Overlay technique for direct detection and identification of haemolytic Listeria on selective plating medium. Comparison of five media

An overlay technique is proposed for the identification and counting of haemolytic Listeria colonies directly on selective plating media. The technique was applied to different Listeria-selective plating media. In pure culture studies with collection strains, the overlay technique was more efficient and reliable for detection haemolytic Listeria species compared with the incorporation of blood into the agar. The efficacy of the overlay technique for the direct detection of haemolytic colonies of Listeria from raw milk samples was related to agar selectivity. The best results were obtained with Listeria-selective agar medium modified (LSAMM). Catalase assay, together with reactions for aesculin and tellurite, were useful and reliable criteria for the identification of Listeria. All colonies on LSAMM which were positive for catalase, tellurite and aesculin while those displaying typical haemolysis corresponded in most cases to L. monocytogenes.     

Preenrichment versus direct selective agar plating for the detection of Salmonella Enteritidis in shell eggs

The relative effectiveness of two methods for the recovery of Salmonella Enteritidis (SE) from jumbo and medium shell eggs was compared. The first method used in the comparison consisted of a preenrichment of the sample, and the second method was developed by the U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS). Three bulk lots of blended, pooled eggs, each containing 220 liquid whole eggs that were thoroughly mixed manually were artificially inoculated with different levels of SE cells between approximately 10(0) and 10(3) CFU/ml. Twenty samples containing the contents of approximately 10 eggs each (by weight) were withdrawn from each of the inoculated bulk lots and incubated for 4 days at room temperature (ca. 23 degrees C). For the APHIS method, each sample was cultured by direct plating onto brilliant green (BG), brilliant green with novobiocin (BGN), xylose lysine desoxycholate (XLD), and xylose lysine agar Tergitol 4 (XLT4) agars. For the preenrichment method, 25-g portions from each pool were enriched in modified tryptic soy broth with 30 mg/liter of FeSO4. After 24 h of incubation, the preenrichments were subcultured to tetrathionate and Rappaport-Vassiliadis broths, and streaked to BG, BGN, bismuth sulfite, XLD, and XLT4 agar plates. SE isolates were confirmed biochemically and serologically. In all of the experiments, the preenrichment method recovered significantly more SE isolates (P < 0.05) of all the phage types and inoculum levels than did the APHIS method. From a total of 539 jumbo egg test portions analyzed, 381 (71%) were SE-positive by the preenrichment method and 232 (43%) were positive by the APHIS method. From a total of 360 medium egg test portions analyzed, 223 (62%) were SE-positive by the preenrichment method and 174 (48%) were positive by the APHIS method. The preenrichment method provided greater sensitivity for the isolation of SE in contaminated egg slurries than did the APHIS method.     

Efficacy of direct plating media for recovering Listeria monocytogenes from foods

Studies were done to evaluate 14 direct plating media for their suitability to recover Listeria monocytogenes strain Scott A from pasteurized whole milk, chocolate ice cream mix, Brie cheese and raw cabbage. Healthy cells were inoculated into foods to achieve viable populations of 10(2), 10(4), or 10(6) cells/ml(g). Bind's Acriflavine Agar, Trypaflavine Nalidixic Acid Serum Agar, Listeria Transport Enrichment Agar, Doyle and Schoeni Selective Enrichment Agar (DSSEA) and Modified DSSEA were not suitable for recovering L. monocytogenes from milk and Brie cheese and were therefore not evaluated as direct plating media for recovering the organism from ice cream mix and cabbage. McBride Listeria Agar (MLA), Gum Base Nalidixic Acid Tryptone Soya Agar (GBNTSA), Modified Despierres Agar (MDA) and Modified MLA (MMLA) performed best for recovering all inoculum populations from milk and ice cream mix. Enumeration of L. monocytogenes on several test media was complicated by the growth of large numbers of background microflora present in cabbage and Brie cheese, especially at the lowest test inoculum (10(2)/g). Generally, complete recovery of L. monocytogenes from Brie cheese and cabbage was attained on media when the inoculum population was greater than or equal to 10(4) cells/g. For Brie cheese, MLA, MDA, MMLA and Dominguez Rodriguez Isolation Agar were superior for recovering L. monocytogenes, while GBNTSA, DLEA, MDA and MMLA were best for recovering the organism from cabbage. Results of this experiment indicate that direct-plating procedures, without prior enrichment, can successfully be utilized for recovering L. monocytogenes from foods such as pasteurized milk and ice cream mix which contain low populations of background microflora. However, recovery of L. monocytogenes from foods such as raw cabbage and Brie cheese, which contain high populations of other microorganisms, was not satisfactory using the direct-plating procedures evaluated in this investigation.     

Preparation of Listeria monocytogenes specimens for molecular detection and identification

Listeria monocytogenes is a common foodborne pathogen that has the capacity to cause severe clinical illness in vulnerable human population groups. The availability of rapid and specific laboratory tests to identify this bacterium is essential for preventing an otherwise easily treated malaise from developing into a life-threatening disease. To this end, a variety of rapid, sensitive and precise nucleic acid-based assays have been developed, contributing to the improved diagnosis of listeriosis. Nonetheless, since many molecular assays rely on enzymatic reaction for template amplification, which is liable to interference from inhibitory substances present in clinical, food and environmental specimens, they often require purified nucleic acids as starting material for test consistency. As a consequence, considerable efforts have been directed toward the development of innovative and efficient sample handling procedures that reduce and eliminate inhibitory elements present in the specimens. By reviewing the recent progresses in the sample preparation methods that have been described for enhanced molecular detection and identification of L. monocytogenes, including rapid procedures for cultured isolates, more elaborate techniques for processing clinical, food and environmental samples, and specific considerations in preparing samples for quantitative PCR analysis, this article highlights further research requirement in the specimen processing protocols that form the basis for continued improvement in the overall performance of molecular assays for listeriosis.     

Improved plating media for simplified,quantitative detection of Listeria monocytogenes in foods

Moxalactam, nalidixic acid and bacitracin were found to be an effective combination of selective agents that permitted growth of Listeria while suppressing most foodborne genera. These selective agents were used to formulate Modified Vogel Johnson Agar which permitted the quantitative detection of Listeria from foods. Listeria were characteristically tellurite⁺/mannitol⁻ and could be readily differentiated without the need to view the colonies with oblique reflected light. The combination of selective agents was also effective when employed in conjunction with Modified McBride Agar. Modified Vogel Johnson Agar proved highly effective for isolation of Listeria and should significantly enhance its detection in foods.     

Persistence of Listeria monocytogenes in yogurt as determined by direct plating and enrichment methods

Listeria Selective Isolation Agar (LSI) and Modified Acriflavin Ceftazidime Esculin Agar (MACE) were compared to McBride Listeria Agar minus Blood (MLA-B) for ability to recover Listeria monocytogenes Scott A cells inoculated into commercial yogurt, pH 4.1, Yogurt was stored at 5 degrees C and sampled periodically over a 12 day period. LSI, MACE and MLA-B inhibited the growth of the two yogurt organisms but LSI and MACE gave better inhibition of other separately tested Gram-positive bacteria likely to be present in other fermented foods. Acid-stressed Listeria monocytogenes Scott A cells were optimally recovered by enrichment at 5 degrees C for 5-18 days in 0.1 M phosphate buffer, pH 7.2, followed by transfer to tryptic soy broth +0.5% yeast extract at 30 degrees C for 2 days. At low inoculum levels (10(2) cells/g yogurt), they were not detectable by direct plating or enrichment of yogurt after day 0. At high inoculum levels (10(7) cells/g yogurt), they were detectable up to day 6 but not at day 9 by direct plating on MLA-B, LSI or MACE with log counts per gram of yogurt being about 10 fold higher on LSI than on MACE or MLA-B. The above enrichment procedure permitted recovery on MLA-B, LSI, or MACE of viable Listeria cells from the day 9 samples found negative by direct plating.     

New chromogenic plating media for detection and enumeration of pathogenic Listeria spp.--an overview

In recent years a number of selective chromogenic plating media for pathogenic Listeria spp. have been developed and marketed. Their advantages are direct detection and enumeration of pathogenic Listeria spp. utilizing cleavage of substrates by the virulence factor phosphatidylinositol-phospholipase C (PI-PLC) and, to a lesser extent, by phosphatidylcholin-phospholipase C (PC-PLC). There are two groups of such media: the first utilizes cleavage by PI-PLC of L-alpha-phosphatidyl-inositol, forming a white precipitation zone around the colony, combined with the chromogenic substrate 5-bromo-4-chloro-3-indoxyl-beta-D-glucopyranoside for detection of beta-d-glucosidase, which occurs in all Listeria spp. All Listeria spp. produce turquoise colonies on these media which include ALOA , CHROMagar Listeria, BBL CHROMagar Listeria, and OCLA. The second group of media utilizes 5-bromo-4-chloro-3-indoxyl-myoinositol-1-phosphate, forming blue-turquoise colonies of pathogenic Listeria spp. and white colonies of non-pathogenic Listeria spp. BCM trade mark Listeria monocytogenes plating medium, Rapid'L.mono and LIMONO-Ident-Agar belong to this group. Selective chromogenic L. monocytogenes plating media offer the attraction of rapid economic detection and enumeration of pathogenic Listeria spp. within 24 or 48 h of incubation at 36+/-1 degrees C. This overview summarises the characteristics of these chromogenic plating media, reviews important evaluations, and focuses on replacement of conventional by these chromogenic plating media, particularly for applications in the food industry.     

Evaluation of standard and new chromogenic selective plating media for isolation and identification of Bacillus cereus

In this study, the performance of two new chromogenic plating media (CBC and BCM) was compared with two standard selective plating media (PEMBA and MYP) recommended by food authorities for isolation, identification and enumeration of Bacillus cereus. The four media types were challenged with a strain set comprising 100 B. cereus isolates from different origins and with different toxigenic potentials (40 food isolates, 40 isolates from food borne outbreaks and 20 clinical isolates). Additionally, the performance of the plating media for analysis of complex samples was assessed using naturally contaminated foods. Our survey showed that the new chromogenic media represent a good alternative to the conventional standard media. Especially, if laboratory staff are not highly trained in identification of B. cereus, the conventional media could lead to substantial misidentification and underestimation of food borne illness caused by B. cereus. However, there are some B. cereus strains that could not even be detected with this new type of chromogenic media. After the fatal misidentification of a highly toxic strain, other methods for a conclusive identification of B. cereus are needed. Sequence analysis of the plcR gene, a pleiotropic regulator of various virulence factors and B. cereus specific enzymes, revealed a significant correlation between atypical colony appearance and specific variances within the plcR gene sequences of those strains. The current concept of selective plating media, utilising PlcR regulated enzyme activities for differentiation purposes, should therefore be reconsidered and research should be geared towards culture independent methods.     

PCR-RFLP方法检测和鉴别五种李斯特菌

目的 建立快速检测和鉴别单增李斯特菌、绵羊李斯特菌、英诺克李斯特菌、威尔李斯特菌和格氏李斯特菌等常见李斯特菌的方法。方法 采用PCR-RFLP技术,首先通过引物“Lis1A-Lis1B”对李斯特菌属iap基因进行扩增,扩增产物大小约1.4 kb,然后用限制性内切酶DdeⅠ对PCR产物进行酶切,电泳观察具有种间特异性的酶切谱带进行鉴别。结果 上述5种李斯特菌的PCR扩增产物经内切酶DdeⅠ消化后,得到片段大小不同的,具有种间差异的特异性酶切图谱。结论 本实验建立的PCR-RFLP方法可以用于上述5种常见李斯特菌的快速检测和鉴定。     

A selective and diagnostic medium for use in the enumeration of Listeria spp. in foods

A new medium, called RAPAMY agar, has been elaborated for the isolation from and the enumeration of Listeria spp. in foods. It is based on Ralovich's nalidixic acid-trypaflavin-agar with the following modifications: (i) the slight inhibitory properties of that medium were overcome by the use of Columbia Blood agar base instead of tryptose agar and the addition of 0.05% ferric ammonium citrate and 2.5% egg yolk emulsion; (ii) selectivity was improved by the addition of 0.25% 2-phenyl ethanol and incubation under microaerobic conditions; (iii) the medium was provided with two diagnostic traits by the addition of (a) aesculin + ferric ammonium citrate; and (b) D-mannitol and phenol red. The growth of Enterococcus spp., the only organisms other than Listeria spp. which grow on RAPAMY agar, was not inhibited by the addition of 20 microgram.ml-1 Cefoxitin (Moxolactam). Higher levels inhibited some Listeria spp., but not the enterococci. The medium recovered Listeria spp. quantitatively and allowed recovery from foods colonized by Enterococcus spp. at levels upto 10(2) per g.     

Evaluation of selective plating media for the detection of heat- or freeze-injured Staphylococcus aureus

The efficacy of Baird-Parker (BP) agar, mannitol-salt-egg yolk (MSEY) agar and mannitol salt (MS) agar in detecting Staphylococcus aureus FRI-100 heated at 52 degrees C for 20 min in 100 mmol/L potassium phosphate buffer was determined. Brain heart infusion agar with 1% pyruvate (BHIP agar) supported the highest recovery of injured cells and was used as the control medium. Of the three selective media, significantly higher recovery of heat-injured cells was observed on BP agar than MSEY agar, and the poorest recovery was observed on MS agar (p < 0.05). Low recovery of unheated cells was obtained for MS compared with other media (p < 0.05). A reduction in populations occurred gradually in reagent-grade water stored for 14 days at -20 degrees C. There was no significant difference between BHIP agar and MS agar in the number of freeze-injured cells recovered from 1 to 14 days.     

利用选择性培养基快速初步鉴定食品中的致病微生物

目的 建立快速区分鉴别某种(类)微生物的方法。方法 选择几种常用的培养基, 将不同的菌株接种在培养基上, 通过培养基成分对不同微生物的选择性、显色反应及不同菌株在选择性培养基上具有的特征性菌落形态, 初步并快速判定微生物, 尤其是致病类微生物。结果 大肠埃希氏菌会与其他致病菌混淆, 可以通过结晶紫中性红胆盐(crystal violet neutral red bile salt, VRBA)琼脂将其区分; 志贺氏菌在培养基上菌落基本均为半透明, 体现的均为培养基本身的颜色; 亚硫酸铋(sulfurous acid bismuth, BS)琼脂的选择性较强, 但鼠伤寒沙门氏菌在该培养基上长势良好。结论 该方法可有效提高鉴别致病菌的效率。     

Liquid and solid selective differential media for the detection and enumeration of L. monocytogenes and other Listeria spp

A selective and differential medium (PALCAM agar) was elaborated for the isolation and enumeration of Listeria monocytogenes. PALCAM is based on Columbia agar with 0.05% glucose made selective by the addition of 0.001% polymyxin B, 0.0005% acriflavin, 1.5% lithium chloride and 0.002% ceftazidime. The diagnostic traits were attained by the incorporation of (i) 0.08% aesculin and 0.05% ferric salt; and (ii) 1% mannitol plus 0.008% phenol red. PALCAM recovered test strains of L. monocytogenes and other Listeria spp quantitatively and suppressed most other bacteria of common occurrence in fresh food. L. monocytogenes colonies were approximately 2 mm grey-green with a black sunken centre and a black halo on a cherry-red background. The occasional Enterococcus or Staphylococcus strains developing on the medium gave rise to grey colonies with a brown-green halo or yellow colonies with a yellow halo. PALCAM was the preferred medium out of 13 tested Listeria selective agars in current use. A similar differential enrichment broth, L-PALCAMY was developed based on peptone yeast extract broth with 2.5% egg yolk emulsion. The diagnostic traits and inhibitors used in this medium were the same as in PALCAM agar, through in different concentrations. Growth rate and cellcrop of L. monocytogenes in L-PALCAMY were of the same order as in Columbia broth. The growth of the majority of other bacteria of common occurrence in fresh foods was inhibited. The medium recovered L. monocytogenes more effectively from severely contaminated food than other current enrichment media.     

Different enrichment procedures for recovery of Listeria monocytogenes from raw chicken samples can affect the results of detection (by chromogenic plating or real-time PCR) and lineage or strain identification

This study aimed to evaluate the effect of different enrichment procedures on the detection of Listeria monocytogenes in food, by a comparison of subculture onto chromogenic agar with real-time PCR. Two different culture media, the primary and secondary enrichment broths of the U.S. Food Safety and Inspection Service (FSIS) method used for PCR detection of L. monocytogenes, were compared for the primary enrichment of retail ground chicken samples. L. monocytogenes was detected after the completion of each enrichment procedure in 63% (complete FSIS procedure) and 60% (plain FSIS secondary enrichment broth incubated for 48 h) of the samples by both culture and PCR, whereas a combination of the results for the two enrichment procedures revealed 86% of the samples to be positive. Most of the samples analyzed contained a mixture of lineage I and II strains, and their ratio varied for each enrichment procedure. This mixture could have a significant effect on the result of detection of L. monocytogenes for each individual sample, explaining the increase in positive samples when the results of the two enrichment procedures were combined. The use of different isolation procedures can affect the specific samples identified as positive and the specific strains isolated.     

A chromogenic plating medium for the isolation and identification of Enterobacter sakazakii from foods, food ingredients, and environmental sources

A chromogenic agar, R&F Enterobacter sakazakii chromogenic plating medium (ESPM), was developed for isolating presumptive colonies of E. sakazakii from foods and environmental sources. ESPM contains two chromogenic substrates (5-bromo-4-chloro-3-indoxyl-alpha-D-glucopyranoside and 5-bromo-4-chloro-3-indoxyl-beta-D-cellobioside), three sugars (sorbitol, D-arabitol, and adonitol), a pH indicator, and inhibitors (bile salts, vancomycin, and cefsulodin), which all contribute to its selectivity and differential properties. On ESPM, 79 pure culture strains of E. sakazakii (10 clinical isolates and others from food and environmental sources) yielded blue-black (three strains were blue-gray) raised colonies, 1 to 2 mm in diameter with and without halos after 24 h at 35 degrees C. Other enteric organisms plus Pseudomonas aeruginosa yielded white, yellow, green, or clear colonies with and without clear halos. Of these genera, only Shigella sonnei and one Pantoea strain produced blue-black to blue-gray colonies. ESPM was used to isolate E. sakazakii from a variety of foods: corn, wheat, and rice flours; powdered infant formula; dairy products (dried milk, whey, and caseinates); cereals; and environmental sources. Most false-positive results on ESPM were eliminated by observing acid production on either sucrose or melibiose after 6 h at 35 degrees C on a R&F E. sakazakii screening medium (ESSM) biplate. In an analysis of 240 samples, the number of samples positive for E. sakazakii by the ESPM-ESSM method and the U.S. Food and Drug Administration protocols (violet red bile glucose agar and tryptic soy agar) were 27 and 16, respectively, with sensitivity and specificity values of 100.0 and 96.9% versus 59.3 and 43.7%, respectively. These data support the fact that E. sakazakii confirmation should be based on more than one confirmation system. Both the API 20E and Biolog Microlog3 4.20 systems should be used for confirmation of E. sakazakii isolates.     

The VIT technology for rapid detection of Listeria monocytogenes and other Listeria spp

Detection of Listeria monocytogenes is generally performed in a two-step cultural enrichment process and takes on average 1 week until the biochemical identification of a L. monocytogenes suspicious colony is completed. However, food processing companies depend increasingly on test methods, which attempt to generate results comparable to standard methods but in reduced time-frame and which allow to release produced batches dependent on such results. In the present study, the vermicon identification technology (VIT), a rapid commercial test system using fluorescently labelled gene probes, was compared to a cultural standard method. In total, 298 naturally contaminated samples were analysed. The sensitivity and the specificity of the VIT system were 100% for the detection of L. monocytogenes and 97.1% and 100%, respectively, for the detection of the genus Listeria.     

Peptide nucleic acid fluorescence in situ hybridization for identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii

A fluorescent in situ hybridization (FISH) method in conjunction with fluorescin-labeled peptide nucleic acid (PNA) probes (PNA-FISH) for detection of Listeria species was developed. In silico analysis showed that three PNA probes Lis-16S-1, Lm-16S-2 and Liv-16S-5 were suitable for specific identification of Listeria genus, Listeria monocytogenes and Listeria ivanovii, respectively. These probes were experimentally verified by their reactivity against 19 strains of six Listeria species (excluding newly described species Listeria marthii and Listeria rocourtiae) and eight other bacterial species. The PNA-FISH method was optimized as 30 min of hybridization with 0.2% Triton X-100 in the solution and used to identify 85 Listeria strains from individual putative Listeria colonies on PALCAM agar plates streaked from selectively enriched cultures of 780 food or food-related samples. Of the 85 Listeria strains, thirty-seven were identified as L. monocytogenes with the probe Lm-16S-2 and two as L. ivanovii with the probe Liv-16S-5 which was in agreement with the results obtained by the API LISTERIA method. Thus, the PNA-FISH protocol has the potential for identification of pathogenic Listeria spp. from food or food-related samples.     

SPA-ELISA用于单核细胞增生性李斯特菌检验的应用研究

创造性地将SPA-ELISA应用于食品中单核细胞增生性李斯特菌(简称LM)的检验中,建立了快速检测该菌的ELISA方法。试验表明:该方法可特异性检测肉品中的LM,样品中最低检出量为5cfu/10g,经增菌后的培养液最低检测限可达到105cfu/mL,并可在48h内报告结果,比常规分离培养鉴定方法快5￣6d,适于LM的快速筛选。     

Development of a selective broth medium for the detection of injured Campylobacter jejuni by capacitance monitoring

The purpose of these studies was to develop a conductimetric method for the rapid detection of Campylobacter jejuni. Numerous basal medium components were analyzed to develop a growth-enhancing broth medium for detection of freeze-injured Campylobacter cells using a conductimetric system. The final medium was composed of a modified Campy-Line agar from which the agar and triphenyltetrazolium chloride were removed and the amino acid, L-arginine was added. Pure isolates of C. jejuni. (frozen and thawed to produce stressed cells) were utilized to test the detection methodology. Monitoring of significant changes in the capacitance signal was found suitable for detection of Campylobacter proliferation. Using stressed pure cultures, Campylobacter growth was repeatedly detected at very low inoculum levels (about one cell per well). There was a direct linear relationship between detection times (DTs) and the initial inoculum level. For example, using a single strain, the mean DT (n = 20) at the 10 CFU/ml inoculum level was 28.6 h, with 100% of the inoculated wells detecting. The mean DTs at the 100, 1,000, and 10,000 CFU/ml inoculum levels were 24.9, 21.4, and 17.0 h, respectively. This study demonstrates that conductimetric methods can be utilized for the rapid detection of C. jejuni.     

Rapid detection of Listeria monocytogenes using fluorescence immunochromatographic assay combined with immunomagnetic separation technique

To ensure the safety and quality of food ingredients, especially meat and dairy products, a high‐throughput, rapid and sensitive method to detect Listeria monocytogenes (LM) is always on a high demand. In this work, a specific induction method to enrich and detect LM based on fluorescence immunochromatographic assay (FICA) combined with immunomagnetic separation (IMS) techniques has been developed. The immunomagnetic‐beads (IM) beads were obtained through functionalised magnetic microspheres and the conjugated reaction between the carboxyl on beads surface and amino groups of antibody. The prepared IM‐beads could be used for rapidly enriching pathogenic bacteria with fewer steps, resulting in a high specificity for four pathogenic serotypes and about a 40‐fold improvement of detection limit compared with FICA only. In addition, this method was successfully applied in LM detection in sausage, pork and milk samples with a potential for further application in rapid on‐site detection of pathogenic bacteria.