The most cited articles published since 2010, extracted from Scopus
Volume 80, Issue 1, January 2010, Pages 49-55
John T. Heap | Sarah A. Kuehne | Muhammad Ehsaan | Stephen T. Cartman | Clare M. Cooksley | Jamie C. Scott | Nigel P. Minton
The recent development of the ClosTron Group II intron directed mutagenesis tool for Clostridium has advanced genetics in this genus, and here we present several significant improvements. We have shown how marker re-cycling can be used to construct strains with multiple mutations, demonstrated using FLP/FRT in Clostridium acetobutylicum; tested the capacity of the system for the delivery of transgenes to the chromosome of Clostridium sporogenes, which proved feasible for 1.0 kbp transgenes in addition to a marker; and extended the host range of the system, constructing mutants in Clostridium beijerinckii and, for the first time, in a B1/NAP1/027 'epidemic' strain of Clostridium difficile. Automated intron design bioinformatics are now available free-of-charge at our website http://clostron.com; the out-sourced construction of re-targeted intron plasmids has become cost-effective as well as rapid; and the combination of constitutive intron expression with direct selection for intron insertions has made mutant isolation trivial. These developments mean mutants can now be constructed with very little time and effort for the researcher. Those who prefer to construct plasmids in-house are no longer reliant on a commercial kit, as a mixture of two new plasmids provides unlimited template for intron re-targeting by Splicing by Overlap Extension (SOE) PCR. The new ClosTron plasmids also offer blue-white screening and other options for identification of recombinant plasmids. The improved ClosTron system supersedes the prototype plasmid pMTL007 and the original method, and exploits the potential of Group II introns more fully. © 2009 Elsevier B.V. All rights reserved.
Volume 81, Issue 2, May 2010, Pages 127-134
Anne Salonen | Janne Nikkilä | Jonna Jalanka-Tuovinen | Outi Immonen | Mirjana Rajilić-Stojanović | Riina A. Kekkonen | Airi M. Palva | Willem Meindert de Vos
Several different protocols are used for fecal DNA extraction, which is an integral step in all phylogenetic and metagenomic approaches to characterize the highly diverse intestinal ecosystem. We compared four widely used methods, and found their DNA yields to vary up to 35-fold. Bacterial, archaeal and human DNA was quantified by real-time PCR, and a compositional analysis of different extracts was carried out using the Human Intestinal Tract Chip, a 16S rRNA gene-based phylogenetic microarray. The overall microbiota composition was highly similar between the methods in contrast to the profound differences between the subjects (Pearson correlations >0.899 and 0.735, respectively). A detailed comparative analysis of mechanical and enzymatic methods showed that despite their overall similarity, the mechanical cell disruption by repeated bead beating showed the highest bacterial diversity and resulted in significantly improved DNA extraction efficiency of archaea and some bacteria, including Clostridium cluster IV. By applying the mechanical disruption method a high prevalence (67%) of methanogenic archaea was detected in healthy subjects (n=24), exceeding the typical values reported previously. The assessment of performance differences between different methodologies serves as a concrete step towards the comparison and reliable meta-analysis of the results obtained in different laboratories. © 2010 Elsevier B.V.
Volume 83, Issue 2, November 2010, Pages 89-105
Tom Coenye | Hans J C F Nelis
Biofilm formation is often considered the underlying reason why treatment with an antimicrobial agent fails and as an estimated 65-80% of all human infections is thought to be biofilm-related, this presents a serious challenge. Biofilm model systems are essential to gain a better understanding of the mechanisms involved in biofilm formation and resistance. In this review a comprehensive overview of various in vitro and in vivo systems is presented, and their advantages and disadvantages are discussed. © 2010 Elsevier B.V.
Volume 81, Issue 2, May 2010, Pages 121-126
Anja Klančnik | Saša Piskernik | Barbara Jeršek | Sonja Smole Možina
The aim of this study was to evaluate diffusion and dilution methods for determining the antibacterial activity of plant extracts and their mixtures. Several methods for measurement of the minimal inhibitory concentration (MIC) of a plant extract are available, but there is no standard procedure as there is for antibiotics. We tested different plant extracts, their mixtures and phenolic acids on selected gram-positive (Staphylococcus aureus, Bacillus cereus, and Listeria monocytogenes) and gram-negative bacteria (Escherichia coli O157:H7, Salmonella Infantis, Campylobacter jejuni, Campylobacter coli) with the disk diffusion, agar dilution, broth microdilution and macrodilution methods. The disk diffusion method was appropriate only as a preliminary screening test prior to quantitative MIC determination with dilution methods. A comparison of the results for MIC obtained by agar dilution and broth microdilution was possible only for gram-positive bacteria, and indicated the latter as the most accurate way of assessing the antimicrobial effect. The microdilution method with TTC (2,3,5-triphenyl tetrazolium chloride) or INT (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl tetrazolium chloride) to indicate the viability of aerobic bacteria was found to be the best alternative approach, while only ATP determination was appropriate for microaerophilic Campylobacter spp. Using survival curves the kinetics of bacterial inactivation on plant extract exposure was followed for 24. h and in this way the MIC values determined by the microdilution method were confirmed as the concentrations of extracts that inhibited bacterial growth. We suggest evaluation of the antibacterial activity of plant extracts using the broth microdilution method as a fast screening method for MIC determination and the macrodilution method at selected MIC values to confirm bacterial inactivation. Campylobacter spp. showed a similar sensitivity to plant extracts as the tested gram-positive bacteria, but S. Infantis and E. coli O157:H7 were more resistant. © 2010 Elsevier B.V.
Volume 85, Issue 2, May 2011, Pages 124-130
Maria Adela Yáñez | Andreas Nocker | Elena Soria-Soria | Raquel Múrtula | Lorena Martínez | Vicente Catalán
One of the greatest challenges of implementing fast molecular detection methods as part of Legionella surveillance systems is to limit detection to live cells. In this work, a protocol for sample treatment with propidium monoazide (PMA) in combination with quantitative PCR (qPCR) has been optimized and validated for L. pneumophila as an alternative of the currently used time-consuming culture method. Results from PMA-qPCR were compared with culture isolation and traditional qPCR. Under the conditions used, sample treatment with 50μM PMA followed by 5. min of light exposure were assumed optimal resulting in an average reduction of 4.45 log units of the qPCR signal from heat-killed cells. When applied to environmental samples (including water from cooling water towers, hospitals, spas, hot water systems in hotels, and tap water), different degrees of correlations between the three methods were obtained which might be explained by different matrix properties, but also varying degrees of non-culturable cells. It was furthermore shown that PMA displayed substantially lower cytotoxicity with Legionella than the alternative dye ethidium monoazide (EMA) when exposing live cells to the dye followed by plate counting. This result confirmed the findings with other species that PMA is less membrane-permeant and more selective for the intact cells. In conclusion, PMA-qPCR is a promising technique for limiting detection to intact cells and makes Legionella surveillance data substantially more relevant in comparison with qPCR alone. For future research it would be desirable to increase the method's capacity to exclude signals from dead cells in difficult matrices or samples containing high numbers of dead cells. © 2011 Elsevier B.V.
Volume 91, Issue 2, November 2012, Pages 276-289
Mariana Fittipaldi | Andreas Nocker | Francesc Codony
The ideal scenario in most applications of microbial diagnostics is that only viable cells are detected. Bacteria were traditionally considered viable when they could be cultured, whereas today's viability concept tends to be alternatively based on the presence of some form of metabolic activity, a positive energy status, responsiveness, detection of RNA transcripts that tend to degrade rapidly after cell death, or of an intact membrane. The latter criterion, although conservative, was the focus of one of the most successful recent approaches to detect viable cells in combination with DNA amplification techniques. The technology is based on sample treatment with the photoactivatable, and cell membrane impermeant, nucleic acid intercalating dyes ethidium monoazide (EMA) or propidium monoazide (PMA) followed by light exposure prior to extraction of DNA and amplification. Light activation of DNA-bound dye molecules results in irreversible DNA modification and subsequent inhibition of its amplification. Sample pretreatment with viability dyes has so far been mainly used in combination with PCR (leading to the term viability PCR, v-PCR), and increasingly with isothermal amplification method. The principle is not limited to bacteria, but has also successfully been applied to fungi, protozoa and viruses. Despite the success of the method, some practical limitations have been identified, especially when applied to environmental samples. In part they can be minimized by choice of experimental parameters and conditions adequate for a particular sample. This review summarizes current knowledge and presents aspects which are important when designing experiments employing viability dyes. © 2012 Elsevier B.V.
Volume 84, Issue 1, January 2011, Pages 81-87
Minseok Kim | Mark D. Morrison | Zhongtang Yu
Operational taxonomic units (OTUs) are conventionally defined at a phylogenetic distance (0.03-species, 0.05-genus, 0.10-family) based on full-length 16S rRNA gene sequences. However, partial sequences (700. bp or shorter) have been used in most studies. This discord may affect analysis of diversity and species richness because sequence divergence is not distributed evenly along the 16S rRNA gene. In this study, we compared a set each of bacterial and archaeal 16S rRNA gene sequences of nearly full length with multiple sets of different partial 16S rRNA gene sequences derived therefrom (approximately 440-700. bp), at conventional and alternative distance levels. Our objective was to identify partial sequence region(s) and distance level(s) that allow more accurate phylogenetic analysis of partial 16S rRNA genes. Our results showed that no partial sequence region could estimate OTU richness or define OTUs as reliably as nearly full-length genes. However, the V1-V4 regions can provide more accurate estimates than others. For analysis of archaea, we recommend the V1-V3 and the V4-V7 regions and clustering of species-level OTUs at 0.03 and 0.02 distances, respectively. For analysis of bacteria, the V1-V3 and the V1-V4 regions should be targeted, with species-level OTUs being clustered at 0.04 distance in both cases. © 2010 Elsevier B.V.
Volume 86, Issue 1, July 2011, Pages 42-51
Leandro N. Lemos | Roberta Roberta Fulthorpe | Eric W. Triplett | Luiz Fernando Würdig Roesch
The analysis of amplified and sequenced 16S rRNA genes has become the most important single approach for microbial diversity studies. The new sequencing technologies allow for sequencing thousands of reads in a single run and a cost-effective option is split into a single run across many samples. However for this type of investigation the key question that needs to be answered is how many samples can be sequenced without biasing the results due to lack of sequence representativeness? In this work we demonstrated that the level of sequencing effort used for analyzing soil microbial communities biases the results and determines the most effective type of analysis for small and large datasets. Many simulations were performed with four independent pyrosequencing-generated 16S rRNA gene libraries from different environments. The analysis performed here illustrates the lack of resolution of OTU-based approaches for datasets with low sequence coverage. This analysis should be performed with at least 90% of sequence coverage. Diversity index values increase with sample size making normalization of the number of sequences in all samples crucial. An important finding of this study was the advantage of phylogenetic approaches for examining microbial communities with low sequence coverage. However, if the environments being compared were closely related, a deeper sequencing would be necessary to detect the variation in the microbial composition. © 2011 Elsevier B.V.
Volume 86, Issue 3, September 2011, Pages 351-356
Tristano Bacchetti De Gregoris | Nick Aldred | A. S. Clare | James Grant Burgess
Mapping the distribution of phylogenetically distinct bacteria in natural environments is of primary importance to an understanding of ecological dynamics. Here we present a quantitative PCR (qPCR) assay for the analysis of higher taxa composition in natural communities that advances previously available methods by allowing quantification of several taxa during the same qPCR run. Existing primers targeting the 16S rRNA gene specific for Firmicutes, Actinobacteria, Bacteroidetes and for the α and γ subdivisions of the Proteobacteria were improved by largely increasing the coverage of the taxon they target without diminishing their specificity. The qPCR assay was validated in vitro testing artificial mixtures of 16S rRNA sequences and used to characterise the composition of natural communities developing in young marine biofilms. The possible contribution of the proposed technique in revealing ecological dynamics affecting higher bacterial taxa is discussed. © 2011 Elsevier B.V.
Volume 81, Issue 2, May 2010, Pages 187-193
Norbert Stoppacher | Bernhard Kluger | Susanne Zeilinger | Rudolf Rudolf Krska | Rainer Schuhmacher
In the present study we describe a method, which is based on solid phase microextraction (SPME) coupled to gas chromatography-mass spectrometry (GC-MS) and which can be used for the profiling of microbial volatile organic compounds (MVOCs) in the headspace (HS) of cultures of filamentous fungi. The method comprises the following successive steps: 1. growth of the fungus on a solid culture medium directly in headspace vials, 2. measurement of volatiles by HS-SPME-GC-MS, 3. deconvolution of mass spectra, 4. identification of volatiles by comparison of measured, deconvoluted mass spectra and linear temperature programmed retention indices (LTPRI) on two stationary GC phases with database entries and LTPRI published in the literature, and 5. profiling of the identified MVOCs.The developed method was successfully applied to cultures of the biocontrol fungus Trichoderma atroviride. An in-house library consisting of mass spectra and LTPRI values of fungal VOCs was established and used to study the profiles of MVOCs of this fungus. In total, 25 different MVOCs were identified by applying strict criteria (spectral match factor at least 90% and a maximum relative deviation of LTPRI of ± 2% from literature values). The MVOCs were assigned to the compound classes of alcohols, ketones, alkanes, furanes, pyrones (mainly the bioactive 6-pentyl-alpha-pyrone), mono- and sesquiterpenes, 13 of which have never been reported to be produced by Trichoderma spp. before. Eleven of these volatiles have been additionally confirmed using authentic standards. Finally, time course experiments and cultivation of T. atroviride in the presence of the mycotoxin fusaric acid demonstrated the potential of the method to study the dynamics of MVOC profiles as well as the effect of different environmental/biological conditions on the expression of MVOCs of filamentous fungi. © 2010 Elsevier B.V.
Volume 80, Issue 2, February 2010, Pages 219-221
Jeffrey C. Lord | Kris L. Hartzer | Michelle J. Toutges | Brenda S. Oppert
To investigate gene expression in Tribolium castaneum exposed to Beauveria bassiana, reference genes for qPCR were evaluated. Of these, β-actin, α-tubulin, and RPS6 were not stable. The most stable were ribosomal protein genes, RPS3, RPS18, and RPL13a. Syntaxin1, syntaxin6, and E-cadherin may be appropriate for some experimental systems.
Volume 80, Issue 1, January 2010, Pages 25-43
Lars Bogø Jensen | Lourdes García-Migura | Adriana J S Valenzuela | Mille Løhr | Henrik Hasman | Frank Møller Aarestrup
A classification system for plasmids isolated from enterococci and other Gram-positive bacteria was developed based on 111 published plasmid sequences from enterococci and other Gram-positive bacteria; mostly staphylococci. Based on PCR amplification of conserved areas of the replication initiating genes (rep), alignment of these sequences and using a cutoff value of 80% identity on both protein and DNA level, 19 replicon families (rep-families) were defined together with several unique sequences. The prevalence of these rep-families was tested on 79 enterococcal isolates from a collection of isolates of animal and human origin. Difference in prevalence of the designed rep-families were detected with rep9 being most prevalent in Enterococcus faecalis and rep2 in Enterococcus faecium. In 33% of the tested E. faecium and 32% of the tested E. faecalis no positive amplicons were detected. Furthermore, conjugation experiments were performed obtaining 30 transconjugants when selecting for antimicrobial resistance. Among them 19 gave no positive amplicons indicating presence of rep-families not tested for in this experimental setup. © 2009 Elsevier B.V. All rights reserved.
Volume 82, Issue 1, July 2010, Pages 64-70
Arnaud Bridier | Florence Dubois-Brissonnet | Abdelkader Boubetra | Vincent K. Thomas | Romain Briandet
This study proposes a high throughput method based on Confocal Laser Scanning Microscopy (CLSM) combined with the use of 96-wells microtiter plates compatible with high resolution imaging for the study of biofilm formation and structure. As an illustration, the three-dimensional structures of biofilms formed by 60 opportunistic pathogens were thus observed and quantified. The results revealed the diversity of biofilm architectures. Specific spatial arrangement such as the mushroom-like structures already described for Pseudomonas aeruginosa was observed. Other features, such as hollow voids in microcolonies of Salmonella enterica strain Agona, were identified for the first time. The combined use of microplates and confocal imaging proved to be a good alternative to the other high throughput methods commonly used as it enables the direct, in situ, qualitative and quantitative characterization of biofilm architecture. This high content method should lead to a clearer understanding of the structure-function relationships implicated in biofilms traits. © 2010 Elsevier B.V.
Volume 80, Issue 3, March 2010, Pages 281-286
Laïd Douidah | Lieven De Zutter | Peter A R Vandamme | Kurt Houf
A multiplex-PCR assay with seven primers was developed for the identification of the five human and mammal related species of the emerging foodborne pathogen Arcobacter. The assay was validated using 58 reference and 358 collection strains isolated from humans and mammals. The selected primers on the 23 S RNA gene amplify a 2061. bp fragment from A. butzleri, a 1590. bp fragment from A. thereuis, a 1125. bp fragment from A. cibarius and an A. skirrowii specific fragment of 198. bp. For A. cryaerophilus, a primer set on the gyrA gene amplified a specific fragment of 395. bp. No PCR product was generated for closely related bacteria including Campylobacter and Helicobacter species. Furthermore, examination of the 23 S RNA gene of A. cryaerophilus revealed, besides large heterogeneity, the presence of intervening sequences ranging from 87 to 196. bp. © 2010 Elsevier B.V.
Volume 81, Issue 3, June 2010, Pages 226-231
Christian Spangler | Alex P Wim Böhm | Urs Jenal | Roland W. Seifert | Volkhard Kaever
Cyclic di-guanosine monophosphate (c-di-GMP) represents an important ubiquitous second messenger in bacteria. It controls the transition between a sessile and a motile lifestyle of bacteria and, hence, affects the formation of biofilms which are highly resistant to antimicrobial treatment. c-di-GMP is synthesized by di-guanylate cyclases (DGCs) and degraded by specific phosphodiesterases (PDEs), two highly abundant protein families in bacteria. We have established a robust and highly sensitive high performance liquid chromatography-coupled tandem mass spectrometry (HPLC-MS/MS) based method for the quantitation of c-di-GMP and investigated various method performance parameters such as limit of detection (LOD), lower limit of quantitation (LLOQ), linearity, accuracy, recovery and analyte stability. As a proof of principle we used this method to accurately measure the activity of the prototype DGC PleD* from Caulobacter crescentus in vitro. In addition the methodology was successfully applied to determine in vivo levels of c-di-GMP in bacterial extracts of E. coli at different stages of bacterial growth. This demonstrates that our method is suitable for the sensitive and specific quantitation of c-di-GMP in bacterial cell extracts. © 2010 Elsevier B.V.
Volume 82, Issue 1, July 2010, Pages 1-10
Shilpakalasainath Rao | Ketha V Krishna Mohan | Chintamani D. Atreya
Bacillus anthracis is a Gram-positive, spore-forming bacterium representing the etiological agent of acute infectious disease anthrax, a lethal but rare disease of animals and humans in nature. With recent use of anthrax as a bioweapon, a number of techniques have been recently developed and evaluated to facilitate its rapid detection of B. anthracis in the environment as well as in point-of-care settings for humans suspected of exposure to the pathogen. Complex laboratory methods for B. anthracis identification are required since B. anthracis has similarities with other Bacillus species and its existence in both spore and vegetative forms. This review discusses current challenges and various improvements associated with anthrax agent detection. © 2010.
Volume 85, Issue 1, April 2011, Pages 16-21
Alexander Schriewer | A. Wehlmann | Stefan Wuertz
Quantitative PCR is becoming the method of choice for the detection of pathogenic microorganisms and other targets in the environment. A major obstacle when amplifying DNA is the presence of inhibiting substances like humic acids that decrease the efficiency of PCR. We combined the polymeric adsorbent Supelite™ DAX-8 with a large-volume (10mL) nucleic acid extraction method to decrease the humic acid content prior to qPCR quantification in water samples. The method was tested by spiking with humic acid standards and the bacterial surrogate Acinetobacter baylyi ADP1. Improvements in qPCR detection of ADP1 after application of DAX-8 resin (5 and 10 w/v%) were compared with the effects of added bovine serum albumin (BSA) (50, 100 and 200ng/μL). Both additions improved detection of ADP1 by counteracting inhibitory effects. There were no changes in mean cycle threshold difference (δCT) after application of DAX-8 compared to the control despite some loss of DNA, whereas significant increases occurred for BSA, irrespective of BSA concentration applied. The use of DAX-8 leads to an increase in qPCR amplification efficiency in contrast to BSA. The commonly used method to calculate genomic sample concentrations by comparing measured CT values relative to standard curves is only valid if amplification efficiencies of both are sufficiently similar. DAX-8 can provide this efficiency by removing humic acids permanently from nucleic acid extracts and has the potential to significantly increase the reliability of reported non-detects and measured results obtained by qPCR in environmental monitoring. © 2011 Elsevier B.V.
Volume 80, Issue 2, February 2010, Pages 212-214
Monica Imperi | Marco Pataracchia | Giovanna Alfarone | Lucilla Baldassarri | Graziella Orefici | Roberta Creti
A multiplex PCR assay for the identification of serotypes Ia to IX of Streptococcus agalactiae was developed. By using a single PCR reaction containing a mix of 19 primers the assay identified each serotype by the analysis of the unique two or three bands pattern on agarose gel. © 2009 Elsevier B.V. All rights reserved.
Volume 84, Issue 1, January 2011, Pages 12-18
Stephan Gantner | Anders F. Andersson | Laura Alonso-Sáez | Stefan A. Bertilsson
Next generation sequencing technologies for in depth analyses of complex microbial communities rely on rational primer design based on up-to-date reference databases. Most of the 16S rRNA-gene based analyses of environmental Archaea community composition use PCR primers developed from small data sets several years ago, making an update long overdue. Here we present a new set of archaeal primers targeting the 16S rRNA gene designed from 8500 aligned archaeal sequences in the SILVA database. The primers 340F-1000R showed a high archaeal specificity (< 1% bacteria amplification) covering 93 and 97% of available sequences for Crenarchaeota and Euryarchaeota respectively. In silico tests of the primers revealed at least 38% higher coverage for Archaea compared to other commonly used primers. Empirical tests with clone libraries confirmed the high specificity of the primer pair to Archaea in three biomes: surface waters in the Arctic Ocean, the pelagic zone of a temperate lake and a methanogenic bioreactor. The clone libraries featured both Euryarchaeota and Crenarchaeota in variable proportions and revealed dramatic differences in the archaeal community composition and minimal phylogenetic overlap between samples. © 2010 Elsevier B.V.
Volume 84, Issue 1, January 2011, Pages 71-73
G. Geojith | Sakthivel Dhanasekaran | Salesh P. Chandran | John Kenneth
Current methods of TB diagnosis are time consuming and less suited for developing countries. The LAMP (loop mediated isothermal amplification) is a rapid method more suitable for diagnosis in resource limited settings and has been proposed as a viable test requiring further evaluation for use as a laboratory method as well. We evaluated two LAMP assays, using culture lysates of clinical sputum samples (from Southern India) and compared it to a proprietary multiplex PCR reverse-hybridization line probe assay ('GenoType MTBC' from HAIN Lifescience GmbH, Germany). The LAMP procedure was modified to suit the local conditions. The Mycobacterium tuberculosis specific LAMP assay ('MTB LAMP') showed sensitivity and specificity, of 44.7% and 94.4% respectively in a 60 min format, 85.7% and 93.9% respectively in a 90. min format and 91.7%, and 90.9% respectively in a 120. min format. The Mycobacteria universal LAMP assay ('Muniv LAMP') showed a sensitivity of 99.1%. The LAMP was shown to be a rapid and accessible assay for the laboratory identification of M. tuberculosis isolates. Initial denaturation of template was shown to be essential for amplification in unpurified/dilute samples and longer incubation was shown to increase the sensitivity. The need for modification of protocols to yield better efficacy in this scenario needs to be addressed in subsequent studies. © 2010 Elsevier B.V.
Volume 87, Issue 2, November 2011, Pages 150-153
Roger Stephan | Nicole Cernela | Dominik Matthew Ziegler | Valentin Pflüger | Mauro Tonolla | Damiana F. Ravasi | Maria Fredriksson-Ahomaa | Herbert P. Hächler
Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000. Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level. © 2011 Elsevier B.V.
Volume 82, Issue 1, July 2010, Pages 85-89
Jianfa Bai | Xiaorong Shi | Tiruvoor G. Nagaraja
A multiplex PCR procedure that detects six major virulence genes, fliC, stx1, stx2, eae, rfbE, and hlyA, in Escherichia coli O157:H7 was developed. Analyses of the available sequences of the six major virulence genes and the published primers allowed us to develop the six-gene, multiplex PCR protocol that maintained the specificity of each primer pair. The resulting six bands for fliC, stx1, stx2, eae, rfbE, and hlyA were even and distinct with product sizes of 949, 655, 477, 375, 296, and 199bp, respectively. The procedure was validated with a total of 221 E. coli strains that included 4 ATCC, 84 cattle, and 57 human E. coli O157:H7 strains as well as 76 non-O157 cattle and human E. coli strains. The results of all 221 strains were similar to the results generated by established multiplex PCR methods that involved two separate reactions to detect five virulence genes (stx1, stx2, eae, fliC, and hlyA). Specificity of the O antigen was indicated by amplification of only O157, and not O25, O26, O55, O78, O103, O111, O127, and O145 E. coli serotypes. Sensitivity tests showed that the procedure amplified genes from a fecal sample spiked with a minimum of 104CFU/g (10 cells/reaction) of E. coli O157. After a 6-h enrichment of E. coli O157-spiked samples, a sensitivity level of 10CFU/g was achieved. © 2010 Elsevier B.V.
Volume 91, Issue 1, October 2012, Pages 80-88
Andrew S. Whiteley | Sasha N. Jenkins | Ian S. Waite | Nina Kresoje | Hugh G. Payne | Bruce P. Mullan | Richard James Nigel Allcock | Anthony Gerard O'Donnell
Here we demonstrate a cost effective and scalable microbial ecology sequencing platform using the Ion Torrent Personal Genome Machine (PGM). We assessed both PCR amplified 16S rRNA and shotgun metagenomic approaches and generated 100,000. + to 1,000,000. + reads using 'post-light' based sequencing technology within different sized semi-conductor chips. Further development of Golay barcoded Ion Tags allowed multiplex analyses of microbial communities with substantially reduced costs compared with platforms such as 454/GS-FLX. Using these protocols we assessed the bacterial and archaeal dynamics within covered anaerobic digesters used to treat piggery wastes. Analysis of these sequence data showed that these novel methanogenic waste treatment systems are dominated by bacterial taxa, in particular Clostridium, Synergistia and Bacteroides that were maintained as a stable community over extended time periods. Archaeal community dynamics were more stochastic with the key methanogenic taxa more difficult to resolve, principally due to the poor congruence seen between community structures generated either by nested PCR or metagenomic approaches for archaeal analyses. Our results show that for microbial community structure and function analyses, the PGM platform provides a low cost, scalable and high throughput solution for both Tag sequencing and metagenomic analyses. © 2012 Elsevier B.V.
Volume 80, Issue 1, January 2010, Pages 86-92
Daqing Xu | Yanzhen Tan | Xiaojing Huan | Xiaoqing Hu | Xiaoyuan Wang
Brevibacterium flavum has been developed to produce amino acids l-valine, l-lysine and l-threonine. However, there are not enough vectors available for the research on metabolic engineering in Brevibacterium flavum. Here we have constructed a shuttle expression vector pDXW-8 between Escherichia coli and B. flavum. The vector harbors an origin oriE for replicating in E. coli, a second origin oriC for replicating in B. flavum, a large multiple cloning site including 11 single restriction enzyme sites and suitable for cloning multiple genes or large DNA fragments, a tac promoter and a lacIPF104 fragment which tightly controls the tac promoter. The applicability of pDXW-8 was confirmed by the expression of the vhb gene in B. flavum. The vector pDXW-8 will be very useful for research on metabolic engineering in corynebacteria. © 2009 Elsevier B.V. All rights reserved.
Volume 82, Issue 3, September 2010, Pages 214-222
Emiel B M Spuesens | Theo Hoogenboezem | Marcel Sluijter | Nico G. Hartwig | Annemarie M C Van Rossum | Cornelis Vink
The first choice antibiotics for treatment of Mycoplasma pneumoniae infections are macrolides. Several recent studies, however, have indicated that the prevalence of macrolide (ML)-resistance, which is determined by mutations in the bacterial 23S rRNA, is increasing among M. pneumoniae isolates. Consequently, it is imperative that ML-resistance in M. pneumoniae is rapidly detected to allow appropriate and timely treatment of patients. We therefore set out to determine the utility of pyrosequencing as a convenient technique to assess ML-resistance. In addition, we studied whether pyrosequencing could be useful for molecular typing of M. pneumoniae isolates. To this end, a total of four separate pyrosequencing assays were developed. These assays were designed such as to determine a short genomic sequence from four different sites, i.e. two locations within the 23S rRNA gene, one within the MPN141 (or P1) gene and one within the MPN528a gene. While the 23S rRNA regions were employed to determine ML-resistance, the latter two were used for molecular typing. The pyrosequencing assays were performed on a collection of 108 M. pneumoniae isolates. The ML-resistant isolates within the collection (n=4) were readily identified by pyrosequencing. Moreover, each strain was correctly typed as either a subtype 1 or subtype 2 strain by both the MPN141 and MPN528a pyrosequencing test. Interestingly, two recent isolates from our collection, which were identified as subtype 2 strains by the pyrosequencing assays, were found to carry novel variants of the MPN141 gene, having rearrangements in each of the two repetitive elements (RepMP4 and RepMP2/3) within the gene. In conclusion, pyrosequencing is a convenient technique for ML-resistance determination as well as molecular typing of M. pneumoniae isolates. © 2010 Elsevier B.V.