FDA Bacteriological Analytical Manual Research Articles

Recalls of tattoo and permanent makeup inks in the United States and a follow-up microbiological survey of inks with a previous recall history.

In this study, we collected voluntary recall records of tattoo and permanent makeup ink from the U.S. Food and Drug Administration (US FDA) Enforcement Report Database. The recall records contain information, such as recall date, manufacturer, ink color, reason for recall, and the microorganisms detected from the ink samples. Between 2003 and 2021, a total of 15 voluntary tattoo ink recalls occurred in the U.S. market, involving over 200 tattoo inks marketed by 13 manufacturers and one distributor. Fourteen recalls were due to microbial contamination, and one recall was due to allergic reaction. As follow-up, a microbiological survey of 28 tattoo inks of new batches from seven manufacturers having products that were previously recalled was conducted. Aerobic plate count (APC) and enrichment culture methods based on the FDA's Bacteriological Analytical Manual (BAM) were used to detect microbial contamination. The results revealed that six out of 28 tattoo inks were contaminated with bacteria and were produced by two manufacturers. The level of microbial contamination was less than 250 CFU/g in three of the tattoo inks and between 1 × 103 and 1 × 105 CFU/g in the other three inks. Eleven bacterial isolates were identified, including spore-forming Bacillus-related species and potentially pathogenic species. Overall, this study shows that some tattoo ink products produced by manufacturers with a recall history continue to be contaminated with microorganisms. This highlights the need for ongoing monitoring and quality control of such products.

Multi-laboratory validation study of a real-time PCR method for detection of Salmonella in baby spinach

The FDA Bacteriological Analytical Manual (BAM) Salmonella culture method takes at least 3 days for a presumptive positive result. The FDA developed a quantitative PCR (qPCR) method to detect Salmonella from 24-h preenriched cultures, using ABI 7500 PCR system. The qPCR method has been evaluated as a rapid screening method for a broad range of foods by single laboratory validation (SLV) studies. The present multi-laboratory validation (MLV) study was aimed to measure the reproducibility of this qPCR method and compare its performance with the culture method. Sixteen laboratories participated in two rounds of MLV study to analyze twenty-four blind-coded baby spinach test portions each. The first round yielded ∼84% and ∼82% positive rates across laboratories for the qPCR and culture methods, respectively, which were both outside the fractional range (25%–75%) required for fractionally inoculated test portions by the FDA's Microbiological Method Validation Guidelines. The second round yielded ∼68% and ∼67% positive rates. The relative level of detection (RLOD) for the second-round study was 0.969, suggesting that qPCR and culture methods had similar sensitivity (p > 0.05). The study demonstrated that the qPCR yields reproducible results and is sufficiently sensitive and specific for the detection of Salmonella in food.

MICROBIOLOGICAL QUALITY OF LEAFY GREEN VEGETABLES GROWN IN THE TOLUCA VALLEY

The intake of fresh fruits and vegetables has increased in recent years, as have outbreaks of foodborne illnesses associated with these products. The risk of microbiological hazards in vegetables can occur from the field to the table. The main sources of contamination are agricultural soil and irrigation water, which can affect product quality and be a reservoir of foodborne pathogenic bacteria. This research analysed the microbiological quality of green leafy vegetable crops: spinach (Spinacia oleracea L.), lettuce (Lactuca sativa L.) and coriander (Coriandrum sativum L.), grown in the valley of Toluca. The presence of microorganisms indicating microbiological contamination (mesophilic bacteria, total coliforms, faecal coliforms and Escherichia coli, Salmonella and Listeria) was evaluated in plants, water and soil. Sampling, processing, isolation and bacterial identification were performed in accordance with Mexican Official Standards and the FDA Bacteriological Analytical Manual. The recovered bacteria were subjected to biochemical tests, serotyping and PCR. Microbial counts were present in 100 % of the samples tested. The spinach culture had higher recovery of mesophilic bacteria and total coliforms, while the lettuce culture presented higher values for the faecal coliform group. The results indicated higher microbiological contamination in water and soil > 250 CFU for mesophilic bacteria, total coliforms and faecal coliforms, respectively. The microbiological counts of the three cultures were found within the maximum limits established by Mexican regulations. However, the presence of faecal coliforms, which included E. coli bacteria of serotype O105 ab flagellar, compromises the quality of the product and poses a risk to the health of the consumer.

Development and validation of an improved method for the detection of Salmonella in cinnamon bark and oregano leaves using the adsorbent beta zeolite in the pre-enrichment media

The detection of Salmonella in spices is challenging due to the presence of antibacterial components. In this study, we evaluated the use of an adsorbent beta zeolite in pre-enrichment media to improve the recovery of Salmonella from cinnamon bark and oregano leaves. Samples (25 g) were spiked with varying levels of S. Montevideo or S. Senftenberg. After 2 weeks of stabilization at RT, betazeolite was added to cinnamon and oregano samples prior to the addition of 225 mL or 475 mL of pre-enrichment media, respectively. Detection sensitivity and rate of the test method were compared to the FDA Bacteriological Analytical Manual (BAM) method which requires the use of 2.5 L pre-enrichment broth. While Salmonella could not be detected in the test method using the reduced volume of pre-enrichment media alone, the addition of beta zeolite resulted in a positivity rate of 62% and 72.6% for cinnamon bark and oregano leaves respectively (all spike levels and both serovars combined). Furthermore, while there were differences in the LOD50 compared to the BAM method, there was no significant difference in the minimum level of detection between the betazeolite and the BAM methods. Our results demonstrate that the use of betazeolite in the pre-enrichment media offers a method with reduced media volumes without compromising on the sensitivity or efficiency of Salmonella detection in cinnamon bark and oregano leaves.

Prevalence of Listeria Species on Food Contact Surfaces in Washington State Apple Packinghouses.

The 2014 caramel apple listeriosis outbreak was traced back to cross-contamination between food contact surfaces (FCS) of equipment used for packing and fresh apples. For Washington state, the leading apple producer in the United States with 79% of its total production directed to the fresh market, managing the risk of apple contamination with Listeria monocytogenes within the packing environment is crucial. The objectives of this study were to determine the prevalence of Listeria spp. on FCS in Washington state apple packinghouses over two packing seasons and to identify those FCS types with the greatest likelihood to harbor Listeria spp. Five commercial apple packinghouses were visited quarterly over two consecutive year-long packing seasons. A range of 27 to 50 FCS were swabbed at each facility to detect Listeria spp. at two sample times, (i) postsanitation and (ii) in-process (3 h of packinghouse operation), following a modified protocol of the FDA's Bacteriological Analytical Manual method. Among 2,988 samples tested, 4.6% (n = 136) were positive for Listeria spp. Wax coating was the unit operation from which Listeria spp. were most frequently isolated. The FCS that showed the greatest prevalence of Listeria spp. were polishing brushes, stainless steel dividers and brushes under fans/blowers, and dryer rollers. The prevalence of Listeria spp. on FCS increased throughout apple storage time. The results of this study will aid apple packers in controlling for contamination and harborage of L. monocytogenes and improving cleaning and practices for sanitation of the FCS on which Listeria spp. are the most prevalent.IMPORTANCE Since 2014, fresh apples have been linked to outbreaks and recalls associated with postharvest cross-contamination with the foodborne pathogen L. monocytogenes These situations drive both public health burden and economic loss and underscore the need for continued scrutiny of packinghouse management to eliminate potential Listeria niches. This research assesses the prevalence of Listeria spp. on FCS in apple packinghouses and identifies those FCS most likely to harbor Listeria spp. Such findings are essential for the apple-packing industry striving to further understand and exhaustively mitigate the risk of contamination with L. monocytogenes to prevent future listeriosis outbreaks and recalls.

Modifications of the U.S. food and drug administration validated method for detection of Cyclospora cayetanensis oocysts in prepared dishes: Mexican-style salsas and guacamole

Although multiple outbreak clusters of Cyclospora cayetanensis have been traced back to consumption of dishes in Mexican-style restaurants, the FDA Bacteriological Analytical Manual (BAM) does not currently provide methods to detect C. cayetanensis in dishes that contain multiple produce ingredients, such as salsas and guacamole. These complex food matrices also may contain high levels of fats, which can interfere with the detection. Several modifications to the BAM Chapter 19b method (washing produce, DNA extraction, and a TaqMan real-time PCR assay targeting the 18S rRNA gene of C. cayetanensis) were assessed with the goal to detect as few as 5 oocysts of C. cayetanensis in 25 g samples of commercial salsa/pico de gallo, guacamole, and salsa verde. Both freshly prepared and frozen versions of these foods were seeded with 5, 10 and 200 oocysts. For salsa samples, using a gentler washing step than recommended by BAM, we achieved detection of 5 oocysts in the samples (81.8%, n = 11). Increasing the amount of Alconox® in the wash solution to 1%, rather than the 0.1% used in BAM, and adjusting the DNA extraction protocol to process large wash pellets, enabled detection of 5 oocysts in guacamole. To reach the desired level of detection in salsa verde, two types of modifications were necessary: gentler washing and DNA extraction modifications. The use of these same method modifications on previously frozen food samples, provided levels of detection similar to those achieved with fresh dishes. Our modifications enabled robust and reproducible detection of C. cayetanensis in multi-ingredient Mexican dishes, detecting as few as 5 oocysts in 25 g samples. Validating and deploying effective methods to detect C. cayetanensis in high risk fresh produce and prepared dishes are critically important for prevalence studies and outbreak investigations of this parasite.

Utility of rapid tests to assess the prevalence of indicator organisms (Aerobic plate count, Enterobacteriaceae, coliforms, Escherichia coli, and Listeria spp.) in apple packinghouses

The 2014 listeriosis outbreak caused by caramel-coated apples was linked to apples cross-contaminated within an apple packing facility. This outbreak has increased the focus on effective cleaning and sanitation methods that must be validated and monitored during apple packing. Thus, rapid and reliable testing methods are necessary for assessing cleanliness in the apple packing industry. The objectives of this study were to assess the prevalence of common indicator organisms [Aerobic plate count (APC), Enterobacteriaceae, coliforms, Escherichia coli, and Listeria spp.] on food contact surfaces (zone 1) in apple packinghouses and to evaluate the utility and accuracy of currently used rapid tests (ATP and glucose/lactose residue swabs). Food contact surfaces were sampled over a 100 cm2 area in five commercial apple packinghouses to evaluate populations of indicator organisms APC, Enterobacteriaceae, coliforms, E. coli (n = 741), and rapid test readings (n = 659). Petrifilm plates were used for the quantification of APC, Enterobacteriaceae, and coliform/E. coli. Rapid tests [ATP swabs (UltraSnap) and glucose/lactose residue swabs (SpotCheck Plus)] were processed on-site. A larger area (0.93 m2) was sampled for the detection of Listeria spp. (n = 747), following a modified protocol of the FDA's Bacteriological Analytical Manual method, and confirmed with PCR and gel electrophoresis via the iap gene. No significant association was found between either rapid test and populations of APC, Enterobacteriaceae, coliforms, E. coli, and Listeria spp. detection. However, recovery of APC (log CFU/100 cm2) was higher with a failed glucose/lactose residue swab surface hygiene result (3.1) than a passed result (2.9) (p = 0.03).Populations of APC, Enterobacteriaceae, and coliforms were significantly different at each unit operation during the packing process (p ≤ 0.05). This study concluded that ATP and glucose/lactose residue rapid tests were poorly suited for determining microbial load since they were not related to populations of any common indicator organisms or the detection of Listeria spp. These findings emphasize the need to utilize a rapid test, which can be a good indicator of residual matter on a surface, along with traditional microbiological methods to assess cleaning and sanitation practices in apple packinghouses.

Performance Comparison of Rapid Petrifilm™ Test Methods vs. Conventional Test Methods for Microbiological Testing of Dietary Supplements

Abstract Objectives The aim of this study is to show suitability of 3M™ Petrifilm™ across various types of common multi ingredient finished dietary supplement products. Methods In present investigation, parallel analysis and suitability was conducted using gold standard methods prescribed under U.S. Pharmacopeia general chapter dietary supplement ⟨2021⟩/⟨2022⟩, US FDA's Bacteriological Analytical Manual (BAM), and AOAC using 3M™ Petrifilm™. Five commonly used tests in food and dietary supplement quality testing were deployed in this study, namely: Total Aerobic Microbial Count (TAMC), Total Yeast and Mold Count (TYMC), Escherichia coli, Staphylococcus aureus, and Coliforms. Dietary supplements selected for this investigation are from most common types of dietary supplements categories sold in US market i.e., multivitamin and mineral, protein, multi mineral, Prenatal, Vitamin D, and Omega 3 Fish oil. Three individual lots of each of the five products were tested to increase robustness of data. Acceptance criteria was set same as USP ⟨2021⟩/⟨2022⟩ requirements, that is to demonstrate a greater than 70% recovery in comparison to a control. Results AOAC using 3M™ Petrifilm™ results show recovery of >70% compared to control for all five test Total Aerobic Microbial Count (TAMC), Total Yeast and Mold Count (TYMC), Escherichia coli, Staphylococcus aureus, and Coliforms: Multivitamin and mineral product recovery ranges were between 79%–111%, Protein product recovery ranges were between 94%–104%, Multi mineral product recovery ranges were between 94%–107%, Prenatal product recovery ranges were between 74%–117%, Vitamin D product recovery ranges were between 93%–123%, and Omega 3 fish oil product recovery ranges were between 83%–101%. Conclusions We found that AOAC based 3M™ Petrifilm™ methods are suitable for selected major categories of dietary supplement finished products. All results from AOAC using 3M™ Petrifilm™ microbiological testing achieved >70% bioburden recovery in comparison to a control. We also found that AOAC using 3M™ Petrifilm™ provides consistent and comparable results to USP ⟨2021⟩/⟨2022⟩/FDA's BAM results for the same samples as well. Funding Sources Reliance Vitamin.

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.

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.

Evaluation of 3M Molecular Detection System and ANSR Pathogen Detection System for rapid detection of Salmonella from egg products

Isothermal amplification assay is a novel simple detection technology that amplifies DNA with high speed, efficiency, and specificity under isothermal conditions. The objective of this study was to evaluate the effectiveness of the 3M Molecular Detection System (MDS) and ANSR Pathogen Detection System (PDS) for the detection of Salmonella in egg products as compared to the Food and Drug Administration's Bacteriological Analytical Manual (BAM) culture method and a modified culture method (3M MDS and ANSR PDS preferred method). Two Salmonella ser. Enteritidis (18579, PT4; CDC_2010K_1441, PT8), one Salmonella ser. Heidelberg (607310-1), and one Salmonella ser. Typhimurium (0723) isolates were used in this study. Seven wet egg products and 13 dry egg products were inoculated with these strains individually at 1 to 5 CFU/25 g. One set of test portions was prepared following FDA BAM procedures [with lactose broth (LB) as pre-enrichment broth]. Another set of test portions was prepared using buffered peptone water (BPW) as pre-enrichment broth, as instructed by the 2 detection systems. Results from 3M MDS and ANSR PDS were 100% in agreement with their BPW-based culture method results. When LB was used as pre-enrichment broth, the number of Salmonella positive test portions (80 tested), identified with the BAM, 3M MDS, and ANSR PDS, were 63, 61, and 60, respectively. In conclusion, both 3M MDS and ANSR PDS Salmonella assays were as effective as their BPW based culture methods and were equivalent to the BAM culture method for the detection of Salmonella in egg products. These sensitive isothermal assays can be used as rapid detection tools for Salmonella in egg products provided that BPW is used as pre-enrichment broth.

Detection of Salmonella Typhimurium on Spinach Using Phage-Based Magnetoelastic Biosensors.

Phage-based magnetoelastic (ME) biosensors have been studied as an in-situ, real-time, wireless, direct detection method of foodborne pathogens in recent years. This paper investigates an ME biosensor method for the detection of Salmonella Typhimurium on fresh spinach leaves. A procedure to obtain a concentrated suspension of Salmonella from contaminated spinach leaves is described that is based on methods outlined in the U.S. FDA Bacteriological Analytical Manual for the detection of Salmonella on leafy green vegetables. The effects of an alternative pre-enrichment broth (LB broth vs. lactose broth), incubation time on the detection performance and negative control were investigated. In addition, different blocking agents (BSA, Casein, and Superblock) were evaluated to minimize the effect of nonspecific binding. None of the blocking agents was found to be superior to the others, or even better than none. Unblocked ME biosensors were placed directly in a concentrated suspension and allowed to bind with Salmonella cells for 30 min before measuring the resonant frequency using a surface-scanning coil detector. It was found that 7 h incubation at 37 °C in LB broth was necessary to detect an initial spike of 100 cfu/25 g S. Typhimurium on spinach leaves with a confidence level of difference greater than 95% (p < 0.05). Thus, the ME biosensor method, on both partly and fully detection, was demonstrated to be a robust and competitive method for foodborne pathogens on fresh products.

Color features as an approach for the automated screening ofSalmonellastrain

We present the implementation of a feature extraction approach for the automated screening of Salmonella sp., a task visually carried out by a microbiologist, where the resulting color characteristics of the culture media plate indicate the presence of this strain. The screening of Salmonella sp. is based on the inoculation and incubation of a sample on an agar plate, allowing the isolation of this strain, if present. This process uses three media: Xylose lysine deoxycholate, Salmonella Shigella, and Brilliant Green agar plates, which exhibit specific color characteristics over the colonies and over the surrounding medium for a presumed positive interpretation. Under a controlled illumination environment, images of plates are captured and the characteristics found over each agar are processed separately. Each agar is analyzed using statistical descriptors for texture, to determine the presence of colonies, followed by the extraction of color features. A comparison among the color features seen over the three media, according to the FDA Bacteriological Analytical Manual, determines the presence of Salmonella sp. on a given sample. The implemented process proves that the task addressed can be accomplished under an image processing approach, leading to the future validation and automation of additional screening processes.

English

Antimicrobial resistance is becoming an extremely serious global problem. The goal of this study was to determine the prevalence and phenotypic and genotypic characteristics of Staphylococcus aureus isolated from milk and milk product samples in Ethiopia and also to determine the presence of methicillin resistant Staphylococcus aureus (MRSA). A total of 577 milk and milk product samples were collected from central Ethiopia and Staphylococcus spp. were isolated using the method described in FDA Bacteriological Analytical Manual (BAM). Resistance of S. aureus isolates to 12 antimicrobials was determined by using the Kirby-Bauer disk diffusion method. PCR detection of mecA and nuc gene was also conducted. To determine the clonal relatedness of S. aureus isolates, DNA fingerprinting of selected isolates was performed by PFGE. Of the 577 milk and milk product samples investigated, S. aureus isolates were recovered from 120 (21%) of the sample. In addition, coagulase negative Staphylococcus species were also isolated from 361/577 (63%) of the samples. The highest frequency of resistance was observed for penicillin (83%) and the lowest was noted for amoxicillin/clavulanic acid (3%) and gentamicin (3%). Fourteen (14) isolates (13%) recovered from raw milk were found to be susceptible to all the tested antimicrobials while 57% of the isolates were resistant to more than one of the antimicrobials. All the isolates were susceptible to vancomycin and none were found to be methicillin resistant S. aureus based on mecA gene carriage. PFGE analysis of 39 S. aureus isolates identified three separate clonal clusters and also several sporadic isolates. S. aureus isolates in this study were found to be resistant to multiple antimicrobials. This warrants a larger representative study to fully understand the extent of the problem and design better strategies for regulation of antimicrobial use in both the medical and veterinary sectors in central Ethiopia. Key words: Bovine milk, Ethiopia, genotypic resistance, Staphylococcus aureus.

Effect of Listeria seeligeri or Listeria welshimeri on Listeria monocytogenes detection in and recovery from buffered Listeria enrichment broth

The presence of multiple species of Listeria in regulated food products is not uncommon and can complicate the recovery of Listeria monocytogenes particularly on a non-differentiating medium. The potential complications of Listeria seeligeri and Listeria welshimeri on the recovery of L. monocytogenes from inoculated food test samples using the U.S. Food and Drug Administration's (FDA) selective enrichment procedure was investigated. Post-enrichment enumeration, in the absence of food product, indicates that some L. seeligeri and L. monocytogenes pairings may have population differentials as great as 2.7 ± 0.1 logs with L. seeligeri being the predominant species. A similar observation was noted for L. welshimeri and L. monocytogenes pairings which resulted in population differentials as large as 3.7 ± 0.2 logs with L. welshimeri being the predominant species. Select strain pairings were used to inoculate guacamole, crab meat, broccoli, and cheese with subsequent recovery by the FDA Bacteriological Analytical Manual (BAM) method with 10 colonies per sample selected for confirmation. The presence of L. seeligeri had little effect on the recovery of L. monocytogenes. The presence of L. welshimeri resulted in the failure to recover L. monocytogenes in three out of the four food matrices. This work extends the observation that non-pathogenic species of Listeria can complicate the recovery of L. monocytogenes and that competition during selective enrichment is not limited to the presence of just Listeria innocua.

Comparison of eight different agars for the recovery of clinically relevant non-O157 Shiga toxin-producing Escherichia coli from baby spinach, cilantro, alfalfa sprouts and raw milk

The FDA Bacteriological Analytical Manual (BAM) Chapter 4a recommends several agars for isolating non-O157 Shiga toxin-producing Escherichia coli (STEC); not all have been thoroughly tested for recovering STECs from food. Using E. coli strains representing ten clinically relevant O serogroups (O26, O45, O91, O103, O104, O111, O113, O121, O128, O145) in artificially-contaminated fresh produce – bagged baby spinach, alfalfa sprouts, cilantro, and raw milk – we evaluated the performance of 8 different agars. Performance was highly dependent upon strain used and the presence of inhibitors, but not necessarily dependent on food matrix. Tellurite resistant-negative strains, O91:–, O103:H6, O104:H21, O113:H21, and O128, grew poorly on CHROMagar STEC, Rainbow agar O157, and a modified Rainbow O157 (mRB) agar. Although adding washed sheep's blood to CHROMagar STEC and mRB agars improved overall performance; however, this also reversed the inhibition of non-target bacteria provided by original formulations. Variable colony coloration made selecting colonies from Rainbow agar O157 and mRB agars difficult. Study results support a strategy using inclusive agars (e.g. L-EMB, SHIBAM) in combination with selective agars (R & F E. coli O157:H7, CHROMagar STEC) to allow for recovery of the most STECs while increasing the probability of recovering STEC in high bacterial count matrices.

Diversity and antimicrobial resistance of Salmonella enterica isolates from surface water in Southeastern United States.

A study of prevalence, diversity, and antimicrobial resistance of Salmonella enterica in surface water in the southeastern United States was conducted. A new scheme was developed for recovery of Salmonella from irrigation pond water and compared with the FDA's Bacteriological Analytical Manual (8th ed., 2014) (BAM) method. Fifty-one isolates were recovered from 10 irrigation ponds in produce farms over a 2-year period; nine Salmonella serovars were identified by pulsed-field gel electrophoresis analysis, and the major serovar was Salmonella enterica serovar Newport (S. Newport, n = 29), followed by S. enterica serovar Enteritidis (n = 6), S. enterica serovar Muenchen (n = 4), S. enterica serovar Javiana (n = 3), S. enterica serovar Thompson (n = 2), and other serovars. It is noteworthy that the PulseNet patterns of some of the isolates were identical to those of the strains that were associated with the S. Thompson outbreaks in 2010, 2012, and 2013, S. Enteritidis outbreaks in 2011 and 2013, and an S. Javiana outbreak in 2012. Antimicrobial susceptibility testing confirmed 16 S. Newport isolates of the multidrug resistant-AmpC (MDR-AmpC) phenotype, which exhibited resistance to ampicillin, chloramphenicol, streptomycin, sulfamethoxazole, and tetracycline (ACSSuT), and to the 1st, 2nd, and 3rd generations of cephalosporins (cephalothin, amoxicillin-clavulanic acid, and ceftriaxone). Moreover, the S. Newport MDR-AmpC isolates had a PFGE pattern indistinguishable from the patterns of the isolates from clinical settings. These findings suggest that the irrigation water may be a potential source of contamination of Salmonella in fresh produce. The new Salmonella isolation scheme significantly increased recovery efficiency from 21.2 (36/170) to 29.4% (50/170) (P = 0.0002) and streamlined the turnaround time from 5 to 9 days with the BAM method to 4 days and thus may facilitate microbiological analysis of environmental water.

Comparison of a novel strategy for the detection and isolation of Salmonella in shell eggs with the Food and Drug Administration Bacteriological Analytical Manual method

The current FDA Bacteriological Analytical Manual (BAM) method for the detection of Salmonella in eggs requires 2 wk to complete. The objective of this project was to improve the BAM method for the detection and isolation of Salmonella in whole shell eggs. A novel protocol, using 1,000 g of liquid eggs for direct preenrichment with 2 L of tryptic soy broth (TSB) followed by enrichment using Rappaport-Vassiliadis and Tetrathionate broths, was compared with the standard BAM method, which requires 96 h room temperature incubation of whole shell egg samples followed by preenrichment in TSB supplemented with FeSO4. Four Salmonella ser. Enteritidis (4 phage types) and one Salmonella ser. Heidelberg isolates were used in the study. Bulk inoculated pooled liquid eggs, weighing 52 or 56 kg (approximately 1,100 eggs) were used in each trial. Twenty 1,000-g test portions were withdrawn from the pooled eggs for both the alternative and the reference methods. Test portions were inoculated with Salmonella at 1 to 5 cfu/1,000 g eggs. Two replicates were performed for each isolate. In the 8 trials conducted with Salmonella ser. Enteritidis, the alternative method was significantly (P < 0.05) more productive than the reference method in 3 trials, and significantly (P < 0.05) less productive than the reference method in 1 trial. There were no significant (P < 0.05) differences between the 2 methods for the other 4 trials. For Salmonella ser. Heidelberg, combined data from 2 trials showed the alternative method was significantly (P < 0.05) more efficient than the BAM method. We have concluded that the alternative method, described herein, has the potential to replace the current BAM culture method for detection and isolation of Salmonella from shell eggs based on the following factors: 1) the alternative method is 4 d shorter than the reference method; 2) it uses regular TSB instead of the more complicated TSB supplemented with FeSO4; and 3) it was equivalent or superior to the reference method in 9 out of 10 trials for the detection of Salmonella in shell eggs.

Comparison of different sample preparation procedures for the detection and isolation of Escherichia coli O157:H7 and Non-O157 STECs from leafy greens and cilantro

The FDA Bacteriological Analytical Manual (BAM) method for the detection/isolation of Shiga toxin-producing Escherichia coli (STEC) involves enrichment of produce rinses, blended homogenates or stomached homogenates. However, the effectiveness of rinsing produce to remove attached bacteria is largely unknown. Moreover, PCR inhibitors can be released under physical treatment. The study objective was to determine the relative effectiveness of recovery methods for STEC contaminated produce. Spinach, lettuce, and cilantro were contaminated with E. coli O157:H7 or a non-O157 STEC, subjected to both the BAM method and a soak method, and tested by real-time PCR and cultural methods. For O157:H7 and non-O157:H7 STECs, the soak method was significantly more productive than leafy green rinses. Of 320 test portions, PCR of recovered colonies confirmed 148 were positive by rinsing and 271 were positive by soaking (an 83% increase in sensitivity). For recovery of O157:H7 from cilantro, of 60 test portions, positives were 38 by soaking, 41 by stomaching, and 28 by blending. Soaking and stomaching were significantly more productive than blending, although soaking was only arithmetically superior to stomaching. Based upon these results, it is recommended that a soak method replace the current BAM procedures.