Bacteriological Analytical Manual Research Articles

Etiology and Antimicroba Susceptibility of Udder Pathogens from Cases of Subclinical Mastitis in Dairy Ettawa Crosbread Goat (PE) in Kulonprogo Yogyakarta

Subclinical mastitis in Ettawa crosbreeds (PE) is an inflammatory disease that no clinical symptoms, but there is an increase the number of somatic cells and causes decrease milk production which economically detrimental. The aim of these study was to isolation of bacteria that causing subclinical mastitis in PE goats and their susceptibility with antimicrobial. A total of 37 PE goats from 5 farms in Kulonprogo were tested by California Mastitis Test (CMT). PE goats were said subclinical mastitis if the CMT test positive (++) or (++). Isolation and identification bacteria from PE goat subclinical mastitis by bacteriological analytical manual. Coagulase test with tube coagulation and antimicroba susceptibility with agar diffusion. Subclinical mastitis in PE goats at Kulonprogo farm is 10/37 (27%) which caused by S. intermedius positive coagulase 4/4 (100%), S. aureus negative coagulase 7/10 (70%), S. aureus positive coagulase 3/10 (30%), and E. coli 1/10 (10%). S. intermedius positive coagulase was resistant to ampicillin, tetracycline, and sulfamethoxazole 2/4 (50%) respectively. S. aureus negative coagulase was resistant to ampicillin 2/7 (28.6%), penicillin 1/7 (14.3%), and sulfamethoxazole 1/7 (14.3%). S. aureus positive coagulase was resistant to ampicillin 1/3 (33.3%), penicillin 3/3 (100%), sulfametoxazole 2/3 (66.7%), and tetracycline group 1/3 (33.3%). Subclinical mastitis PE goats in Kulonprogo was caused by S. intermedius positive coagulase and S. aureus negative coagulase which resistant to penicillin and sulfamethoxazole.

Effect of the Number of ME Elements on Capture Rate of a Small Number of Pathogens in the Prototype of Phage Filter with a Comparison of Rocking Method

Foodborne illness is a frequent public health problem because there is potential for food to be contaminated by pathogens at any point from farms to the dinner tables. Each year, the cost of foodborne illness in the U.S. is enormous. The direct and indirect costs associated with foodborne illness are more than $15.6 billion per year. For rapid detection of foodborne pathogens, Magnetoelastic (ME) biosensor is one of the methods to detect pathogens on food surface and liquids. As the multistate outbreak caused by Romaine lettuce contamination in 2018, current microbiological testing methods, required by Food and Drug Administration (FDA)’s Bacteriological Analytical Manual (BAM), shows shortages. According to the requirement, a new method, named 3D bio-molecular filter, was developed to capture, concentrate, and isolate a small number of pathogens in large volume of liquid streams. The filter consists of ME filter elements, a pair of permanent magnetic plates, and testing glass tube. ME filter elements are magnetically held and arrayed in a testing glass tube. The target pathogen in a large volume of liquid will be capture by ME filter elements when a large volume of liquid passes through. A prototype of a compact edition was developed in this paper to support this method. The device consists of high magnetic plates, which provide uniform high magnetic force to hold ME filter elements, and a square glass tube to pass through the liquid with target pathogen. In this work, E2 phage is immobilized on ME filter elements to capture, concentrate, and isolate Salmonella Typhimurium in processing water. Two different methods to capture Salmonella, rocking method and 3D biomolecular filter method, are compared. The capture rate and effects of the number of ME filter elements on Salmonella capture efficiency will be discussed.

Independent Validation for the Polyskope 1.0 Multiplex Pathogen Detection Assay for the Detection of Shiga Toxin-Producing Escherichia coli Non-O157 STEC, Escherichia coli O157, Listeria monocytogenes, and Salmonella Species.

The Polyskope 1.0 Multiplex Assay is a novel test to simultaneously detect Escherichia coli O157, non-O157 Shiga Toxin-Producing E. coli (STEC), Listeria monocytogenes, and Salmonella species in a single enrichment using real-time PCR. A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. This assay was evaluated in an unpaired independent validation study compared with reference methods according to AOAC INTERNATIONAL validation guidelines. Polyskope 1.0 evaluated raw ground beef (25 g), deli turkey (25 g), baby spinach (25 g), and stainless-steel environmental surface sponges (4 × 4 in. test area) after inoculation with a suspension of the three target microorganisms. All matrices were compared with appropriate reference methods from the U.S. Food and Drug Administration Bacteriological Analytical Manual, U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, or International Organization for Standardization standards. Polyskope 1.0 demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for three food matrices and one environmental surface. Results from inclusivity and exclusivity evaluations indicated the test method can accurately detect the target analytes and excluded all nontarget organisms. No differences were observed with the stability or lot-to-lot evaluations. Polyskope 1.0 demonstrated robustness by remaining unaffected by small variations in method parameters, which had no statistically significant effect on the results for all eight variations. Conclusions and Highlights: Polyskope 1.0 was shown to be a specific, highly accurate, and robust method for the detection of Listeria monocytogenes, Salmonella species, non-O157 STECs, and E. coli O157 across four matrices.

Evaluation of the MC-Media PadTM Yeast and Mold Device for the Enumeration of Yeast and Mold: Collaborative Study, First Action 2018.02.

Background: The MC-Media Pad™ Yeast and Mold (YM) is a ready-to use culture device that combines a test pad coated with medium and water-absorption polymers that is designed for the rapid quantification of yeast and mold in food products. Objective: The MC-Media Pad YM was compared with the U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) for the enumeration of yeast and mold in frozen orange juice concentrate. Methods: The candidate method was evaluated using a paired study design in a multilaboratory collaborative study following the current AOAC Validation Guidelines. Three target contamination levels (low, 10-100 CFU/g; medium, 100-1000 CFU/g; and high 1000-10 000 CFU/g) and an uninoculated control level (0 CFU/g) were evaluated. MC-Media Pad YM devices were enumerated after 48 and 72 h of incubation. Results: Plate count obtained by both methods were log-transformed and the difference of means (including 95% confidence intervals), repeatability SD, and SD were determined for each contamination level. Conclusions: No statistical difference was observed between the MC-Media Pad YM (for both 48 and 72 h) and the FDA BAM for each contamination level. Highlights: The new method offers a convenient alternative to the reference method (FDA BAM) for detection of yeast and mold contamination in food products, yielding reliable and comparable results in 48 h compared to 5 days for the reference method.

Comparison of TEMPO® BC with Spiral Plating Methods for the Enumeration of Bacillus cereus in Cosmetic Products Either Naturally Preserved or Preserved with Phenoxyethanol.

Background: The U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) uses Bacillus cereus rapid agar (BACARA) and Mannitol-yolk-polymyxin (MYP) agar for the enumeration of the members of B. cereus group. Objective: The automated TEMPO Most Probable Number system was compared with the FDA BAM method for the detection of B. cereus group members in cosmetic products. Methods: We inoculated a range of cosmetic products with pure B. cereus spore suspensions (density = 0.5 McFarland) at high (6 log CFU/mL), medium (5 log CFU/mL), and low (4 log CFU/mL) levels. Test portions were aged for 72 h. Five replicates per sample were analyzed; uninoculated test portions served as controls. We also evaluated whether TEMPO BC erroneously detected non-B. cereus or other adulterant organisms. Results: No significant differences (P > 0.05) were found among the TEMPO BC and the BAM spiral plating methods. Correlations between TEMPO BC - BACARA and TEMPO BC - MYP were 0.895 and 0.893 for powder type products, 0.834 and 0.846 for cream and oil-based products, and 0.929 and 0.923 for liquid products, respectively. Non-B. cereus strains were not detected by TEMPO BC. Conclusions: The TEMPO BC method can be used for the detection of B. cereus in cosmetic products without preservatives, or those preserved with either phenoxyethanol or other organic substances.

Application of an Environmental Phage-Based Assay (Sample6 Detect HT/L) for the Detection of Listeria spp. in Ice Cream.

Background: Dairy products are common sources of Listeria outbreaks, and early detection of the pathogen is critical to prevent outbreaks of illnesses and financial losses for dairy producers. Objective: This study aimed to evaluate Sample6 Detect HT/L for effective detection of Listeria monocytogenes and L. innocua in ice cream. Methods: Performance of the Sample6 DETECT HT/L was compared with U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 method for detection of Listeria spp. in ice cream using an unpaired study design. Results: R2-enriched samples tested with Sample6 Detect HT/L performed as well as the reference method at all time points tested from 15 to 24 h. R2 is a proprietary blend for use with the test kit that helps with early detection. All the dPODC values (Sample6 Detect HT/L presumptive and confirmed results) equaled zero, indicating 100% concordance between the methods. Both Sample6 Detect HT/L and FDA BAM results showed low dPODC values, with confidence intervals indicating no significant differences between Sample6 Detect HT/L and reference method results. Conclusions: Sample6 Detect HT/L is suitable to detect Listeria spp. in ice cream, even with a 12 h enrichment. Sample6 Detect HT/L demonstrated equivalent detection of L. monocytogenes and L. innocua from R2-enriched samples as expected with 15 and 18 h enrichment when compared with the 24 h FDA BAM method for L. monocytogenes. Highlights: These results indicate that Sample6 Detect HT/L, primarily developed for environmental samples, can be used to detect Listeria spp. in ice cream with less incubation time, resulting in faster detection.

Prevalence of resistant Salmonella spp. isolated from raw meat and liver of cattle in the Wa Municipality of Ghana

This study was carried out to determine the prevalence of resistant Salmonella spp. isolated from meat (beef) and liver of cattle in the Wa, Municipality of Ghana. Isolation of Salmonella spp. was done according to the USA-FDA Bacteriological Analytical Manual. Antibiotic susceptibility test was performed using the disc diffusion method and the results interpreted using the CLSI guidelines. Antibiotic residues determination was carried out using Premi®Test Kit. Liver samples 32%(16/50) were more contaminated with Salmonella spp. as compared to beef samples 30% (15/50). The Salmonella spp. were highly resistant to teicoplanin (96.77%) but susceptible to chloramphenicol (100%), ciprofloxacin (100%), tetracycline (100%), suphamethoxazole/trimethoprim (100%), amoxycillin/clavulanic acid (93.55%), ceftriaxone (93.55%) and gentamicin (83.87%). The Salmonella spp. exhibited 5 antibiotic resistant patterns with the pattern (Tec) being the commonest. Multiple antibiotic index ranged from 0.11 to 0.33. A total of 35 samples (20 beef and 15 liver samples) were examined for antibiotic residues, and 7 (20%) were positive for antibiotic residues; 6 (17%) were found in the liver while 1 (3%) was in the beef. This study revealed that salmonellae are present in beef and liver samples examined and showed high resistance to teicoplanin. Some of the beef and kidney samples harbored antibiotic residues.

Validation of Solus One Salmonella from Select Food Matrixes and Stainless-Steel and Plastic Environmental Surfaces.

Background: Solus One Salmonella is designed to accurately detect Salmonella species (Salmonella enterica subspecies enterica, salamae, arizonae, diarizonae, houtenae, indica, and Salmonella bongori) from select food matrixes and stainless-steel and plastic environmental surfaces. Solus One Salmonella uses an antibody-based technology test system that is paired with media and our proprietary media supplement, the Solus One Salmonella supplement combined with a manual or automated sample preparation method. Objective: Solus One Salmonella was evaluated for inclusivity and exclusivity, and a matrix comparison study was done for six food matrixes (raw beef trim, pasteurized liquid egg, raw salmon, cheddar cheese, Romaine lettuce, nonfat dry milk) and two environmental surfaces (stainless steel and polystyrene). Methods: Solus One Salmonella was compared with the U.S. Food and Drug Administration Bacteriological Analytical Manual Chapter 5: Salmonella (July 2018) and the U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Manual, 4.09 (January 2017) in the matrix study. Both the manual and automated sample preparation methods were performed for cheddar cheese and stainless-steel environmental surfaces. Results: For the inclusivity and exclusivity evaluation, Solus One Salmonella correctly detected all 108 target organism isolates and correctly excluded all 35 nontarget strains that were analyzed. Conclusions: In the method comparison study, both Solus One Salmonella manual and automated sample preparation methods demonstrated no significant differences based on probability of detection (POD) statistical analysis between presumptive and confirmed results or between candidate and reference method results for the six food matrixes after 20-22 h and two environmental surfaces after 16-20 h of enrichment time. POD analysis of Solus One Salmonella method robustness, product consistency, and stability studies using the automated sample preparation method demonstrated no statistically significant differences.

Independent Validation for the Polyskope 1.0 Multiplex Pathogen Detection Assay for the Detection of Shiga Toxin-Producing Escherichia coli Non-O157 STEC, Escherichia coli O157, Listeria monocytogenes, and Salmonella Species

Background: The Polyskope 1.0 Multiplex Assay is a novel test to simultaneously detect Escherichia coli O157, non-O157 Shiga Toxin-Producing E. coli (STEC), Listeria monocytogenes, and Salmonella species in a single enrichment using real-time PCR. Objective: A Performance Tested MethodSM study was conducted to validate Polyskope 1.0 for inclusivity and exclusivity as well as a matrix comparison study. Method: This assay was evaluated in an unpaired independent validation study compared with reference methods according to AOAC INTERNATIONAL validation guidelines. Polyskope 1.0 evaluated raw ground beef (25 g), deli turkey (25 g), baby spinach (25 g), and stainless-steel environmental surface sponges (4 × 4 in. test area) after inoculation with a suspension of the three target microorganisms. All matrices were compared with appropriate reference methods from the U.S. Food and Drug Administration Bacteriological Analytical Manual, U.S. Department of Agriculture Food Safety and Inspection Service Microbiology Laboratory Guidebook, or International Organization for Standardization standards. Results: Polyskope 1.0 demonstrated no statistically significant differences between candidate and reference method results or between presumptive and confirmed results for three food matrices and one environmental surface. Results from inclusivity and exclusivity evaluations indicated the test method can accurately detect the target analytes and excluded all nontarget organisms. No differences were observed with the stability or lot-to-lot evaluations. Polyskope 1.0 demonstrated robustness by remaining unaffected by small variations in method parameters, which had no statistically significant effect on the results for all eight variations. Conclusions and Highlights: Polyskope 1.0 was shown to be a specific, highly accurate, and robust method for the detection of Listeria monocytogenes, Salmonella species, non-O157 STECs, and E. coli O157 across four matrices.

Multi-Laboratory Validation of a Loop-Mediated Isothermal Amplification Method for Screening Salmonella in Animal Food.

Loop-mediated isothermal amplification (LAMP) has gained wide popularity in the detection of Salmonella in foods owing to its simplicity, rapidity, and robustness. This multi-laboratory validation (MLV) study aimed to validate a Salmonella LAMP-based method against the United States Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 5 Salmonella reference method in a representative animal food matrix (dry dog food). Fourteen independent collaborators from seven laboratories in the United States and Canada participated in the study. Each collaborator received two sets of 24 blind-coded dry dog food samples (eight uninoculated; eight inoculated at a low level, 0.65 MPN/25 g; and eight inoculated at a high level, 3.01 MPN/25 g) and initiated the testing on the same day. The MLV study used an unpaired design where different test portions were analyzed by the LAMP and BAM methods using different preenrichment protocols (buffered peptone water for LAMP and lactose broth for BAM). All LAMP samples were confirmed by culture using the BAM method. BAM samples were also tested by LAMP following lactose broth preenrichment (paired samples). Statistical analysis was carried out by the probability of detection (POD) per AOAC guidelines and by a random intercept logistic regression model. Overall, no significant differences in POD between the Salmonella LAMP and BAM methods were observed with either unpaired or paired samples, indicating the methods were comparable. LAMP testing following preenrichment in buffered peptone water or lactose broth also resulted in insignificant POD differences (P > 0.05). The MLV study strongly supports the utility and applicability of this rapid and reliable LAMP method in routine regulatory screening of Salmonella in animal food.

Preliminary results on prevalence and persistence of Listeria monocytogenes in different dairy and meat processing plants in Central Italy

Purpose: Listeria monocytogenes is a relevant food - borne pathogen able to cause severe and fatal infections in humans. Understanding correlations among different Listeria monocytogenes strains and evaluating Listeria monocytogenes isolates capacity to persist in processing plant environments is an essential tool for food surveillance. Most outbreaks of human listeriosis seem to be linked to strains able to persist in the environment and to replicate in foods. The aim of this study was to investigate prevalence and persistence of Listeria monocytogenes strains subtypes in different dairy and meat processing plants in the Abruzzo and Molise Region. Methods & Materials: Totally 14 food processing plants have been selected for the study (7 for dairy products – DP - and 7 for meat products - MP). Sampling was performed in 4 session from March to October 2017, from 30 to 35 environmental sampling have been sampled in all the division of the plants, both from food contact surfaces (FCS) and non food contact surfaces (NFCS). Sampling sites were selected as niches for Listeria monocytogenes presence. Samples were tested with FSIS MLG8-10:2017 method. Listeria monocytogenes strains were serotyped using the method described in the U.S. FDA Bacteriological Analytical Manual (Bennett and Weaver, 2001). PFGE subtyping was performed according to the PulseNet protocol (Pulsenet USA, 2013) and macrorestriction patterns were analyzed using the BioNumerics 7.6 (Applied Maths, Kortrijk, Belgium). Results: Results are based on 1772 environmental samples tested, 135 (7.6%) samples were found positive for Listeria monocytogenes (94 NFCS and 41 FCS) in 4 DP and 6 MP. PFGE showed indistinguishable persistent strains in 2 DP and 3 MP in all sampling sessions and in one MP in 3 sampling sessions. Conclusion: The persistence of Listeria monocytogenes underlines the ability to persist in food producing plants, in favourable niches inside the processing environment. To reduce Listeria monocytogenes prevalence, Good Manufacturing Practices and Good Hygiene Practices are needed. Analysis are still ongoing to model the contamination path, NGS on isolated strains will be carried out to confirm the persistence of the strains and to find the presence of gene devoted to resistance and persistence.

Effects of Different Broth Enrichment upon Phage Magnetoelastic Biosensor for Fast Detecting Low <i>Salmonella</i> Counts on Problematic Produce

According to the FDA Bacteriological Analytical Manual (BAM) for Salmonella identification in produce, two pre-enrichment steps with 48 hours of incubation are the golden procedures. Lactose broth is recommended for the first pre-enrichment step medium for leafy greens, and the universal pre-enrichment (UP) broth is for tomatoes. However, the suggested broths were evaluated to have the maximum performance using the culture-dependent methods, and may not be applied to other methods, such as biosensor detection platform. A wireless bacteriophage magnetoelastic (ME) biosensor has been recently developed for real-time or rapid detection of food-borne pathogens in various foods. This affinity-based biosensor utilizes a phage oligonucleotide as the probe to capture target bacteria. In this study, the efficiencies of different pre-enrichment media for early detection of low Salmonella on spinach leaves and tomatoes use ME biosensors to shorten detection time. Four broths of modified peptone water, Lennox broth (LB), lactose broth, and UP broth were selected in this study. Various pre-enrichment times for ME biosensor detection were investigated. After spiking 4 cfu/g Salmonella on the tomatoes surfaces, the phage biosensor was able to detect Salmonella within 5 hours of pre-enrichment comparing to 24 hours in the FDA procedures. For Salmonella spiked spinach leaves, the same medium showed Salmonella positive within 7 hours. This study demonstrated that LB broth is the best medium to shorten pre-enrichment time to pass Salmonella number detection thresholds for ME biosensor detection in spinach and tomatoes when comparing to FDA procedures.

Method Modification to Extend the Matrix Claim of the Thermo Scientific RapidFinder Salmonella species, Typhimurium, and Enteritidis Multiplex PCR Kit.

Background: The Thermo Scientific RapidFinder™ Salmonella species, Typhimurium and Enteritidis Multiplex PCR Kit is a real-time multiplex PCR assay for the detection and differentiation of Salmonella species, Salmonella Typhimurium, and S. Enteritidis from poultry, pork, and environmental samples. The method has previously been granted certification as Performance Tested Method SM (PTM) 081701, validated according to the AOAC Research Institute (RI) PTM program for poultry (chicken thighs with skin, chicken wings with skin, and chicken nuggets), raw pork sausage matrixes, and stainless steel environmental surface sponges. Objective: This report details the method modification study to validate ground turkey (375 g sample size), chicken carcass rinse, and shell egg matrixes. Methods: The candidate method was compared with the U.S. Food and Drug Administration's Bacteriological Analytical Manual Chapter 5 for shell eggs and the U.S. Department of Agriculture Food Safety and Inspection Service's Microbiology Laboratory Guidebook 4.09 for ground turkey (375 g) and chicken carcass rinse matrixes. Results: The statistically significant differences found between the candidate and reference methods upon analysis by probability of detection were in favor of the candidate method. Inclusivity and exclusivity testing demonstrated that the RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Kit was able to detect all the major groups of Salmonella. All exclusivity isolates were correctly excluded. Conclusions: The data presented in this report show that the candidate is suitable for the detection and differentiation of Salmonellae from shell egg, chicken carcass rinse, and ground turkey (375 g) matrixes. Highlights: Thermo Scientific RapidFinder Salmonella species, Typhimurium and Enteritidis Multiplex PCR Kit (candidate method) matrix claims extended to include ground turkey (375 g), chicken carcass rinse and shell egg samples.

Carbohydrate Ligands on Magnetic Nanoparticles for Centrifuge-Free Extraction of Pathogenic Contaminants in Pasteurized Milk

Rapid detection of bacterial contamination in the food supply chain is critically important for food safety monitoring. Reliable extraction and concentration of bacteria from complex matrices is required to achieve high detection sensitivity, especially in situations of low contamination and infective dose. Carbohydrate ligands that attach to microbial cell-surface epitopes are promising economical and biocompatible substitutes for cell-targeting ligands and antibodies. Two different carbohydrate ligands immobilized onto magnetic nanoparticles (MNPs) were easily suspended in liquid food (milk) and allowed expedient extraction of microbes within minutes, without the need for centrifugation or loss in capture capacity. In this pilot study, 25-mL samples of undiluted milk were spiked with 5 mg of MNPs and artificially contaminated with bacteria at 3 to 5 log CFU/mL. MNPs and bacteria formed MNP-cell complexes, which were rapidly separated from the milk matrix with a simple magnet to allow supernatant removal. MNP-cell complexes were then concentrated by resuspension in 1 mL of fresh milk and plated per Bacteriological Analytical Manual procedures. Capture was carried out in vitamin D, 2% reduced fat, and fat-free milk spiked with Salmonella Enteritidis, Escherichia coli O157:H7, and Bacillus cereus for a combined total of 18 experiments (three replicates each). An additional eight experiments were conducted to investigate the effect of competitive bacteria on capture. All experiments were carried out over several months to account for environmental variations. Capture efficiency, on a log basis, for all combinations of milk and bacteria was 73 to 90%. Long-term exposure of the MNPs to milk did not markedly affect capture efficiency. These carbohydrate-functionalized MNPs have potential as nonspecific receptors for rapid extraction of bacteria from complex liquids, opening the door to discovery of biocompatible ligands that can reliably target pathogens in our food.

Prevalence, genotyping, serotyping, and antibiotic resistance of isolated Salmonella strains from industrial and local eggs in Iran

AbstractThis present study aimed to evaluate prevalence and characterization of serotypes, antimicrobial resistance (AMR) and genotypic profiles of Salmonella isolates in industrial and local eggs from Zanjan province of Iran. A total of 120 egg samples were collected and processed according to bacteriological analytical manual to isolate Salmonella strains from both eggs surface and contents. PCR method and genotyping were used to verify, identification and classification of isolates by screening invA genes and 16 s‐rRNA gene sequencing. Salmonella contamination rate in eggshell and contents of industrial and local eggs were 0% and 1.66%, respectively. High degree of AMR was observed to nalidixic acid and erythromycin. Gene sequencing for each isolates showed more similarity with Salmonella enterica subsp. enterica serovar Enteritidis and Salmonella enterica subsp. enterica serovar Typhimurium strains.Practical applicationsSalmonella contamination in eggs and the role of healthy chickens as reservoir and distributer of Salmonella in poultry farms can be a common food safety concern for public health. Therefore, Continuous monitoring of presence Salmonella and their antimicrobial resistance is of critical importance. Present study gives an insight of the current statues of egg's contamination with Salmonella isolates, their antimicrobial resistance, genotupic profile, and epidemiological relationship in Iran.

Rapid Detection of Listeria in Ice Cream in 13 Hours Using the Roka Listeria Detection Assay.

Background: Listeria contamination is a major concern in the ice cream industry; therefore, early and accurate detection is vital. Current detection methods require about a 24 h enrichment period for detection. Objective: Enhance the early detection of Listeria in ice cream using the highly sensitive isothermal ribosomal RNA-based Roka/Atlas Listeria Detection Assay. Methods: The R2 Medium was developed for Listeria enrichment by Molecular Epidemiology, Inc. (Seattle, WA). Comparative growth curve studies were performed on the new R2 Medium for Listeria and the currently validated media for the Roka Listeria Detection Assay. Subsequently, a method comparison between the Roka Listeria Detection Assay and the U.S. Food and Drug Administration's (FDA) Bacteriological Analytical Manual (BAM) Chapter 10 reference method on ice cream was carried out. Results: The R2 Medium supports the growth of L. monocytogenes better than Buffered Listeria Enrichment Broth, Demi-Fraser broth, and Modified University of Vermont Broth, as indicated by the faster growth rate of the organism. When used as an enrichment medium in a method comparison study of ice cream, the results showed that R2 Medium-enriched samples tested with the Roka Listeria Detection Assay gave an equivalent performance compared with the 24 h FDA-BAM reference method at 10 and 18 h post-enrichment for Listeria. Conclusions: The results from this study indicate that the new R2 Medium and the highly sensitive Roka Listeria Detection Assay allowed for the rapid detection of Listeria species in ice cream in 13 h. Highlights: The Roka Listeria Detection Assay, in conjunction with a new media formulation (R2 Medium), allowed for the early detection of Listeria in ice cream and may be applied in other food matrixes and environmental samples.

Romer Labs RapidChek®Listeria monocytogenes Test System for the Detection of L. monocytogenes on Selected Foods and Environmental Surfaces.

The Romer Labs RapidChek® Listeria monocytogenes test system (Performance Tested Method 011805) was validated against the U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook (USDA-FSIS/MLG), U.S. Food and Drug Association Bacteriological Analytical Manual (FDA/BAM), and AOAC Official Methods of Analysis (AOAC/OMA) cultural reference methods for the detection of L. monocytogenes on selected foods including hot dogs, frozen cooked breaded chicken, frozen cooked shrimp, cured ham, and ice cream, and environmental surfaces including stainless steel and plastic in an unpaired study design. The RapidChek method uses a proprietary enrichment media system, a 44-48h enrichment at 30 ± 1°C, and detects L. monocytogenes on an immunochromatographic lateral flow device within 10min. Different L. monocytogenes strains were used to spike each of the matrixes. Samples were confirmed based on the reference method confirmations and an alternate confirmation method. A total of 140 low-level spiked samples were tested by the RapidChek method after enrichment for 44-48h in parallel with the cultural reference method. There were 88 RapidChek presumptive positives. One of the presumptive positives was not confirmed culturally. Additionally, one of the culturally confirmed samples did not exhibit a presumptive positive. No difference between the alternate confirmation method and reference confirmation method was observed. The respective cultural reference methods (USDA-FSIS/MLG, FDA/BAM, and AOAC/OMA) produced a total of 63 confirmed positive results. Nonspiked samples from all foods were reported as negative for L. monocytogenes by all methods. Probability of detection analysis demonstrated no significant differences in the number of positive samples detected by the RapidChek method and the respective cultural reference method.

Evaluation of Roka Atlas Salmonella method for the detection of Salmonella in egg products in comparison with culture method, real-time PCR and isothermal amplification assays

With the increasing focus on the food safety, rapid methods for the detection of Salmonella are crucial for both food industry and regulatory agencies. Recently, many molecular methodologies with diverse technologies have been introduced. Roka Atlas®Salmonella Assay (SEN) is a molecular method that uses ribosomal RNA as target for detection, which is theoretically more sensitive than PCR or isothermal amplification methods that target the DNA sequences of single genes. In this study, SEN assay was compared with four PCR- and isothermal amplification-based assays and a culture method, such as the MicroSEQ®Salmonella spp. Detection kit (MicroSEQ), 3M™ Molecular Detection Assay (MDA) Salmonella, ANSRTMSalmonella Assay (ANSR), and Pro-AmpRTTM SALM spp. Kit (Pro-AmpRT). Food samples were prepared and analyzed according to the current U. S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Salmonella culture method. A total of 155 bacterial isolates (121 for Salmonella inclusivity and 34 for Salmeonlla exclusivity) and 200 egg product samples inoculated at a level of 1–5 CFU/25 g were analyzed. The study also estimated the limit of detection of these molecular methods, and illustrated their advantages and disadvantages. For exclusivity, all 34 non-Salmonella isolates were negative by all 5 molecular methods studied. For inclusivity, all 121 Salmonella isolates were positive by MDA, ANSR, and Pro-AmpRT methods. However, the SEN and MicroSEQ results were negative for 9 samples inoculated with Salmonella bongori. The detection limit of the 5 molecular methods ranged from 1.76 to 3.76 log CFU/mL pre-enrichment culture, with the SEN assay being the most sensitive (1.76 – 2.64 log CFU/mL). The results indicated that the SEN assay was as effective and sensitive in detecting Salmonella enterica in egg products as was the FDA BAM culture method and the 4 other isothermal amplification and PCR methods evaluated in the study.

Bacteriological Evaluation of Essential‐Oil Based Products from a Local Small Business

All‐natural, essential‐oil based products are becoming a significant segment of local small business. In fact, an increasing number of consumers are choosing organic plant‐based alternatives over the cosmetics currently available. Independently of the source of ingredients, cosmetics should be tested before becoming available for purchase. It has been recognized that microorganisms can grow and reproduce in cosmetic products, and may cause spoilage or chemical changes in these products, which can result in injury to the user. Standards for the analyses of food and cosmetics have been established by the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM). The present study followed these analytical guidelines to ensure the quality and safety of All‐Natural products (defined as such by the business owner) from a local small business. The tested products included a lotion, a diaper balm, and a bug spray. Two bottles of each product from different batches were randomly chosen and used for testing. Each bottle of each product was tested separately using the Aerobic Plate Count (APC) method and the total coliform analysis according to the BAM. After incubation in the appropriate agar plates, no colonies were found. Therefore, all plate readings from all products were below detectable limits of these methods. These results indicate that the products meet the FDA's requirements for distribution to, and safe use of, the consumer.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Validation of Modifications to the ANSR for Listeria Method for Improved Internal Positive Control Performance.

A study was conducted to validate a minor reagent formulation change to the ANSR for Listeria method, Performance Tested MethodSM 101202. This change involves increasing the master mix volume prelyophilization by 40% and addition of salmon sperm DNA (nontarget DNA) to the master mix. These changes improve the robustness of the internal positive control response and reduce the possibility of obtaining invalid results due to weak-positive control curves. When three foods (hot dogs, Mexican-style cheese, and cantaloupe) and sponge samples taken from a stainless steel surface were tested, no significant differences in performance between the ANSR and U.S. Food and Drug Administration Bacteriological Analytical Manual or U.S. Department of Agriculture-Food Safety and Inspection Service Microbiology Laboratory Guidebook reference culture procedures were observed for any of the matrixes as determined by probability of detection analysis. Inclusivity and exclusivity testing yielded 100% expected results for target and nontarget bacteria. Accelerated stability testing was carried out over a 7 week period and showed no decrease in assay performance over time.