Bacteriological Analytical Manual Research Articles

Occurrence and Phenotypic Antimicrobial Resistance of <i>Escherichia Coli</i> and <i>Salmonella Enterica</i> as Well as Coliform Load Recovered from Healthy Dogs in Tamale Metropolis, Ghana

Salmonella enterica and Escherichia coli are vital bacteria associated with infections in both humans and animals. Their presence in dogs expose humans to the risk of infections. This study determined the occurrence and phenotype antimicrobial resistance of Salmonella enterica and Escherichia coli recovered from healthy dogs in the Tamale Metropolis, Ghana. The study also examined coliform loads in these dogs. A total of 120 samples from five different parts (anus, mouth, nose, inner ear and outer ear) of apparently 24 healthy dogs were examined. Isolation and antimicrobial resistance of Salmonella enterica and Escherichia coli were determined according to the Bacteriological Analytical Manual and the Disc Diffusion methods, respectively. The occurrence of Salmonella enterica was highest at 41.7% and lowest at 8.33% while Escherichia coli in the dogs was highest at 62.5% and lowest at 16.7%. The coliform load was highest at 3.7 log cfu/cm2 and lowest at 3.1 log cfu/cm2 . The Salmonella enterica isolates were highly resistant to teicoplanin (100%) and tetracycline (89.5%), but susceptible to gentamicin (68.4%). The MAR index ranged from 0.1 to 0.7 and the resistance pattern TecTeCCro (resistant to teicoplanin, tetracycline, chloramphenicol and ceftriaxone) was the most common. For Escherichia coli, they were highly resistant to teicoplanin (84.6%) and tetracycline (73.1%), but susceptible to gentamicin (80.8%), ceftriaxone (88.5%) and chloramphenicol (92.3%). The MAR index ranged from 0.0 to 0.6 and the resistannce pattern Tec (resistant to only tetracycline) was the most common. In conclusion, this showed that apparently healthy dogs were sources of Salmonella enterica and Escherichia coli. The Salmonella enterica and Escherichia coli isolates showed varied resistances to antibiotics.

Comparison of microbiological loads of enteral products at two different ambient room temperatures.

Despite the high risk of bacterial contamination, no studies have been found that evaluate the optimal hanging time of open-system enteral sets under room temperature and intensive care conditions. This study aims to determine the differences in microbial loads of solutions taken from open-system enteral feeding sets hanging for 24 h under room temperature and intensive care conditions. This study is a descriptive type of research. The study sample consisted of a total of 40 samples, with 20 samples under room temperature (25.2-23.3°C) and 20 samples under intensive care conditions (21.1-24.2°C). A simple random sampling method was used for sample selection. The assessment of microbial quality was based on the US Food and Drug Administration online Bacteriological Analytical Manual. In this study, it was determined that 5% of the samples taken from intensive care and 15% of the samples taken at room temperature exceeded the accepted limit for aerobic mesophilic bacteria count. The count of catalase and coagulase-negative Staphylococcus spp was 5% in intensive care samples and 40% in room temperature samples. The total coliform count was not detected in intensive care samples but was 35% in room temperature samples. Escherichia coli was not detected in either group. Differences in microbial loads were observed when commercial enteral solutions were administered using open-system enteral feeding sets under room and intensive care conditions for 24 h. Therefore, large-scale studies are needed to determine the safe use of open-system enteral feeding sets under room temperature conditions.

Innovative Multiplex PCR Assay for Detection of tlh, trh, and tdh Genes in Vibrio parahaemolyticus with Reference to the U.S. FDA's Bacteriological Analytical Manual (BAM).

Vibrio parahaemolyticus is an important foodborne bacterium that causes severe gastroenteritis following the consumption of contaminated seafood. To identify V. parahaemolyticus and determine its pathogenicity, the U.S. Food and Drug Administration (FDA)'s Bacteriological Analytical Manual (BAM) recommends a multiplex polymerase chain reaction (PCR) protocol to simultaneously detect the species-specific thermolabile hemolysin (tlh) gene and the pathogenic thermostable-related hemolysin (trh) and thermostable-direct hemolysin (tdh) genes. However, this assay has shown two limitations: difficulty in separating the amplicons of the trh (486 bp) and tlh (450 bp) genes due to their highly similar sizes, and the weaker band exhibited by the tdh gene amplicon (270 bp). The present study aimed to improve the BAM's multiplex PCR assay by separating the three amplicons with similar intensity. A new primer set was applied for the tlh gene (369 bp) alongside the existing primers for the trh and tdh genes. The amplicons for the three genes were effectively separated by electrophoresis on a 2% tris-borate-EDTA (TBE) agarose gel within 45 min. Primer concentrations of 0.25 µM for three genes produced a significant amount of amplicons among various combinations of primer concentrations with 35 PCR cycles. This assay exhibited a detection limit of 10 pg of bacterial DNA, demonstrating its high sensitivity. It did not display amplicons from nine Vibrio species known to be human pathogens or from 18 well-documented foodborne pathogens. Therefore, the present multiplex PCR protocol could help overcome the limitations of existing assays and provide a more reliable method for detecting the three genes of V. parahaemolyticus.

Development of a Rapid and Sensitive CANARY Biosensor Assay for the Detection of Shiga Toxin 2 from Escherichia coli.

Shiga-toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). The current Food Safety Inspection Service (FSIS) testing methods for STEC use the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) protocol, which includes enrichment, cell plating, and genomic sequencing and takes time to complete, thus delaying diagnosis and treatment. We wanted to develop a rapid, sensitive, and potentially portable assay that can identify STEC by detecting Shiga toxin (Stx) using the CANARY (Cellular Analysis and Notification of Antigen Risks and Yields) B-cell based biosensor technology. Five potential biosensor cell lines were evaluated for their ability to detect Stx2. The results using the best biosensor cell line (T5) indicated that this biosensor was stable after reconstitution with assay buffer covered in foil at 4 °C for up to 10 days with an estimated limit of detection (LOD) of ≈0.1-0.2 ng/mL for days up to day 5 and ≈0.4 ng/mL on day 10. The assay detected a broad range of Stx2 subtypes, including Stx2a, Stx2b, Stx2c, Stx2d, and Stx2g but did not cross-react with closely related Stx1, abrin, or ricin. Additionally, this assay was able to detect Stx2 in culture supernatants of STEC grown in media with mitomycin C at 8 and 24 h post-inoculation. These results indicate that the STEC CANARY biosensor developed in this study is sensitive, reproducible, specific, rapid (≈3 min), and may be applicable for surveillance of the environment and food to protect public health.

Validation of rapid detection methods for Salmonella enterica in green chile.

The objective of this study is to validate the US Food and Drug Administration (FDA) rea-time polymerase chain reaction (qPCR) assay, the Neogen Amplified Nucleic Single Temperature Reaction (ANSR) assay, and the Vitek ImmunoDiagnostic Assay System (VIDAS) SLM procedure against the FDA cultural procedure for Salmonella detection in green chile pepper. Green chile was artificially contaminated with Salmonella according to the FDA guidelines (FDA. Guidelines for the Validation of Microbiological Methods for the FDA Foods Program, 3rd Edition. 2019. www.fda.gov/media/83812/download?attachment (17 March 2024, date last accessed)) at a fractional recovery level (where 50%-25% tests positive and at a level+1 log greater for each organism tested). Enriched samples were tested directly by the ANSR Salmonella test and by qPCR, and were subcultured into Rappaport-Vassiliadis and tetrathionate brilliant green broth for cultural detection and qPCR. For the VIDAS-SLM assay, the selective enrichments were further cultured in M broth before testing. Presumptive salmonellae were confirmed with biochemical tests, serology, and qPCR. All three rapid assays were compared favorably with the FDA-BAM (Bacteriological Analytical Manual) method. No significant differences at P<.05 were found between the procedures using McNemar's χ2 test. The three procedures were found to be rapid and reliable alternatives to cultural detection of Salmonella enterica in green chile.

Bacteriological Contamination Level of Foods and Water Sold With Escherichia Coli, Salmonella spp, Staphylococcus Aureus, Coliforms and Vibrio Cholera in Food Establishments in Nairobi City Kenya

Purpose: Food borne illnesses are major health burden leading to high morbidity and mortality. It is a growing public health concerns worldwide resulting from food and water contaminated by pathogenic microorganism, toxins or chemical hazards. It is estimated that 10 to 20% of food borne illness are contracted from food establishments. The main aim was to determine bacteriological contamination levels of foods and water sold with Escherichia coli, Salmonella spp, Staphylococcus aureus, coliforms and Vibrio cholerae in food establishments in Embakasi South Nairobi City County Kenya.&#x0D; Methodology: The study design was descriptive analytical design. The Samples were collected in selected food establishments (Cafeteria, Hotels, Restaurants and food Kiosks) in Embakasi South Sub county Nairobi City County. The study collected 274 samples of food and water randomly sampled and collected using sterile food bags and water bottles within selected establishments and transported to the laboratory in cool boxes packed with ice packs. The samples were analyzed within 6 hours after collection. Microbiological analysis of food and water were borrowed from WHO and bacteriological analytical manual of foods to identify and isolate coliforms, Staphylococcus aureus, Vibrio cholera, Escherichia coli and Salmonella spp.&#x0D; Results: It was found that some foods and water sold and consumed in the selected food establishments was contaminated with food borne microorganisms. Escherichia coli isolated in 137 food samples were at 24.1%, Vibrio cholera at 23.4%, Staphylococcus aureus at 32.8%, and Salmonella spp at 4.4%. Total coliforms detected in 137 samples of water were at 43.8% where 32.8% of them were Escherichia coli.&#x0D; Unique Contribution to Theory, Practice and Policy: The study concluded there was high level of bacterial contamination of foods and water consumed in selected foods establishments. Nairobi City County health officers should enhance regular sampling of foods and water for microbial quality, health education on sources of food contamination at food eateries and establishments to prevent food and water contamination which later lead to food borne illness outbreak.&#x0D;

ISOLATION, IDENTIFICATION, AND ANTIBIOTIC SENSITIVITY AGAINST Salmonella sp. FROM HATCHERY KUB CHICKEN IN YOGYAKARTA [Isolasi, Identifikasi, dan Sensitivitas Antibiotik Terhadap Salmonella sp. dari Penetasan Ayam KUB di Yogyakarta

Salmonella sp. is responsible bacteria that causes salmonellosis in hatchery-produced local chicken (KUB) in Yogyakarta. The objective of this study was to determine of character Salmonella sp. from hatchery KUB chicken in Yogyakarta. This study was carried out in KUB chicken hatcheries in Yogyakarta province, specifically in the Sleman district (n = 5), Bantul (n = 3), Kulonprogo (n = 3), and Gunungkidul (n=2). Isolation and identification of Salmonella sp. were done from eggshells (n=17) and chick fluff of KUB chicken (n = 13) based on conventional methods with the bacteriological analytical manual (BAM). Sensitivity of Salmonella sp. against antibiotics is determined by antimicrobial diffusion agar with a paper disc antibiotic. A total of 3 isolates of Salmonella sp. were isolated from eggshells (n=2) and chick fluff from KUB chicken (n=1). Salmonella sp. was sensitive to tetracycline, oxytetracycline, enrofloxacin, sulfamethoxazole and resistant to ampicillin and erythromycin. The study showed that the hatchery of KUB chicken in Yogyakarta was contaminated with Salmonella sp. and had resistance to several antibiotics. Therefore, desinfection in incubator hatcheries using KUB chicken should continue after the egg hatches.

Microbial Quality of Some Fresh-Cut Ready-to-Eat Fruits Sold in Some Markets and Bus Terminals in the Kumasi Metropolis, Ghana

The consumption of fruits is an important source of vitamins and micronutrients. Despite their health benefits, fruits have been linked to food-borne infections, particularly when untreated animal excreta are used in their cultivation and not properly washed before consumption. This study aimed to evaluate the parasitological, fungal, and bacteriological quality of some fresh-cut ready-to-eat fruits sold in markets and bus terminals in the Kumasi Metropolis, Ghana. A total of 270 fresh-cut ready-to-eat fruits, including pineapples, watermelon, pawpaw, sugarcane, and tiger nuts, were sampled from both stationary vendors and hawkers from six different locations in the Kumasi Metropolis. Samples were analysed for the presence of bacteria, fungi, and intestinal parasites using the Microbial Methods in the Bacteriological Analytical Manual (BAM) of the United States Food and Drugs Administration (2001). The pour plate technique was used to culture and grow bacteria and fungi, while the most probable number (MPN) table was employed for bacteria and fungi count. The total coliform counts ranged from 2.32 to 3.15 log cfu/g; faecal coliform counts ranged from 1.58 to 2.58 log cfu/g; enterococci count ranged from 1.06 to 1.90 log cfu/g; and fungal count ranged from 2.57 to 3.73 log cfu/g. There was a significant difference in the mean faecal coliform and enterococci counts for all fruit samples. Tiger nut had the most bacteria, while watermelon was the most fungi contaminated fruit. Shigella spp. was the commonest bacteria contaminates of fresh-cut ready-to-eat fruits sold in Kumasi. Giardia lamblia was the most abundant parasite detected. Fresh-cut ready-to-eat fruits and vegetables sold in markets and bus terminals in the Kumasi Metropolis, Ghana, are contaminated with bacteria, parasites, and fungi. These findings emphasize the need for public awareness and proper hygiene practices to reduce the risk of food-borne illnesses associated with the consumption of fresh-cut fruits.

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.

Modeling Preharvest Cyclospora cayetanensis Sampling and Testing for Various Water and Produce Sampling Plans

As of August 2023, the two U.S. Food and Drug Administration (FDA) official detection methods for C. cayetanensis are outlined in the FDA Bacteriological Analytical Manual (BAM) Chapters 19b (produce testing) and 19c (agricultural water testing). These newly developed detection methods have been shown to not always detect contamination when present at low levels. Yet, industry and regulators may choose to use these methods as part of their monitoring and verification activities while detection methods continue to be improved. This study uses simulation to better understand the performance of these methods for various produce and water sampling plans. To do so, we used published FDA test validation data to fit a logistic regression model that predicts the methods’ detection rate given the number of oocysts present in a 10-L agricultural water or 25 g produce sample. By doing so, we were able to determine contamination thresholds at which different numbers of samples (n = 1, 2, 4, 8, 16, and 32) would be adequate for detecting contamination. Furthermore, to evaluate sampling plans in use cases, a simulation was developed to represent C. cayetanensis contamination in agricultural water and on cilantro throughout a 45-day growth cycle. The model included uncertainty around the contamination sources, including scenarios of unintentionally contaminated irrigation water or in-field contamination. The results demonstrate that in cases where irrigation water was the contamination source, frequent water testing proved to be more powerful than produce testing. In scenarios where contamination occurred in-field, conducting frequent produce testing or testing produce toward the end of the season more reliably detected contamination. This study models the power of C. cayetanensis detection methods to understand the sampling plan performance and how these methods can be better used to monitor this emerging food safety hazard.

Microbiological safety and quality of ceviche, poke, and sushi dishes sold at retail outlets in Orange County, CA

Raw, ready-to-eat (RTE) seafood products, such as ceviche, poke, and sushi, have experienced growing demand globally; however, these products have the potential to be contaminated with foodborne pathogens. The objective of this study was to determine the prevalence of Escherichiacoli/coliforms, Salmonella, and Listeria in ceviche, poke, and sushi dishes sold at the retail level in Orange County, CA, USA. Additional organisms detected during testing were also considered in the results. A total of 105 raw, RTE samples of ceviche, poke, and sushi were collected from restaurants and grocery stores in Orange County, CA. Samples were tested for Salmonella and Listeria utilizing methods from the Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM). E. coli and total coliforms were enumerated utilizing 3 M Petrifilm plates. Overall, two samples (1.9%) were positive for generic E. coli, with a range of 5–35 CFU/g. Coliforms were detected in 85 samples (81%), with a range of 5–1710 CFU/g. The average coliform levels in ceviche samples (259 CFU/g) were significantly higher than the levels in sushi samples (95 CFU/g), according to a Kruskal-Wallis H test followed by the Dunn test (p < 0.05). The coliform levels in poke samples (196 CFU/g) were not significantly different from those in ceviche or sushi. All levels of E. coli and coliforms were considered acceptable or satisfactory/borderline according to standards for RTE seafood. None of the samples tested positive for Salmonella or Listeria monocytogenes; however, other microorganisms were detected in 17 samples, including Listeria spp., Proteus mirabilis, Providencia rettgeri, and Morganella morganii. The results of this study are novel in that they present data on the microbiological safety and quality of ceviche, poke, and sushi dishes sold at retail in the United States, as well as provide a comparison across the three categories of raw, RTE seafood.

Validation of the Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay for the Detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in Seafood Matrixes: AOAC Performance Tested MethodsSM 022301.

The Thermo Scientific™ SureTect™ Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus PCR Assay method is a real-time PCR method for the multiplex detection of Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus in seafood. The Thermo Scientific SureTect Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus Assay was evaluated for AOAC Performance Tested MethodsSM certification. Inclusivity/exclusivity, matrix, product consistency/stability, and robustness studies were conducted to assess the method's performance. For the matrix study, the method was validated using the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR Food Safety Instrument and the Applied Biosystems™ 7500 Fast Real-Time PCR Food Safety Instrument against the U.S. Food and Drug Administration Bacteriological Analytical Manual, Chapter 9 (2004), Vibrio and ISO 21872-1:2017 Microbiology of the food chain-Horizontal method for the determination of Vibrio spp.-Part 1: Detection of potentially enteropathogenic Vibrio parahaemolyticus, Vibrio cholerae, and Vibrio vulnificus reference methods. Matrix studies showed equivalent or superior performance of the candidate method compared to the reference method and, overall, no difference between presumptive and confirmed results, except for one matrix due to high background flora. The inclusivity/exclusivity study correctly identified/excluded all strains analyzed. Robustness testing showed no statistically significant differences in assay performance under varied test conditions. Product consistency and stability studies demonstrated no statistically significant differences between assay lots with different expiration dates. The data presented show that the assay constitutes a rapid and reliable workflow for the detection of V. cholerae, V. parahaemolyticus, and V. vulnificus in seafood matrixes. The SureTect PCR Assay method allows for fast, reliable detection of stipulated strains in seafood matrixes with results obtained in as little as 80 min post-enrichment.

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.

Suitability of Culture Broth and Conditions for Escherichia coli Growth and Gas Production as a Test for Food Additives in EC Broth

The growth and gas production test for Escherichia coli in the microbiological examination of food additives is stipulated in the ninth edition of Japan's Specifications and Standards for Food Additives (JSFA) and described as a part of the "Confirmation Test for Escherichia coli" in "Microbial Limit Tests" in the same manuscript. The growth and gas production test for E. coli indicated that the positive or negative of "gas production and/or turbidity" in EC broth should be confirmed after incubating at 45.5±0.2℃ for 24±2 h. If both gas production and turbidity are negative, the culture is additionally incubated up to 48±2 h to determine E. coli contamination. The internationally referenced Bacteriological Analytical Manual of the U.S. FDA had revised the incubation temperature in tests for coliforms and E. coli from 45.5±0.2℃ to 44.5±0.2℃ in 2017. Therefore, we conducted research in anticipation of this temperature change being reflected in the microbiological examination of the JSFA. We used seven EC broth products and six food additives across eight products that are available in Japan in order to compare the growth and gas production at temperatures of 45.5±0.2℃ and 44.5±0.2℃ of E. coli NBRC 3972, which is designated as the test strain in JSFA. Both with/without food additives, the number of EC broth products in which medium turbidity and gas production by the strain were positive in three out of three tubes at all test times was greater at 44.5±0.2℃ than at 45.5±0.2℃. These results suggest that the growth and gas production test for E. coli could be more appropriately conducted by incubation at 44.5±0.2℃ in the "Confirmation Test for Escherichia coli" for E. coli in the JSFA in comparison to 45.5±0.2℃. Furthermore, there were differences in the growth and gas production of E. coli NBRC 3972 depending on the EC broth product used. Therefore, the importance of "Media growth promotion test" and "Method suitability test" in the ninth edition of the JSFA should be emphasized.

Microbiological assessment of raw milk available in the metropolitan city of Sindh, Karachi - Pakistan

Currently, milk quality is judge on its health and hygienic parameters equally as its composition. Raw milk is a rich medium for the growth and proliferation of different pathogenic microorganisms such food-safety hazard may incorporate into the dairy chain, not only affect its safety and quality but also cause various milk borne diseases. Objective: To evaluate the microbiological assessment of raw milk accessible in Karachi with the intention to develop risk tracking strategies and its mitigation plan in future. Methods: Total 270 raw milk samples were collected from different sites throughout the dairy supply chains in each district of the city. Microbiological assessment of raw milk was carried out by following the bacteriological analytical manual (BAM, FDA) protocol. Results: The raw milk safety and quality at the dairy farms were satisfied, except raw milk storage tanks samples. The bacterial count boosted in almost all the samples collected during its transportation, higher aerobic and pathogenic load was observed in the retail shop’s samples however, maximum counts were noticed in the selling point milk samples. None of the milk samples contained other pathogens like Listeria or Salmonella. Conclusions: The milk quality of the supply chain, retail shops and storage tanks at dairy farms were un-satisfied based on different microbiological parameters.

Toward the Adoption of Loop-Mediated Isothermal Amplification for Salmonella Screening at the National Antimicrobial Resistance Monitoring System's Retail Meat Sites.

The National Antimicrobial Resistance Monitoring System (NARMS) is a One Health program in the United States that collects data on antimicrobial resistance in enteric bacteria from humans, animals, and the environment. Salmonella is a major pathogen tracked by the NARMS retail meat arm but currently lacks a uniform screening method. We evaluated a loop-mediated isothermal amplification (LAMP) assay for the rapid screening of Salmonella from 69 NARMS retail meat and poultry samples. All samples were processed side by side for culture isolation using two protocols, one from NARMS and the other one described in the U.S. Food and Drug Administration's Bacteriological Analytical Manual (BAM). Overall, 10 (14.5%) samples screened positive by the Salmonella LAMP assay. Of those, six were culture-confirmed by the NARMS protocol and six by the BAM method with overlap on four samples. No Salmonella isolates were recovered from samples that screened negative with LAMP. These results suggested 100% sensitivity for LAMP in reference to culture. Antimicrobial susceptibility testing and whole-genome sequencing analysis confirmed identities of these isolates. Using the BAM protocol, all Salmonella isolates were recovered from samples undergoing Rappaport-Vassiliadis medium selective enrichment and presumptive colonies (n = 130) were dominated by Hafnia alvei (44.6%), Proteus mirabilis (22.3%), and Morganella morganii (9.9%) based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. This method comparison study clearly demonstrated the benefit of a rapid, robust, and highly sensitive molecular screening method in streamlining the laboratory workflow. Fourteen NARMS retail meat sites further verified the performance of this assay using a portion of their routine samples, reporting an overall specificity of 98.8% and sensitivity of 90%. As of July 2022, the vast majority of NARMS retail meat sites have adopted the Salmonella LAMP assay for rapid screening of Salmonella in all samples.

3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate Method for the Enumeration of Aerobic Bacteria on Selected Surfaces: AOAC Official MethodSM 2015.13.

The 3M™ Petrifilm™ Rapid Aerobic Count (RAC) Plate is a sample-ready culture medium system designed to facilitate colony enumeration in 24 h (48 h for dry powders) for selected food and environmental surfaces. The objective of this study is to evaluate the 3M Petrifilm RAC Plate in a matrix extension study for the enumeration of aerobic bacteria on stainless steel, sealed concrete, and rubber surfaces. The 3M Petrifilm RAC Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 3 (January 2001): Aerobic Plate Count in a paired matrix study for enumeration of aerobic bacteria on stainless steel, sealed concrete, and rubber environmental surfaces. The 3M Petrifilm RAC Plate showed no statistically significant differences when compared to the reference method for enumeration of aerobic bacteria from stainless steel, sealed concrete, and rubber environmental surfaces. There were no significant statistical differences between the 3M Petrifilm RAC Plate and reference method results for the three matrixes evaluated. The 3M Petrifilm RAC Plate is an effective plating method for aerobic plate count when analyzing stainless steel, sealed concrete, and rubber surfaces. The 3M Petrifilm RAC Plate is robust, quick, and simple to perform, providing enumerable colonies in 22 to 26 h after incubation when compared to the 48 h of the reference method. The small size of the Petrifilm Plate allows for less space to be occupied by plates in the incubators. The visual biochemical and enzymatic indicators make enumeration of colonies a simple task by presenting colonies in either a blue or red color.

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 &gt; 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.

3M™ Petrifilm™ Rapid Yeast and Mold Count Plate Method for the Enumeration of Yeast and Mold on Selected Surfaces: AOAC Official Method 2014.05.

The 3M™ Petrifilm™ Rapid Yeast and Mold Count (RYM) Plate contains nutrients supplemented with antibiotics, a cold-water-soluble gelling agent, and an indicator system that facilitates yeast and mold enumeration in 48-60 h. The objective of this study is to evaluate the 3M Petrifilm RYM Plate in a matrix extension study for the enumeration of yeast and mold on stainless steel, sealed concrete, and rubber surfaces. The 3M Petrifilm RYM Plate was compared to the U.S. Food and Drug Administration Bacteriological Analytical Manual, Ch. 18: Yeasts, Molds and Mycotoxins in a paired matrix study for enumeration of yeast and mold on stainless steel, sealed concrete, and rubber environmental surfaces. The 3M Petrifilm RYM Plate demonstrated equivalent performance to the reference method for enumeration of yeast and mold from stainless steel, sealed concrete, and rubber environmental surfaces. There were no significant statistical differences between the 3M Petrifilm RYM Plate and reference method results for the three matrixes evaluated. The 3M Petrifilm RYM Plate is an effective plating method for enumerating yeast and mold when analyzing stainless steel, sealed concrete, and rubber surfaces. The 3M Petrifilm RYM Plate method allows the user to obtain accurate results within 48-60 h in the matrices evaluated for the presence of yeast and mold when incubated at 25 ± 1°C or 28 ± 1°C. Interpretation and colony counting was straightforward and the 3M Petrifilm RYM Plate method required no additional agar or Petri dishes, creating an easier workflow by cutting down on supplies, sample plating time, and most noticeably, occupying less space in the incubator.

Phenotypic Resistance of Staphylococcus aureus to Antibiotics in Dogs of Tamale Metropolis, Ghana

Introduction: Staphylococcus aureus is an important bacterium which induces a wide range of diseases. Its presence in dogs and resistance to antibiotics is a threat to public health due to the close association of humans with dogs. The objective of the present study was to determine the phenotypic resistance of Staphylococcus aureus (S. aureus) to antibiotics in dogs without any clinical manifestation of diseases in Tamale Metropolis, Ghana. The current study also examined microbial load in these dogs. Materials and methods: A total of 120 samples from various parts of dogs, including the mouth, nose, anus, inner ear, and outer ear, were examined. Isolation and antibiotic resistance of S. aureus were determined using the USA Bacteriological Analytical Manual and the Disc Diffusion method, respectively.&#x0D; Results: The presence of S. aureus in the dogs ranged from 8.3% (anus) to 58.3% (nose), averaging 40%. The microbial load also ranged from 2.9 log cfu/cm2 (mouth) to 3.4 log cfu/cm2 (outer ear) with an average of 3.2 log cfu/cm2. There were significant differences among the examined samples regarding the presence of S. aureus, but not the microbial load. The overall resistance, intermediate resistance, and susceptibility of S. aureus were 46.2%, 12.9%, and 42.2%, respectively. The S. aureus was highly resistant to teicoplanin (88.0%) and susceptible to chloramphenicol (72.0%). The multiple antibiotic indexes ranged from 0 to 0.9, and 89.1% of the isolates exhibited multidrug resistance.&#x0D; Conclusion: The findings of the current study revealed that healthy dogs in Tamale Metropolis, Ghana, were carriers of S. aureus as well as other bacteria, and the S. aureus exhibited different resistance patterns to antibiotics.