Comparison of classical isolation protocols with a 24 h screen to detect viable salmonellas in faeces

A 24 h screen which detects three viable salmonella cells per g of faeces was compared with classical isolation procedures for their ability to identify salmonella-positive samples from a pig rearing unit. The screen involved an overnight enrichment in Muller-Kauffmann tetrathionate (MK) broth, subculture for 4 h in M broth containing 10 μg ml⁻¹ novobiocin, followed by detection of the presence of salmonellas by BacTrace and Salmonella-tek ELISAs. The classical protocols were: (1) an overnight and 48 h incubation in MK or selenite cysteine broth; or (2) overnight incubation in buffered peptone water and 24 h subculture in Rappaport-Vassiliadis broth (BPW-RV). Salmonellas were isolated from the broth cultures on xylose lysine deoxycholate and brilliant green agars. Thirty four of 100 samples were positive for salmonellas but no single isolation protocol identified all of them. The best of the classical isolation protocols, 48 h incubation in MK broth, identified 27 (79%) of the 34 positive samples whilst the screen identified 26 (76%) of the 34 positive samples. False-positive results were obtained from all isolation protocols except BPW-RV.     

Method to detect only viable cells in microbial ecology

Propidium monoazide can limit the analysis of microbial communities derived from genetic fingerprints to viable cells with intact cell membranes. However, PMA treatment cannot completely suppress polymerase chain reaction (PCR) amplification when the targeted gene is too short. PMA treatment in combination with two-step nested PCR was designed to overcome this problem. Four experiments were performed to determine the limitation of PMA treatment and to evaluate the suitability of the method by applying the following samples: (1) pure cultures of Escherichia coli O157:H7, Enterobacter aerogenes, and Alcaligenes faecalis; (2) pond water samples spiked with heat-killed E. coli O157:H7 and E. aerogenes; (3) anaerobic sludge samples exposed to increasing heat stress; and (4) selected natural samples of estuarine sediment and lake mud. Results from the first two experiments show that PMA treatment cannot efficiently suppress dead cells from PCR amplification when the targeted gene is as short as 190 bp, however, the two-step nested PCR can overcome this problem. The last two experiments indicate the method that PMA treatment in combination with two-step nested PCR is useful for viable cells detection in microbial ecology.     

基于EMA-qPCR的茄科青枯菌活体检测技术的建立

【目的】利用特异性核酸染料叠氮溴乙锭(Ethidium monoazide bromide, EMA)与实时荧光定量PCR技术相结合, 建立一种能有效区分青枯菌死活细胞的检测方法。【方法】样品DNA制备前经EMA渗透预处理, 再进行实时荧光定量PCR特异扩增菌体DNA。【结果】终浓度为2.0 mg/L的EMA能有效排除1.0×107 CFU/mL灭活青枯菌细胞DNA的扩增, 对活细胞和不可培养状态(Viable but non-culturable, VBNC)活菌的DNA扩增均没有影响。当每个定量PCR反应体系中的活细胞在5.0×100?5.0×104 CFU范围内时, 扩增Ct值与定量PCR反应体系中活细胞CFU对数值呈良好的负相关性(R2=0.992 5)。比较EMA-qPCR法和平板计数法对经过不同温度短期保存的青枯菌检测结果发现, 待检样品可在24 °C与4 °C冷藏条件下短期保存。【结论】本研究建立的EMA-qPCR方法能有效检测青枯菌VBNC细胞和有效区分死活菌, 避免或减少青枯菌PCR检测的假阳性和假阴性。     

PMA-qPCR法检测冷冻基质中非可培养状态(VBNC)副溶血性弧菌

【背景】副溶血性弧菌为冰鲜产品及肉制品中常见的污染微生物,致病性强,危害严重,出入境运输及加工的肉食品常采取冷冻冷藏的处理手段来防止微生物污染及生长,以保持食物新鲜。而残留的部分副溶血性弧菌会进入活的非可培养状态(Viable but non-culturable state,VBNC),从而构成潜在的风险隐患。【目的】建立可用于冷冻食品中VBNC副溶血性弧菌的快速检测方法,并探讨其适用性。【方法】将大西洋鲑鱼1:10匀浆,加入终浓度为6.6×10~5 CFU/mL的副溶血性弧菌,-20°C分别诱导10、20、30和50 d。建立实时荧光PCR技术(qPCR)方法,测定其特异性、灵敏度及稳定性。利用PMA-qPCR法对不同冷冻时期样品中的副溶血性弧菌进行检测,同时与qPCR、平板培养法进行比较。【结果】建立的qPCR方法特异性好,与其他阴性参考株无交叉反应;灵敏度高,检测限为19.8 CFU/mL;重复性好,C_q值的变异系数(CV)均在1.5%以下;标准曲线为y=-3.272x+45.310,线性回归系数R~2为0.996,定量范围为1×10~2–1×10~9 CFU/mL。在低温诱导10-50 d后,qPCR法的C_q值在26.32-27.34之间,与诱导前相比几乎没有变化;叠氮溴化丙锭(PMA)-qPCR法的C_q值则从诱导前的26.43逐步上升到38.84,呈现明显的上升趋势,表明死菌的数量在显著上升。经过比较及统计,PMA-qPCR检测的活菌数均高于平板培养法测出的数量,差异显著(P0.05)。【结论】PMA-qPCR特异性及灵敏度高,能有效抑制对死菌的扩增,同时能克服传统平板培养法对VBNC的漏检缺陷,可方便、快捷地用于冷冻食品中受损致病微生物,尤其是进入VBNC状态的细菌检测。     

A rapid method for determination of viable sulphate-reducing bacteria in human faeces

Adenosine phosphosulphate reductase (APS reductase) (E.C.1.8.99.2), viable counts of sulphate-reducing bacteria and rates of sulphate reduction were determined in 20 human samples of faeces. The activity of APS reductase, in contrast to sulphate reduction rates, correlated well with viable counts ( r = 0.987) and may therefore be used rapidly to quantify sulphate-reducing bacteria in human gut contents.     

Solid phase cytometry as a tool to detect viable but non-culturable cells of Campylobacter jejuni

Solid phase cytometry (SPC) in conjunction with fluorescent viability staining has been investigated as a tool to detect viable but non-culturable Campylobacter jejuni in drinking water. Inoculated water samples were filtered over a polyester membrane filter and the retained cells were stained using a carboxyfluorescein ester as a substrate for intracellular esterases. The number of green fluorescent bacteria was automatically counted by an Ar laser scanning device (ChemScan) in 3 min. In parallel, the plate count was determined on Columbia Blood Agar. The number of culturable cells decreased below the detection limit of plate counting in less than 50 days. In contrast, the number of fluorescent bacteria remained at its initial level for at least 85 days. The discrepancy between the two results can be attributed to the transition of culturable C. jejuni cells into VBNC C. jejuni cells. Furthermore, as SPC can distinguish between low numbers of dividing and non-dividing cells of Campylobacter it has the potential to monitor attempts to resuscitate VBNC cells.     

The use of fluorogenic esters to detect viable bacteria by flow cytometry

The ability of flow cytometry (FCM) to detect viable bacteria after staining with a range of fluorogenic esters was investigated with several bacterial species. The dyes studied were the fluorescein diacetate (FDA) derivatives carboxyfluorescein diacetate, 2',7'-bis-(2-carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester and calcein acetoxymethyl ester, as well as ChemChrome B, a commercially-available stain for the detection of viable bacteria in suspension. No one dye was found to be universal but ChemChrome B dye stained the widest number of Gram-positive and Gram-negative species, whereas the FDA derivatives preferentially stained Gram-positive bacteria. The use of ChemChrome B to detect viable bacteria in environmental samples was investigated further by studying the survival of Klebsiella pneumoniae in lakewater. During survival studies, a higher number of viable bacteria were detected both by direct viable counts and FCM after staining with rhodamine 123 and ChemChrome B than by colony-forming units, suggesting the presence of viable but nonculturable cells. These results demonstrate the potential use of FCM to enumerate viable bacteria in natural waters.     

A targeted screen to detect recessive mutations that have quantitative effects

A key problem in mutagenesis research is developing methods that are sufficiently sensitive to detect a wide range of abnormal phenotypes. Major variants may be easy to identify, but it can be difficult to detect mutations that have subtle effects, particularly on a complex genetic background. This paper describes a targeted mutagenesis protocol with enough sensitivity to detect recessive mutations that have modest quantitative effects. The procedure relies on consomic inbred strains of mice—strains in which one homologous pair of chromosomes of an inbred strain has been replaced with the corresponding pair from a donor strain. Mice that carry the desired donor chromosome—the target of the screen—are mutagenized and bred back to the original recipient strain. The first-generation progeny (G1) that are heterozygous only for the donor chromosome are also bred back to the recipient strain. G2 animals that inherit nonrecombinant donor chromosomes are identified by genotyping. These animals may be backcrossed repeatedly to the recipient strain to dilute off-target mutations, but ultimately, nonrecombinant G2 animals are bred to each other. Their G3 progeny are genotyped at markers spaced at 5- to 10-cM intervals to identify mating pairs that are homozygous for shared segments of the mutagenized donor chromosome. Entire litters of G4 progeny that are homozygous for defined intervals are screened. By comparing phenotypes within and among litters of nearly isogenic G4 animals, mutations can be verified and simultaneously mapped with a precision of 5–10 cM. This method has the potential to consistently detect mutations that have effects on trait means of well under one standard deviation. Received: 29 December 1998 / Accepted: 17 March 1999     

Enzymatic and hormonal responses following a 24 h endurance run and a 10 h triathlon race

Muscle cell leakage and hormonal changes were compared immediately after and during the 3 days following a 24 h endurance run (R24h) in 8 subjects, and a 10 h triathlon non-competitive race (T10h) in 6 subjects. The study showed three main differences: 1) plasma enzyme increases were considerably more significant in R24h than in T10h: compared with resting levels, creatine kinase increased x 120 after R24h but only x 2 after T10h; lactic dehydrogenase x 4, as opposed to x 1.5; and transaminases only showed an increase after R24h. The plasma myoglobin increase after R24h was double that found after T10h; 2) for the same magnitude of plasma aldosterone and cortisol after R24h and T10h (3 times the resting levels), a highly significant decrease in urinary Na+ (p less than 0.001) and an increase in urinary K+ (p less than 0.01) were found only after R24h; and 3) the plasma free noradrenaline level increased significantly after R24h (x 2.6) whereas it was unchanged after T10h. In contrast, the plasma level of conjugated dopamine increased only after T10h (x 3.7, p less than 0.05). These results suggest that long-distance running causes more muscular lesions than the triathlon, and that important factors other than aldosterone are probably involved in the regulation of urinary electrolyte excretions during T10h.     

Immunomagnetic capture PCR to detect viable Cryptosporidium parvum oocysts from environmental samples.

A method to detect viable Cryptosporidium parvum oocysts was developed. Polyclonal immunoglobulin G against C. parvum oocyst and sporozoite surface antigens was purified from rabbit immune serum, biotinylated, and bound to streptoavidin-coated magnetic particles. C. parvum oocysts were captured by a specific antigen-antibody reaction and magnetic separation. The oocysts were then induced to excyst, and DNA was extracted by heating at 95 degrees C for 10 min. A 452-bp fragment of C. parvum DNA was amplified by using a pair of C. parvum-specific primers in PCR. The method detected as few as 10 oocysts in purified preparations and from 30 to 100 oocysts inoculated in fecal samples. The immunomagnetic capture PCR (IC-PCR) product was identified and characterized by a nested PCR that amplified a 210-bp fragment, followed by restriction endonuclease digestion of the IC-PCR and nested-PCR products at the StyI site and a nonradioactive hybridization using an internal oligonucleotide probe labeled with biotin. PCR specificity was also tested, by using DNAs from other organisms as templates. In the control experiments, inactivated oocysts were undetectable, indicating the ability of this method to differentiate between viable and nonviable oocysts. Thus, this system can be used to specifically detect viable C. parvum oocysts in environmental samples with great sensitivity, providing an efficient way to monitor the environment for C. parvum contamination.     

24 h urinary creatinine excretion during pregnancy and its application in appropriate estimation of 24 h urinary iodine excretion

BackgroundUrinary creatinine can be used to adjust urinary iodine to evaluate iodine nutritional status during pregnancy. However, the reference intervals and impact factors of urinary creatinine are unknown.Methods24 h urine creatinine concentration (24 hUCr) and spot UCr at four different time periods of the day of pregnant women from Part 1 (n = 743) were measured. Linear regression analysis was performed to identify the impact factors of 24 h urinary creatinine excretion (24 hUCrE) and obtain the estimated 24 h urinary creatinine excretion (24 hUCrE_est). Then measured urinary iodine concentration (UIC) of 24 h and at fasting of pregnant women from Part 2 (n = 325), used spot urinary iodine to creatinine concentration ratio (UIC/UCr) and 24 hUCrE_est to calculate the estimated 24 h urinary iodine excretion (24 hUIE_est), finally checked the consistency and correlation of 24 hUIE_est and 24 h urinary iodine excretion (24 hUIE).ResultsIn Part 1, the median 24 hUCrE was 1.24(IQR0.98–1.76)g, and the reference interval was 0.61−2.93 g. The median 24 hUCr was 0.76 (IQR0.57−1.01)g/L, and the reference interval was 0.36−1.88 g/L. Multiple linear regression results showed that pregnancy weight was an influencing factor to 24 hUCrE after adjusting by gestational weeks, age, pre-pregnancy BMI, and percentage of body fat (F = 45.029, p＜0.001). In Part 2, there was no statistically significant difference between 24 hUIE_est and 24 hUIE (Z =−0.767, p = 0.443). Using 24hUIE as the gold standard, the relative average difference in 24hUIE_est was 4.2 %, the relative average differences for UIC and UIC/UCr were 32.4 % and 37.2 %. The reference interval of 24 hUIE and 24 hUIE_est were 88.43–585.90 μg and 50.97–700.39 μg, respectively.ConclusionsThe reference intervals of 24 hUCrE, spot UCr, 24 hUIE, and 24 hUIE_est during pregnancy were established. 24 hUCrE has important application value in iodine nutrition evaluation to gain more lead time for pregnant women with iodine nutrition-related diseases.     

Simultaneous DSC-FTIR microspectroscopy used to screen and detect the co-crystal formation in real time

We first report that the formation of pharmaceutical co-crystal can be synchronously screened and confirmed by a simultaneous DSC-FTIR microspectroscopic system, in which this combined unique system giving spectroscopic and thermodynamic information could provide a easy and direct way for one step screening and qualitative detection of the co-crystal formation in real time.     

Most-Probable-Number Rapid Viability PCR method to detect viable spores of Bacillus anthracis in swab samples

A comparison of Most-Probable-Number Rapid Viability (MPN RV) PCR and traditional culture methods for the quantification of Bacillus anthracis Sterne spores in macrofoam swabs from a multi-center validation study was performed. The purpose of the study was to compare environmental swab processing methods for recovery, detection, and quantification of viable B. anthracis spores from surfaces. Results show that spore numbers provided by the MPN RV-PCR method were typically within 1-log of the values from a plate count method for all three levels of spores tested (3.1 × 10⁴, 400, and 40 spores sampled from surfaces with swabs) even in the presence of debris. The MPN method tended to overestimate the expected result, especially at lower spore levels. Blind negative samples were correctly identified using both methods showing a lack of cross contamination. In addition to detecting low levels of spores in environmental conditions, the MPN RV-PCR method is specific, and compatible with automated high-throughput sample processing and analysis protocols, enhancing its utility for characterization and clearance following a biothreat agent release.     

Development of a highly sensitive bead-ELISA to detect bacterial protein toxins

A highly sensitive sandwich enzyme-linked immunosorbent assay to detect bacterial toxins was developed. Fab' of anti-toxin IgG was conjugated with horseradish peroxidase by the maleimide method and tetramethylbenzidine was used as substrate. As the solid phase, a 6.5 mm diameter polystyrene bead was used and this was coated with the anti-toxin IgG. The entire assay could be completed within 3.5 hr. The sensitivity of this bead-ELISA was found to be quite high with various bacterial toxins: less than 20 pg/ml for thermostable direct hemolysin of Vibrio parahaemolyticus, less than 60 pg/ml for Shiga toxin, less than 20 pg/ml for VT2 (Shiga-like toxin II) of Escherichia coli, less than 200 pg/ml for heat-labile enterotoxin of E. coli, and less than 6 pg/ml for cholera enterotoxin.     

Development of a molecular method to detect and quantify Aphanomyces euteiches in soil

A real-time PCR assay using 136F/211R primers and 161T TaqMan probe for the detection and quantification of Aphanomyces euteiches in soil is presented. The specificity of primers was tested on 105 different A. euteiches isolates, mainly from France. A calibration curve was established with a plasmid pHS1 resulting from the target region cloned into the pCR4 Topo vector (Invitrogen). The target copy number was evaluated and was constant whatever the isolate. A DNA-based method was able to discriminate between different artificial infestation levels in soil with small SDs thus validating the relevance of the extraction and amplification method in soil samples. Furthermore, a good correlation was observed between inoculum quantity in soil estimated by qPCR and root rot severity in plant evaluated by bioassays. These steps are essential when considering the feasibility of using a DNA-based method as a fast and accurate way to evaluate inoculum quantity in soil.     

Development of a rapid phage-based method for the detection of viable Mycobacterium avium subsp. paratuberculosis in blood within 48 h

The aim of this study was to develop a methodology to rapidly detect viable Mycobacterium avium subsp. paratuberculosis (MAP) in clinical blood samples. MAP cells spiked into commercially available blood were recovered using optimised peptide-mediated magnetic separation (PMMS) and detected using a phage-based method, and the identity of the cells detected confirmed using nested-PCR amplification of MAP signature sequences (IS900). The limit of detection was determined to be 10 MAP cells per ml of blood and was used to detect MAP present in clinical bovine blood samples. Using the PMMS-phage method there was no difference when detecting MAP from whole blood or from isolated buffy coat. MAP was detected in animals that were milk-ELISA positive (15 animals) by PMMS-phage and no MAP was detected in blood samples from an accredited Johne's disease free herd (5 animals). In a set of samples from one herd (10 animals) that came from animals with variable milk ELISA status, the PMMS-phage results agreed with the positive milk-ELISA results in all but one case. These results show that the PMMS-phage method can detect MAP present in naturally infected blood. Total assay time is 48 h and, unlike PCR-based detection tests, only viable cells are detected. A rapid method for detecting MAP in blood could further the understanding of disseminated infection in animals with Johne's disease.     

Influence of a 24 h fast on high intensity cycle exercise performance in man

The influence of a 24 h fast on endurance performance and the metabolic response to maximal cycle exercise was investigated in 6 healthy men (mean +/- SD: age = 27 +/- 7 years; weight = 73 +/- 10 kg; VO2max = 46 +/- 10 ml.kg-1.min-1). Subjects performed in randomised order two exercise bouts to exhaustion separated by one week. Test rides were performed in fasted (F) and post-absorptive (normal-diet, ND) conditions on an electrically braked cycle ergometer at a workload equivalent to 100% of VO2max. Acid-base status and selected metabolites were measured on arterialised venous blood at rest prior to exercise and at intervals for 15 mins following exercise. Exercise time to exhaustion was shorter after F compared with ND (p less than 0.01). Pre-exercise blood bicarbonate (HCO3-) concentration, PCO2 and base excess (BE) were lower after F compared with ND (p less than 0.05). Prior to exercise, circulating concentrations of free fatty acids (FFA), beta-hydroxybutyrate (B-HB) and glycerol were higher after F compared with ND (p less than 0.01) but blood glucose and lactate concentration were not different. On the F treatment, after exercise, blood pH, HCO3-, and BE were all significantly higher (p less than 0.01) than on ND; blood lactate concentration was significantly lower for the whole of the post-exercise period after F compared with ND (p less than 0.01). Circulating levels of FFA and B-HB after exercise on the F treatment fell but levels of these substrates were not altered by exercise after ND.(ABSTRACT TRUNCATED AT 250 WORDS)