Research on the Biofilm Formation of Staphylococcus aureus after Cold Stress

Staphylococcus aureus is a common food pathogen and has a strong tolerance to environmental stress. Here, the biofilm formation of S. aureus strains after cold stress for 24 weeks were investigated. It was found that the biofilm formation of S. aureus CICC 21600, CICC 22942, W1, W3, and C1 cells was enhanced after cold stress for 20 weeks. What is more, the mRNA levels of the clfA, icaA, icaB, icaC or icaD genes in these strains were increased for >2-fold. The increased gene transcription levels were consistent with the increase in the polysaccharide content in the biofilm matrix of these S. aureus strains after cold stress. Meanwhile, hydrophobicity and the adhesion proteins also played a role in the formation of biofilms. The biofilm of S. aureus cells can be effectively degraded by snailase and proteinase K (125 µg/mL + 20 µg/mL) mixture. In summary, S. aureus frozen at −20 °C for 12 to 20 weeks is still a potential hazard. Food factory equipment should be cleaned in a timely manner to avoid outbreaks of foodborne pathogenic bacteria due to contamination.

DNAFoil, a novel technology for the rapid detection of food pathogens: Preliminary validation on Salmonella and Listeria monocytogenes

Over the past decades, several tools have been developed for food pathogen detection, including proteomics, metabolomics, immunological, biosensor, and nucleic acid-based approaches. Although these techniques are reliable and precise, they are time-consuming, technically challenging, and costly. Hence, it is necessary to develop rapid techniques for food pathogen detection, which can be performed at the household level. DNAFoil mechanism is a portable, completely self-administered, on-site DNA test that does not need expensive instru-ments or settings to confirm food pathogen detection in as little as 30 min. DNAFoil was used successfully for detecting food contamination and adulteration with pork derivatives (down to 0.1%) and vegetal components (down to 0.01%), respectively. In this study, initial validation experiments of DNAFoil were investigated to detect Listeria monocytogenes and Salmonella contamination. To confirm the specificity of the proposed method toward Salmonella, 18 different Salmonella strains, 6 non-Salmonella bacteria, and 2 fungi were investigated; also, in the case of Listeria monocytogenes, five bacterial strains, two fungi, and Listeria monocytogenes were investigated. The results stated that the Swiss Decode Salmonella and L. monocytogenes solutions can detect as few as 1 and 10 copies of DNA per microliter, respectively. The results also showed that the accuracy of our method ranges between 92 and 100%, while the precision value ranged between 88 and 100%. In terms of quality control applicability, DNAFoil Salmonella and Listeria monocytogenes reactions could be visually detected with the naked eye using a lateral flow strip, which could be used for in-place quality control during manufacturing and also can be used for more lab tests. In terms of cost, DNAFoil is usually much cheaper than the traditional detection methods. Therefore, DNAFoil is proposed as a promising and universal detection technology for food pathogens.

Utilizing the Food–Pathogen Metabolome to Putatively Identify Biomarkers for the Detection of Shiga Toxin-Producing E. coli (STEC) from Spinach

Shiga toxigenic E. coli (STEC) are an important cause of foodborne disease globally with many outbreaks linked to the consumption of contaminated foods such as leafy greens. Existing methods for STEC detection and isolation are time-consuming. Rapid methods may assist in preventing contaminated products from reaching consumers. This proof-of-concept study aimed to determine if a metabolomics approach could be used to detect STEC contamination in spinach. Using untargeted metabolic profiling, the bacterial pellets and supernatants arising from bacterial and inoculated spinach enrichments were investigated for the presence of unique metabolites that enabled categorization of three E. coli risk groups. A total of 109 and 471 metabolite features were identified in bacterial and inoculated spinach enrichments, respectively. Supervised OPLS-DA analysis demonstrated clear discrimination between bacterial enrichments containing different risk groups. Further analysis of the spinach enrichments determined that pathogen risk groups 1 and 2 could be easily discriminated from the other groups, though some clustering of risk groups 1 and 2 was observed, likely representing their genomic similarity. Biomarker discovery identified metabolites that were significantly associated with risk groups and may be appropriate targets for potential biosensor development. This study has confirmed that metabolomics can be used to identify the presence of pathogenic E. coli likely to be implicated in human disease.

Utilizing the Food-Pathogen Metabolome to Putatively Identify Biomarkers for the Detection of Shiga Toxin-Producing E. coli (STEC) from Spinach

Shiga toxigenic E. coli (STEC) are an important cause of foodborne disease globally with many outbreaks linked to the consumption of contaminated foods such as leafy greens. Existing methods for STEC detection and isolation are time-consuming. Rapid methods may assist in preventing contaminated products from reaching consumers. This proof-of-concept study aimed to determine if a metabolomics approach could be used to detect STEC contamination in spinach. Using untargeted metabolic profiling, the bacterial pellets and supernatants arising from bacterial and inoculated spinach enrichments were investigated for the presence of unique metabolites that enabled categorization of three E. coli risk groups. A total of 109 and 471 metabolite features were identified in bacterial and inoculated spinach enrichments, respectively. Supervised OPLS-DA analysis demonstrated clear dis-crimination between bacterial enrichments containing different risk groups. Further analysis of the spinach enrichments determined that pathogen risk groups 1 and 2 could be easily discriminated from the other groups, though some clustering of risk groups 1 and 2 was observed, likely representing their genomic similarity. Biomarker discovery identified metabolites that were significantly associated with risk groups and may be appropriate targets for potential biosensor development. This study has confirmed that metabolomics can be used to identify the presence of pathogenic E. coli likely to be implicated in human disease.

Plasmid Dynamics of mcr-1-Positive Salmonella spp. in a General Hospital in China

Salmonella is an important food pathogen that can cause severe gastroenteritis with more than 600,000 deaths globally every year. Colistin (COL), a last-resort antibiotic, is ineffective in bacteria that carry a functional mcr-1 gene, which is often spread by conjugative plasmids. Our work aimed to understand the prevalence of the mcr-1 gene in clinical isolates of Salmonella, as the frequency of occurrence of the mcr-1 gene is increasing globally. Therefore, we analyzed 689 clinical strains, that were isolated between 2009 and late 2018. The mcr-1 gene was found in six strains, which we analyzed in detail by whole genome sequencing and antibiotic susceptibility testing, while we also provide the clinical information on the patients suffering from an infection. The genomic analysis revealed that five strains had plasmid-encoded mcr-1 gene located in four IncHI2 plasmids and one IncI2 plasmid, while one strain had the chromosomal mcr-1 gene originated from plasmid. Surprisingly, in two strains the mcr-1 genes were inactive due to disruption by insertion sequences (ISs): ISApl1 and ISVsa5. A detailed analysis of the plasmids revealed a multitude of ISs, most commonly IS26. The IS contained genes that meditate broad resistance toward most antibiotics underlining their importance of the mobile elements, also with respect to the spread of the mcr-1 gene. Our study revealed potential reservoirs for the transmission of COL resistance and offers insights into the evolution of the mcr-1 gene in Salmonella.

Development and on-site evaluation of an easy-to-perform and low-cost food pathogen diagnostic workflow for low-resource communities

Food-borne diseases are a leading cause of illness and death in many developing countries and thus, there is a real need to develop affordable and practical technologies that can help improve food safety in these countries. The ability to efficiently identify food pathogens is essential to allow national regulatory authorities to monitor food quality and implement safety protocols. In this study, we have developed a simple, low-cost ($0.76 (USD)) complete food pathogen diagnostic workflow ideally suited for deployment in low-resource environments that uses a simple four step process (sample enrichment, cell lysis, DNA amplification, and naked-eye readout). The minimal number of steps and equipment involved in our diagnostic workflow, as well as the simplicity of the yes/no flocculation readout, allows non-technical personnel to perform and interpret the assay. To evaluate the system’s performance, we tested the entire system on fresh produce samples collected from local farms and markets in Cambodia for the presence of the E. coli O157 O-antigen polymerase, wzy. Although this was a proof-of-concept study, our system successfully revealed a clear correlation between the origin and condition of the produce collected and their likelihood of contamination. In conclusion, we believe that our easy-to-perform diagnostic system can have a significant impact on improving food quality and human health if adopted by regulatory authorities in developing countries due to the assay’s simplicity, affordability, and adaptability.

In Vitro Effect of the Common Culinary Herb Winter Savory (Satureja montana) against the Infamous Food Pathogen Campylobacter jejuni

The culinary herb Satureja montana, known as winter savory, is an ingredient of traditional dishes known in different parts of the world. As an ingredient of foods it has the potential to improve their safety. In this study, the herb’s activity was investigated against Campylobacter jejuni, the leading cause of the most prevalent bacterial gastroenteritis worldwide. The ethanolic extract and essential oil of the herb were chemically characterized and six pure compounds—carvacrol, thymol, thymoquinone, p-cymene, γ-terpinene, and rosmarinic acid—were chosen for further analysis. The antimicrobial activity of the ethanolic extract (MIC 250 mg/L) was 4-fold higher compared to the essential oil. Carvacrol, thymol and thymoquinone had the strongest antimicrobial effect (MIC 31.25 mg/L) and a strong synergistic activity between carvacrol and thymol was determined (FICi 0.2). Strong inhibitory effect on C. jejuni efflux pumps (2-fold inhibition) and disruption of membrane integrity (> 80% disruption) of the herb were determined as modes of action. For resistance against the herb, C. jejuni need efflux pumps, although increased resistance against this herb does not co-occur with increased efflux pump activity, as for antibiotics. This study shows the potential of a common culinary herb for the reduction of the food pathogen C. jejuni without increasing resistance.