile of N. meningitidis in a time course experiment of ex vivo bacteremia by incubating bacteria in human whole blood and then recovering RNA at different time points. The application
of a newly developed bioinformatic tool to the tiling array data set allowed the identification of new transcripts—small intergenic
RNAs, cis-encoded antisense RNAs, mRNAs with extended 5′ and 3′ untranslated regions (UTRs), and operons—differentially expressed in
human blood. Here, we report a panel of expressed small RNAs, some of which can potentially regulate genes involved in bacterial
metabolism, and we show, for the first time in N. meningitidis, extensive antisense transcription activity. This analysis suggests the presence of a circuit of regulatory RNA elements
used by N. meningitidis to adapt to proliferate in human blood that is worthy of further investigation.
Background
Enteric fever is a major public health problem, causing an estimated 21million new cases and 216,000 or more deaths every year. Current diagnosis of the disease is inadequate. Blood culture only identifies 45 to 70% of the cases and is time-consuming. Serological tests have very low sensitivity and specificity. Clinical samples obtained for diagnosis of enteric fever in the field generally have <1 organism/ml of blood, so that even PCR-based methods, widely used for detection of other infectious diseases, are not a straightforward option in typhoid diagnosis. We developed a novel method to enrich target bacterial DNA by selective removal of human DNA from blood samples, enhancing the sensitivity of PCR tests. This method offers the possibility of improving PCR assays directly using clinical specimens for diagnosis of this globally important infectious disease.
Methods
Blood samples were mixed with ox bile for selective lysis of human blood cells and the released human DNA was then digested with addition of bile resistant micrococcal nuclease. The intact Salmonella Typhi bacteria were collected from the specimen by centrifugation and the DNA extracted with QIAamp DNA mini kit. The presence of Salmonella Typhi bacteria in blood samples was detected by PCR with the fliC-d gene of Salmonella Typhi as the target.
Results
Micrococcal nuclease retained activity against human blood DNA in the presence of up to 9% ox bile. Background human DNA was dramatically removed from blood samples through the use of ox bile lysis and micrococcal nuclease for removal of mammalian DNA. Consequently target Salmonella Typhi DNA was enriched in DNA preparations and the PCR sensitivity for detection of Salmonella Typhi in spiked blood samples was enhanced by 1,000 fold.
Conclusions
Use of a combination of selective ox-bile blood cell lysis and removal of human DNA with micrococcal nuclease significantly improves PCR sensitivity and offers a better option for improved typhoid PCR assays directly using clinical specimens in diagnosis of this globally important infection disease which we believe could be of importance in improving clinical care and providing effective evaluation of novel vaccines.
In cases of bacteraemia, a rapid species identification of the causal agent directly from positive blood culture broths could assist clinicians in the timely targeting of empirical antimicrobial therapy. For this purpose, we evaluated the direct identification of micro-organisms from BacT/ALERT (bioMérieux) anaerobic positive blood cultures without charcoal using the Microflex matrix-assisted laser desorption/ionization (MALDI) time of flight MS (Bruker), after bacterial extraction by using two different methods: the MALDI Sepsityper kit (Bruker) and an in-house saponin lysis method. Bruker's recommended criteria for identification were expanded in this study, with acceptance of the species identification when the first three results with the best matches with the MALDI Biotyper database were identical, whatever the scores were. In total, 107 monobacterial cultures and six polymicrobial cultures from 77 different patients were included in this study. Among monomicrobial cultures, we identified up to the species level 67 and 66 % of bacteria with the MALDI Sepsityper kit and the saponin method, respectively. There was no significant difference between the two extraction methods. The direct species identification was particularly inconclusive for Gram-positive bacteria, as only 58 and 52 % of them were identified to the species level with the MALDI Sepsityper kit and the saponin method, respectively. Results for Gram-negative bacilli were better, with 82.5 and 90 % of correct identification to the species level with the MALDI Sepsityper kit and the saponin method, respectively. No misidentifications were given by the direct procedures when compared with identifications provided by the conventional method. Concerning the six polymicrobial blood cultures, whatever the extraction method used, a correct direct identification was only provided for one of the isolated bacteria on solid medium in all cases. The analysis of the time-to-result demonstrated a reduction in the turnaround time for identification ranging from 1 h 06 min to 24 h 44 min, when performing the blood culture direct identification in comparison with the conventional method, whatever the extraction method.
Neisseria meningitidis is a nasopharyngeal commensal of humans which occasionally invades the blood to cause septicaemia. The transcriptome of N. meningitidis strain MC58 grown in human blood for up to 4 hours was determined and around 10% of the genome was found to be differentially regulated. The nuo, pet and atp operons, involved in energy metabolism, were up-regulated, while many house-keeping genes were down-regulated. Genes encoding protein chaperones and proteases, involved in the stress response; complement resistant genes encoding enzymes for LOS sialylation and biosynthesis; and fHbp (NMB1870) and nspA (NMB0663), encoding vaccine candidates, were all up-regulated. Genes for glutamate uptake and metabolism, and biosynthesis of purine and pyrimidine were also up-regulated. Blood grown meningococci are under stress and undergo a metabolic adaptation and energy conservation strategy. The localisation of four putative outer membrane proteins encoded by genes found to be up-regulated in blood was assessed by FACS using polyclonal mouse antisera, and one (NMB0390) showed evidence of surface expression, supporting its vaccine candidacy.
With rising rates of drug-resistant infections, there is a need for diagnostic methods that rapidly can detect the presence of pathogens and reveal their susceptibility to antibiotics. Here we propose an approach to diagnosing the presence and drug-susceptibility of infectious diseases based on direct detection of RNA from clinical samples. We demonstrate that species-specific RNA signatures can be used to identify a broad spectrum of infectious agents, including bacteria, viruses, yeast, and parasites. Moreover, we show that the behavior of a small set of bacterial transcripts after a brief antibiotic pulse can rapidly differentiate drug-susceptible and -resistant organisms and that these measurements can be made directly from clinical materials. Thus, transcriptional signatures could form the basis of a uniform diagnostic platform applicable across a broad range of infectious agents.
A major inhibitor of diagnostic PCR in human plasma was identified and the mechanism of inhibition was characterized. Human blood was divided by centrifugation into buffy coat, plasma, platelets, and erythrocytes. All these blood fractions were found to be highly inhibitory to a standardized PCR mixture containing the thermostable DNA polymerase AmpliTaq Gold. PCR inhibitors in human plasma were purified by chromatographic procedures and were characterized by a process of elimination, so that the PCR-inhibitory effects of plasma fractions were tested after each purification step. The major inhibitor in human plasma, as determined by size-exclusion chromatography, anion-exchange chromatography, and chromatofocusing, was found to be immunoglobulin G (IgG) on the basis of N-terminal amino acid sequencing and electrophoretic analysis of the purified polypeptide. When different concentrations of purified plasma IgG (PIgG) were added to PCR mixtures containing 11 different thermostable DNA polymerases and 1 ng of Listeria monocytogenes DNA as template DNA, the only polymerase that resisted inhibition was rTth. The inhibitory effect was reduced when PIgG was heated at 95 degrees C before it was added to PCR or after the addition of excess nontarget DNA to the PCR mixture. However, heating of PIgG together with target DNA at 95 degrees C was found to block the amplification. Inhibition by PIgG may be due to an interaction with single-stranded DNA, which makes the target DNA unavailable for 10 of the DNA polymerases tested. The results show the danger of using boiling as a method of sample pretreatment or using a hot start prior to PCR. The effect of plasma PCR inhibition could be removed by mixing plasma with DNA-agarose beads prior to amplification, while plasma PCR inhibitors were found to bind to the DNA-agarose beads.
During infection Neisseria meningitidis (Nm) encounters multiple environments within the host, which makes rapid adaptation a crucial factor for meningococcal survival. Despite the importance of invasion into the bloodstream in the meningococcal disease process, little is known about how Nm adapts to permit survival and growth in blood. To address this, we performed a time-course transcriptome analysis using an ex vivo model of human whole blood infection. We observed that Nm alters the expression of ≈30% of ORFs of the genome and major dynamic changes were observed in the expression of transcriptional regulators, transport and binding proteins, energy metabolism, and surface-exposed virulence factors. In particular, we found that the gene encoding the regulator Fur, as well as all genes encoding iron uptake systems, were significantly up-regulated. Analysis of regulated genes encoding for surface-exposed proteins involved in Nm pathogenesis allowed us to better understand mechanisms used to circumvent host defenses. During blood infection, Nm activates genes encoding for the factor H binding proteins, fHbp and NspA, genes encoding for detoxifying enzymes such as SodC, Kat and AniA, as well as several less characterized surface-exposed proteins that might have a role in blood survival. Through mutagenesis studies of a subset of up-regulated genes we were able to identify new proteins important for survival in human blood and also to identify additional roles of previously known virulence factors in aiding survival in blood. Nm mutant strains lacking the genes encoding the hypothetical protein NMB1483 and the surface-exposed proteins NalP, Mip and NspA, the Fur regulator, the transferrin binding protein TbpB, and the L-lactate permease LctP were sensitive to killing by human blood. This increased knowledge of how Nm responds to adaptation in blood could also be helpful to develop diagnostic and therapeutic strategies to control the devastating disease cause by this microorganism.
Infection with uropathogenic Escherichia coli (UPEC), the causative agent of most uncomplicated urinary tract infections, proceeds in an ascending manner and, if left untreated, may result in bacteremia and urosepsis. To examine the fate of UPEC after its entry into the bloodstream, we developed a murine model of sublethal bacteremia. CBA/J mice were inoculated intravenously with 1 × 10 ⁶ CFU of pyelonephritis strain E. coli CFT073 carrying a bioluminescent reporter. Biophotonic imaging, used to monitor the infection over 48 h, demonstrated that the bacteria disseminated systemically and appeared to localize at discrete sites. UPEC was recovered from the spleen, liver, kidneys, lungs, heart, brain, and intestines as early as 20 min postinoculation, peaking at 24 h postinoculation. A nonpathogenic E. coli K-12 strain, however, disseminated at significantly lower levels ( P < 0.01) and was cleared from the liver and cecum by 24 h postinoculation. Isogenic mutants lacking type 1 fimbriae, P fimbriae, capsule, TonB, the heme receptors Hma and ChuA, or particularly the sialic acid catabolism enzyme NanA were significantly outcompeted by wild-type CFT073 during bacteremia ( P < 0.05), while flagellin and hemolysin mutants were not.
IMPORTANCE E. coli is the primary cause of urinary tract infections. In severe cases of kidney infection, bacteria can enter the bloodstream and cause systemic disease. While the ability of E. coli to cause urinary tract infection has been extensively studied, the fate of these bacteria once they enter the bloodstream is largely unknown. Here we used an imaging technique to develop a mouse model of E. coli bloodstream infection and identify bacterial genes that are important for the bacteria to spread to and infect various organs. Understanding how urinary tract pathogens like E. coli cause disease after they enter the bloodstream may aid in the development of protective and therapeutic treatments.
Protein and messenger RNA (mRNA) copy numbers vary from cell to cell in isogenic bacterial populations. However, these molecules
often exist in low copy numbers and are difficult to detect in single cells. We carried out quantitative system-wide analyses
of protein and mRNA expression in individual cells with single-molecule sensitivity using a newly constructed yellow fluorescent
protein fusion library for Escherichia coli. We found that almost all protein number distributions can be described by the gamma distribution with two fitting parameters
which, at low expression levels, have clear physical interpretations as the transcription rate and protein burst size. At
high expression levels, the distributions are dominated by extrinsic noise. We found that a single cell’s protein and mRNA
copy numbers for any given gene are uncorrelated.
Neonatal sepsis is difficult to diagnose and pathogens cannot be detected from blood cultures in many cases. Development of a rapid and accurate method for detecting pathogens is thus essential. The main purpose of this study was to identify etiological agents in clinically diagnosed neonatal sepsis using bacterial ribosomal RNA-targeted reverse transcription-quantitative PCR (BrRNA-RT-qPCR) and to conduct comparisons with the results of conventional blood culture. Since BrRNA-RT-qPCR targets bacterial ribosomal RNA, detection rates using this approach may exceed those using conventional PCR.
Subjects comprised 36 patients with 39 episodes of suspected neonatal sepsis who underwent BrRNA-RT-qPCR and conventional blood culture to diagnose sepsis. Blood samples were collected aseptically for BrRNA-RT-qPCR and blood culture at the time of initial sepsis evaluation by arterial puncture. BrRNA-RT-qPCR and blood culture were undertaken using identical blood samples, and BrRNA-RT-qPCR was performed using 12 primer sets.
Positive rate was significantly higher for BrRNA-RT-qPCR (15/39, 38.5%) than for blood culture (6/39, 15.4%; p = 0.0039). BrRNA-RT-qPCR was able to identify all pathogens detected by blood culture. Furthermore, this method detected pathogens from neonates with clinical sepsis in whom pathogens was not detected by culture methods.
This RT-PCR technique is useful for sensitive detection of pathogens causing neonatal sepsis, even in cases with negative results by blood culture.
The purpose of this study was to evaluate the clinical utility of bacterial rRNA-targeted reverse transcription-quantitative PCR (BrRNA RT-qPCR) assays for identifying the bacterial pathogens that cause fever with neutropenia in pediatric cancer patients, by comparing the bacterial detection rate of this technique with that of blood culture. One milliliter of blood was collected from pediatric patients who developed fever with neutropenia following cancer chemotherapy. BrRNA RT-qPCR was performed using 16 primer sets, each designed for a specific type of bacteria. The entire BrRNA RT-qPCR procedure took less than 5 h. Blood culture was performed at the same time, following the standard institutional procedure. Blood from 13 patients was collected during 23 febrile neutropenic episodes. Of these samples, bacteria were identified in 16 by BrRNA RT-qPCR (69.6%) and in 4 by blood culture (17.4%, P<0.001). In all 4 blood culture-positive samples, BrRNA RT-qPCR detected the same type of bacteria as that identified by culture. In 9 samples, more than 4 types of bacteria were identified simultaneously by BrRNA RT-qPCR, most of which were anaerobic bacteria known to be part of the gut flora. We conclude that BrRNA RT-qPCR could be useful in the diagnosis of fever with neutropenia, given its high bacterial detection rate, short turnaround time, and the small blood sample required compared with the standard blood culture techniques. Our findings also indicate that anaerobic intestinal bacteria, which are difficult to detect by standard culture techniques, may be responsible for some cases of febrile neutropenia.
Extraction of DNA, RNA, and protein is the basic method used in molecular biology. These biomolecules can be isolated from any biological material for subsequent downstream processes, analytical, or preparative purposes. In the past, the process of extraction and purification of nucleic acids used to be complicated, time-consuming, labor-intensive, and limited in terms of overall throughput. Currently, there are many specialized methods that can be used to extract pure biomolecules, such as solution-based and column-based protocols. Manual method has certainly come a long way over time with various commercial offerings which included complete kits containing most of the components needed to isolate nucleic acid, but most of them require repeated centrifugation steps, followed by removal of supernatants depending on the type of specimen and additional mechanical treatment. Automated systems designed for medium-to-large laboratories have grown in demand over recent years. It is an alternative to labor-intensive manual methods. The technology should allow a high throughput of samples; the yield, purity, reproducibility, and scalability of the biomolecules as well as the speed, accuracy, and reliability of the assay should be maximal, while minimizing the risk of cross-contamination.
As one of human pathogens, the genome of Uropathogenic Escherichia coli strain CFT073 was sequenced and published in 2002, which was significant in pathogenetic bacterial genomics research. However, the current RefSeq annotation of this pathogen is now outdated to some degree, due to missing or misannotation of some essential genes associated with its virulence. We carried out a systematic reannotation by combining automated annotation tools with manual efforts to provide a comprehensive understanding of virulence for the CFT073 genome.
The reannotation excluded 608 coding sequences from the RefSeq annotation. Meanwhile, a total of 299 coding sequences were newly added, about one third of them are found in genomic island (GI) regions while more than one fifth of them are located in virulence related regions pathogenicity islands (PAIs). Furthermore, there are totally 341 genes were relocated with their translational initiation sites (TISs), which resulted in a high quality of gene start annotation. In addition, 94 pseudogenes annotated in RefSeq were thoroughly inspected and updated. The number of miscellaneous genes (sRNAs) has been updated from 6 in RefSeq to 46 in the reannotation. Based on the adjustment in the reannotation, subsequent analysis were conducted by both general and case studies on new virulence factors or new virulence-associated genes that are crucial during the urinary tract infections (UTIs) process, including invasion, colonization, nutrition uptaking and population density control. Furthermore, miscellaneous RNAs collected in the reannotation are believed to contribute to the virulence of strain CFT073. The reannotation including the nucleotide data, the original RefSeq annotation, and all reannotated results is freely available via http://mech.ctb.pku.edu.cn/CFT073/.
As a result, the reannotation presents a more comprehensive picture of mechanisms of uropathogenicity of UPEC strain CFT073. The new genes change the view of its uropathogenicity in many respects, particularly by new genes in GI regions and new virulence-associated factors. The reannotation thus functions as an important source by providing new information about genomic structure and organization, and gene function. Moreover, we expect that the detailed analysis will facilitate the studies for exploration of novel virulence mechanisms and help guide experimental design.
Two novel preanalysis sample treatment tools were evaluated in combination with four DNA extraction kits for the selective
isolation of bacterial DNA from whole blood. The combination of performing a preanalysis sample treatment and using a larger
sample volume increased the detection limit to 50 CFU per ml.
To enhance understanding of how Streptococcus agalactiae (group B streptococcus, GBS) adapts during invasive infection, we performed a whole-genome transcriptome analysis after incubation with whole human blood. Global changes occurred in the GBS transcriptome rapidly in response to blood contact following shift from growth in a rich laboratory medium. Most (83%) of the significantly altered transcripts were down-regulated after 30 minutes of incubation in blood, and all functional categories of genes were abundantly represented. We observed complex dynamic changes in the expression of transcriptional regulators and stress response genes that allow GBS to rapidly adapt to blood. The transcripts of relatively few proven virulence genes were up-regulated during the first 90 minutes. However, a key discovery was that genes encoding proteins involved in interaction with the host coagulation/fibrinolysis system and bacterial-host interactions were rapidly up-regulated. Extensive transcript changes also occurred for genes involved in carbohydrate metabolism, including multi-functional proteins and regulators putatively involved in pathogenesis. Finally, we discovered that an incubation temperature closer to that occurring in patients with severe infection and high fever (40 degrees C) induced additional differences in the GBS transcriptome relative to normal body temperature (37 degrees C). Taken together, the data provide extensive new information about transcriptional adaptation of GBS exposed to human blood, a crucial step during GBS pathogenesis in invasive diseases, and identify many new leads for molecular pathogenesis research.
In a controlled evaluation of 6,010 blood cultures, the yield of clinically significant microorganisms was greater from a lysis-centrifugation system (Isolator, Du Pont Co.) than from a nonvented vacuum bottle containing tryptic soy broth with sodium polyanetholesulfonate and CO2 and a vented bottle containing biphasic brain heart infusion medium with sodium polyanetholesulfonate. The Isolator significantly increased the frequency of isolation of Staphylococcus aureus and Candida spp. and significantly decreased the time required for the detection of S. aureus, Pseudomonas aeruginosa, and Candida spp.; however, anaerobic bacteria were recovered significantly more frequently from nonvented bottles with tryptic soy broth, and pneumococci were recovered significantly more frequently from both bottle systems. Contamination of cultures was significantly greater with the Isolator system than with either bottle system. Regardless of the number of blood cultures obtained per septic episode, the Isolator detected microbiologically proven bacteremia or fungemia in a significantly greater number of patients and significantly decreased the time required for detection.
The CpxA/R two-component signal transduction system of Escherichia coli can combat a variety of extracytoplasmic protein-mediated toxicities. The Cpx system performs this function, in part, by increasing the synthesis of the periplasmic protease, DegP. However, other factors are also employed by the Cpx system for this stress-combative function. In an effort to identify these remaining factors, we screened a collection of random lacZ operon fusions for those fusions whose transcription is regulated by CpxA/R. Through this approach, we have identified a new locus, cpxP, whose transcription is stimulated by activation of the Cpx pathway. cpxP specifies a periplasmic protein that can combat the lethal phenotype associated with the synthesis of a toxic envelope protein. In addition, we show that cpxP transcription is strongly induced by alkaline pH in a CpxA-dependent manner and that cpxP and cpx mutant strains display hypersensitivity to growth in alkaline conditions.
We studied two of the possible factors which can interfere with specific DNA amplification in a peripheral-blood PCR assay used for the diagnosis of human brucellosis. We found that high concentrations of leukocyte DNA and heme compounds inhibit PCR. These inhibitors can be efficiently suppressed by increasing the number of washings to four or five and decreasing the amount of total DNA to 2 to 4 microg, thereby avoiding false-negative results.
Blood-stream infections (BSI) remain a major health challenge, with an increasing incidence worldwide and a high mortality rate. Early treatment with appropriate antibiotics can reduce BSI-related morbidity and mortality, but success requires rapid identification of the infecting organisms. The rapid, culture-independent diagnosis of BSI could be significantly facilitated by straightforward isolation of highly purified bacteria from whole blood. We present a microfluidic-based, sample-preparation system that rapidly and selectively lyses all blood cells while it extracts intact bacteria for downstream analysis. Whole blood is exposed to a mild detergent, which lyses most blood cells, and then to osmotic shock using deionized water, which eliminates the remaining white blood cells. The recovered bacteria are 100 % viable, which opens up possibilities for performing drug susceptibility tests and for nucleic-acid-based molecular identification.
Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum β-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.
Copyright © 2015 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.
Modulation of Chemical Composition and Other Parameters of the Cell at Different Exponential Growth Rates, Page 1 of 2
Abstract
This review begins by briefly presenting the history of research on the chemical composition and other parameters of cells of E. coli and S. enterica at different exponential growth rates. Studies have allowed us to determine the in vivo strength of promoters and have allowed us to distinguish between factor-dependent transcriptional control of the promoter and changes in promoter activity due to changes in the concentration of free functional RNA polymerase associated with different growth conditions. The total, or bulk, amounts of RNA and protein are linked to the growth rate, because most bacterial RNA is ribosomal RNA (rRNA). Since ribosomes are required for protein synthesis, their number and their rate of function determine the rate of protein synthesis and cytoplasmic mass accumulation. Many mRNAs made in the presence of amino acids have strong ribosome binding sites whose presence reduces the expression of all other active genes. This implies that there can be profound differences in the spectrum of gene activities in cultures grown in different media that produce the same growth rate. Five classes of growth-related parameters that are generally useful in describing or establishing the macromolecular composition of bacterial cultures are described in detail in this review. A number of equations have been reported that describe the macromolecular composition of an average cell in an exponential culture as a function of the culture doubling time and five additional parameters: the C- and D-periods, protein per origin (PO), ribosome activity, and peptide chain elongation rate.
Absolute neutrophil count (ANC) is used clinically to monitor physiological dysfunctions such as myelosuppression or infection. In the research laboratory, ANC is a valuable measure to monitor the evolution of a wide range of disease states in disease models. Flow cytometry (FCM) is a fast, widely used approach to confidently identify thousands of cells within minutes. FCM can be optimised for absolute counting using spiked-in beads or by measuring the sample volume analysed. Here we combine the 1A8 antibody, specific for the mouse granulocyte protein Ly6G, with flow cytometric counting in straightforward FCM assays for mouse ANC, easily implementable in the research laboratory. Volumetric and Trucount™ bead assays were optimized for mouse neutrophils, and ANC values obtained with these protocols were compared to ANC measured by a dual-platform assay using the Orphee Mythic 18 veterinary haematology analyser. The single platform assays were more precise with decreased intra-assay variability compared with ANC obtained using the dual protocol. Defining ANC based on Ly6G expression produces a 15% higher estimate than the dual protocol. Allowing for this difference in ANC definition, the flow cytometry counting assays using Ly6G can be used reliably in the research laboratory to quantify mouse ANC from a small volume of blood. We demonstrate the utility of the volumetric protocol in a time-course study of chemotherapy induced neutropenia using four drug regimens. © 2014 International Society for Advancement of Cytometry
ABSTRACT: Bacterial bloodstream infections (BSI) and ensuing sepsis are important causes of morbidity and mortality. Early diagnosis and rapid treatment with appropriate antibiotics are vital for improving outcome. Nucleic acid amplification of bacteria directly from whole blood has the potential of providing a faster means of diagnosing BSI than automated blood culture. However, effective DNA extraction of commonly low levels of bacterial target from whole blood is critical for this approach to be successful. This study compared the Molzyme MolYsis™ Complete5 DNA extraction method to a previously described organic bead-based method for use with whole blood. A well-characterized S. aureus-induced pneumonia model of sepsis in canines was used to provide clinically relevant whole blood samples. DNA extracts were assessed for purity and concentration and analyzed for bacterial rRNA gene targets using PCR and sequence-based identification. Both extraction methods yielded relatively pure DNA with median A260/280 absorbance ratios of 1.71 (MolYsis™) and 1.97 (bead-based). The organic bead-based extraction method yielded significantly higher average DNA concentrations (P < 0.05) at each time point throughout the experiment, closely correlating with changes observed in white blood cell (WBC) concentrations during this same time period, while DNA concentrations of the MolYsis™ extracts closely mirrored quantitative blood culture results. Overall, S. aureus DNA was detected from whole blood samples in 70.7% (58/82) of MolYsis™ DNA extracts, and in 59.8% (49/82) of organic bead-based extracts, with peak detection rates seen at 48 h for both MolYsis™ (87.0%) and organic bead-based (82.6%) methods. In summary, the MolYsis™ Complete5 DNA extraction kit proved to be the more effective method for isolating bacterial DNA directly from extracts made from whole blood.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-DeltaDeltaCr) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-DeltaDeltaCr) method. In addition, we present the derivation and applications of two variations of the 2(-DeltaDeltaCr) method that may be useful in the analysis of real-time, quantitative PCR data. (C) 2001 Elsevier science.
We demonstrate a novel assay for physicochemical extraction and isotachophoresis-based purification of 16S rRNA from whole human blood infected with Pseudomonas putida . This on-chip assay is unique in that the extraction can be automated using isotachophoresis in a simple device with no moving parts, it protects RNA from degradation when isolating from ribonuclease-rich matrices (such as blood), and produces a purified total nucleic acid sample that is compatible with enzymatic amplification assays. We show that the purified RNA is compatible with reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and demonstrate a clinically relevant sensitivity of 0.03 bacteria per nanoliter using RT-qPCR.
Polymerase chain reaction (PCR) has been used with increasing frequency to diagnose infectious and genetic diseases. In this study, the effects of heparin on PCR were investigated, because heparinized blood may sometimes be used in PCR studies. HLA-DQA1 gene amplification was used as a model. PCR was clearly interfered with when heparinized blood was used as a source of template DNA, and the degree of interference was affected by the following three factors; (1) type of Taq DNA polymerase; (2) leukocyte count in blood; and (3) concentration of heparin contained. When additional tests were conducted with additions of definite heparin concentrations to a PCR reaction mixture, specimens with large amounts of DNA tended to exhibit less interference by heparin. The addition of ≥ 0.1 to 0.0016 U of heparin per reaction mixture (50 μl) suppressed DNA amplification in a dose-dependent fashion. We therefore concluded that much care should be taken when heparinized blood is used as a PCR material. J. Clin. Lab. Anal. 13:133–140, 1999. © 1999 Wiley-Liss, Inc.
Escherichia coli encounter numerous different stresses during their growth, survival, and infection. These stresses are relevant to survival in foods and food processing environments. E. coli and other bacteria respond to stress conditions by activating small or large groups of genes under the control of common regulator proteins. Stress conditions result in the accumulation of these regulator proteins and subsequent transcription of many genes allows cells to cope with specific stress situations, conferring stress tolerance and survival. In addition, induced stress tolerance of cells is attributed to enhanced virulence and enhanced tolerance to other stresses (cross-protection). In this review, regulation of stress and the stress tolerance response of E. coli to heat, acid, starvation, and cold stresses that are commonly used in food preservation and food production will be addressed. The effect of different stress on survival, adaptation, and cross-protection of E. coli studied using laboratory media, and real foods will be briefly summarized. Finally, the relationship of stress response and subsequent virulence and cross-protection will also be discussed.
The emerging technique of microfluidic digital PCR (dPCR) offers a unique approach to real-time quantitative PCR for measuring nucleic acids that may be particularly suited for low-level detection. In this study, we evaluated the quantitative capabilities of dPCR when measuring small amounts (<200 copies) of DNA and investigated parameters influencing technical performance. We used various DNA templates, matrixes, and assays to evaluate the precision, sensitivity and reproducibility of dPCR, and demonstrate that this technique can be highly reproducible when performed at different times and when different primer sets are targeting the same molecule. dPCR exhibited good analytical sensitivity and was reproducible outside the range recommended by the instrument manufacturer; detecting 16 estimated targets with high precision. The inclusion of carrier had no effect on this estimated quantity, but did improve measurement precision. We report disagreement when using dPCR to measure different template types and when comparing the estimated quantities by dPCR and UV spectrophotometry. Finally, we also demonstrate that preamplification can impose a significant measurement bias. These findings provide an independent assessment of low copy molecular measurement using dPCR and underline important factors for consideration in dPCR experimental design.
Clin Microbiol Infect 2011; 17: 451–458
The Health Protection Agency in England operates a voluntary surveillance system that collects data on bacteraemias reported by over 90% of laboratories in England. Trends in causative microorganisms reported between 2004 and 2008 were analyzed using a generalized linear model with a log link function for Poisson distribution. In 2008, 101 276 episodes of bacteraemia were reported; a rate of 189 per 100 000 population. More than one-half occurred in those aged over 65 years and males. The most common organisms reported were Escherichia coli (23%), coagulase-negative staphylococci (CNS) (16.9%) and Staphylococcus aureus (11.4%). Between 2004 and 2008, E. coli bacteraemia increased by 33% (p <0.001); the species now accounts for more than 30% of bacteraemia in those aged over 75 years. There also were significant increases in bacteraemia caused by other Gram-negative pathogens and marked seasonal variation. Bacteraemia caused by S. aureus increased until 2005, with a decline after 2006 (p <0.001) entirely due to methicillin-resistant strains. CNS bacteraemia have declined significantly since 2007. The renewed dominance of Gram-negative pathogens as major causes of bacteraemia in England is of particular concern because they are associated with a high morbidity and increasing resistance to antibiotics. Further investigation of the underlying causes and prevention strategies is a public health priority. Recent declines in methicillin-resistant S. aureus bacteraemia have not been reflected in other pathogens, including methicillin-susceptible S. aureus.
microRNAs are small regulatory RNAs that are currently emerging as new biomarkers for cancer and other diseases. In order for biomarkers to be useful in clinical settings, they should be accurately and reliably detected in clinical samples such as formalin fixed paraffin embedded (FFPE) sections and blood serum or plasma. These types of samples represent a challenge in terms of microRNA quantification. A newly developed method for microRNA qPCR using Locked Nucleic Acid (LNA)-enhanced primers enables accurate and reproducible quantification of microRNAs in scarce clinical samples. Here we show that LNA-based microRNA qPCR enables biomarker screening using very low amounts of total RNA from FFPE samples and the results are compared to microarray analysis data. We also present evidence that the addition of a small carrier RNA prior to total RNA extraction, improves microRNA quantification in blood plasma and laser capture microdissected (LCM) sections of FFPE samples.
The purpose of the study was to determine the independent risk factors on mortality in patients with community-acquired severe sepsis and septic shock.
A single-site prospective cohort study was carried out in a medical-surgical intensive care unit in an academic tertiary care center. One hundred twelve patients with community-acquired bloodstream infection with severe sepsis and septic shock were identified. Clinical, microbiologic, and laboratory parameters were compared between hospital survivors and hospital deaths.
One-hundred twelve patients were included. The global mortality rate was 41.9%, 44.5% in septic shock and 34.4% in severe sepsis. One or more comorbidities were present in 66% of patients. The most commonly identified bloodstream pathogens were Escherichia coli (25%) and Staphylococcus aureus (21.4%). The proportion of patients receiving inadequate antimicrobial treatment was 8.9%. By univariate analysis, age, Acute Physiology and Chronic Health Evaluation II score, at least 3 organ dysfunctions, and albumin, but neither microbiologic characteristics nor site of infection, differed significantly between survivors and nonsurvivors. Acute Physiology and Chronic Health Evaluation II (odds ratio, 1.13; 95% confidence interval, 1.06-1.21) and albumin (odds ratio, 0.34; 95% confidence interval, 0.15-0.76) were independent risk factors associated with global mortality in logistic regression analysis.
In addition to the severity of illness, hypoalbuminemia was identified as the most important prognostic factor in community-acquired bloodstream infection with severe sepsis and septic shock.
The phage shock protein (psp) operon of Escherichia coli is strongly induced in response to heat, ethanol, osmotic shock, and infection by filamentous bacteriophages. The operon contains at least four genes--pspA, pspB, pspC, and pspE--and is regulated at the transcriptional level. We report here that psp expression is controlled by a network of positive and negative regulatory factors and that transcription in response to all inducing agents is directed by the sigma-factor sigma 54. Negative regulation is mediated by both PspA and the sigma 32-dependent heat shock proteins. The PspB and PspC proteins cooperatively activate expression, possibly by antagonizing the PspA-controlled repression. The strength of this activation is determined primarily by the concentration of PspC, whereas PspB enhances but is not absolutely essential for PspC-dependent expression. PspC is predicted to contain a leucine zipper, a motif responsible for the dimerization of many eukaryotic transcriptional activators. PspB and PspC, though not necessary for psp expression during heat shock, are required for the strong psp response to phage infection, osmotic shock, and ethanol treatment. The psp operon thus represents a third category of transcriptional control mechanisms, in addition to the sigma 32- and sigma E-dependent systems, for genes induced by heat and other stresses.
Bacterial counts in blood of E. coli (number of viable organisms per milliliter) are reported for 30 neonates whowere cultured because of suspected septicemia. Fifteen of the infants died; 13 of the deaths occurred within 48 hours after cultures were obtained and appropriate antibiotic therapy had been initiated. Thirty-five positive cultures were obtained from the 30 neonates; in 11 (31%) of the cultures the colony counts were in excess of 1,000/ml; in 5 (14%), the counts ranged from 50 to 1,000/ml; in 11 (31%), from 5 to 49/ml; in 8 (23%), from 0 to 4/ml. Seventy-three per cent (8/11) of the neonates with colony counts greater than 1,000/ml died.
A range of techniques for efficient removal of damaged cells, cell debris and erythroid cells from lymphoid cell suspensions is presented. They are simple, reproducible, and give almost complete recovery of intact lymphoid cells with maintenance of biological activity. The procedures are a zonal setting and spinning procedure for removal of very fine and very coarse debris; density separations in albumin media for elimination of damaged cells (at pH 5.1) or damaged cells and erythroid cells (pH 7.2); NH4Cl treatment for elimination of erythroid cells alone. Other applications are discussed including a simple procedure for separation of cells of any given density.
In 206 cases of bacterial endocarditis with positive blood cultures, 95% of 789 blood cultures were positive for the causative microorganisms. In streptococcal endocarditis, the first blood culture was positive in 96% of the cases, and one of the first two blood cultures was positive in 98% of the cases. In endocarditis caused by microorganisms other than streptococci, the first blood culture was positive in 82% of the cases, and one of the first two blood cultures was positive in all of the cases. The incidence of positive cultures was slightly reduced in those cases in which the patient had received an antimicrobial agent within two weeks prior to blood culture. The bacteremia in the 206 cases of bacterial endocarditis was persistent and of low magnitude.
A novel cationic surfactant solution is used to lyse blood cells and precipitate RNA and DNA. The RNA is recovered by extracting the pellet with a small volume of guanidinium isothiocyanate or formamide.
The heme compound found in deoxyribonucleic acid (DNA) extracted from bloodstains, which is regarded as a major inhibitor of polymerase chain reaction (PCR), was characterized in comparison with alkaline and acid hematin, histidine and ammonia hemochromogens, and globin and serum albumin hemochromogens digested by proteinase K. Alkaline and acid hematin were almost completely removed by phenol/chloroform treatment and ethanol precipitation, so as not to be copurified with DNA from the specimens. Spectrophotometric results indicated that the contaminant was likely to be the product of proteinase K digestion of some heme-blood protein complex, which was not completely extracted by organic solvents and remained in the ethanol precipitates of DNA. The results of polyacrylamide gradient gel electrophoresis and intensity of the inhibition of PCR suggested that the ligand of the contaminant was a somewhat large molecule, resistant to the proteolysis by proteinase K. The addition of bovine serum albumin to the reaction mixture prevented the inhibition of PCR by the heme compounds, probably by binding to the heme. This showed that the inhibition was not due to the irreversible inactivation of the enzyme.
The objectives of this study were to (1) describe the epidemiology and microbiology of community-acquired bacteremia; (2) determine the crude mortality associated with such infections; and (3) identify independent predictors of mortality. All patients with clinically significant community-acquired bacteremia admitted to a university-affiliated Veterans Affairs medical center from January 1994 through December 1997 were evaluated. During the study period, 387 bacteremic episodes occurred in 334 patients. Staphylococcus aureus, Escherichia coli, and coagulase-negative staphylococci were the most commonly isolated organisms; the most frequent sources were the urinary tract and intravascular catheters. Approximately 14% of patients died. Patient characteristics independently associated with increased mortality included shock (OR 3.7, p = 0.02) and renal failure (OR 4.0, p = 0.003). The risk of death was also higher in those whose source was pneumonia (OR 6.3, p = 0.03) or an intra-abdominal site (OR 10.7, p = 0.02), or if multiple sources were identified (OR 13.4, p = 0.003). Community-acquired bacteremia is often device-related and may be preventable. Strategies that have been successful in preventing nosocomial device-related bacteremia should be adapted to the outpatient setting.
The two most commonly used methods to analyze data from real-time, quantitative PCR experiments are absolute quantification and relative quantification. Absolute quantification determines the input copy number, usually by relating the PCR signal to a standard curve. Relative quantification relates the PCR signal of the target transcript in a treatment group to that of another sample such as an untreated control. The 2(-Delta Delta C(T)) method is a convenient way to analyze the relative changes in gene expression from real-time quantitative PCR experiments. The purpose of this report is to present the derivation, assumptions, and applications of the 2(-Delta Delta C(T)) method. In addition, we present the derivation and applications of two variations of the 2(-Delta Delta C(T)) method that may be useful in the analysis of real-time, quantitative PCR data.
Much of the information available about factors that affect mRNA decay in Escherichia coli, and by inference in other bacteria, has been gleaned from study of less than 25 of the approximately 4,300 predicted E. coli messages. To investigate these factors more broadly, we examined the half-lives and steady-state abundance of known and predicted E. coli mRNAs at single-gene resolution by using two-color fluorescent DNA microarrays. An rRNA-based strategy for normalization of microarray data was developed to permit quantitation of mRNA decay after transcriptional arrest by rifampicin. We found that globally, mRNA half-lives were similar in nutrient-rich media and defined media in which the generation time was approximately tripled. A wide range of stabilities was observed for individual mRNAs of E. coli, although approximately 80% of all mRNAs had half-lives between 3 and 8 min. Genes having biologically related metabolic functions were commonly observed to have similar stabilities. Whereas the half-lives of a limited number of mRNAs correlated positively with their abundance, we found that overall, increased mRNA stability is not predictive of increased abundance. Neither the density of putative sites of cleavage by RNase E, which is believed to initiate mRNA decay in E. coli, nor the free energy of folding of 5' or 3' untranslated region sequences was predictive of mRNA half-life. Our results identify previously unsuspected features of mRNA decay at a global level and also indicate that generalizations about decay derived from the study of individual gene transcripts may have limited applicability.
Accurate quantification of mRNA in whole blood is made difficult by the simultaneous degradation of gene transcripts and unintended gene induction caused by sample handling or uncontrolled activation of coagulation. This study was designed to compare a new blood collection tube (PAXgene Blood RNA System) and a companion sample preparation reagent set with a traditional sample collection and preparation method for the purpose of gene expression analysis.
We collected parallel blood samples from healthy donors into the new sample collection tubes and control EDTA tubes and performed serial RNA extractions on samples stored for 5 days at room temperature and for up to 90 days at 4 and 20 degrees C. Samples were analyzed by Northern blot analysis or reverse transcription-PCR (RT-PCR).
Specific mRNA concentrations in blood stored in EDTA tubes at any temperature changed substantially, as determined by high-precision RT-PCR. These changes were eliminated or markedly reduced when whole blood was stored in PAXgene tubes. Loss of specific mRNAs, as measured by RT-PCR, reflected total RNA depletion as well as specific mRNA destruction demonstrated by Northern blot analysis. The salutary effects of PAXgene on mRNA stabilization extended to blood samples from eight unrelated donors.
Compared with whole blood collected in EDTA tubes and extracted by an organic method, the PAXgene Blood RNA System reduced RNA degradation and inhibited or eliminated gene induction in phlebotomy whole blood samples. Storage of whole blood samples in PAXgene tubes can be recommended for clinically related blood samples that will be analyzed for total or specific RNA content.