Journal: Journal of microbiological methods
Serotyping of Shiga toxin-producing Escherichia coli (STEC) has been contingent upon the availability of antisera. Here we describe a 7-plex microbead-based immunoassay to simultaneously serotype seven STEC (i.e., belonging to serogroups O26, O45, O103, O111, O121, O145, and O157) by the Luminex xMAP® technology. This technology presents many advantages: Its multiplexed format (up to 100 analytes) saves time, reagents, and test sample, and many regulatory agencies currently utilize this platform for other assays. In this study, a total of seventy-nine STEC strains belonging to the 7 different serogroups of interest were tested. These strains had been previously serotyped and their serogroup confirmed by PCR. Except for one strain belonging to the O111 serogroup, nearly all strains (i.e., 98.7%; 78/79) were correctly identified on the Bio-Plex 100 instrument in less than 4 h. This newly developed microbead-based immunoassay could be extended to include other STEC serogroups, virulence factors, and/or bacterial species.
β-Galactosidase encoded by lacZ remains a popular reporter enzyme. Here, we present three fast and convenient tools that facilitate rapid construction of reporter lacZ fusions. The first enables the simple generation of lacZ (slacZ)-based chromosomally encoded reporter fusions within the lac operon in Escherichia coli using Red®/ET® recombination. The slacZ tool is based on rpsL counter-selection in combination with homologous recombination catalyzed by the λ Red recombinase, and blue/white screening. This permits construction of transcriptional and translational reporter lacZ fusions within a day. The second tool allows the introduction of lacZ reporter fusions into the chromosome by a single-crossover method. The strategy relies on the γ-origin-based suicide vector pNPTS138-R6KT, which can only replicate in λpir E. coli strains. The third tool comprises four pBBR1-based broad-host-range vectors for transcriptional and translational lacZ fusions. The functionality of our toolbox was confirmed by the K(+)-dependent activation of kdp promoter-lacZ fusions in vivo.
Onions (Allium cepa L.) are plagued by a number of bacterial pathogens including Pantoea ananatis, P. agglomerans, Burkholderia cepacia, Enterobacter cloacae, Pectobacterium carotovorum subsp. carotovorum, Xanthomonas axonopodis pv. axonopodis and several Pseudomonas spp. We developed a semi-selective medium, termed onion extract medium (OEM), to selectively and rapidly isolate bacteria pathogenic to and associated with onions and onion-related samples including bulbs, seeds, sets, transplant seedlings, soil and water. Most strains of interest grow sufficiently on OEM in 24h at 28°C for tentative identification based on colony morphology, facilitating further characterization by microbiological and/or molecular means.
Here we determine an optimal technique for the visualization of extracellular DNA in bacterial biofilms using the fluorescent eDNA stain TOTO-1 and the counterstain SYTO 60. This technique allows for more sensitive eDNA visualization than other fluorescent staining methods currently in use.
Rapid, low-cost, and user-friendly strategies are urgently needed for early disease diagnosis and timely treatment, particularly for on-site screening of pathogens in aquaculture. In this study, we successfully developed a real-time fluorogenic loop-mediated isothermal amplification assay integrated on a microfluidic disc chip (on-chip LAMP), which was capable of simultaneously detecting 10 pathogenic bacteria in aquatic animals, i.e., Nocardia seriolae, Pseudomonas putida, Streptococcus iniae, Vibrio alginolyticus, Vibrio anguillarum, Vibrio fluvialis, Vibrio harveyi, Vibrio parahaemolyticus, Vibrio rotiferianus, and Vibrio vulnificus. The assay provided a nearly-automated approach, with only a single pipetting step per chip for sample dispensing. This technique could achieve limits of detection (LOD) ranging from 0.40 to 6.42 pg per 1.414 μL reaction in less than 30 minutes. The robust reproducibility was demonstrated by little variation among duplications for each bacterium with the coefficient of variation (CV) for time to positive (Tp) value less than 0.10. The clinical sensitivity and specificity of this on-chip LAMP assay in detecting field samples was 96.2% and 93.8% by comparison with conventional microbiological methods. Compared with other well-known techniques, on-chip LAMP assay provides low sample and reagent consumption, ease-of-use, accelerated analysis, multiple bacteria and on-site detection, and high reproducibility, indicating that such a technique would be applicable for on-site detection and routine monitoring of multiple pathogens in aquaculture.
Listeria monocytogenes 15G01, a strain belonging to the persistent pulsotype 5132, was isolated from a seafood processing plant in New Zealand. Simple monoculture assays using crystal violet staining showed good biofilm formation for this strain and it was therefore chosen to be further investigated in regard to its biofilm forming ability. To evaluate its behaviour in different conditions commonly encountered in food processing environments, biofilm assays and growth studies were performed using common laboratory media under a range of temperatures (20°C, 30°C and 37°C). Furthermore, the effects of incubation time and different environmental conditions including static, dynamic and anaerobic incubation on biofilm formation were investigated. Changes in the environmental conditions resulted in different biofilm phenotype of L. monocytogenes 15G01. We demonstrated that increasing temperature and incubation time led to a higher biofilm mass and that dynamic incubation has little effect on biofilm formation at 37°C but encourages biofilm formation at 30°C. Biofilm production at 20°C was minimal regardless of the medium used. We furthermore observed that anaerobic environment led to reduced biofilm mass at 30°C for all tested media but not at 37°C. Biofilm formation could not be narrowed down to one factor but was rather dependent on multiple factors with temperature and medium having the biggest effects.
This study analyzed the RYP1 gene as a target for the molecular diagnosis of histoplasmosis. This assay detected fungal DNA in 13/13 blood samples from HIV/AIDS-patients with histoplasmosis. Therefore, the detection of RYP1 gene in whole blood sample is a quick and sensitive test to diagnose histoplasmosis.
Chlamydiae are obligate intracellular bacteria with two distinct morphological stages, the infectious elementary bodies (EBs) and non-infectious reticulate bodies (RBs). Here we describe a rapid and straightforward protocol for the purification of EBs and RBs involving special density gradients. It has been successfully applied to three chlamydial species.
According to the Human Microbiome Project, 90% of the cells in a healthy adult body are microorganisms. What happens to these cells after human host death, defined here as the thanatomicrobiome (i.e., thanatos-, Greek defn., death), is not clear. To fill the void, we examined the thanatomicrobiome of the spleen, liver, brain, heart and blood of human cadavers. These organs are thought to be devoid of microorganisms in a healthy adult host. We report that the thanatomicrobiome was highly similar among organ tissues from the same cadaver but very different among the cadavers possibly due to differences in the elapsed time since death and/or environmental factors. Isolation of microbial DNA from cadavers is known to be a challenge. We compared the effectiveness of two methods by amplifying the 16S rRNA genes and sequencing the amplicons from four cadavers. Paired comparisons revealed that the conventional DNA extraction method (bead-beating in phenol/chloroform/bead-beating followed by ethanol precipitation) yielded more 16S rRNA amplicons (28 of 30 amplicons) than a second method (repeated cycles of heating/cooling followed by centrifugation to remove cellular debris) (19 of 30 amplicons). Shannon diversity index of the 16S rRNA genes revealed no significant difference by extraction method. The present report provides a proof of principle that the thanatomicrobiome may be an efficient biomarker to study postmortem transformations of cadavers.
Culture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large-scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain high reproducibility and repeatability, which is mostly attained through robust amplification. We aimed to assess the reproducibility of saliva microbiota by comparing triplicate samples. The microbiota was produced with simplified in-house 16S amplicon assays taking advantage of large number of barcodes. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-nt internal index. All amplification protocols produced consistent microbial profiles for the same samples. Although, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 ± 0.097 and 13.56 ± 1.634 respectively). Large-scale profiling of microbiota can consistently be produced by all 16S amplicon assays. The TS-tailed-1S dual index protocol is preferred since it provides repeatable profiles on the HiSeq platform and are less labour intensive.