Rappaport Vassiliadis Research Articles

Rapid detection and characterization of Salmonella enterica serovars by multiplex polymerase chain reaction assay

Multiplex polymerase chain reaction (PCR) was used for molecular typing of Salmonella enterica serovars in Egypt. During the summer of 2010, a total of 1075 samples were collected from cattle, sheep and poultry farms to be subjected for isolation of Salmonella (290 rectal swabs from cattle, 335 rectal swabs from sheep and 450 cloacal swabs from poultry). Bacteriological examination revealed the isolation of 68 Salmonella belonging to 13 different Salmonella serovars. The most common serovars were Salmonella Typhimurium (16 isolates), Salmonella Enteritidis (13 isolates), Salmonella Kentucky (eight isolates) and Salmonella Arizona (seven isolates). Other serovars typed were Salmonella Heidelberg (four), Salmonella Cerro (four), Salmonella Gallinarum (three), Salmonella Virginia (three), Salmonella Paratyphi-A (three), Salmonella Dublin (two), Salmonella Agona (two), Salmonella Hadar (two) and Salmonella Bardo (one). The results of molecular techniques highlight the usefulness of the multiplex PCR for the rapid detection of the two serotypes of Salmonella from field samples especially after pre-enrichment on Rappaport-Vassiliadis (RV) media. Moreover, detecting S. Typhimurium and S. Enteritidis by this assay was carried out within two days as opposed to five to six days by the bacteriological and serological methods. Key words: Multiplex polymerase chain reaction (PCR), Salmonella Enteritidis, Salmonella Typhimurium , Egypt.

鶏ミンチ肉および油糧種子を用いた市販Rappaport–Vassiliadis Brothの評価

Rappaport–Vassiliadis broth (RV) has been available in Japan. However the composition of its broth was found to be varied among the RV product manufactures. We evaluated 9 commercially available RV products by using 2 selective plate media (DHL and CAS) for the Salmonella isolation from ground chicken meat and oilseed. The results showed that the high percentages were yielded by RV product H (42.1%), followed by product G (40.8%) and D (39.5%) respectively. On the contrary, the low isolation percentages were yielded by RV product B and E (30.3%) each. In addition, RV products which isolated high percentage (RV product D, G and H) and low percentage product (RV product B and E) contained the same 29 g/L in the magnesium chloride concentrations. Although certain magnesium chloride concentration effectively inhibits contaminated bacteria other than Salmonella, no relationships between the magnesium chloride concentration level and the isolation percentage of Salmonella were confirmed in this study. On the other hand, it was found that only the lowest percentage of product E is used casein peptone. This results suggested that the kinds of peptone might be influenced on isolation percentage of Salmonella. In conclusion, this study exhibited that the isolation percentage of Salmonella were varied among RV products regarding of the selective plate media. Therefore, using multiple RV products with different compositions can be a key factor for isolating of Salmonella from various samples.

Rapid detection of Salmonella enterica in food of animal origins collected from Riyadh, King Saudi Arabia

The present study is aimed to investigate Salmonella species in food of animal origin collected from Riyadh, King Saudi Arabia (KSA) using conventional methods and polymerase chain reaction (PCR), targeting fimA gene specific for members of genus Salmonella. Salmonellaisolation revealed 20 Salmonella serovars (8%) out of 250 examined samples. Nine strains (6, 92%) were recovered from 130 examined minced meats and 5 Salmonella strains (8.33%) were recovered from 60 local frozen chickens. Moreover, 6 Salmonella strains (10%) were isolated from 60 examined local chicken cuts. PCR using selective broth culture, Rappaport-Vassiliadis (RV) was used for the detection of different Salmonella species, targeting the fimAgene. All samples revealed positive results with bacteriological examination were positive by PCR-RV, and amplification of 120 bp fragments specific for fimA gene were observed, in addition, to 4 samples (1.6%) previously identified as negative samples with bacteriological examination were positive with PCR using the two primer pairs. The results revealed that the PCR-RV using primers specific for fimA gene could detect more positive samples ofSalmonella species than conventional methods for rapid detection of food borne pathogens. Key words: Salmonella species, fimA gene, polymerase chain reaction- Rappaport- Vassiliadis (PCR-RV), frozen chickens, minced meats.

Development and evaluation of DNA and RNA real-time assays for food analysis using the hilA gene of Salmonella enterica subspecies enterica

The objective of this study was the development of DNA and RNA real-time PCR methods for detection of food-borne Salmonella sp. as rapid alternatives to the traditional cultural method (ISO 6579, 2004) in fresh meat carcasses and processed meat samples. These PCR methods were based on the hilA sequence, with primers and hybridisation probes designed against this gene target. The primers and probes were evaluated for their efficiency and dynamic range and subsequently the specificity of the assay was tested using 106 Salmonella enterica subspecies enterica strains and 30 non-salmonellae strains. An internal amplification control (IAC) was also developed for incorporation. The optimum copy number of IAC was determined to be 500 copies per reaction. A complementary enrichment protocol was adapted from the existing standard ISO 6579:2004 and consisted of enrichment in Buffered Peptone Water (BPW) 22 ± 2 h and a second selective enrichment for 6 h in Rappaport Vassiliadis with Soya (RVS). The DNA and RNA-based real-time PCR protocols, were applied to meat samples inoculated with Salmonella enterica subspecies enterica strains, including swabs from meat carcasses and minced beef samples which were heat treated or frozen. The developed methods have the potential as useful alternatives to the standard ISO 6579:2004 method for the detection of Salmonella enterica subspecies enterica on carcass swabs and raw meat using hilA as a target. The DNA assay is a useful tool for the screening of meat samples in the abattoir within 3 days of slaughter or in a food production process and the RNA-based assay has the potential to detect viable Salmonella enterica subspecies enterica in ready-to-eat products.

Application of PCR for rapid detection and serotyping of Salmonella spp. from porcine carcass swabs following enrichment in semi-solid agar

A new procedure for the detection and serotyping of Salmonella spp. based on PCR was developed and applied to porcine carcass swabs. Detection was based on a two-step enrichment using Buffered Peptone Water (BPW) and Modified Semi-solid Rappaport Vassiliadis (MSRV) agar followed by real time PCR targeting the genus specific hilA gene. In addition, pUC19 plasmid DNA was included as an extraction and internal amplification control (IAC). Serotyping consisted of multiplex PCR and capillary electrophoresis (CE) based on the method of Leader, Frye, Hu, Fedorka-Cray, & Boyle, 2009. To validate this method, 271 porcine carcass swabs were examined in tandem by the conventional culture method using MSRV (ISO 6579:2002; Annex D) followed by serotyping using slide agglutination and classification according to the White–Kauffmann–Le Minor scheme. Results showed 100% agreement between the conventional culture method and PCR, with 27 samples positive for Salmonella spp. by both methods.Using slide agglutination, full antigenic formulas were obtained for 25 of the 27 isolates (14 S. Typhimurium, 6 S. Infantis, 3 S. Derby, 1 S. Virchow and 1 S. Livingstone), while two isolates were not fully typeable and were designated S. Unnamed. PCR serotyping discriminated 7 different molecular code patterns, of which 2 were identical to those described by Leader et al. (2009), namely S. Typhimurium (ST) and S. Virchow (SV), found in 16 and one, respectively, of the 27 isolates. Both S. Unnamed isolates were identified as ST by the PCR method, therefore improving the identification of ST over the conventional method. The remaining 10 isolates shared four PCR patterns and although consistent codes were generally observed, discrepancies were observed compared to Leader et al. (2009). MSRV-PCR followed by serotyping using multiplex PCR and CE is rapid with final results obtained in 2days from receipt of samples compared with 6–9days using the conventional methods. The inclusion of the pUC19 plasmid DNA as an extraction and IAC demonstrated the robustness of the PCR procedure, with consistent Ct values obtained for both the hilA and pUC19 targets. This procedure could be particularly useful for surveillance and outbreak investigations by tracking sources of S. Typhimurium contamination, thus improving Salmonella control activities.

Development and preliminary validation of a real-time RT-PCR based method targeting tmRNA for the rapid and specific detection of Salmonella

In this study, a real-time reverse transcriptase PCR (real-time RT-PCR) assay for the detection of Salmonella was designed and developed. The real-time RT-PCR assay targeted the multi-copy RNA, tmRNA, had an inclusivity and exclusivity of 100% and a sensitivity of ≤1 cell equivalent. The assay was combined with culture enrichment and an initial validation was performed in accordance with ISO 16140: 2003. Culture enrichment required 18h primary enrichment in buffered peptone water (BPW) and 6h selective enrichment in Rappaport–Vassiliadis Soya Peptone Broth (RVS). The combined culture enrichment/real-time RT-PCR method had a relative specificity of 100% when compared to the traditional culture method. When compared to an assay targeting the ssrA gene, which encodes for tmRNA, an approximate 1000-fold increase in sensitivity of detection was observed. Targeting the multi-copy tmRNA transcript rather than its encoding gene improves assay sensitivity which should enable reduction in turn-around time of this alternative method for Salmonella testing.

Evaluation of Culture Media for Selective Enrichment and Isolation of Salmonella in Seafood

Seafood, including fish, shrimp, clam, crab, mussel, oyster, lobster, squid, octopus, and cuttlefish samples, was used to compare the recovery of Salmonella serovars by different selective enrichment and isolation media. The samples were selectively enriched in Rappaport-Vassiliadis (RV) broth and tetrathionate broth (TT), followed by selective isolation on Hektoen enteric (HE) agar, xylose lysine desoxycholate (XLD) agar, bismuth sulfite (BS) agar, and Brilliant Green (BG) agar media. Of 443 seafood samples analyzed, 108 were found to be contaminated with Salmonella. The role of selective enrichment in Salmonella spp. recovery with RV medium was distinctly high (70%) compared to TT broth (30%). The selective enrichment in RV broth followed by selective isolation on XLD, HE, BS, and BG agar recovered Salmonella at levels of 56, 41, 28, and 16%, respectively. Similarly, after enrichment in TT broth, XLD and HE agars recovered 27 and 23% respectively. The recovery of Salmonella with enrichment in TT followed by isolation on BS and BG was abysmally low at 4.6 and 5%, respectively. There was no significant difference (P > 0.05) in the recovery of Salmonella using the combinations of XLD and HE media with selective enrichment in RV broth. However, performance difference (P < 0.05) was observed in the recovery when BS and BG with RV, and XLD, HE, BS, and BG agars with TT broth were used. The present study showed that the combination of RV with XLD was the most efficient media for isolation of Salmonella from seafood when compared to other isolation media combinations.

Validation of a Duplex Real-Time PCR for the Detection of Salmonella spp. in Different Food Products

A 5′ nuclease duplex real-time polymerase chain reaction (PCR) assay was developed and validated with various food products for the specific and fast detection of Salmonella spp. in food. The assay used previously published primers in combination with a newly developed probe targeting the invA gene. An internal amplification control, which is coamplified in a duplex PCR, was included in the assay. The analysis of 1,934 natural food samples with real-time PCR and the cultural method in parallel resulted in a relative accuracy of 100% and 99.84% respectively, depending on the enrichment procedure in which buffered peptone water and selective enrichment in Rappaport–Vassiliadis (RV) broth were employed. The duplex real-time PCR assay has proven to be a specific, sensitive and fast screening method for Salmonella spp. in food. The overall analysis time of the PCR method was approximately 28 h, in contrast to 4 to 5 days with conventional Salmonella diagnostics. The developed assay has been shown to be a reliable diagnostic tool for use in routine analysis. It has been validated thoroughly and has become an official method in Germany for the detection of Salmonella spp. in food.

English

&nbsp; The current study was aimed to investigate the incidence of different&nbsp;Salmonellaserovars in chicken products either from local or imported source. A total of 152 samples of chicken and chicken products were collected from different retail establishment markets in Riyadh, KSA including 38 local whole frozen chickens, 62 imported whole frozen chickens, 22 whole poultry eggs and 30 local chicken cuts samples and examined by standard microbiological techniques (SMT).&nbsp;Salmonellaisolation revealed a total percentage of 5.92%; chicken cuts revealed a high incidence among the examined samples (10%), followed by local frozen chickens and imported frozen chicken samples with incidence of 7.89 and 4.83%, respectively. For this experiment, the whole chicken eggs were negative forSalmonella&nbsp;species by SMT.&nbsp;Salmonella enteritidis&nbsp;was dominating among the recovered&nbsp;Salmonella&nbsp;serovars, followed by&nbsp;Salmonella typhimurium, while only two strains of&nbsp;Salmonella agona&nbsp;and&nbsp;Salmonella newport&nbsp;were isolated. The PCR assay combined with Rappaport- Vassiliadis (RV) selective broth (PCR-RV) for the detection of&nbsp;Salmonella&nbsp;species in the collected field samples revealed the same positive samples directly from the imported frozen chickens and whole chicken eggs which gave negative results by SMT. Thus PCR-RV technique is rapid, time saving and applicable to detect&nbsp;Salmonella&nbsp;serovars directly from chicken samples. &nbsp; Key words:&nbsp;Frozen chickens,&nbsp;Salmonella&nbsp;serovars, diagnosis, enrichment, selective, polymerase chain reaction.

Evaluation of PCR inhibitory effect of enrichment broths and comparison of DNA extraction methods for detection of Salmonella Enteritidis using real-time PCR assay

The best enrichment broth and DNA extraction scheme was determined for rapid and sensitive detection of Salmonella Enteritidis in steamed pork using real-time PCR. The inhibitory effect of commonly used Salmonella enrichment broths, Rappaport-Vassiliadis (RV) and Muller-Kauffmann tetrathionate with novobiocin (MKTTn), on real-time PCR was confirmed. The inhibition of PCR was statistically significant (p < 0.05) in RV and MKTTn, as compared with buffered peptone water (BPW) or phosphate-buffered saline. The inhibitory effect of the selective enrichment media was successfully removed by using a modified DNA extraction, PrepMan Ultra Reagent with an additional washing step or the DNeasy Tissue Kit. In three experiments, when applied to detection of Salmonella Enteritidis in steamed pork, the real-time PCR coupled with single 24 h enrichment with BPW performed better than double 48 h enrichment with BPW plus RV or MKTTn. The simple real-time PCR assay using BPW proved to be a rapid and sensitive test for detection of low concentrations of Salmonella Enteritidis in steamed pork samples as compared with the conventional culture method.

6084 Efficacy and safety of bevacizumab-based combination regimens in patients with metastatic colorectal cancer: final results from a randomised phase II study of bevacizumab + FOLFIRI vs. bevacizumab + XELIRI (FNCLCC ACCORD 13/0503 study)

A method is presented for the detection of Salmonella in food, which produces definitive results on the third day after the sample collection. The method is equivalent to ISO 6579 in terms of the same detection limit of 100 cfu/25 g and 100% relative accuracy. The method involves pre-enrichment in buffered peptone water (16–20 h), two parallel selective enrichments in Rappaport–Vassiliadis and selenite–cystine medium, respectively (7 h), post-enrichment in Luria–Bertani medium (14–16 h), cell lysis by boiling in 1% Triton X-100, PCR using primers ST11, ST15 and a mimic internal control, and agarose gel electrophoresis. At the evaluation of the method on model food samples artificially contaminated with decimal dilutions of a Salmonella serotype Panama culture (cream cakes, ice cream, mayonnaise, meat products; 12 samples altogether), a detection limit of 100 cfu/25 g was determined. This detection limit was confirmed by the evaluation of the method on the mentioned model food samples artificially contaminated with reference materials containing on average 5 cfu of S. Panama. At the evaluation of the method on naturally contaminated food samples (cream cakes, ice cream, mayonnaise, egg melange, minced and separated meat, ham and salami; 44 samples altogether), identical results (9 positives) as with the reference method were obtained.

Comparison of three plating media for the isolation ofSalmonellafrom poultry environmental samples in Great Britain using ISO 6579:2002 (Annex D)

To evaluate the performance of three Salmonella plating media (Rambach, Xylose Lysine Deoxycholate agar and modified Brilliant Green Agar plus Novobiocin) as part of the ISO 6579: 2002 (Annex D) on poultry environmental samples. The samples analysed were those for the European Union Salmonella baseline surveys of laying (N = 3087), broiler (N = 1550), turkey fattening (N = 1540) and turkey breeding (N = 580) flocks for Great Britain. Results were considered separately for Rambach (including and excluding pale orange colonies) and for growth on selective media [Modified semi-solid Rappaport Vassiliadis (MSRV)] after 24 and 48 h of incubation. Overall, Rambach was the most sensitive medium, provided that pale orange colonies were checked. In all cases, an increase in the sensitivity of detection was obtained by plating growth on MSRV after 48 h of incubation. In broilers and laying flocks, the specificity significantly improved when Rambach only was used. The use of Rambach results in considerable savings compared with the two-plate method prescribed by ISO 6579:2002 (Annex D) without compromising sensitivity. Salmonella isolation protocols should be reviewed in terms of their efficiency and cost.

Letter to the Editor

To Dr. Donnelly, Scientific Editor, Food Microbiology and Safety Section, Consumer demand for healthy, additive-free foods has led to increased consumption of raw salad vegetables in developed countries. Availability of prepared bagged salads has increased, and so have the number of studies on the microbiological quality of minimally processed vegetables ready for consumption. The initial microbial load of vegetables, especially leafy ones, plays a crucial role in the microbiological content of pre-packed salads, yet few studies have been published to this day on microorganisms of fecal origin isolated from raw/untreated vegetables. In 1980, an article, authored by the late Professor J.A. Papadakis of the Athens School of Hygiene and myself (National Salmonella-Shigella Reference Centre of Greece), was published in ‘Hippocrates’ scientific journal of the Medical School of the Univ. of Athens Greece, discussing isolation techniques for the qualitative detection of Salmonella from fresh vegetables (Papadakis and Efstratiou 1980). In Greece the practice of consuming with every meal a side plate of salad made of uncooked fresh vegetables is a widely applied practice. Irrigation with wastewater might result in vegetable contamination with fecal microorganisms. Indeed it had been published (Papavassiliou and others 1967) that vegetables from the central market of Athens were found contaminated with coli-aerogenes bacteria (86.8%) and E. coli (22.3%). In the article by Papadakis and Efstratiou, 538 samples of 7 vegetables (lettuce, tomatoes, green peppers, parsley, spinach, celery, chicory) were examined for the presence of Salmonella spp. The vegetables were locally produced and were bought from green-grocery stores in the Athens area. 300gr of each vegetable was homogenized in 300ml buffered peptone water in a stomacher and incubated at 37 °C for 16 to 20 h. Tomatoes were ‘washed’ in buffered peptone water for 3 to 5 min. Following this pre-enrichment step, enrichment was carried out by inoculation in 3 broths: Muller-Kauffmann tetrathionate broth (1 ml of inoculum in 10 ml broth), Mannitol Selenite F broth (1 ml of inoculum in 10 ml broth), and Rappaport broth modified by Vassiliadis and others (1978) (0.1 ml of inoculum in 10 ml broth). All enrichment media were incubated for 24 h and 48 h. They were streaked out on SS (Salmonella Shigella) agar (Difco) and on brilliant green agar (Oxoid 329 with the addition of 0.25% sodium desoxycholate). The plates were incubated at 37 °C for 24 h, and 4 suspect colonies from each plate, if present, were inoculated into Kligler Iron agar slants. Tubes exhibiting the characteristics of Salmonella were further examined by standard biochemical and serological methods at the National Salmonella-Shigella Reference Centre of Greece. Salmonellae were isolated from six (1.1%) of the examined samples. Only lettuce (2.0%) and parsley (8.6%) were positive. All 3 positive lettuce samples yielded Salmonella Montevideo, although they were purchased 1 wk apart each other, but from the same retailer. The 3 positive parsley samples contained Salmonella Newport, Abony, and Orion. All 6 Salmonella strains were isolated from the modified Rappaport enrichment broth. One strain was isolated from Muller-Kauffmann tetrathionate broth; no Salmonella was isolated from Mannitol Selenite F broth. The conclusion was reached that enrichment in Rappaport medium as modified by Vassiliadis was more appropriate for the isolation of salmonellae from fresh vegetables. Superiority of Rappaport-Vassiliadis enrichment broth in detecting salmonellae in food products and polluted water led to its commercial preparation as dehydrated medium ‘Rappaport-Vassiliadis (RV) enrichment broth’ (Oxoid CM669 and other manufacturers). Should any of your readers be interested in of the original article, which is in English, I would be happy to post a copy if contacted at efstratiou@aegean.gr Sincerely,

A comparative study of cultural methods for the detection of Salmonella in feed and feed ingredients

BackgroundAnimal feed as a source of infection to food producing animals is much debated. In order to increase our present knowledge about possible feed transmission it is important to know that the present isolation methods for Salmonella are reliable also for feed materials.In a comparative study the ability of the standard method used for isolation of Salmonella in feed in the Nordic countries, the NMKL71 method (Nordic Committee on Food Analysis) was compared to the Modified Semisolid Rappaport Vassiliadis method (MSRV) and the international standard method (EN ISO 6579:2002). Five different feed materials were investigated, namely wheat grain, soybean meal, rape seed meal, palm kernel meal, pellets of pig feed and also scrapings from a feed mill elevator. Four different levels of the Salmonella serotypes S. Typhimurium, S. Cubana and S. Yoruba were added to each feed material, respectively. For all methods pre-enrichment in Buffered Peptone Water (BPW) were carried out followed by enrichments in the different selective media and finally plating on selective agar media.ResultsThe results obtained with all three methods showed no differences in detection levels, with an accuracy and sensitivity of 65% and 56%, respectively. However, Müller-Kauffmann tetrathionate-novobiocin broth (MKTTn), performed less well due to many false-negative results on Brilliant Green agar (BGA) plates. Compared to other feed materials palm kernel meal showed a higher detection level with all serotypes and methods tested.ConclusionThe results of this study showed that the accuracy, sensitivity and specificity of the investigated cultural methods were equivalent. However, the detection levels for different feed and feed ingredients varied considerably.

Comparison of culture media for enrichment and isolation of Salmonella spp. from frozen Channel catfish and Vietnamese basa fillets

Frozen fillets of Channel catfish and Vietnamese basa fish were used to compare Salmonella spp. recovery effectiveness of selective enrichment in Rappaport–Vassiliadis (RV) broth and tetrathionate broth (TT) and selective isolation on Hekteon enteric (HE) agar, xylose lysine deoxycholate (XLD) agar, and bismuth sulfite (BS) agar. Isolate confirmation was through fatty acid methyl ester analysis. Of 60 samples analyzed, 25 were found contaminated with Salmonella (42% incidence). Salmonella spp. recovery after enrichment in RV medium was 35% on HE agar, 30% on XLD agar, and 42% on BS agar. Similarly, after enrichment in TT broth, HE and XLD agars recovered 22% each and BS agar recovered 37%. No performance difference ( p > 0.05) was observed in the recovery of Salmonella using the combinations of BS, HE, and XLD agars with RV broth and BS agar with TT broth. The combination of selective enrichment in RV and selective isolation on BS gave numerically greatest isolation of Salmonella from Channel catfish and Vietnamese basa fish compared to other isolation combinations.

SEL, a selective enrichment broth for simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes.

Multipathogen detection on a single-assay platform not only reduces the cost for testing but also provides data on the presence of pathogens in a single experiment. To achieve this detection, a multipathogen selective enrichment medium is essential to allow the concurrent growth of pathogens. SEL broth was formulated to allow the simultaneous growth of Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes. The results were compared to those obtained with the respective individual selective enrichment broths, Rappaport-Vassiliadis (RV) for S. enterica, modified E. coli broth with 20 mg of novobiocin/liter for E. coli O157:H7, and Fraser broth for L. monocytogenes, and a currently used universal preenrichment broth (UPB). The growth of each pathogen in SEL inoculated at 10(1) or 10(3) CFU/ml was superior to that in the respective individual enrichment broth, except in the case of RV, in which Salmonella cells inoculated at both concentrations grew equally well. In mixed-culture experiments with cells of the three species present in equal concentrations or at a 1:10:1,000 ratio, the overall growth was proportional to the initial inoculation levels; however, the growth of L. monocytogenes was markedly suppressed when cells of this species were present at lower concentrations than those of the other two species. Further, SEL was able to resuscitate acid- and cold-stressed cells, and recovery was comparable to that in nonselective tryptic soy broth containing 6% yeast extract but superior to that in the respective individual selective broths. SEL promoted the growth of all three pathogens in a mixture in ready-to-eat salami and in turkey meat samples. Moreover, each pathogen was readily detected by a pathogen-specific immunochromatographic lateral-flow or multiplex PCR assay. Even though the growth of each pathogen in SEL was comparable to that in UPB, SEL inhibited greater numbers of nontarget organisms than did UPB. In summary, SEL was demonstrated to be a promising new multiplex selective enrichment broth for the detection of the three most prominent food-borne pathogens by antibody- or nucleic acid-based methods.

Comparação da eficiência dos caldos de enriquecimento seletivo no isolamento de Salmonella Dublin

The aim of this study was to compare three different selective enrichment broths: Rappaport-Vassiliadis (RV), selenite cystine (SC) and Muller-Kauffmann tetrathionate (MKT) for Salmonella Dublin isolation from faecal samples of calf experimentally infected. The bacteriological procedure involved pre-enrichment stages in Hajna-GN broth (only for the samples inoculated in RV broth), selective enrichment, culture in modified brilliant green agar (BGA), presumptive biochemistry tests (using triple-sugar-iron agar and lysine-agar) and slide agglutination test with poli-O and poli-H Salmonella antiserum. The effects of enrichment temperatures using RV broth were also evaluated (37oC and 42oC). SC broth was significantly more efficient in the isolation of Salmonella Dublin (P<0,05), whereas RV broth incubated at 42oC had a lower efficiency in the microbiological isolation.

Detection of Group D Salmonellae Including Salmonella Enteritidis in Eggs by Polymyxin-Based Enzyme-Linked Immunosorbent Assay

A high-throughput, rapid method was devised for the detection of Salmonella Enteritidis in egg products. For each target organism, preenrichment in nutrient broth was followed by selective enrichment in Rappaport-Vassiliadis soya peptone and tetrathionate brilliant green broths or by plating on modified semisolid Rappaport Vassiliadis (MSRV) agar medium. The presence of Salmonella Enteritidis was determined by subjecting portions of the selective broth cultures or swarming growth on MSRV medium to an enzyme-linked immunosorbent assay (ELISA) procedure using polymyxin immobilized in the wells of a microtiter plate as a high-affinity adsorbent for lipopolysaccharide (LPS) antigens. Sample extracts were reacted with polymyxin-coated microwells, and captured LPS antigens were detected immunoenzymatically with a commercially available Salmonella factor O9–specific antibody. The polymyxin-ELISA was 100% sensitive and 100% specific for Salmonella strains bearing the O9 antigen. When the ELISA was combined with enrichment using either the selective broths or plating on MSRV medium, the system was an effective means for detection of Salmonella Enteritidis in artificially inoculated egg products. The polymyxin-ELISA is a simple and inexpensive assay for group D salmonellae (including Salmonella Enteritidis) in a convenient 96-well microtiter plate format, making this system ideally suited for processing large numbers of samples.

The Effect of Preenrichment and Selective Enrichment Media on Recovery of Salmonella Typhi from the Tropical Fruit Mamey

The Bacteriological Analytical Manual (BAM) Salmonella culture method did not detect Salmonella Typhi in mamey, the tropical fruit that was implicated in a 1999 typhoid outbreak. The relative effectiveness of BAM's nonselective preenrichment and selective media for the recovery of S. Typhi from mamey was examined to determine if the BAM's preenrichment/selective enrichment strategy was the cause of the method's failure with this food. The preenrichment media were lactose broth, buffered peptone water (BPW), and universal preenrichment (UP) broth. The selective enrichment media were selenite cystine (SC) broth, tetrathionate (TT) broth, and Rappaport-Vassiliadis (RV) medium. UP broth was significantly more effective (P < 0.05) than either lactose broth or BPW for the recovery of 2 different S. Typhi strains from mamey. Of 120 test portions tested, 105 S. Typhi-positive test portions were recovered using UP broth, whereas only 1 S. Typhi-positive test portion was recovered using BPW, and no S. Typhi-positive test portions were recovered using lactose broth. SC and TT broths were both significantly more effective (P < 0.05) than RV medium. Of 105 S. Typhi-positive test portions, SC broth recovered 80, TT broth recovered 67, and RV medium recovered 9. After the above comparison, an incomplete UP (UPI) broth formulation was found to be significantly more effective (P < 0.05) than the complete formulation (UPC). Of 80 total positive test portions, UPI recovered 71, whereas UPC recovered only 48. The following UP broth formulations were compared to determine if any of the components of the UP broth formulation were inhibitory to S. Typhi: UPC, UP broth without sodium pyruvate (UPS), UP broth without ferric ammonium citrate (UPF), UP broth without MgSO4 (UPM), and UPI. It was found that none of the ingredients were inhibitory to S. Typhi and that, out of 140 total test portions, UPI and UPF, with 108 and 103 positive test portions, respectively, were significantly more effective (P < 0.05) than the other UP broth formulations (82 for UPC, 74 for UPS, and 60 for UPM). Rather than being inhibitory to the growth of S. Typhi, it appears that ferric ammonium citrate enhanced the growth of competitors which suppressed the growth of S. Typhi. These results demonstrate that UPI and UPF are effective for the recovery of S. Typhi from mamey.

A comparison of standard cultural methods for the detection of foodborne Salmonella species including three new chromogenic plating media

In this study the draft of the horizontal method for the detection of Salmonella species from human food and animal feed (ISO 6579:2002) was compared to the European gold standard (DIN EN 12824:1998), including the three new chromogenic plating media AES Salmonella Agar Plate (ASAP), Oxoid Salmonella Chromogen Media (OSCM) and Miller–Mallinson agar (MM). First the growth and appearance of 36 bacterial type strains ( Salmonella and other 21 species) on ASAP, OSCM and MM were compared to those on the three traditional agars Brilliant Green Agar according to Edel and Kampelmacher (BGA), Xylose Lysine Deoxycholate Agar (XLD) and Xylose Lysine Tergitol 4 Agar (XLT4). Only on MM agar, did all of 36 tested type strains produce typical colonies, especially strains of S. Senftenberg, Salmonella arizonae, S. Dublin and S. Derby. Artificial inoculation experiments using raw pork ground meat ( n = 92) were subsequently conducted. A shortened incubation time of 24 h in RVS broth yielded a Salmonella species recovery of 100% from spiked meat samples. Finally, 286 naturally contaminated raw porcine and bovine minced meat samples and raw poultry meat samples were investigated. Forty-three strains from a total of 39 Salmonella-positive samples were found. S. Typhimurium ( n = 21), with DT 104 L, DT 012 and RDNC being the most prevalent subtypes isolated. d-tartrate-positive S. Paratyphi B ( n = 2) and S. Saint-Paul ( n = 3) were also recovered. They were cultured from poultry meat and were multi-resistant against antibiotics including nalidixic acid. Rappaport Vassiliadis broth with soypeptone (RVS) yielded the highest recovery of Salmonella spp. (97,4%) compared to Tetrathionate broth with Novobiocin according to Muller and Kauffman (MKTTn, 94,9%) and Selenite Cystine broth (SC, 38,5%). However, no significant difference was obtained by comparing the ISO 6579:2002 draft to the gold standard.