Prevalence of Salmonella spp. in meat, seafood, and leafy green vegetables from local markets and vegetable farms in Phnom Penh, Cambodia.

Salmonella is a major bacterial concern for public health globally. Although there are limited documentation on the prevalence of Salmonella species in Cambodia's food chain, some reports indicate that salmonellosis is a severe gastrointestinal infection in its population and especially in children. To investigate the presence of Salmonella spp., 285 food samples (75 meat, 50 seafood, and 160 leafy green vegetable samples) were randomly collected from various local markets in Phnom Penh capital and nearby farms in Cambodia. Concurrently, field observations were conducted to collect data on food hygiene and practices among the relevant actors. All food samples were analyzed using bacterial culture and plate counts, and the findings were confirmed serially with biochemical, serological, and PCR tests. The observational data on food hygiene and practices from farm to market revealed that the spread of Salmonella in the food-value chain from farm to market could pose health risks to consumers. The overall prevalence of Salmonella spp. was 48.4% (138/285), while the prevalence in meat, seafood, and vegetables was 71% (53/75), 64% (32/50), and 33% (53/160), respectively. Mean Salmonella plate count ranged from 1.2 to 7.40 log10 CFU/g, and there was no significant difference in bacterial counts between meat, seafood, and vegetable samples (p > 0.05). The most common serogroups among the isolated Salmonella spp. were B and C. These results suggest that a large proportion of meat, seafood, and vegetable products sold at local markets in Phnom Penh are contaminated with Salmonella spp. This is likely linked to inadequate hygiene and sanitation practices, including handling, storage, and preservation conditions. Observations on farms suggested that the prevalence of Salmonella in vegetables sold at the market could be linked to contamination relating to agricultural practices. Thus, controlling the spread of foodborne salmonellosis through the food-value chain from farms and retailers to consumers is warranted to enhance food safety in Cambodia.

Microbiological Quality and Safety of Brazilian Mozzarella Cheese During Production Stages.

This study assessed the microbiological quality and safety of mozzarella during various production stages in northern Tocantins, Brazil, by identifying critical biological points in the industrial environment within a tropical climatic region. Batches of mozzarella were evaluated, from raw milk to primary packaging, with a shelf life of 120 d at 4°C. Indicator microorganisms were quantified, and through microbiological and biomolecular approaches, Salmonella spp. and Listeria monocytogenes were identified. In addition, the toxigenic potential of coagulase-positive staphylococci (CPS) was characterized. Results indicated that the raw milk used for mozzarella production had low microbiological quality; pasteurization of raw milk effectively eliminated all identified pathogens and reduced microbiological counts (p > 0.05). An increase in bacterial counts (>2 log colony-forming unit [CFU]/g) and recontamination with Salmonella spp. and CPS, which potentially produce staphylococcal enterotoxin B, were observed during milk coagulation and curd draining. Stretching of the fermented curd reduced the enterobacteria, total coliforms, and Escherichia coli median values by 2.56, 2.64, and 2.3 log CFU/mL, respectively. Similarly, brining the pieces by immersion reduced the quantity of enterobacteria and total coliforms by 2.3 and 1.6 log CFU/mL, respectively. Of interest, in the freshly finished product, Salmonella spp. was present but L. monocytogenes was absent; however, after the shelf-life period, L. monocytogenes was present but Salmonella spp. was absent. Considering the environmental conditions that can promote the multiplication and preservation of pathogens and spoilage of dairy products in tropical climates, it is necessary to review operational hygiene procedures, particularly in milk coagulation vats and fermentation tables. This will ensure the production of high-quality mozzarella cheese with a reduced consumption risk.

Assessment of the Microbiological Quality and Effect of Public Health of Ready-to-Eat Salad Samples in Isparta.

Salmonella spp. and Citrobacter spp. are among the microorganisms causing important foodborne outbreaks. In this study, it was tried to determine the presence and rate of Salmonella spp. and Citrobacter spp. in salad samples collected from certain regions of province of Isparta in Türkiye. A total of 50 salad samples were analyzed. Classical culture technique was used for microbiological analysis of salad samples. Suspected isolates obtained were identified using the VITEK-2 system. Although no negative visual changes were observed in the salad samples used in the study, it was determined that the number of Gram-negative microorganisms was very high and six salad samples were not suitable for public health. In 50 salad samples, 2% Salmonella and 4% Citrobacter freundii were detected. In addition, it was determined that the Salmonella strain isolated from the salad sample was resistant to three different antibiotics and Citrobacter was resistant to two different antibiotics. Salmonella spp. and Citrobacter spp. are considered very dangerous to public health because they are associated with foodborne outbreaks and can develop antibiotic resistance very quickly. Salad producers should try to reduce the possibility of microbial contamination by using different technologies.

A New Peptide Nucleic Acid Fluorescence In Situ Hybridization Probe for the Specific Detection of Salmonella Species in Food Matrices.

Salmonella spp. is among the most central etiological agents in foodborne bacterial disorders. To identify Salmonella spp., numerous new molecular techniques have been developed conversely to the traditional culture-based methods. In this work, a new peptide nucleic acid fluorescence in situ hybridization (PNA-FISH) method was developed for the specific detection of Salmonella species, allowing a faster analysis compared with the traditional methods (ISO 6579-1: 2017). The method was optimized based on a novel PNA probe (SalPNA1692) combined with a blocker probe to detect Salmonella in food samples through an assessment of diverse-rich and selective enrichment broths. Our findings indicated that the best outcome was obtained using a 24-h pre-enrichment step in buffered peptone water, followed by RambaQuick broth selective enrichment for 16 h. For the enrichment step performance validation, fresh ground beef was artificially contaminated with two ranges of concentration of inoculum: a low level (0.2-2 colony-forming units [CFUs]/25 g) and a high level (2-10 CFUs/25 g). The new PNA-FISH method presented a specificity of 100% and a detection limit of 0.5 CFU/25 g of food sample, which confirms the great potential of applying PNA probes in food analysis.

Development and Application of Four Foodborne Pathogens by TaqMan Multiplex Real-Time PCR.

A TaqMan multiplex real-time PCR (mRT-PCR) was developed to detect simultaneously Salmonella spp., Escherichia coli O157, Staphylococcus aureus, and Listeria monocytogenes in food samples. The method involves four sets of primers and probes tailored to the unique DNA sequences found in the invA, nuc, rfbE, and hly genes of each pathogen. The generated standard curves, correlating gene copy numbers with Ct values, demonstrated high accuracy (R2 > 0.99) and efficiency (92%-104%). Meanwhile, the limit of detection was 100 CFU/mL for the four target bacteria in artificially contaminated food samples after 6-8 h of enrichment. The assay's effectiveness was further verified by testing 80 naturally contaminated food samples, showing results largely in agreement with traditional culture methods. Overall, this newly developed TaqMan mRT-PCR, inclusive of a pre-enrichment step, proves to be a dependable and effective tool for detecting single or multiple pathogens in diverse food items, offering significant potential for in vitro diagnostics.

Microbiology proficiency testing in fish and fishery products: detection of Listeria monocytogenes and Salmonella spp.

This paper presents the results of two proficiency testing (PT) rounds conducted by the Export Inspection Agency (EIA) Chennai laboratory in 2021 for food testing laboratories in India. The PT program was designed in accordance with ISO/TS 22117, a standard for proficiency testing in food microbiology, and targeted Listeria monocytogenes and Salmonella spp as the organisms of focus. The samples were found to be stable and recoverable during the analysis, and all PT sample packages were delivered to participant laboratories in good condition. The participant laboratories reported high sensitivity rates of 100% for PT round 061021 M and 96.49% for PT round 050721 M. The accuracy rate in PT round 061021 M was 91.89% and 92.10% in case of PT round 050721 M. However, there were some false positive and false negative results reported by some participant laboratories in both PT rounds, which may have been caused by operational errors or inconsistencies in analysis. During the PT round 061021 M, out of a total of 38 participant laboratories, five laboratories reported false positive results and one laboratory reported a false negative result. Similarly, during the PT round 050721 M, six laboratories reported false positive results which resulted in their results being deemed unsatisfactory.

A multiplex real-time RT-PCR system to simultaneously diagnose 16 pathogens associated with swine respiratory disease.

AIMS: Swine respiratory disease (SRD) is a major disease complex in pigs that causes severe economic losses. SRD is associated with several intrinsic and extrinsic factors such as host health status, viruses, bacteria, and environmental factors. Particularly, it is known that many pathogens are associated with SRD to date, but most of the test to detect those pathogens can be normally investigated only one pathogen while taking time and labor. Therefore, it is desirable to develop rapidly and efficiently detectable methods those pathogens to minimize the damage caused by SRD. METHODS AND RESULTS: We designed a multiplex real-time RT-PCR (RT-qPCR) system to diagnose simultaneously 16 pathogens, including nine viruses and seven bacteria associated with SRD, on the basis of single qPCR and RT-qPCR assays reported in previous studies. Multiplex RT-qPCR system we designed had the same ability to single RT-qPCR without significant differences in detection sensitivity for all target pathogens at minimum to maximum genomic levels. Moreover, the primers and probes used in this system had highly specificity because the sets had not been detected pathogens other than the target and its taxonomically related pathogens. Furthermore, our data demonstrated that this system would be useful to detect a causative pathogen in the diagnosis using oral fluid from healthy pigs and lung tissue from pigs with respiratory disorders collected in the field. CONCLUSIONS: The rapid detection of infected animals from the herd using our system will contribute to infection control and prompt treatment in the field.

A Pilot Study to Detect Viable Salmonella spp. in Diarrheal Stool Using Viability Real-Time PCR as a Culture-Independent Diagnostic Tool in a Clinical Setting.

Frontline laboratories are adopting culture-independent diagnostic testing (CIDT) such as nucleic acid amplification tests (NAATs) due to numerous advantages over culture-based testing methods. Paradoxically, the viability of pathogens, a crucial factor determining active infections, cannot be confirmed with current NAATs alone. A recent development of viability PCR (vPCR) was introduced to mitigate this limitation associated with real-time PCR (qPCR) by using a DNA-intercalating dye to remove residual and dead cell DNA. This study assessed the applicability of the vPCR assay on diarrheal stools. Eighty-five diarrheal stools confirmed for Salmonellosis were tested via qPCR and vPCR using in-house primers and probe targeting the invA gene. vPCR-negative stools (Ct cut off > 31) were enriched in mannitol selenite broth (MSB) to verify low bacterial loads. vPCR assay showed ~89% sensitivity (qPCR- and vPCR-positive stools: 76/85). vPCR-negative stools (9/85; qPCR-positive: 5; qPCR-negative: 4) were qPCR- and culture-positive post-MSB-enrichment and confirmed the presence of low viable bacterial loads. Random sampling error, low bacterial loads, and receiving stools in batches could contribute to false negatives. This is a pilot study and further investigations are warranted to explore vPCR to assess pathogen viability in a clinical setting, especially when culture-based testing is unavailable.

Class 1 and 2 integrons and antibiotic resistance profile in Salmonella spp. from San Cristobal River, Laguna, Philippines.

Infection with multidrug resistant bacteria is a significant public health concern. Bacteria culture of water samples (n=120) collected in San Cristobal River, Philippines, showed that half (n=60) were positive for Salmonella spp. Screening of all isolates (n=179) for susceptibility to antibiotics showed that most (76.4%; n=113) were positive for class 1 integrons, of which one isolate was also positive for the class 2 integron. The presence of class 1 integrons was associated with resistance to antibiotics (p<0.05). Sequencing of class 1 integron variable regions (VRs) differeciated 11 gene cassettes: dfrA1 or dfrA17; aadA1 or aadA2; blaCTX-M-2 or bla-OXA-1; SmdAB; CmlA1 and aaC 3-Id. However, sequencing of class 2 integron VR differenciated estX, sat2, and aadA1. These results provide insights into evolutionary changes within bacterial multidrug resistant cassettes, more accurately to estimate heath risk associated with the river water. .

Comprehensive evaluation of abattoirs with no Hazard Analysis Critical Control Point plan in Tucumán, Argentina.

This work focused on the comprehensive study of two provincial transit abattoirs in Tucumán, Argentina, with no Hazard Analysis Critical Control Point (HACCP) plan. Visits (n=20) were conducted between 2016 and 2018 during the operational and post-operational processes. Risk was estimated and the bacteriological analysis of carcass and environmental samples was performed. Risk estimation showed the predominance of high risk in both abattoirs. The main deviations from the HACCP plan were: deficient building conditions, deficient workflow, lack of sectorization of changing rooms and bathrooms, lack of implementation of Standardized Sanitary Operational Procedures, and no food safety training of workers. The counts of indicator microorganisms from both abattoirs were not significant. Salmonella spp. was isolated from 7.5% carcass and 7.3% environmental samples. The Salmonella serovars identified were Cerro, Corvallis, Havana and Agona. Shiga toxin (stx) genes were detected in 24.4% carcass and 30.9% environmental samples. The isolates were characterized as Escherichia coli O8:H7/stx1, O116:H49/stx2 and O136:H40/stx2. Based on these results, it would be possible to implement an improvement plan in Tucumán abattoirs together with the local health authorities. Still, the need to work jointly with the sanitary authority in search of a unique sanitary standard for Argentina remains unaddressed.

Public Health Risk of Foodborne Pathogens in Edible African Land Snails, Cameroon.

In tropical countries, land snails are an important food source; however, foodborne disease risks are poorly quantified. We detected Campylobacter spp., Yersinia spp., Listeria spp., Salmonella spp., or Shiga-toxigenic Escherichia coli in 57%-86% of snails in Cameroon. Snail meat is a likely vector for enteric diseases in sub-Saharan Africa countries.

A trait-based framework for predicting foodborne pathogen risk from wild birds.

Recent foodborne illness outbreaks have heightened pressures on growers to deter wildlife from farms, jeopardizing conservation efforts. However, it remains unclear which species, particularly birds, pose the greatest risk to food safety. Using >11,000 pathogen tests and 1565 bird surveys covering 139 bird species from across the western United States, we examined the importance of 11 traits in mediating wild bird risk to food safety. We tested whether traits associated with pathogen exposure (e.g., habitat associations, movement, and foraging strategy) and pace-of-life (clutch size and generation length) mediated foodborne pathogen prevalence and proclivities to enter farm fields and defecate on crops. Campylobacter spp. were the most prevalent enteric pathogen (8.0%), while Salmonella and Shiga-toxin producing Escherichia coli (STEC) were rare (0.46% and 0.22% prevalence, respectively). We found that several traits related to pathogen exposure predicted pathogen prevalence. Specifically, Campylobacter and STEC-associated virulence genes were more often detected in species associated with cattle feedlots and bird feeders, respectively. Campylobacter was also more prevalent in species that consumed plants and had longer generation lengths. We found that species associated with feedlots were more likely to enter fields and defecate on crops. Our results indicated that canopy-foraging insectivores were less likely to deposit foodborne pathogens on crops, suggesting growers may be able to promote pest-eating birds and birds of conservation concern (e.g., via nest boxes) without necessarily compromising food safety. As such, promoting insectivorous birds may represent a win-win-win for bird conservation, crop production, and food safety. Collectively, our results suggest that separating crop production from livestock farming may be the best way to lower food safety risks from birds. More broadly, our trait-based framework suggests a path forward for co-managing wildlife conservation and food safety risks in farmlands by providing a strategy for holistically evaluating the food safety risks of wild animals, including under-studied species.

Agricultural practices in Brazilian organic farms and microbiological characteristics of samples collected along the production chain.

AIMS: To gather data on agricultural practices in organic farms in Sao Paulo, Brazil, and evaluate their relationship with the microbiological characteristics of samples collected along the production chain. METHODS AND RESULTS: Practices data were based on field observations and interviews with farmers in 10 selected organic lettuce producing farms. Counts of Enterobacteriaceae and surveys for Salmonella were performed in samples of lettuce (before and after washing), fertilizers, irrigation and washing water, all collected in the same farm. Water samples were also tested for total coliforms and generic Escherichia coli. Isolated Enterobacteriaceae were identified by MALDI-TOF MS. Contamination of lettuce was influenced by some agricultural practices: chicken manure-based fertilization resulted in higher Enterobacteriaceae counts in lettuce when compared to other types of manure, whereas pre-washed lettuces presented lower microbial counts than non-pre-washed samples. Salmonella was detected in one lettuce sample by qPCR. Escherichia coli was detected in all irrigation water samples. All sample types contained Enterobacteriaceae species commonly reported as opportunistic human pathogens. CONCLUSIONS: The data highlight the need for improvement in the good agricultural practices in the studied farms. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information on agricultural practices and microbiological characteristics of organic lettuce, contributing to the development of more accurate risk assessments.

Hygienic assessment of digestate from a high solids anaerobic co-digestion of sewage sludge with biowaste by testing Salmonella Typhimurium, Escherichia coli and SARS-CoV-2.

Anaerobic digestion is a consolidated technology to convert sewage sludge and other organic wastes into biogas and a nutrient-rich fertilizer (i.e. digestate). The origin of sewage sludge does not exclude the potential presence of pathogens (e.g. Salmonella spp. and SARS-CoV-2) in mature digestate that hence could represent a source of sanitary concerns when it is spread on soil for agriculture purpose. Therefore, an experimental study aimed at proving the sanitizing effect of a full scale thermophilic high solids anaerobic digestion process was conducted by monitoring the hygienic characteristics of mature digestate. Although Salmonella spp. was detected in the sewage sludge fed to the full scale plant, the anaerobic digestion treatment demonstrated sanitization capacity since the monitored pathogens were never found in the mature digestate over the entire duration of the monitoring survey. Furthermore, tests on the regrowth of Salmonella Typhimurium and Escherichia coli, artificially inoculated on mature digestate, were also conducted under both anaerobic and aerobic conditions with the aim to assess the effectiveness of mature digestate as microbial growth medium. Concentrations of Salmonella Typhimurium and Escherichia coli were drastically reduced after a short time of incubation under anaerobic process and the two microorganisms already resulted undetectable after 24-48 h, whereas, under aerobic conditions, two microorganisms' concentrations were stably high for longer than 10 days. The combination of no free oxygen, high temperature, anaerobic metabolites (e.g. total ammonium nitrogen, and volatile fatty acids) production, bacteria competition and lack of nutritional elements in mature digestate considerably reduced in 24-48 h the sanitary risks associated to accidently contaminated digestate. Furthermore, a SARS-CoV-2 monitoring survey on mature digestate during 13 months, resulted in the absence of the virus RNA in the analyzed digestate.

Dual-species biofilms formation between dominant microbiota isolated from a meat processing industry with Listeria monocytogenes and Salmonella enterica: Unraveling their ecological interactions.

Alternatives to combat the persistence of pathogens need to consider the microbiota established on industrial surfaces as they can influence the protection or replacement (i.e. reduction/inhibition) of pathogens. The objective of the present study was to determine the ecological interactions established in dual-species biofilms between Listeria monocytogenes and Salmonella enterica as target pathogens, and isolates recovered from a meat processing facility (i.e.Pseudomonas fluorescens, Pseudomonas fragi, Bacillus safensis, Bacillus megaterium, and Candida zeylanoides). Results showed different ecological relations in biofilms depending on the species evaluated. Pseudomonas spp. did not influence the growth of either pathogen, although tested species tended to protect the pathogens in the structures generated. B. megaterium and C. zeylanoides affected the two pathogens differently, demonstrating a reduction of L. monocytogenes adhered cells within the formed biofilm. B. safensis reduced or presented non-influence on S. enterica depending on the incubation conditions. Contrarily, B. safensis was the microorganism that demonstrated the highest replacement capacity for L. monocytogenes, reducing its growth by up to 4 log CFU/cm2. The in vitro study of bispecies biofilms is important for the food industry, helping to understand how they behave and to find an effective way to eliminate them.

Fluorescent on-site detection of multiple pathogens using smartphone-based portable device with paper-based isothermal amplification chip.

The development of cost-effective, portable, and ease-of-use sensing system for on-site genetic diagnostics is highly desirable for pathogen screening and infectious disease diagnosis. This study develops (1) a paper-based biochip which is able to integrate the loop-mediated isothermal amplification (LAMP) protocols for simultaneous detection of Escherichia coli O157:H7, Salmonella spp., and Staphylococcus aureus, and (2) a stand-alone smartphone-based portable device which can control exactly 65 °C for isothermal amplification as well as collect and analyze the thus generated fluorescence signals. The reported sensing system has been successfully demonstrated for foodborne pathogen detection with a limit of detection of 2.8 × 10-5 ng µL-1. Spiked milk samples with concentration as low as 10 CFU mL-1 were successfully determined within 4 h, demonstrating the practicality of the reported sensing system in the fields. The reported sensing system featuring simplicity and reliability is ideally suited for genetic diagnostics in low resource settings.

The Antimicrobial Peptide 1018-K6 Interacts Distinctly with Eukaryotic and Bacterial Membranes, the Basis of Its Specificity and Bactericidal Activity.

Since penicillin was discovered, antibiotics have been critical in the fight against infections. However, antibiotic misuse has led to drug resistance, which now constitutes a serious health problem. In this context, antimicrobial peptides (AMPs) constitute a natural group of short proteins, varying in structure and length, that act against certain types of bacterial pathogens. The antimicrobial peptide 1018-K6 (VRLIVKVRIWRR- NH2) has significant bactericidal and antibiofilm activity against Listeria monocytogenes isolates, and against different strains and serotypes of Salmonella. Here, the mechanism of action of 1018-K6 was explored further to understand the peptide-membrane interactions relevant to its activity, and to define their determinants. We combined studies with model synthetic membranes (liposomes) and model biological membranes, assessing the absorption maximum and the quenching of 1018-K6 fluorescence in aqueous and lipid environments, the self-quenching of carboxyfluorescein, as well as performing lipid sedimentation assays. The data obtained reflect the differential interactions of the 1018-K6 peptide with eukaryotic and prokaryotic membranes, and the specific interactions and mechanisms of action in the three prokaryotic species studied: Salmonella Typhimurium2GN, Escherichia coli3GN, and Staphylococcus aureus3GP. The AMP 1018-K6 is a candidate to prevent (food preservation) or treat (antibiotic use) infections caused by certain pathogenic bacteria, especially some that are resistant to current antibiotics.

Improving the detection limit of Salmonella colorimetry using long ssDNA of asymmetric-PCR and non-functionalized AuNPs.

A colorimetric sensor based on gold nanoparticles (AuNPs) and single-stranded DNA (ssDNA) is a simple and rapid method for detecting foodborne pathogens. However, the colorimetric method employed in previous studies involved short ssDNA (<100 nucleotides), including the aptamer and PCR products, resulting in the high detection limit of this technique. In this study, a colorimetric sensor was developed based on long ssDNA of asymmetric PCR (aPCR) and non-functionalized AuNPs for detecting Salmonella Typhimurium (S. Typhimurium). In the presence of S. Typhimurium, the long ssDNA (547 nt) amplified by aPCR-protected AuNPs from NaCl-induced aggregation, while the solution retained a red color. After optimizing parameters, the limit of detection (LOD) of the colorimetric sensor was 2.56 CFU/mL with high specificity. Recovery studies showed its feasibility for detecting S. Typhimurium (102 CFU/mL, 104 CFU/mL, and 106 CFU/mL) in spiked lettuce samples. This colorimetric sensor provides new opportunities for the highly sensitive detection of bacteria in real food samples.

Colorimetric method for Salmonella spp. detection based on peroxidase-like activity of Cu(II)-rGO nanoparticles and PCR.

Development of a rapid and sensitive method for Salmonella spp. detection is of great importance for ensuring food product safety due to its low infective dose. In this study, a colorimetric method based on the peroxidase-like activity of Cu(II)-modified reduced graphene oxide nanoparticles (Cu2+-rGO NPs) and PCR was successfully developed to detect Salmonella spp. in milk. Under optimal conditions, the developed colorimetric method exhibited high sensitivity and strong specificity for Salmonella spp. detection. The limit of detection was 0.51 CFU/mL with a linear range from 1.93 × 101 to 1.93 × 105 CFU/mL. A specificity study demonstrated that this method can specifically distinguish Salmonella typhimurium and Salmonella enteritidis from other foodborne pathogens. The application of the proposed method for milk sample detection was also validated, and the recovery rates of S. typhimurium in spiked milk sample ranged from 102.84% to 112.25%. This colorimetric sensor exhibits enormous potential for highly sensitive detection of bacteria in milk sample.

Development of a genomic DNA reference material for Salmonella enteritidis detection using polymerase chain reaction.

Several rapid methods based on nucleic acids can detect foodborne pathogens, such as Salmonella spp. However, a common reference that enables metrological traceability among measurement results is not available. Reference materials (RM) are thus key to guarantee methodological comparability. This study developed a candidate genomic DNA reference material for Salmonella enteritidis quantification to establish performance conditions and reference values for normalized RM production. The growth of Salmonella enteritidis ATCC® 13076 in Rappaport Vassiliadis selective medium was characterized, and we optimized a method of DNA extraction using cetrimonium bromide (CTAB) and LiCl. In a first stage six concentrations of DNA were prepared with and without yeast RNA (40 ng/µL) to evaluate its effect as a stabilizer in terms of homogeneity and short-term stability. Based on the findings, in a second stage two DNA concentrations were prepared and a reference value with its uncertainty was assigned based on the results of characterization, homogeneity, and stability studies using digital polymerase chain reaction and the gene targets, invA, ttr, and hilA. The material was stable for 9 months at 4 °C, with a expanded uncertainty contribution range of 11%-14%. The novel candidate RM is the first to be developed nationwide and will improve the quality of measurements in the area of food safety.