Pre-enrichment Media Research Articles

Evaluation of universal preenrichment broth and comparison of rapid molecular methods for the detection of Salmonella from spent sprout irrigation water (SSIW)

Sprouts and spent sprout irrigation water (SSIW) present unique challenges for the development of a Salmonella detection method in food matrices. This study aimed to compare universal preenrichment broth (UPB) and lactose broth (LB) as preenrichment media for cultural and rapid screening methods and to compare their abilities to recover Salmonella in SSIW samples from different sprout varieties (i.e., alfalfa, broccoli, and mung bean sprouts). The associated co-enriched microbiota with different sprout varieties using different preenrichment media were also examined using a quasi-metagenomic approach. The performance of media and detection methods was compared using the relative level of detection (RLOD) value, as recommended by ISO 16140-2:2016. The level of detection (LOD) for Salmonella culture method with UPB was similar to that with LB in low aerobic plate count (APC) background samples (the relative LOD, i.e., RLOD, was nearly 1 after adjusting for the effects of SSIW variety and serovar), but significantly lower than that with LB in high APC background samples (RLOD = 0.32). The LOD for Salmonella with selected rapid methods was comparable to each other (RLOD from 0.97 to 1.50) and to the culture method (RLOD from 0.69 to 1.03), and no significant difference was detected between preenrichment broths in low APC background samples with RLOD values between 0.76 and 1.04. In samples with a high APC background, however, a drastic difference in LOD was observed between methods and between preenrichment broths for each method. The RLOD ranged from 0.03 to 0.32 when UPB was compared to LB as preenrichment broth. The composition and relative abundance (RA) of co-enriched microbiota was affected by multiple factors including food matrices, preenrichment media and Salmonella contamination. Altogether, this study validated UPB as a better preenrichment broth than LB for the detection of Salmonella enterica from SSIW. This study also suggested UPB may also be an optimal preenrichment medium for rapid screening methods when APC level is high. The observation of potential exclusion of Salmonella in preenrichment through the overgrowth of competitive microflora from the quasi-metagenomic study provided novel information that may be used to further optimize preenrichment formulations.

Multi-locus Sequencing Typing of Bartonella henselae isolates reveals coinfection with different variants in domestic cats from Midwestern Brazil

Bartonella henselae is a zoonotic pathogen responsible for causing Cat Scratch Disease (CSD) and other clinical manifestations in humans. Domestic cats are the main reservoirs of this Bartonella species. Previous studies have suggested that certain genotypes of B. henselae seem to be more associated with human infections. The present study aimed to genotype B. henselae isolates from domestic cats’ blood samples in the state of Goiás, midwestern Brazil. The association of quantitative real-time PCR (qPCR) based on the nuoG gene from Bartonella spp. of blood samples, before and after incubation in pre-enrichment liquid medium (BAPGM) and isolation on chocolate agar, showed a positivity frequency of 42% (42/100) for Bartonella spp. Twelve B. henselae isolates obtained on agar chocolate from six cats’ blood samples (two isolates from each animal) were characterized by Multi-locus Sequencing Typing (MLST) and revealed to belong to Sequence Types ST1 and ST5. One of the cats (1/6) presented both STs, demonstrating that domestic cats can be coinfected with different variants of B. henselae. The STs detected in this study are distributed worldwide and have already been detected in humans with clinical manifestations of bartonellosis. This is the first report of the zoonotic variants ST1 and ST5 of B. henselae in domestic cats from Brazil.

Detection of eae Gene as A Marker Strain EPEC (Enteropathogenic Escherichia coli) in Chicken Meat Sold at Some Traditional Markets in Surabaya

This study aimed to detect the presence of eae gene in E. coli from chicken meat sold at the traditional market Surabaya. Identification of bacteria was carried out in several stages, inoculated on pre-enrichment media using Buffered Peptone Water (BPW), planting on Levine’s Eosin Methylene Blue Agar (L-EMB) media, suspected colonies on L-EMB were subjected to Triple Sugar Iron Agar (TSIA) and Indol test for the confirmation of E. coli. The confirmed E. coli isolates were tested by Polymerase chain reaction (PCR) to determine the presence of the eae gene. The results showed that E. coli could be identified in 28 out of 100 (28%) chicken meat samples, and eae gene was detected in three E. coli isolates (11%).

Evaluation of the tris phosphate carbonate Salmonella pre-enrichment medium for poultry feed and feed ingredients

Summary Detecting Salmonella in poultry feed is challenging. Studies have shown that pre-enrichment media lack the buffering capacity to prevent an acidic pH, which results in cell injury, death, or altered biochemical pathways preventing Salmonella detection. A tris phosphate carbonate (TPC) pre-enrichment medium has been developed to maintain a near-neutral pH during incubation of feed, but its utility has not been confirmed for a wide variety of feed and ingredients. Five-gram samples of feed ingredients (8 types) and corn- or wheat-based poultry feeds (10 types) were individually added to 45 mL of each of 5 different pre-enrichment broths: lactose broth (LB), buffered peptone water (BPW), double-strength buffered peptone water (2xBPW), universal pre-enrichment broth (UPB), and TPC. After incubation for 0, 18, 24, and 48 h at 37° C, the pH of each pre-enrichment was measured (5 replicates/treatment). Data were analyzed by the ANOVA and least significant difference T-test. The initial pH of the LB, BPW, 2xBPW, and UPB ranged from 6 to 7 for all ingredients and feeds. The initial pH of ingredients and feed in TPC was 8.0-8.4. After 24 h of incubation, the pH had decreased to 4.6-5.5 for LB and BPW. Variability was observed among feed types incubated in 2xBPW and UPB, where the pH ranged from 4.8 to 6.4 at 24 h. Tris phosphate carbonate was less variable than 2xBPW or UPB with the pH ranging from 6.5 to 7.4 at 24 h. Results suggest that pre-enrichment of a variety of feed in TPC provides a near-neutral pH throughout incubation and may improve the ability to recover Salmonella.

Detection of Salmonella Typhimurium and Listeria monocytogenes biofilm cells exposed to different drying and pre-enrichment times using conventional and rapid methods

The capacity of real-time PCR (RT-PCR), the VIDAS immunoassay system, and the conventional count method for detecting Salmonella enterica serovar Typhimurium and Listeria monocytogenes biofilm cells was evaluated in this study. After biofilm formation, tests were performed under different drying times (0, 6, 12, 24, and 72 h) and pre-enrichment times (0, 6, 18, and 25 h). The direct epifluorescence microscopic results demonstrated that Salmonella Typhimurium and L. monocytogenes biofilm cells can remain viable for 72 h under drying conditions. Pre-enrichment time and type of medium played an essential role in the detection of both microorganisms after drying. Furthermore, RT-PCR was more sensitive than VIDAS and the conventional method for detecting Salmonella Typhimurium and L. monocytogenes cells at different drying times and without pre-enrichment (0 h), with a detection range between 102 and 107 CFU/mL. TSBYE-T80 used as a pre-enrichment medium was effective for detecting both bacteria and was more effective than Demi Fraser-T80 medium for detecting L. monocytogenes. Therefore, pre-enrichment is recommended to avoid false positives and false negatives due to the presence of dead cells or a very low initial concentration of cells after drying.

Mutu Bakteriologis Pindang Tongkol (Euthynnus affinis) yang Berasal dari Beberapa Pasar di Yogyakarta

Food poisoning cases need to be examinated intensively. There have been five poisoning cases reported between 2003-2004 in Sleman area. Three of them were caused by contaminated tuna fish. The objective of this research was to determine bacteriological quality of salted tuna fish from several markets in Yogyakarta. Salted tuna fish sample were collected from five markets, namely Beringharjo, Condongcatur, Demangan, Kranggan and Sentul. In each market, four vendors were selected. Bacteriological test were done including total microbe cells utilizing PCA medium, Coliform test utilizing VRBA medium. Identification and isolation of Vibrio parahaemolyticus use pre-enrichment selective medium of Alkaline Petone Water and selective Chromogenic medium agar for Vibrio. Salmonella test utilizing Chromogenic agar continued by utilizing TSI and LIA medium. The result showed that bacteriological quality of salted tuna fish from markets in Yogyakarta did not fulfill the requirements based on SNI 01-2717-1992. It was shown that total microbial count were high, i.e. 2.4 x 105 – 5.27 x 107 CFU/ g. Coliform count ranged from < 2.5 x 101 - > 6.5 x 106 CFU/g. From 20 samples that were brought to analyzed, 60% was positively presumed contaminated by V. parahaemolyticus and 50% was positively presumed contaminated by Salmonella.

Assessment of a multiplex detection method for Salmonella enterica, Escherichia coli O157:H7, and Listeria monocytogenes in cow milk

Raw cow milk is considered one of the most important vehicles for pathogenic bacteria like Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes. These three bacteria are responsible for foodborne diseases. Routine microbiological methods to detect these microorganisms in cow milk can be complicated and time consuming. The aim of this work was to evaluate a method to simultaneously detect Salmonella spp., Escherichia coli O157:H7, and Listeria monocytogenes in experimentally contaminated cow milk. The assessed method combined a standard microbiological culture step, using a pre-enrichment medium that favors the growth of the three focal microorganisms: SEL broth, followed by a single PCR assay. A total of 43 interference bacterial strains were used to evaluate the method’s specificity. The detection rate for the microbiological method with standard culture media was 10 UFC/mL, and that of the PCR detection, following pre-enrichment in SEL broth, was 10 UFC/mL for S. enterica and L. monocytogenes and between 1 and 5 UFC/mL for E. coli O157:H7. The PCR method showed specificity for the reference strains. Simultaneous detection by multiple PCR using SEL broth was successful for the detection of S. enterica, E. coli O157:H7, and L. monocytogenes in samples of experimentally contaminated cow milk, featuring both a high detection rate and a high specificity. This approach promises to be a feasible routine procedure when testing milk samples in industry and public health control setups.

The effect of environmental poultry samples on the pH of typical Salmonella pre-enrichment and enrichment media following incubation

The first step to detect Salmonella in feed and other dry contaminated samples is a pre-enrichment broth that can assist in the recovery of small numbers of stressed Salmonella cells. A previous study demonstrated that incubation of feed and feed ingredients in commonly used pre-enrichment media resulted in a low pH that injured or killed the Salmonella. The objective of this study was to determine which environmental samples and pre-enrichment and selective broths could interfere with the accuracy of Salmonella detection by allowing pH to drop. Samples were collected from commercial poultry operations. Triplicate 10-g subunits were dispensed into sterile 18-oz Whirl Pak bags and 90 mL of each of the media [lactose broth (LB), buffered peptone water (BPW), Universal Pre-enrichment (UP), minimal salts (M-9), tetrathionate broth (TT) and Rappaport-Vassiliadis broth (RV)] were added to the bags and incubated 24 h at 37°C (or 42°C for RV and TT). The pH was measured after incubation. With turkey litter, fluff, and eggshells, regardless of media, the pH never went below 5.7. Regardless of sample type, the lowest pH was 6.1, 6.2, and 6.4 for UP, M-9, and BPW, respectively. For the pre-enrichment medium commonly used in the US, (LB) the pH dropped to 4.7 to 4.9 with broiler litter and 4.2 for boot covers used in turkey or broiler houses. Many researchers testing these sample types are unaware of this potential for pH change during pre-enrichment and may be underestimating the presence of Salmonella.

Detection of Salmonella enterica subsp. enterica Serovar Cubana from Naturally Contaminated Chick Feed

Because some significant outbreaks of human salmonellosis have been traced to contaminated animal feed, the rapid and efficient detection of Salmonella in feed is essential. However, the current U.S. Food and Drug Administration Bacteriological Analytical Manual (BAM) culture method that uses lactose broth as a preenrichment medium has not reliably supported the results of real-time PCR assays for certain foods. We evaluated the BAM culture method and a quantitative real-time PCR (qPCR) assay using two preenrichment media, modified buffered peptone water and lactose broth, to detect Salmonella enterica subsp. enterica serovar Cubana in naturally contaminated chick feed. After 24 h of incubation, the qPCR method was as sensitive as the culture method when modified buffered peptone water was used as the preenrichment medium but less sensitive than culture when lactose broth was used. After 48 h of incubation, detection of Salmonella Cubana by qPCR and by culture in either preenrichment medium was equivalent. We also compared the performance of the traditional serotyping method, which uses pure cultures of Salmonella grown on blood agar, to two molecular serotyping methods. The serotyping method based on whole genome sequencing also requires pure cultures, but the PCR-based molecular serotyping method can be done directly with the enriched culture medium. The PCR-based molecular serotyping method provided simple and rapid detection and identification of Salmonella Cubana. However, whole genome sequencing allows accurate identification of many Salmonella serotypes and highlights variations in the genomes, even in tight genomic clusters. We also compared the genome of the chick feed isolate with 58 Salmonella Cubana strains in GenBank and found that the chick feed isolate was very closely related to an isolate from a foodborne outbreak involving alfalfa sprouts.

Prevalence and Characterization of Salmonella Isolates from Poultry Farms in Ilorin, Nigeria

Poultry birds are very important source of essential proteins in developing countries, but also play an important role in transmission of Salmonella to human and nonhuman. Exposure to this pathogen also occurs through use of poultry droppings as manure for crop and vegetable production. This study investigated prevalence of Salmonella in feces of poultry farms in Ilorin, Nigeria. Salmonella isolated were further characterized by molecular method. Fecal samples were cultured in pre-enrichment medium, Selenite F medium and finally sub-cultured on Salmonella-Shigella agar. None lactose fermenting colonies with black center were picked for presumptive identification using biochemical tests and confirmed by serological test by method previously described. Molecular characterization was carried out using 16S rRNA gene sequencing following standard procedure. Out of 170 samples collected and examined, 8 (4.7%) gave biochemical characteristics that resembled Salmonella, but only 6 (3.5%) were confirmed as Salmonella by polyvalent antisera. Molecular characterization revealed that serovars isolated were Salmonella Enteritidis 3 (1.8%) and Salmonella Paratyphi 3 (1.8%). Phylogenetic tree constructed by neighbor-joining method as derived from analysis of the 16S rRNA gene sequences showed close relationship to Salmonella Paratyphi strain FB0015 16S ribosoma RNA gene partial sequence to our Salmonella Paratyphi isolates. The detection of 3.5% prevalence rate of Salmonella serovar from feces of laying poultry birds my serve as potential source of transmission of this pathogen to human through chicken meat, egg or use of poultry dropping as manure in crop or vegetable production.

Injury and death of various Salmonella serotypes due to acidic conditions

Acid injury could prevent detection of Salmonella in feed and feed-type samples. A previous study showed that after incubation in commonly used pre-enrichment media, mixed feeds and feed ingredients reached a pH (4.0 to 5.0), capable of injuring or killing Salmonella. Approximately 105 colony forming units (CFU) of S. Enteritidis (SE), S. Heidelberg (SH), S. Kentucky (SK), or S. Typhimurium (ST) were individually placed into 50 mL of a citrate buffer at pH of 4, 4.5, or 5.0 for 6 or 24 h at 37°C. After holding at 37°C, each serotype was serially diluted in sterile physiological saline and plated onto xylose lysine tergitol 4 [for injury] and nutrient agar (NA) [for death]. These plates were enumerated and percent injury and/or death determined. The injury and death confirmed that pH 4.0 was detrimental to these Salmonella serotypes. After 24 h at pH 4.0, 60% or more of all 4 serotypes were killed with 100% of S. Kentucky killed. At pH 5.0, 65 to 75% of these organisms were uninjured, with death between 15 to 20%. Researchers testing feed/feed ingredients for Salmonella may not be aware of the acidic nature of the pre-enrichment step and the subsequent injury or death of any Salmonella present, whether healthy or stressed. Current protocols that are considered the “gold standard” may not be detecting Salmonella in samples containing fermentable substrates and extraneous microorganisms, which prevent the accurate detection of Salmonella. Injury or death of a few Salmonella cells could reduce the chance of recovery from any sample. More research is needed with additional Salmonella serotypes and strains to fully understand the challenges of isolating Salmonella from feed.

Improving the control of salmonella in meat and meat products by PCR in real time and robotics for the separation matrix of DNA

Traditionally, the control of Salmonella in food products is carried out using classic methods of bacteriological analysis that are sufficiently long and labor-intensive. The authors have demonstrated the possibility of accelerating and automating the analysis and obtain reliable results by using an improved method for control of S. typhimurium in chicken meat and meat of semi-finished products (dumplings) by PCR in real time and robotics for the selection of the template DNA, as well as reducing the time of the sample preparation by growing bacteria in the environment of pre-enrichment medium.

Multiplex detection of nine food-borne pathogens by mPCR and capillary electrophoresis after using a universal pre-enrichment medium.

Routine microbiological quality analyses in food samples require, in some cases, an initial incubation in pre-enrichment medium. This is necessary in order to ensure that small amounts of pathogenic strains are going to be detected. In this work, a universal pre-enrichment medium has been developed for the simultaneous growth of Bacillus cereus, Campylobacter jejuni, Clostridium perfringens, Cronobacter sakazakii, Escherichia coli, Enterobacteriaceae family (38 species, 27 genera), Listeria monocytogenes, Staphylococcus aureus, Salmonella spp. (two species, 13 strains). Growth confirmation for all these species was achieved in all cases, with excellent enrichments. This was confirmed by plating on the corresponding selective agar media for each bacterium. This GVUM universal pre-enrichment medium could be useful in food microbiological analyses, where different pathogenic bacteria must be detected after a pre-enrichment step. Following, a mPCR reaction for detection of all these pathogens was developed, after designing a set of nine oligonucleotide pairs from specific genetic targets on gDNA from each of these bacteria, covering all available strains already sequenced in GenBank for each pathogen type. The detection limits have been 1 Genome Equivalent (GE), with the exception of the Fam. Enterobacteriaceae (5 GEs). We obtained amplification for all targets (from 70 to 251 bp, depending on the bacteria type), showing the capability of this method to detect the most important industrial and sanitary food-borne pathogens from a universal pre-enrichment medium. This method includes an initial pre-enrichment step (18 h), followed by a mPCR (2 h) and a capillary electrophoresis (30 min); avoiding the tedious and long lasting growing on solid media required in traditional analysis (1–4 days, depending on the specific pathogen and verification procedure). An external testing of this method was conducted in order to compare classical and mPCR methods. This evaluation was carried out on five types of food matrices (meat, dairy products, prepared foods, canned fish, and pastry products), which were artificially contaminated with each one of the microorganisms, demonstrating the equivalence between both methods (coincidence percentages between both methods ranged from 78 to 92%).

Evaluation of different buffered peptone water (BPW) based enrichment broths for detection of Gram-negative foodborne pathogens from various food matrices

This study evaluated the effects of changing the composition of the pre-enrichment medium buffered peptone water (BPW) on the growth of stressed and unstressed Gram-negative foodborne pathogens in a one-broth enrichment strategy. BPW supplemented with an available iron source and sodium pyruvate, along with low levels of 8-hydroxyquinoline and sodium deoxycholate (BPW-S) improved the recovery of desiccated Cronobacter spp. from powdered infant formula. Growth of Salmonella and STEC was comparable in all BPW variants tested for different food matrices. In products with high levels of Gram-negative background flora (e.g. sprouts), the target organisms could not be reliably detected by PCR in any of the BPW variants tested unless the initial level exceeded 103cfu/10g of sprouts.Based on these results we suggest BPW-S for a one-broth enrichment strategy of stressed Gram-negative foodborne pathogens from dry products. However, a one-broth enrichment strategy based on BPW variants tested in this evaluation is not recommended for produce with a high level of Gram-negative background flora due to very high detection limits.

Diversidad específica bacteriana en murciélagos de distintos gremios alimenticios en la sierra sur de Oaxaca, México

Specific bacterial diversity in bats of different food guilds in Southern sierra Oaxaca, Mexico. Bats have different ecologic roles in variable ecosystems that have been already described. They have been linked to several zoonoses, however little is known about the relationship between bat microbiota and their diet, and studies on the bacterial ecology of the microbiota in bats are limited. To contribute with the description of this important interaction between microbiota and host, the aim of this work was to characterize the composition and bacterial diversity in the oral and anal regions of 10 species of bats, in relation to food guild. For this monthly samplings were conducted using four mist nets (19:00-24:00h) from February to October 2012; nets were reviewed every 45 minutes. Each captured organism was sampled in the oral and anal cavities with sterile swabs; these were placed in pre-enrichment media and stored at 4°C. Bacterial samples were studied which through selective media, chromogenic and biochemical tests. We obtained samples from 502 frugivorous, 29 hematophagous and 11 nectivorous bats. We found a total of 26 bacterial species, with the predominant phylum Proteobacteria and the family Enterobacteriaceae. Statistically significant differences were observed between oral and anal microhabitats: frugivorous (t = -3.516, g.1 = 14.761, p = 0.003), hematophagous (t = -3.320, g.l = 19.262, p = 0.003), and nectivorous (t = -2.497, g.l = 11.933, p = 0.026), and in some guilds [frugivorous and nectivorous in the anal region (t = 2.274, g.l = 29.660, p = 0.030), hematophagous and nectivorous anal region (t = 2.077, g.l = 29.904, p = 0.049)]. It was also shown that there is bacteria specificity in some guilds such as nectivorous and frugivorous with Bacillus cereus, B. sp. X. sp., as well as, Pseudomonas aeruginosa, Serratia marcescens, Staphylococcus aureus, S. epidermis, Aeromonas hydrophyla in hematophagous. Bacterial presence can be explained by the type of diet and/or by transfer of bacteria from their preys. These bacteria may be indigenous to these bats and play the role of mutual benefit, providing the host with stable growth conditions and supplemental nutrients, while the microbiota contributes to host nutrition, development of the immune system, stabilization of the microbial population and to avoid pathogens colonization. By understanding the importance of the relation- ship between host and its bacterial populations, the conservation efforts being made to protect species such as bats may be improved.

Evaluation and comparison of rapid methods for the detection of Salmonella in naturally contaminated pine nuts using different pre enrichment media

Foodborne outbreaks, involving pine nuts and peanut butter, illustrate the need to rapidly detect Salmonella in low moisture foods. However, the current Bacteriological Analytical Manual (BAM) culture method for Salmonella, using lactose broth (LB) as a pre enrichment medium, has not reliably supported real-time quantitative PCR (qPCR) assays for certain foods. We evaluated two qPCR assays in LB and four other pre enrichment media: buffered peptone water (BPW), modified BPW (mBPW), Universal Pre enrichment broth (UPB), and BAX® MP media to detect Salmonella in naturally-contaminated pine nuts (2011 outbreak). A four-way comparison among culture method, Pathatrix® Auto, VIDAS® Easy SLM, and qPCR was conducted. Automated DNA extraction techniques were compared with manual extraction methods (boiling or InstaGene™). There were no significant differences (P > 0.05) among the five pre enrichment media for pine nuts using the culture method. While both qPCR assays produced significantly (P ≤ 0.05) higher false negatives in 24 h pre enriched LB than in the other four media, they were as sensitive as the culture method in BPW, mBPW, UPB, and BAX media. The VIDAS Easy and qPCR were equivalent; Pathatrix was the least effective method. The Automatic PrepSEQ™ DNA extraction, using 1000 μL of pre enrichment, was as effective as manual extraction methods.

Miniaturized most probable number for the enumeration of Salmonella sp in artificially contaminated chicken meat

Salmonella is traditionally identified by conventional microbiological tests, but the enumeration of this bacterium is not used on a routine basis. Methods such as the most probable number (MPN), which utilize an array of multiple tubes, are time-consuming and expensive, whereas miniaturized most probable number (mMPN) methods, which use microplates, can be adapted for the enumeration of bacteria, saving up time and materials. The aim of the present paper is to assess two mMPN methods for the enumeration of Salmonella sp in artificially-contaminated chicken meat samples. Microplates containing 24 wells (method A) and 96 wells (method B), both with peptone water as pre-enrichment medium and modified semi-solid Rappaport-Vassiliadis (MSRV) as selective enrichment medium, were used. The meat matrix consisted of 25g of autoclaved ground chicken breast contaminated with dilutions of up to 106 of Salmonella Typhimurium (ST) and Escherichia coli (EC). In method A, the dilution 10-5 of Salmonella Typhimurium corresponded to >57 MPN/mL and the dilution 10-6 was equal to 30 MPN/mL. There was a correlation between the counts used for the artificial contamination of the samples and those recovered by mMPN, indicating that the method A was sensitive for the enumeration of different levels of contamination of the meat matrix. In method B, there was no correlation between the inoculated dilutions and the mMPN results.

Comparison of different preenrichment broths, egg:preenrichment broth ratios, and surface disinfection for the detection of Salmonella enterica ssp. enterica serovar Enteritidis in shell eggs

Salmonella enterica ssp. enterica serovar Enteritidis is the leading reported cause of Salmonella infections. Most Salmonella Enteritidis infections are associated with whole shell eggs and egg products. This project attempted to lay the foundation for improving the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella Enteritidis in shell eggs. Two Salmonella Enteritidis isolates were used for comparisons among different preenrichment and enrichment media and for the evaluation of egg:preenrichment broth ratios for the detection of Salmonella Enteritidis in shell eggs. The effect of surface disinfection on the detection of Salmonella Enteritidis in shell eggs was also investigated. The results indicated that tryptic soy broth (TSB) was similar to TSB plus ferrous sulfate, but significantly (α = 0.05) better than nutrient broth, Universal Preenrichment broth, and buffered peptone water when used for preenrichment of Salmonella in shell eggs. Salmonella Enteritidis populations after enrichment with Rappaport-Vassiliadis broth were 0.40 to 1.11 log cfu/mL of culture lower than those in preenrichment cultures. The reduction was statistically significant (α = 0.05). Egg:broth ratios at 1:9 and 1:2 produced significantly (α = 0.05) higher Salmonella Enteritidis populations after preenrichment with TSB with inoculum levels at 4 cfu/100 g of eggs and 40 cfu/1,000 g of eggs than the ratio at 1:1. Salmonella Enteritidis populations in TSB preenrichment cultures of shell eggs surface-disinfected with 70% alcohol:iodine/potassium iodide solution and untreated control were 9.11 ± 0.11 and 9.18 ± 0.05 log cfu/mL, respectively, for SE 13–2, and 9.20 ± 0.04 and 9.16 ± 0.05 log cfu/mL, respectively, for SE CDC_2010K_1543. Surface disinfection of eggs did not reduce the sensitivity of detection of Salmonella Enteritidis in liquid eggs. These results could improve the Food and Drug Administration's current Bacteriological Analytical Manual method for the detection of Salmonella in shell eggs by simplifying the preenrichment medium and changing the sample handling before enrichment.

Comparison of 3 culture methods and PCR assays forSalmonella gallinarum andSalmonella pullorum detection in poultry feed

To detect Salmonella gallinarum or Salmonella pullorum in artificially contaminated poultry feed, 9 culture combinations were compared, including 3 preenrichment/enrichment methods (tryptic soy broth plus ferrous sulfate/tetrathionate Hajna, tryptic soy broth plus ferrous sulfate/selenite cystine broth, and Salmosyst) in combination with 3 selective agars (xylose lysine desoxicholate agar added with tergitol 4, EF-18, and Önöz), respectively. Additionally, a single PCR technique was applied combined with 2 different preenrichment media (tryptic soy broth plus ferrous sulfate and Salmosyst). The specificity and positive predictive value were 1 for all methods. There were some differences among Salmonella strains for sensitivity and accuracy in the culture and Salmosyst-PCR methods. The sensitivity and accuracy values were less than 0.60 and 0.64, respectively, whereas the negative predictive values were between 0.12 and 0.23. Two PCR methods did not show any difference in the parameters of performance evaluated. Kappa coefficients showed good agreement between both methods. None of the culture combinations was able to detect S. gallinarum or S. pullorum when the inoculum was less than 3 × 10² cfu/25 g, except the Salmosyst broth method, which could recover S. gallinarum from 3 × 10¹ cfu/25 g onward. Overall, there were differences in the detection limits among the strains and methods used. In general, the 3 selective plating media did not show any significant difference in the parameters of performance studied for each strain. On the other hand, the agreements were slight to fair when culture methods were compared among them and with both PCR methods. The differences in the detection levels that were obtained using these methods and the difficulty in detecting S. gallinarum or S. pullorum in feed represent a potential problem when a poultry feed sample is considered to be negative. It is highly recommended to use at least 2 methods to increase the chances of detecting S. gallinarum or S. pullorum in poultry feed.

An Optimized EMA-RAPD-PCR for a Reliable Detection of Viable Salmonella spp. in Chicken Products

This study aimed to develop the reliable technique called ethidium bromide monoazide-random amplified polymorphic DNA-PCR (EMA-RAPD-PCR) for detection of only viable Salmonella cells due to PCR cannot distinguish DNA from viable and dead cells. In EMA-RAPD-PCR, EMA was used to intercalate the DNA obtained from 1.2 × 106 cells of viable and heat-killed Salmonella Typhimurium and Salmonella Enteritidis. The optimized conditions of EMA treatment for an effective prevention of DNA amplification from dead cells by RAPD-PCR were as follows: the minimum amount of 3 μg/mL EMA; the suitable light exposure time of at least 5 min; and the optimum light exposure distance of 20 cm. To improve a reliability in specific detection of viable Salmonella cells in food samples, use of an optimized 20-h preenrichment in nutrient broth followed by EMA-RAPD-PCR could inhibit the DNA amplification of the dead cells in all artificially and naturally Salmonella-contaminated chicken products tested. Practical Applications The developed Ethidium Bromide Monoazide-Random Amplified Polymorphic DNA-Polymerase Chain Reaction (EMA-RAPD-PCR) involving 20-h culturing in preenrichment medium is an efficient, reliable and economical procedure to detect only viable Salmonella spp. in food samples. This technique has demonstrated effectiveness in preventing the DNA amplification of dead Salmonella cells. Moreover, the EMA-RAPD-PCR has the potential for use as a rapid, simple and reliable monitoring tool of Salmonella spp. prevalence along the food production chain.