Real-time PCR for the detection of Salmonella spp. in food: An alternative approach to a conventional PCR system suggested by the FOOD-PCR project

A real-time PCR assay using non-patented primers and a TaqMan probe for the detection and quantification of Salmonella spp. is presented. The assay is based on an internationally validated conventional PCR system, which was suggested as a standard method for the detection of Salmonella spp. in the FOOD-PCR project. The assay was sensitive and specific. Consistent detection of 9.5 genome equivalents per PCR reaction was achieved, whereas samples containing an average of 0.95 genome equivalents per reaction were inconsistently positive. The assay performed equally well as a commercially available real-time PCR assay and allowed sensitive detection of Salmonella spp. in artificially contaminated food. After enrichment for 16 h in buffered peptone water (BPW) or universal pre-enrichment broth (UPB) 2.5 CFU/25 g salmon and minced meat, and 5 CFU/25 g chicken meat and 25 ml raw milk were detected. Enrichment in BPW yielded higher numbers of CFU/ml than UPB for all matrices tested. However, the productivity of UPB was sufficient, as all samples were positive with both real-time PCR methods, including those containing less than 300 CFU/ml enrichment broth (enrichment of 5 CFU/25 ml raw milk in UPB).     

Differential expression of InlB and ActA in Listeria monocytogenes in selective and nonselective enrichment broths

Aim: To investigate the effect of selective and nonselective media on the expression of ActA and InlB proteins in Listeria monocytogenes. Methods and Results: Polyclonal antibodies to InlB and ActA were used in western blotting to determine the effect of selective (BLEB, UVM, and FB) or nonselective (BHI and LB) enrichment broths or hotdog exudates. Of the 13 L. monocytogenes serotypes tested, 11 and 12 serotypes showed a strong InlB expression in brain heart infusion (BHI) and Luria‐Bertani (LB), respectively, while only seven and one serotypes showed a strong ActA expression in these two respective broths, and others showed a weaker or no expression. On the contrary, in selective broths, expression of InlB was either very weak or undetectable. However, ActA expression was stronger in 12 serotypes when grown in buffered Listeria enrichment broth (BLEB), 11 in University of Vermont medium (UVM), and 10 in Fraser broth (FB). When tested in hotdog exudates, InlB and ActA were detected in serotypes grown at 37°C but not at 4°C. Transmission electron microscopy, enzyme‐linked immunosorbent assay, and mRNA analysis further supported these observations. Conclusion: Overall, selective enrichment broths promote ActA while nonselective broths promote InlB expression. Significance and Impact of the study: As commonly recommended enrichment broths show differential InlB and ActA expression, proper media must be selected to avoid false results during antibody‐based detection of L. monocytogenes.     

Comparison of selective and non-selective enrichment media in the detection of Listeria monocytogenes from meat containing Listeria innocua

AIMS: This study investigated whether the higher incidence of recovery from meat of Listeria innocua compared with L. monocytogenes could be due to the laboratory media used, leading to an artificially lower detection of the pathogenic species, L. monocytogenes. METHODS AND RESULTS: Minced beef was inoculated with L. monocytogenes, L. innocua, or a mixture of these species, and stored at 0 or 10 degrees C under vacuum or aerobic conditions for up to 28 days. Listeria were recovered from the minced beef using selective (University of Vermont Medium, UVM) and non-selective (Buffered Peptone Water, BPW) enrichment broths after 0, 14, and 28 days of storage. In general, there were no significant differences (P < 0.05) between the numbers of L. monocytogenes recovered from minced beef samples after 24 h enrichment in BPW and the numbers recovered using UVM. In addition, the presence of L. innocua in meat samples containing L. monocytogenes did not significantly (P < 0.05) affect the numbers of L. monocytogenes recovered using either enrichment broth. In most cases there were no significant differences (P < 0.05) between the numbers of L. innocua recovered from minced beef samples after 24 h enrichment in BPW compared with numbers recovered using UVM. CONCLUSION: Listeria innocua was found to have no significant competitive advantage over L. monocytogenes in selective or non-selective enrichment media. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that, in some instances, the use of a selective enrichment broth offers no advantage over a non-selective enrichment broth for the recovery of Listeria species from minced beef.     

Isolation of Salmonella strains from reptile faeces and comparison of different culture media

AIMS: To provide information on epidemiology and isolation of Salmonella strains from reptiles. METHODS AND RESULTS: Ninety-one samples collected from reptiles of the zoo of Rome or belonging to private owners were analysed using a standard protocol for isolation of Salmonella from food. Salmonella strains were tested for susceptibility to 15 antimicrobics by a disc-agar diffusion method. Forty-six samples (50.5%) were positive for Salmonella. Of the 22 strains serotyped, 17 belonged to Salmonella enterica subsp. I, four to the subsp. IIIa and one strain resulted untypeable. Rappaport-Vassiliadis broth (RVB) allowed to recover more Salmonella strains when bacterial growth in buffered peptone water (BPW) was scarce, while selenite cystine broth (SCB) was more efficient, whereas growth in BPW was abundant. The maximum isolation score was obtained by plating onto xylose lysine desoxycholate agar (XLD). The strains exhibited resistance at high percentages to colistin sulphate (58.7%), sulphamethoxazole (55.5%), streptomycin (32.6%), tetracycline (19.6%), ampicillin (17.4%) and nalidixic acid (13.1%). CONCLUSIONS: A high prevalence of Salmonella in reptiles was observed. For isolation, the choice of the enrichment broth depending on the degree of growth in BPW followed by plating onto XLD may be suggested. SIGNIFICANCE AND IMPACT OF THE STUDY: This paper provides epidemiological data on the prevalence of Salmonella and laboratory protocols useful for isolation of Salmonella from faeces of reptiles.     

Chitosan inactivates spoilage yeasts but enhances survival of Escherichia coli O157:H7 in apple juice

AIMS: To develop new measures for controlling both spoilage and pathogenic micro-organisms in unpasteurized apple juice using chitosan. METHODS AND RESULTS: Micro-organisms were isolated and identified from apple juice treated or untreated with chitosan using enrichment, selective media, microscopy, substrate assimilation patterns and ribosomal DNA profiling. Chitosan (0.05-0.1%) delayed spoilage by yeasts at 25 degrees C for up to 12 days but the effect was species specific: Kloeckera apiculata and Metschnikowia pulcherrima were inactivated but Saccharomyces cerevisiae and Pichia spp. multiplied slowly. In challenge experiments at 25 degrees C, total yeast counts were 3-5 log CFU ml(-1) lower in chitosan-treated juices than in the controls for 4 days but the survival of Escherichia coli O157:H7 was extended from 1 to 2 days; at 4 degrees C, chitosan reduced the yeast counts by 2-3 log CFU ml(-1) for up to 10 days but survival of the pathogen was prolonged from 3 to 5 days. The survival of Salmonella enterica serovar Typhimurium was unaffected by chitosan at either temperature. CONCLUSIONS: The addition of chitosan to apple juice delayed spoilage by yeasts but enhanced the survival of E. coli O157:H7. SIGNIFICANCE AND IMPACT OF THE STUDY: The results suggest that the use of chitosan in the treatment of fruit juices may potentially lead to an increased risk of food poisoning from E. coli O157:H7.     

The detection of non-O157 E. coli in food by immunomagnetic separation

AIMS: To compare immunomagnetic separation (IMS) protocols (enrichment media and temperature) for the isolation of Escherichia coli serotypes O26 and O111 from four different foods. METHODS AND RESULTS: Foods (minced beef, cheese, apple juice and pepperoni) spiked with low numbers (<100 g(-1)) of stressed nalidixic mutant E. coli serotypes O26 and O111 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at temperatures of 37 and 42 degrees C to optimize the IMS technique. BPW enrichments gave increased recoveries of both serotypes compared with tryptone soya and EC broths. Elevated temperatures of incubation at 42 degrees C were superior to 37 degrees C. CONCLUSIONS: Positive detection of low numbers of stressed target pathogens in all replicate tests was only possible using BPW enrichments. The majority of tests from alternative enrichments resulted in zero or single colonies recovered post-IMS. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum IMS protocol would improve isolation rates of E. coli O26 and O111 from foods and lead to increased safety for the consumer. Sub-optimal IMS protocols could lead to foods being incorrectly labelled free from these pathogens.     

Comparison of the sensitivity of manual and automated immunomagnetic separation methods for detection of Shiga toxin-producing Escherichia coli O157:H7 in milk

AIM: To determine the sensitivity of methods for detection of injured and uninjured Escherichia coli O157:H7 (E. coli O157) in raw and pasteurized milk. METHODS AND RESULTS: Raw milk, pasteurized milk with 1.5% fat content and pasteurized milk with 3.5% fat content were spiked with E. coli O157 at low levels. The samples were enriched in modified tryptone soya broth with novobiocin (mTSBn) at 37 degrees C. Aliquots of the enriched culture were analysed either by manual immunomagnetic separation (MIMS) and culturing on sorbitol MacConkey agar with or without cefixime and potassium tellurite (SMACct or SMAC), or by automated immunomagnetic separation and integrated ELISA (EiaFosstrade mark). Uninjured E. coli O157 organisms were detected in milk by both methods at 1 cfu 10 ml-1 sample). Injured organisms were detected at levels of about 4 cfu 10 ml-1 sample. Direct enrichment in mTSBn (22 h incubation) showed better sensitivity for injured cells than enrichment in buffered peptone water (BPW, 22 h incubation), or in a two-step enrichment consisting of BPW (6 h, 37 degrees C) and mTSBn (16 h, 37 degrees C), successively. CONCLUSIONS: The methods showed equal sensitivity in that they were both able to detect 1 cfu 10 ml-1 milk sample. Injured organisms can be detected and isolated at a level almost as low as this. A resuscitation step is not recommended for the detection and isolation of injured and non-injured E. coli O157 from milk. SIGNIFICANCE AND IMPACT OF THE STUDY: Due to the dilution of contamination in the bulk tank, analysis of milk for the presence of E. coli O157 requires a very sensitive method. Both methods described here are useful for such analysis.     

Resuscitation of Salmonella enterica serovar typhimurium and enterohemorrhagic Escherichia coli from the viable but nonculturable state by heat-stable enterobacterial autoinducer

Salmonella enterica serovar Typhimurium and enterohemorrhagic Escherichia coli were stressed by prolonged incubation in water microcosms until it was no longer possible to observe colony formation when samples were plated on nonselective medium. Overnight incubation of samples in nutrient-rich broth medium supplemented with growth factors, however, allowed resuscitation of stressed and viable but nonculturable cells so that subsequent plating yielded observable colonies for significantly extended periods of time. The growth factors were (i) the trihydroxamate siderophore ferrioxamine E (for Salmonella only), (ii) the commercially available antioxidant Oxyrase, and (iii) the heat-stable autoinducer of growth secreted by enterobacterial species in response to norepinephrine. Analysis of water microcosms with the Bioscreen C apparatus confirmed that these supplements enhanced recovery of cells in stressed populations; enterobacterial autoinducer was the most effective, promoting resuscitation in populations that were so heavily stressed that ferrioxamine E or Oxyrase had no effect. Similar results were observed in Bioscreen analysis of bacterial populations stressed by heating. Patterns of resuscitation of S. enterica serovar Typhimurium rpoS mutants from water microcosms and heat stress were qualitatively similar, suggesting that the general stress response controlled by the sigma(s) subunit of RNA polymerase plays no role in autoinducer-dependent resuscitation. Enterobacterial autoinducer also resuscitated stressed populations of Citrobacter freundii and Enterobacter agglomerans.     

选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV

本研究通过单因素试验和响应面分析试验建立了能够选择性富集沙门氏菌、副溶血弧菌和霍乱弧菌的共增菌培养基SVV,采用平板计数法及三重荧光PCR技术验证了SVV的增菌效果。结果表明:SVV能同时富集以不同浓度比例混合的3种目标菌,37oC振荡培养18h后,菌体浓度达到105~108CFU/mL;SVV强烈抑制大部分的非目标菌;用荧光PCR方法检测经过37oC振荡培养18h的10份人工接种样品和608份实际样品,结果表明目标菌在SVV中增殖18h后菌量达到检测限以上,SVV联合荧光PCR检测方法的检出率为4.06%,比传统检测方法(3.78%)高,无假阴性和假阳性。SVV可望应用于水产品中沙门氏菌、副溶血弧菌和霍乱弧菌检测前的增菌处理,可简化检测过程,有效克服漏检,提高检出率。     

Growth of Salmonella enterica in model mixed cultures during a two-step enrichment

Growth of Salmonella enterica was studied in model mixed cultures with Citrobacter freundii or Escherichia coli in buffered peptone water (BPW) and in Rappaport-Vassiliadis medium with soya (RVS) with modified concentrations of MgCl2 and malachite green, and at modified incubation temperatures. Selected S. enterica strains were inoculated in BPW (10(0) cfu/ml) together with selected strains of Citrobacter freundii (up to 10(8) cfu/ml) or selected strains of Escherichia coli (up to 10(8) cfu/ml), incubated overnight and then subcultured (1: 100) in RVS variants. Growth of individual bacterial species was followed by the quantitative real-time polymerase chain reaction (PCR). Optimal culture conditions during the second selective step were: MgCl2.6 H2O concentration of 29 g/l, malachite green concentration of 36 mg/1l, and the incubation temperature of 41.5 degrees C. Citr. freundii was found to be a potent competitor and E. coli was a weaker competitor. At optimal culture conditions, competition was reduced and the density of S. enterica cultures reached the level of 10(4) cfu/ml after not later than 2 h of selective enrichment. The results obtained provide a basis for the development of a short two-step enrichment to be used in rapid real-time PCR-based methods for the detection of S. enterica in food and other matrices.     

Pre-Enrichment and Enrichment Methods for Isolating Small Number of Campylobacteria from Contaminating Flora

A method was developed to detect fewer than 100 CFU of campylobacteria from SIFF transport medium to which thawing drip from deep frozen broiler carcasses was added as a source of contamination and which was then stored at room temperature for 20 h. The method was made possible by using pre–enrichment in 1 % buffered peptone water under a microaerophilic atmosphere for 5 h at 43°C before selective enrichment either in brucella enrichment broth and on brucella blood selective agar supplemented with Skirrow antibiotics or in CCD enrichment broth and on blood free CCD selective agar. The other pre–enrichment broth studied was alkaline peptone water with reducing agents (RAPW) and the other enrichment broths and selective agars were Preston broth and agar, THAL broth and alkaline tryptose broth (ATB) and brucella agar with ATB antibiotics. Contaminating flora can be a problem when using enrichment broths and selective agars with limited antibiotic supplementation.     

R-Factor Transfer in Selenite and Tetrathionate Broths

After overnight incubation of R(+)Escherichia coli with R(-)Salmonella typhimurium in selenite and tetrathionate with Brilliant Green (TBG) broths, R-factor transfer was demonstrated in 10 of 12 experiments. R-factor transfer in these enrichment broths occurred at a markedly reduced frequency in comparison to that in Trypticase soy broth, apparently due to an adverse effect either on the viability of the donor E. coli or the conjugation process itself. Transfer of R factors in commonly used enrichment broths may give rise to falsely resistant antibiotic patterns in Salmonella. However, the frequency of R-factor transfer is so low that it is unlikely to affect significantly the interpretation of R-factor studies.     

Thermal tolerance of acid-adapted and unadapted Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice

AIMS: A study was performed to determine D values of acid-adapted and unadapted cells of Salmonella, Escherichia coli O157:H7, and Listeria monocytogenes in cantaloupe juice and watermelon juice. METHODS AND RESULTS: Salmonella enterica serotype Poona, S. enterica serotype Saphra, two strains of E. coli O157:H7, and two strains of L. monocytogenes were grown in tryptic soy broth (TSB) and TSB supplemented with 1% glucose for 24 h at 37 degrees C. Decimal reduction times (D values) of cells suspended in unpasteurized cantaloupe juice and watermelon juice were determined. Acid-adapted cells of Salmonella and E. coli O157:H7, but not L. monocytogenes, had increased thermal tolerance compared with cells that were not acid-adapted. There was no correlation between soluble solids content of the two types of juice and thermal resistance. CONCLUSIONS: Growth of Salmonella and E. coli O157:H7 in cantaloupe juice, watermelon juice, or other acidic milieu, either in preharvest or postharvest environments, may result in cross protection to heat. The pasteurization conditions necessary to achieve elimination of pathogens from these juices would consequently have to be more severe if cells are habituated to acidic environments. SIGNIFICANCE AND IMPACT OF THE STUDY: Insights from this study provide guidance to developing pasteurization processes to eliminate Salmonella, E. coli O157:H7, and L. monocytogenes in cantaloupe juice and watermelon juice.     

Salmonella-TEK, a rapid screening method for Salmonella species in food

A micro-enzyme-linked immunosorbent assay (micro-ELISA) using the Salmonella-TEK screen kit was tested for the detection of Salmonella spp. in pure cultures as well as in 30 artificially contaminated food samples and in 45 naturally contaminated food samples. Different raw, fleshy foods and processed foods were used as test products. The artificially contaminated minced meat samples were preenriched in buffered peptone water, and after incubation, different selective enrichment broths were tested. The micro-ELISA optical density values after enrichment and isolation of the different broths were very analogous. The quickest method to detect Salmonella spp. in different foods is to enrich them with Salmosyst broth, which reduces the total analysis time to 31 h. The Salmonella-TEK kit for Salmonella spp. provides a promising test for the detection of Salmonella antigens in food even when they are present at a low concentration (1 to 5 CFU/25 g). The cross-reaction of the anti-Salmonella antibodies, especially to other gram-negative bacteria, is nil.     

Salmonella-TEK, a rapid screening method for Salmonella species in food.

A micro-enzyme-linked immunosorbent assay (micro-ELISA) using the Salmonella-TEK screen kit was tested for the detection of Salmonella spp. in pure cultures as well as in 30 artificially contaminated food samples and in 45 naturally contaminated food samples. Different raw, fleshy foods and processed foods were used as test products. The artificially contaminated minced meat samples were preenriched in buffered peptone water, and after incubation, different selective enrichment broths were tested. The micro-ELISA optical density values after enrichment and isolation of the different broths were very analogous. The quickest method to detect Salmonella spp. in different foods is to enrich them with Salmosyst broth, which reduces the total analysis time to 31 h. The Salmonella-TEK kit for Salmonella spp. provides a promising test for the detection of Salmonella antigens in food even when they are present at a low concentration (1 to 5 CFU/25 g). The cross-reaction of the anti-Salmonella antibodies, especially to other gram-negative bacteria, is nil.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Evaluation of the ability of primary selective enrichment to resuscitate heat-injured and freeze-injured Listeria monocytogenes cells.

Resuscitation rates of injured Listeria monocytogenes on conventional selective Listeria enrichment broth and nonselective Trypticase soy broth containing 0.6% yeast extract were compared. Cells were heated to 60 degrees C for 5 min or frozen at -20 degrees C for 7 days. Inoculation of Trypticase soy broth-yeast extract with the stressed cells resulted in growth that was superior to that in Listeria enrichment broth. Injured cells were fully recovered at 6 to 8 h.     

Effect of Electropermeabilization by Ohmic Heating for Inactivation of Escherichia coli O157:H7, Salmonella enterica Serovar Typhimurium,and Listeria monocytogenes in Buffered Peptone Water and Apple Juice

The effect of electric field-induced ohmic heating for inactivation of Escherichia coli O157:H7, Salmonella enterica serovar Typhimurium, and Listeria monocytogenes in buffered peptone water (BPW) (pH 7.2) and apple juice (pH 3.5; 11.8 °Brix) was investigated in this study. BPW and apple juice were treated at different temperatures (55°C, 58°C, and 60°C) and for different times (0, 10, 20, 25, and 30 s) by ohmic heating compared with conventional heating. The electric field strength was fixed at 30 V/cm and 60 V/cm for BPW and apple juice, respectively. Bacterial reduction resulting from ohmic heating was significantly different (P < 0.05) from that resulting from conventional heating at 58°C and 60°C in BPW and at 55°C, 58°C, and 60°C in apple juice for intervals of 0, 10, 20, 25, and 30 s. These results show that electric field-induced ohmic heating led to additional bacterial inactivation at sublethal temperatures. Transmission electron microscopy (TEM) observations and the propidium iodide (PI) uptake test were conducted after treatment at 60°C for 0, 10, 20, 25 and 30 s in BPW to observe the effects on cell permeability due to electroporation-caused cell damage. PI values when ohmic and conventional heating were compared were significantly different (P < 0.05), and these differences increased with increasing levels of inactivation of three food-borne pathogens. These results demonstrate that ohmic heating can more effectively reduce bacterial populations at reduced temperatures and shorter time intervals, especially in acidic fruit juices such as apple juice. Therefore, loss of quality can be minimized in a pasteurization process incorporating ohmic heating.     

The optimization of isolation media used in immunomagnetic separation methods for the detection of Escherichia coli O157 in foods

AIMS: To compare media used in immunomagnetic separation (IMS) techniques for the isolation of Escherichia coli O157 from food. METHODS AND RESULTS: Foods, both naturally contaminated and spiked, with low numbers (< 1 g(-1)) of stressed E. coli O157 were enriched in media based on buffered peptone water (BPW), tryptone soya and EC broths incubated at 30, 37, 40 and 42 degrees C. Following immunomagnetic separation, beads were plated on a range of selective agars. CONCLUSION: BPW supplemented with vancomycin (8 mg l(-1)) incubated at 42 degrees C, followed by IMS and subsequent plating of immunobeads onto cefixime tellurite sorbitol MacConkey agar plus either Rainbow or CHROMagar agars, proved optimum for the recovery of spiked, stressed E. coli O157 in minced beef, cheese, apple juice and pepperoni. The same protocol was optimum for recovery from naturally-contaminated minced beef and cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: The optimum protocol would increase isolation rates of E. coli O157 from foods.     

Evaluation of enrichment broths for their ability to recover heat-injured Listeria monocytogenes

J.R. PATEL AND L.R. BEUCHAT. 1995. Listeria selective enrichment broth (LEB), University of Vermont (UVM) broth, modified UVM (MUVM) broth and Fraser broth (FB) were compared for their ability to recover cells of L. monocytogenes from heated tryptose phosphate broth. Three strains of L. monocytogenes were heated at 54C for 30 min, inoculated into enrichment broths supplemented with 400 µg catalase ml⁻¹, and incubated for 8 h at 30°C. After incubation for 4 h, the total viable cell populations either decreased or did not change, whereas the number of healthy (non-injured) cells of all strains increased significantly in all broths except FB inoculated with the LCDC strain. With an increase in incubation time to 8 h, the number of healthy cells of all strains increased in all broths. At 8 h, the difference between populations of total (injured plus healthy cells) and healthy cells detected in LEB inoculated with two strains was not significant. Overall, recovery of heat-treated cells was significantly higher in LEB, followed by MUVM broth, UVM broth and FB. The addition of catalase to enrichment broths significantly enhanced recovery of heat-injured cells. A slight reduction of catalase activity of heated cells of all test strains in all enrichment broths except FB was observed by extending the incubation period from 4 to 8 h. A test strain that produces relatively higher catalase activity compared to the other strains exhibited the greatest resistance to exogenous hydrogen peroxide. Enumeration of viable L. monocytogenes cells in heated foods should be done using LEB supplemented with 400 µg catalase ml⁻¹ to maximize the recovery of injured cells.