Biofilm formation by nontypeable Haemophilus influenzae: strain variability, outer membrane antigen expression and role of pili

Background

Detecting microbial life in extraterrestrial locations is a goal of space exploration because of ecological and health concerns about possible contamination of other planets with earthly organisms, and vice versa. Previously we suggested a method for life detection based on the fact that living entities require a continual input of energy accessed through coupled oxidations and reductions (an electron transport chain). We demonstrated using earthly soils that the identification of extracted components of electron transport chains is useful for remote detection of a chemical signature of life. The instrument package developed used supercritical carbon dioxide for soil extraction, followed by chromatography or electrophoresis to separate extracted compounds, with final detection by voltammetry and tandem mass-spectrometry.

Results

Here we used Earth-derived soils to develop a related life detection system based on direct observation of a biological redox signature. We measured the ability of soil microbial communities to reduce artificial electron acceptors. Living organisms in pure culture and those naturally found in soil were shown to reduce 2,3-dichlorophenol indophenol (DCIP) and the tetrazolium dye 2,3-bis(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide inner salt (XTT). Uninoculated or sterilized controls did not reduce the dyes. A soil from Antarctica that was determined by chemical signature and DNA analysis to be sterile also did not reduce the dyes.

Conclusion

Observation of dye reduction, supplemented with extraction and identification of only a few specific signature redox-active biochemicals such as porphyrins or quinones, provides a simplified means to detect a signature of life in the soils of other planets or their moons.     

Comprehensive screening method for the qualitative detection of narcotics and stimulants using single step derivatisation

A selective and sensitive screening method for the detection of prohibited narcotic and stimulating agents in doping control is described and validated. This method is suitable for the detection of all narcotic agents mentioned on the World Anti-Doping Agency (WADA) doping list in addition to numerous stimulants. The analytes are extracted from urine by a combined extraction procedure using CH(2)Cl(2)/MeOH (9/1, v/v) and t-butylmethyl ether as extraction solvents at pH 9.5 and 14, respectively. Prior to GC-MS analysis the obtained residues are combined and derivatised with MSTFA. The mass spectrometer is operated in the full scan mode in the range between m/z 40 and 550. The obtained limits of detection (LOD) for all components included in this extensive screening method are in the range 20-500 ng/ml, which is in compliance with the requirements set by WADA. Besides narcotic and stimulating agents, this method is also capable of detecting several agents with anti-estrogenic activity and some beta-agonists. As an example, a positive identification of hydroxyl-methoxy-tamoxyfen is shown.     

Multicenter Evaluation of the Bruker MALDI Biotyper CA System for the Identification of Clinical Aerobic Gram-Negative Bacterial Isolates

The prompt and accurate identification of bacterial pathogens is fundamental to patient health and outcome. Recent advances in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) have revolutionized bacterial identification in the clinical laboratory, but uniform incorporation of this technology in the U.S. market has been delayed by a lack of FDA-cleared systems. In this study, we conducted a multicenter evaluation of the MALDI Biotyper CA (MBT-CA) System (Bruker Daltonics Inc, Billerica, MA) for the identification of aerobic gram-negative bacteria as part of a 510(k) submission to the FDA. A total of 2,263 aerobic gram negative bacterial isolates were tested representing 23 genera and 61 species. Isolates were collected from various clinical sources and results obtained from the MBT-CA System were compared to DNA sequencing and/or biochemical testing. Isolates that failed to report as a "high confidence species ID" [log(score) ≥2.00] were re-tested using an extraction method. The MBT-CA System identified 96.8% and 3.1% of isolates with either a "high confidence" or a "low confidence" [log(score) value between 1.70 and <2.00] species ID, respectively. Two isolates did not produce acceptable confidence scores after extraction. The MBT-CA System correctly identified 99.8% (2,258/2,263) to genus and 98.2% (2,222/2,263) to species level. These data demonstrate that the MBT-CA System provides accurate results for the identification of aerobic gram-negative bacteria.     

Modular Spectral Imaging System for Discrimination of Pigments in Cells and Microbial Communities

Here we describe a spectral imaging system for minimally invasive identification, localization, and relative quantification of pigments in cells and microbial communities. The modularity of the system allows pigment detection on spatial scales ranging from the single-cell level to regions whose areas are several tens of square centimeters. For pigment identification in vivo absorption and/or autofluorescence spectra are used as the analytical signals. Along with the hardware, which is easy to transport and simple to assemble and allows rapid measurement, we describe newly developed software that allows highly sensitive and pigment-specific analyses of the hyperspectral data. We also propose and describe a number of applications of the system for microbial ecology, including identification of pigments in living cells and high-spatial-resolution imaging of pigments and the associated phototrophic groups in complex microbial communities, such as photosynthetic endolithic biofilms, microbial mats, and intertidal sediments. This system provides new possibilities for studying the role of spatial organization of microorganisms in the ecological functioning of complex benthic microbial communities or for noninvasively monitoring changes in the spatial organization and/or composition of a microbial community in response to changing environmental factors.     

The zebrafish (Danio rerio) embryo as a model system for identification and characterization of developmental toxins from marine and freshwater microalgae

The zebrafish (Danio rerio) embryo has emerged as an important model of vertebrate development. As such, this model system is finding utility in the investigation of toxic agents that inhibit, or otherwise interfere with, developmental processes (i.e. developmental toxins), including compounds that have potential relevance to both human and environmental health, as well as biomedicine. Recently, this system has been applied increasingly to the study of microbial toxins, and more specifically, as an aquatic animal model, has been employed to investigate toxins from marine and freshwater microalgae, including those classified among the so-called "harmful algal blooms" (HABs). We have developed this system for identification and characterization of toxins from cyanobacteria (i.e. "blue-green algae") isolated from the Florida Everglades and other freshwater sources in South and Central Florida. Here we review the use of this system as it has been applied generally to the investigation of toxins from marine and freshwater microalgae, and illustrate this utility as we have applied it to the detection, bioassay-guided fractionation and subsequent characterization of developmental toxins from freshwater cyanobacteria.     

基质辅助激光解析电离飞行时间质谱在医学真菌领域的应用进展

基质辅助激光解析电离飞行时间质谱（matrix-assisted laser desorption/ionization time-of-flight mass spectrometry,MALDI-TOF MS）是近年来新兴的微生物检测技术,通过核糖体蛋白分析实现对真菌快速、准确鉴定。本文针对MALDI-TOF MS用于致病真菌鉴定、分类、体外抗真菌药物敏感性检测以及临床微生物样本直接检测等方面作一综述。     

An integrated biosensor platform for extraction and detection of nucleic acids

The development of portable systems for analysis of nucleic acids (NAs) is crucial for the evolution of biosensing in the context of future healthcare technologies. The integration of NA extraction, purification, and detection modules, properly actuated by microfluidics technologies, is a key point for the development of portable diagnostic systems. In this paper, we describe an integrated biosensor platform based on a silicon–plastic hybrid lab-on-disk technology capable of managing NA extraction, purification, and detection processes in an integrated format. The sample preparation process is performed by solid-phase extraction technology using magnetic beads on a plastic disk, while detection is done through quantitative real-time polymerase chain reaction (qRT-PCR) on a miniaturized silicon device. The movement of sample and reagents is actuated by a centrifugal force induced by a disk actuator instrument. The assessment of the NA extraction and detection performance has been carried out by using hepatitis B virus (HBV) DNA genome as a biological target. The quantification of the qRT-PCR chip in the hybrid disk showed an improvement in sensitivity with respect to the qRT-PCR commercial platforms, which means an optimization of time and cost. Limit of detection and limit of quantification values of about 8 cps/reaction and 26 cps/reaction, respectively, were found by using analytical samples (synthetic clone), while the results with real samples (serum with spiked HBV genome) indicate that the system performs as well as the standard methods.     

Rapid and Sensitive Detection of Yersinia pestis Using Amplification of Plague Diagnostic Bacteriophages Monitored by Real-Time PCR

Background

Yersinia pestis, the agent of plague, has caused many millions of human deaths and still poses a serious threat to global public health. Timely and reliable detection of such a dangerous pathogen is of critical importance. Lysis by specific bacteriophages remains an essential method of Y. pestis detection and plague diagnostics.

Methodology/Principal Findings

The objective of this work was to develop an alternative to conventional phage lysis tests – a rapid and highly sensitive method of indirect detection of live Y. pestis cells based on quantitative real-time PCR (qPCR) monitoring of amplification of reporter Y. pestis-specific bacteriophages. Plague diagnostic phages ϕA1122 and L-413C were shown to be highly effective diagnostic tools for the detection and identification of Y. pestis by using qPCR with primers specific for phage DNA. The template DNA extraction step that usually precedes qPCR was omitted. ϕA1122-specific qPCR enabled the detection of an initial bacterial concentration of 10³ CFU/ml (equivalent to as few as one Y. pestis cell per 1-µl sample) in four hours. L-413C-mediated detection of Y. pestis was less sensitive (up to 100 bacteria per sample) but more specific, and thus we propose parallel qPCR for the two phages as a rapid and reliable method of Y. pestis identification. Importantly, ϕA1122 propagated in simulated clinical blood specimens containing EDTA and its titer rise was detected by both a standard plating test and qPCR.

Conclusions/Significance

Thus, we developed a novel assay for detection and identification of Y. pestis using amplification of specific phages monitored by qPCR. The method is simple, rapid, highly sensitive, and specific and allows the detection of only live bacteria.     

Clinical validation of integrated nucleic acid and protein detection on an electrochemical biosensor array for urinary tract infection diagnosis

Background

Urinary tract infection (UTI) is a common infection that poses a substantial healthcare burden, yet its definitive diagnosis can be challenging. There is a need for a rapid, sensitive and reliable analytical method that could allow early detection of UTI and reduce unnecessary antibiotics. Pathogen identification along with quantitative detection of lactoferrin, a measure of pyuria, may provide useful information towards the overall diagnosis of UTI. Here, we report an integrated biosensor platform capable of simultaneous pathogen identification and detection of urinary biomarker that could aid the effectiveness of the treatment and clinical management.

Methodology/Principal Findings

The integrated pathogen 16S rRNA and host lactoferrin detection using the biosensor array was performed on 113 clinical urine samples collected from patients at risk for complicated UTI. For pathogen detection, the biosensor used sandwich hybridization of capture and detector oligonucleotides to the target analyte, bacterial 16S rRNA. For detection of the protein biomarker, the biosensor used an analogous electrochemical sandwich assay based on capture and detector antibodies. For this assay, a set of oligonucleotide probes optimized for hybridization at 37°C to facilitate integration with the immunoassay was developed. This probe set targeted common uropathogens including E. coli, P. mirabilis, P. aeruginosa and Enterococcus spp. as well as less common uropathogens including Serratia, Providencia, Morganella and Staphylococcus spp. The biosensor assay for pathogen detection had a specificity of 97% and a sensitivity of 89%. A significant correlation was found between LTF concentration measured by the biosensor and WBC and leukocyte esterase (p<0.001 for both).

Conclusion/Significance

We successfully demonstrate simultaneous detection of nucleic acid and host immune marker on a single biosensor array in clinical samples. This platform can be used for multiplexed detection of nucleic acid and protein as the next generation of urinary tract infection diagnostics.     

Characterization of a methane‐utilizing strain and its application for monitoring methane

Aims: To explore new resources of methane‐utilizing micro‐organism and develop a microbial biosensing system for monitoring methane released from natural and semi‐natural ecosystems. Methods and Results: A methane (CH₄)‐utilizing bacterial strain was isolated from paddy soil using CH₄ as the sole carbon source and identified as Klebsiella sp. ME17 by phenotyping and 16S rDNA sequence analysis. The efficiency of CH₄ utilization of strain ME17 was 83·2% by gas chromatography analysis. A microbial biosensing system for CH₄ detection was developed by combining immobilized cells of strain ME17 with a dissolved oxygen sensor. It was found that response time of the system to CH₄ was <90s. The dissolved O₂ consumption increased with increasing CH₄ from 0% to 16·0% (v/v) demonstrating a positive linear relationship with a low detection limit of 0·2% (v/v). The relative standard deviation is 3·48%. Conclusions: Klebsiella sp. ME17 isolate is capable of utilizing CH₄. The microbial biosensing system of strain ME17 has been successfully applied to measure standard CH₄ sample with satisfactory results. Significance and Impact of the Study: This study suggests that certain strains of Klebsiella genus are capable of utilizing CH₄. Our proposed method appears very attractive for CH₄ measurement in coal mine.     

Fatty Acid Methyl Ester (FAME) Succession in Different Substrates as Affected by the Co-Application of Three Pesticides

Introduction

In intensive agriculture areas the use of pesticides can alter soil properties and microbial community structure with the risk of reducing soil quality.

Materials and Methods

In this study the fatty acid methyl esters (FAMEs) evolution has been studied in a factorial lab experiment combining five substrates (a soil, two aged composts and their mixtures) treated with a co-application of three pesticides (azoxystrobin, chlorotoluron and epoxiconazole), with two extraction methods, and two incubation times (0 and 58 days). FAMEs extraction followed the microbial identification system (MIDI) and ester-linked method (EL).

Results and Discussion

The pesticides showed high persistence, as revealed by half-life (t_1/2) values ranging from 168 to 298 days, which confirms their recalcitrance to degradation. However, t_1/2 values were affected by substrate and compost age down to 8 days for chlorotoluron in S and up to 453 days for epoxiconazole in 12M. Fifty-six FAMEs were detected. Analysis of variance (ANOVA) showed that the EL method detected a higher number of FAMEs and unique FAMEs than the MIDI one, whereas principal component analysis (PCA) highlighted that the monosaturated 18:1ω9c and cyclopropane 19:0ω10c/19ω6 were the most significant FAMEs grouping by extraction method. The cyclopropyl to monoenoic acids ratio evidenced higher stress conditions when pesticides were applied to compost and compost+soil than solely soil, as well as with final time.

Conclusion

Overall, FAMEs profiles showed the importance of the extraction method for both substrate and incubation time, the t_1/2 values highlighted the effectiveness of solely soil and the less mature compost in reducing the persistence of pesticides.     

Determination of morphine in cerebrospinal fluid and plasma by high-performance liquid chromatography with electrochemical detection

Two methods for the extraction of morphine from cerebrospinal fluid or plasma with quantitation by high-performance liquid chromatography with electrochemical detection were compared for accuracy, precision and ease of preparation. One procedure was a standard extraction procedure and the other utilized a commercially available liquid—liquid extraction column. Both methods produced linear calibration curves over the concentration range of 1–200 ng/ml with coefficients of correlation of 0.999. Since the electrochemical detector is capable of detecting 20 pg of morphine, biological samples as small as 0.1 to 0.4 ml can be quantified with an average relative precision of 4.1 ± 3.9% over the concentration range 1–200 ng/ml. The potential clinical importance of the assay is demonstrated using a time course distribution study of morphine in the cerebrospinal fluid and plasma of a Rhesus monkey.     

Roadmap to approval: use of an automated sterility test method as a lot release test for Carticel, autologous cultured chondrocytes

BACKGROUND: In February 2004, FDA approved a supplement to our biologics license for Carticel, autologous cultured chondrocytes, to use the BacT/ALERT microbial detection system as an alternative to the compendial sterility test for lot release. This article provides a roadmap to our approval process. The approval represents more than 4 years of development and validation studies comparing the Steritest compact system to the BacT/ALERT microbial detection system. METHODS: For this study, freshly cultured chondrocytes were prepared from a characterized cell bank. Microbial isolates were prepared from either American Type Culture Collection (ATCC) strains or from in-house contaminants. For each test condition, a suspension of chondrocyte cells and test organisms was inoculated into both aerobic media (SA standard adult culture bottles, FA FAN, tryptic soy broth) and anaerobic media (SN standard adult culture bottles, FN FAN, fluid thioglycollate media) and tested for sterility using the Steritest compact system (Millipore, Bedford, MA, USA) and the BacT/ALERT microbial detection system (bioMerieux, Durham, NC, USA). Negative control bottles were inoculated with chondrocytes and no microorganisms. All bottles were incubated for 14 days and read daily. Bacterial growth was determined by either visual examination of Steritest canisters or detection of a positive by the BacT/ALERT system. A gram stain and streak plate were used to confirm positive bottles and negative bottles after 14 days. RESULTS: The detection of a positive by either the Steritest compact system or the BacT/ALERT system was summarized for each organism in each validation study. Data generated from studies reducing the incubation temperature from 35 degrees C to 32 degrees C improved detection times in the automated method compared with the compendial method. Other improvements included the use of FAN aerobic and anaerobic media to absorb the gentamicin contained in the culture media of prepared chondrocyte samples. Chondrocytes alone did not generate positive results in either the compendial method or the automated method. DISCUSSION: Data from validation studies support the use of the BacT/ALERT microbial detection system as an alternative sterility test for Carticel.     

Use of 16S rRNA Gene for Identification of a Broad Range of Clinically Relevant Bacterial Pathogens

According to World Health Organization statistics of 2011, infectious diseases remain in the top five causes of mortality worldwide. However, despite sophisticated research tools for microbial detection, rapid and accurate molecular diagnostics for identification of infection in humans have not been extensively adopted. Time-consuming culture-based methods remain to the forefront of clinical microbial detection. The 16S rRNA gene, a molecular marker for identification of bacterial species, is ubiquitous to members of this domain and, thanks to ever-expanding databases of sequence information, a useful tool for bacterial identification. In this study, we assembled an extensive repository of clinical isolates (n = 617), representing 30 medically important pathogenic species and originally identified using traditional culture-based or non-16S molecular methods. This strain repository was used to systematically evaluate the ability of 16S rRNA for species level identification. To enable the most accurate species level classification based on the paucity of sequence data accumulated in public databases, we built a Naïve Bayes classifier representing a diverse set of high-quality sequences from medically important bacterial organisms. We show that for species identification, a model-based approach is superior to an alignment based method. Overall, between 16S gene based and clinical identities, our study shows a genus-level concordance rate of 96% and a species-level concordance rate of 87.5%. We point to multiple cases of probable clinical misidentification with traditional culture based identification across a wide range of gram-negative rods and gram-positive cocci as well as common gram-negative cocci.     

Applications of whole-cell matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry in systematic microbiology

In the last few years matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been increasingly studied and applied for the identification and typing of microorganisms. Very recently, MALDI-TOF MS has been introduced in clinical routine microbiological diagnostics with marked success, which is remarkable considering that not long ago the technology was generally seen as being far from practical application. The identification of microbial isolates by whole-cell mass spectrometry (WC-MS) is being recognized as one of the latest tools forging a revolution in microbial diagnostics, with the potential of bringing to an end many of the time-consuming and man-power-intensive identification procedures that have been used for decades. Apart from applications of WC-MS in clinical diagnostics, other fields of microbiology also have adopted the technology with success. In this article, an over-view of the principles of MALDI-TOF MS and WC-MS is presented, highlighting the characteristics of the technology that allow its utilization for systematic microbiology.     

A review: recent advances and future prospects of taxol-producing endophytic fungi

In the urgent search for more effective ways to treat cancer, new extraction methods of taxol from endophytic fungus have demonstrated high potential in increasing the efficiency of taxol extraction for more efficient and sustainable production of taxol and cancer treatment products. This paper summarizes recent advances in taxol-producing endophytic fungi, both in China and abroad, in the following areas: isolation and identification of endophytic fungi types, extraction and detection methods of endophytic taxol in plants, and improved efficiency of the extraction process. With the advancement of science and technology, new techniques in biotechnology, such as fungal strain improvement and recombining technique and microbial fermentation engineering, have increased the extraction yield from taxol-producing fungi, thereby improved the overall efficiency of taxol production.     

Rapid filtration separation-based sample preparation method for Bacillus spores in powdery and environmental matrices

Authorities frequently need to analyze suspicious powders and other samples for biothreat agents in order to assess environmental safety. Numerous nucleic acid detection technologies have been developed to detect and identify biowarfare agents in a timely fashion. The extraction of microbial nucleic acids from a wide variety of powdery and environmental samples to obtain a quality level adequate for these technologies still remains a technical challenge. We aimed to develop a rapid and versatile method of separating bacteria from these samples and then extracting their microbial DNA. Bacillus atrophaeus subsp. globigii was used as a simulant of Bacillus anthracis. We studied the effects of a broad variety of powdery and environmental samples on PCR detection and the steps required to alleviate their interference. With a benchmark DNA extraction procedure, 17 of the 23 samples investigated interfered with bacterial lysis and/or PCR-based detection. Therefore, we developed the dual-filter method for applied recovery of microbial particles from environmental and powdery samples (DARE). The DARE procedure allows the separation of bacteria from contaminating matrices that interfere with PCR detection. This procedure required only 2 min, while the DNA extraction process lasted 7 min, for a total of <10 min. This sample preparation procedure allowed the recovery of cleaned bacterial spores and relieved detection interference caused by a wide variety of samples. Our procedure was easily completed in a laboratory facility and is amenable to field application and automation.     

Development of a novel,rapid processing protocol for polymerase chain reaction-based detection of bacterial infections in synovial fluids

We describe the development of a molecular detection system designed for use with synovial fluid (SF)-based infections. The methodology employs a lysis/extraction procedure that effectively disrupts microorganisms allowing for release of the microbial DNA and its amplification by polymerase chain reaction (PCR). We tested the effectiveness of adding a mixed-bed, ion-exchange resin to the extract to remove PCR inhibitory components present in the SF. After centrifugation to separate the resin, DNA contained in the supernatant is subjected to PCR using oligonucleotide primers designed for broad-spectrum microorganism detection. Amplification products are analyzed by agarose gel electrophoresis and/or DNA hybridization methodology. We report here the detection sensitivity and specificity of the protocol using SF inoculated withEscherichia coli andStaphyloccocus aureus. We have applied this new methodology to clinical SF specimens with results superior to standard laboratory culturing assays.     

Discrepancies in identification of Vibrio cholerae strains as members of Aeromonadaceae and Enterobacteriaceae by automated microbial identification system

Aims: Incidental observation of a discrepancy in identification of Vibrio cholerae prompted a study to understand the ability of an automated microbial identification system to identify this important pathogen. Methods and Results: Twenty clinical isolates of V. cholerae showing difference in genetic profiles by random amplified polymorphic DNA (RAPD) fingerprinting, serologically confirmed as O1, and showing presence of ctxA and tcpA genes in PCR were subjected to analysis by Vitek 2 Compact automated identification system for identification. Vitek 2 Compact detected 10 of 20 isolates correctly, whereas the remaining 10 were identified as various members of Aeromonadaceae and Enterobacteriaceae. Conclusions: Our results indicate that Vitek 2 Compact automated microbial system does not always identify V. cholerae strains correctly. Significance and Impact of Study: These observations should create awareness among end users about possible misidentifications by automated systems and encourage simultaneous use of serology and/or PCR for correct identification at least for V. cholerae, which is one of the most important enteric pathogens.