Occurrence and Characteristics of Toxigenic Staphylococcus aureus in Retail Foods in Iran.

Staphylococcus aureus causes various toxigenic and invasive diseases in humans worldwide. This study examined the prevalence, virulence genes, and antibiotic resistance of S. aureus isolates collected from 894 retail food samples in Ardabil, Iran. Staphylococcal cassette chromosome mec (SCCmec), spa, and multilocus sequence typing methods were employed to further investigate the molecular characteristics of methicillin-resistant S. aureus (MRSA) isolates. The results revealed that 11.18% (n = 100) of food samples exhibited contamination with S. aureus (10.50% methicillin-sensitive S. aureus [MSSA] and 0.67% MRSA). Notably, raw minced meat (29.41%), Faloodeh (25%), and Olivier salad (21.42%) emerged as the most frequently contaminated food items. Among the 100 isolates of S. aureus, 94% were characterized as MSSA, with the remaining 6% identified as MRSA. The highest resistance was observed for penicillin (12%). MRSA isolates exhibited significantly higher resistance rates. Seventy-nine percent of the isolates were positive for sea, 14% for seb, 8% for a sec, and 0% for sed enterotoxin-encoding genes. Sixteen percent of isolates harbored two or more staphylococcal enterotoxin genes, simultaneously. Moreover, 97%, 94%, 24%, and 22% of isolates were positive for hla, hld, tst, and pvl virulence-encoding genes, respectively. No isolate was positive for the exfoliative toxins encoding eta and etb genes. MRSA isolates belonged to CC8 (n = 4) and CC22 (n = 2). Isolates in CC8 belonged to lineage ST239-MRSA-III and spa type t030; the isolates in CC22 belonged to ST22-MRSA-IV and spa types t310 and t223. In conclusion, a relatively high proportion of our retail food samples were contaminated with S. aureus. The high incidence of isolates with toxigenic genes raises serious health concerns. Furthermore, the presence of MRSA lineages linked to humans suggests that retail foods may be contaminated with human origin.

Ready-To-Eat Rocket Salads as Potential Reservoir of Bacteria for the Human Microbiome.

Reportedly, Western-type diets may induce the loss of key microbial taxa within the gastrointestinal microbiota, promoting the onset of noncommunicable diseases. It was hypothesized that the consumption of raw vegetables could contribute to the maintenance of the intestinal microbial community structure. In this context, we explored bacteria associated with commercial rocket salads produced through different farming practices: traditional (conventional, organic, and integrated) and vertical farming. Viable counts of mesophilic bacteria and lactic acid bacteria (LAB) were performed on plate count agar (PCA) and de Man-Rogosa-Sharpe (MRS) agar at pH 5.7, whereas metataxonomics through 16S rRNA gene sequencing was used to profile total bacteria associated with rocket salads. We found that rocket salads from vertical farming had much fewer viable bacteria and had a bacterial community structure markedly different from that of rocket salads from traditional farming. Furthermore, although α- and ß-diversity analyses did not differentiate rocket samples according to farming techniques, several bacterial taxa distinguished organic and integrated from conventional farming salads, suggesting that farming practices could affect the taxonomic composition of rocket bacterial communities. LAB were isolated from only traditional farming samples and belonged to different species, which were variably distributed among samples and could be partly associated with farming practices. Finally, the INFOGEST protocol for in vitro simulation of gastrointestinal digestion revealed that several taxonomically different rocket-associated bacteria (particularly LAB) could survive gastrointestinal transit. This study suggests that commercial ready-to-eat rocket salads harbor live bacteria that possess the ability to survive gastrointestinal transit, potentially contributing to the taxonomic structure of the human gut microbiota. IMPORTANCE Western-type diets are composed of foods with a reduced amount of naturally occurring microorganisms. It was hypothesized that a microbe-depleted diet can favor the alteration of the human intestinal microbial ecosystem, therefore contributing to the onset of chronic metabolic and immune diseases currently recognized as the most significant causes of death in the developed world. Here, we studied the microorganisms that are associated with commercial ready-to-eat rocket salads produced through different farming practices. We showed that rocket salad (a widely consumed vegetal food frequently eaten raw) may be a source of lactic acid bacteria and other microbes that can survive gastrointestinal transit, potentially increasing the biodiversity of the intestinal microbiota. This deduction may be valid for virtually all vegetal foods that are consumed raw.

Public Health Response to Multistate Salmonella Typhimurium Outbreak Associated with Prepackaged Chicken Salad, United States, 2018.

Quantifying the effect of public health actions on population health is essential when justifying sustained public health investment. Using modeling, we conservatively estimated that rapid response to a multistate foodborne outbreak of Salmonella Typhimurium in the United States in 2018 potentially averted 94 reported cases and $633,181 in medical costs and productivity losses.

High Prevalence of Klebsiella pneumoniae in European Food Products: a Multicentric Study Comparing Culture and Molecular Detection Methods.

The Klebsiella pneumoniae species complex (KpSC) is a leading cause of multidrug-resistant human infections. To better understand the potential contribution of food as a vehicle of KpSC, we conducted a multicentric study to define an optimal culture method for its recovery from food matrices and to characterize food isolates phenotypically and genotypically. Chicken meat (n = 160) and salad (n = 145) samples were collected in five European countries and screened for the presence of KpSC using culture-based and zur-khe intergenic region (ZKIR) quantitative PCR (qPCR) methods. Enrichment using buffered peptone water followed by streaking on Simmons citrate agar with inositol (44°C for 48 h) was defined as the most suitable selective culture method for KpSC recovery. A high prevalence of KpSC was found in chicken meat (60% and 52% by ZKIR qPCR and the culture approach, respectively) and salad (30% and 21%, respectively) samples. Genomic analyses revealed high genetic diversity with the dominance of phylogroups Kp1 (91%) and Kp3 (6%). A total of 82% of isolates presented a natural antimicrobial susceptibility phenotype and genotype, with only four CTX-M-15-producing isolates detected. Notably, identical genotypes were found across samples-same food type and same country (15 cases), different food types and same country (1), and same food type and two countries (1)-suggesting high rates of transmission of KpSC within the food sector. Our study provides a novel isolation strategy for KpSC from food matrices and reinforces the view of food as a potential source of KpSC colonization in humans. IMPORTANCE Bacteria of the Klebsiella pneumoniae species complex (KpSC) are ubiquitous, and K. pneumoniae is a leading cause of antibiotic-resistant infections in humans. Despite the urgent public health threat represented by K. pneumoniae, there is a lack of knowledge of the contribution of food sources to colonization and subsequent infection in humans. This is partly due to the absence of standardized methods for characterizing the presence of KpSC in food matrices. Our multicentric study provides and implements a novel isolation strategy for KpSC from food matrices and shows that KpSC members are highly prevalent in salads and chicken meat, reinforcing the view of food as a potential source of KpSC colonization in humans. Despite the large genetic diversity and the low levels of resistance detected, the occurrence of identical genotypes across samples suggests high rates of transmission of KpSC within the food sector, which need to be further explored to define possible control strategies.

Epidemiological investigations identified an outbreak of Shiga toxin-producing Escherichia coli serotype O26:H11 associated with pre-packed sandwiches.

In October 2019, public health surveillance systems in Scotland identified an increase in the number of reported infections of Shiga toxin-producing Escherichia coli (STEC) O26:H11 involving bloody diarrhoea. Ultimately, across the United Kingdom (UK) 32 cases of STEC O26:H11 stx1a were identified, with the median age of 27 years and 64% were male; six cases were hospitalised. Among food exposures there was an association with consuming pre-packed sandwiches purchased at outlets belonging to a national food chain franchise (food outlet A) [odds ratio (OR) = 183.89, P < 0.001]. The common ingredient identified as a component of the majority of the sandwiches sold at food outlet A was a mixed salad of Apollo and Iceberg lettuce and spinach leaves. Microbiological testing of food and environmental samples were negative for STEC O26:H11, although STEC O36:H19 was isolated from a mixed salad sample taken from premises owned by food outlet A. Contamination of fresh produce is often due to a transient event and detection of the aetiological agent in food that has a short-shelf life is challenging. Robust, statistically significant epidemiological analysis should be sufficient evidence to direct timely and targeted on-farm investigations. A shift in focus from testing the microbiological quality of the produce to investigating the processes and practices through the supply chain and sampling the farm environment is recommended.

Survival and growth of Listeria monocytogenes and Staphylococcus aureus in ready-to-eat Mediterranean vegetable salads: Impact of storage temperature and food matrix.

This study aimed to evaluate the survival or growth behavior of L. monocytogenes and S. aureus in ready-to-eat vegetable salads including Arabic salad (tomato-cucumber salad) with or without 0.5% (w/w) salt and 1% (v/w) lemon juice, tahini salad, coleslaw salad, toum sauce salad (aioli) stored at 4, 10, and 24 °C for 5 d. L. monocytogenes showed greater growth or survival in all types of salads at all temperatures tested. L. monocytogenes grew in Arabic salad without additives at 10 °C and tahini salad at 4 and 10 °C. L. monocytogenes survived all preparation and storage conditions used in the current study and its numbers were detectable in all types of salads tested, and with a maximum reduction of 3.0 log CFU/g, except for toum sauce salad at 24 °C, the cells were not detected using enrichment broth. In addition, S. aureus survived in Arabic salad with or without additives and in tahini salad with a maximum reduction of 2.3 log CFU/g. However, S. aureus numbers sharply decreased in coleslaw and toum sauce salad and at 5 d they reached undetectable levels (≥2 log CFU/g) in coleslaw at 24 °C and in toum sauce at 10 and 24 °C. Addition of lemon juice and salt to Arabic salad significantly reduced the numbers of both pathogens.

The Possibilities of Using Ultrasonically Activated Streams to Reduce the Risk of Foodborne Infection from Salad.

In this study, we investigated the effects of an ultrasonically activated stream (UAS) on the removal of microbial contaminants from spinach leaves. The microbial loads on samples cleaned with and without UAS were enumerated using the cell culture method and compared against unwashed samples on day 0 and day 6 after cleaning. The effects of UAS cleaning on leaf quality were also examined through both macroscopic and microscopic inspection, as well as measurement of the electrolyte leakage rate. Results showed that the microbial load on samples cleaned with UAS for 2 min was significantly lower on day 6 after cleaning than on those treated without ultrasound. Comparison between the cleaning effects of UAS for 40 s versus 2 min indicated that a cleaning duration of 2 min allowed sufficient time for UAS to disaggregate and detach the microbial contamination more effectively. In this case, the induction of bacteria into a viable but non-culturable state does not affect the shelf-life test results as much as it does with a 40 s clean. UAS cleaning for 2 min did not produce significant surface damage, which can affect overall leaf quality. These findings highlight the potential of UAS systems in the salad industry to improve the microbiological quality and shelf life of salads.

Ready-to-eat vegetable salads served in Nigerian restaurants: a potential source of multidrug-resistant bacteria.

AIM: Public health promotion of fruit and vegetables as healthier sources of nutrition in Nigeria has led to an increase in consumption of fruit and vegetables, particularly in the form of ready-to-eat (RTE) salads. However, the continual association of drug-resistant bacteria with fruit and vegetables creates an additional challenge for consumer safety. Hence this study was carried out to determine the prevalence of multidrug-resistant bacteria (MRB) in, and characterization of MRB isolated from, RTE salads. METHODS AND RESULTS: RTE vegetable salad samples were randomly selected for bacterial analysis with Wei's Urn iterative randomization technique and a One Way Analysis of Variance performed with Kruskal-Wallis test. Total viable bacteria count (TVC) was performed with pour-plate technique. Bacterial colonies from Petri plates were tested for multidrug resistance with Kirby Bauer disc diffusion test and prevalence/counts of MRB, as well as multiple antibiotic resistance indices (MAR), was/were subsequently deduced. Mean prevalence of MRB in all RTE salad samples was estimated at 54·38% while mean counts of MRB were estimated at 6·83 ± 6·42 log10 CFU per g. Prevalence of bacterial resistance to all antibiotics tested ranged from 5·92 to 100·00%. Mean MAR obtained for all RTE salad samples was estimated at 0·51 (recommended limit = 0·20) indicating that RTE salads were a potential source of MRB, with significant health risk. MRB isolated from RTE salads included Proteus vulgaris strain ATU 243, Bacillus thuringiensis strain AND 236, Citrobacter freundii strain ABC 2 and Serratia marcescens strain ADJ 212. CONCLUSION: This study showed that MRB constitute a significant proportion of the bacterial community present in RTE salads served in Nigerian restaurants. SIGNIFICANCE AND IMPACT OF THE STUDY: Our study provided empirical evidence which showed that RTE salads served in Nigerian restaurants were a potential source of MRB, with significant health risk.

Development of a multiplex real-time PCR assay for the identification and quantification of group-specific Bacillus spp. and the genus Paenibacillus.

Spoilage microorganisms can occur at many points throughout food production systems. Bacillus spp. and Paenibacillus spp. are important aerobic spoilage bacteria in various sectors of the food industry. In this study, we developed a rapid detection and quantification technique for Bacillus group-specific and the genus Paenibacillus by using multiplex quantitative PCR (mqPCR). The 1st was the Bacillus cereus group containing B. cereus and B. weihenstephanensis; the 2nd was the B. subtilis group containing B. subtilis, B. licheniformis, B. safensis, and B. pumilus; the 3rd was the B. simplex group containing B. megaterium and B. simplex; and the 4th was the genus Paenibacillus. Depending on the assays, the detection limit was 10 copy numbers. In addition, mqPCR assays were validated by spiking potato salad and milk samples with four strains; B. weihenstephanensis, B. licheniformis, B. megaterium, and P. lautus. The detection dynamic range for potato salad was 105 CFU/mL-101 CFU/mL with B. weihenstephanensis and B. licheniformis, and 105 CFU/mL-102 CFU/mL with B. megaterium and P. lautus, while, for milk, all strains were 105 CFU/mL-102 CFU/mL. We also stored these food matrices spiked with four bacterial suspensions (approximately 103 CFU/mL) at various temperatures. Results showed that B. weihenstephanensis and B. licheniformis were able to grow in potato salad, whereas, the populations of B. weihenstephanensis, B. licheniformis, and P. lautus increased in milk. Consequently, the mqPCR assays developed here in facilitated the differentiation, quantification, and confirmation of the presence of the psychrophilic and psychrotolerant Bacillus group and Paenibacillus spp.

Outbreaks of Shiga Toxin-Producing Escherichia coli Linked to Sprouted Seeds, Salad, and Leafy Greens: A Systematic Review.

Shiga toxin-producing Escherichia coli (STEC) outbreaks involving ready-to-eat salad products have been described in the scientific literature since 1995. These products typically do not undergo a definitive control step such as cooking to eliminate pathogens. To reduce the number of STEC infections from salad products, efforts will need to focus on preventing and reducing contamination throughout the food chain. We performed a systematic review of STEC outbreaks involving sprouted seeds, salad, or leafy green products to determine whether there were recurrent features, such as availability of microbiological evidence or identification of the contamination event, which may inform future investigations and prevention and control strategies. Thirty-five STEC outbreaks linked to contaminated leafy greens were identified for inclusion. The outbreaks occurred from 1995 to 2018 and ranged from 8 to more than 8,500 cases. Detection of STEC in the food product was rare (4 of 35 outbreaks). For the remaining outbreaks, the determination of leafy greens as the source of the outbreak mainly relied on analytical epidemiology (20 of 35) or descriptive evidence (11 of 35). The traceback investigation in 21 of 32 outbreaks was not able to identify possible routes leading to where the STEC bacteria came from or how the leaves were contaminated. Investigations in eight outbreaks found poor practice during processing that may have contributed to the outbreak, such as insufficient postharvest disinfection of the product. Six outbreak investigations were able to identify the outbreak strain in animal feces near the growing fields; two of these were also able to find it in irrigation water on the farms, providing a likely route of contamination. These results highlight the limitations of relying on microbiological confirmation as a basis to initiate investigations of upstream production to understand the source of contamination. This review also demonstrates the importance of, and difficulties associated with, food-chain traceback studies to inform control measures and future prevention.

Multi-drug resistant extended-spectrum beta-lactamase producing E. coli and Salmonella on raw vegetable salads served at hotels and restaurants in Bharatpur, Nepal.

OBJECTIVE: Antimicrobial resistance among the bacteria present in ready-to-eat foods like vegetable salads is an emerging concern today. The current study was undertaken to investigate the presence of multi-drug resistant extended-spectrum ß-lactamase (ESBL) producing E. coli and Salmonella spp. in raw vegetable salads served at hotels and restaurants in Bharatpur. A total of 216 salad samples were collected from three different grades of hotels and restaurants and examined for the presence of E. coli and Salmonella spp. in Microbiology laboratory of Birendra Multiple Campus by conventional microbiological techniques. RESULTS: Out of 216 samples, 66 samples (35.2%) showed the presence of Salmonella spp. whereas E. coli was recovered from 29 (13.4%) samples of which 3 samples harbored E. coli O157: H7. Antibiotic susceptibility testing revealed that 9 (13.6%) Salmonella and 4 (13.8%) E. coli isolates were detected as multi-drug resistant. Total ESBL producers reported were 5 (7.57%) Salmonella and 4 (13.8%) E. coli. The study also assessed a significant association between occurrence of E. coli and Salmonella with different grades of hotels and restaurants, personal hygiene and literacy rate of chefs and with the type of cleaning materials used to wash knives and chopping boards (p < 0.05). The findings suggest an immediate need of attention by the concerned authorities to prevent the emergence and transmission of food-borne pathogens and infections antimicrobial resistance among them.

Growth of Listeria monocytogenes in ready to eat salads at different storage temperatures and valuation of virulence genes expression.

BACKGROUND: Vegetables are major components of a healthy and balanced diet. However, 25% of foodborne diseases are linked to the consumption of vegetables. STUDY DESIGN: The aim of this work was to assess the microbiological risks associated with consumption of ready to eat salads (RTE). METHODS: Microbiological challenge tests were carried out for the evaluation of the L. monocytogenes growth potential in RTE salads stored at different temperatures. RESULTS: The results indicate that L.monocytogenes was able to grow (δ ≥ 0.5) in all storage conditions considered at the end of shelf life. In order to evaluate the virulence role of L. monocytogenes, the temperature-dependent transcription of major virulence genes was also investigated by RT-PCR. CONCLUSIONS: The microbiological challenge test allowed us to confirm, as also demonstrated by other authors, that RTE salads are able to support the growth of L. monocytogenes strains (d δ≥ 0.5) stored under different temperatures.

Multistate Outbreak of Listeriosis Associated with Packaged Leafy Green Salads, United States and Canada, 2015-2016.

We investigated an outbreak of listeriosis detected by whole-genome multilocus sequence typing and associated with packaged leafy green salads. Nineteen cases were identified in the United States during July 5, 2015-January 31, 2016; isolates from case-patients were closely related (median difference 3 alleles, range 0-16 alleles). Of 16 case-patients interviewed, all reported salad consumption. Of 9 case-patients who recalled brand information, all reported brands processed at a common US facility. The Public Health Agency of Canada simultaneously investigated 14 cases of listeriosis associated with this outbreak. Isolates from the processing facility, packaged leafy green salads, and 9 case-patients from Canada were closely related to US clinical isolates (median difference 3 alleles, range 0-16 alleles). This investigation led to a recall of packaged leafy green salads made at the processing facility. Additional research is needed to identify best practices and effective policies to reduce the likelihood of Listeria monocytogenes contamination of fresh produce.

Prevalence of Listeria monocytogenes in Select Ready-to-Eat Foods-Deli Meat, Soft Cheese, and Packaged Salad: A Systematic Review and Meta-Analysis.

Listeria monocytogenes is the cause of listeriosis, an important foodborne disease. Contaminated ready-to-eat foods are common sources of L. monocytogenes, yet no global estimates exist for prevalence and levels in high-risk ready-to-eat foods. Our objective was to estimate the prevalence and levels of L. monocytogenes in deli meat, soft cheese, and packaged salad. We searched Medline, Web of Science, Agricola, Conference Proceedings Citation Index-Science, Science.gov, ScienceResearch.com , and OpenGREY for studies on L. monocytogenes prevalence and/or levels, with no restriction on publication date. We used a priori study selection, data extraction, and risk of biases processes. Results were synthesized with random-effects meta-analyses and meta-regressions to evaluate heterogeneity between studies. We included in the review 100 studies with a sample size restriction of ≥100, and we estimated L. monocytogenes prevalence in deli meat at 2.9% (95% confidence interval [CI], 2.3 to 3.6%), in soft cheese at 2.4% (95% CI, 1.6 to 3.6%), and in packaged salad at 2.0% (95% CI, 1.2 to 3.1%). High heterogeneity was present in all food groups, and meta-regressions did not reveal consistent explanations for heterogeneity. Pathogen level was not reported consistently or in the format required for synthesis, so meta-analyses of this variable were not performed. The high heterogeneity between studies indicates that use of global summary prevalence estimates for risk assessments are not advisable, but awareness of risk and the heterogeneity of the risk is relevant for education and further risk assessment.

Microbial Contamination of Organically and Conventionally Produced Fresh Vegetable Salads and Herbs from Retail Markets in Southwest Germany.

A total of 189 samples of fresh products (leafy salads, ready-to-eat mixed salads, and fresh herbs) bought in retail in Southwest Germany were investigated for their microbiological quality and the presence of pathogenic bacteria, including Salmonella spp., Listeria monocytogenes, and presumptive Bacillus cereus. Total aerobic mesophilic plate counts (TAC) ranged from 5.5 to 9.6 log colony-forming units (CFUs) per gram. Enterobacteria and pseudomonads were the predominant microorganisms and were detected in all samples with counts between 5.0 and 9.2 log CFU/g. Strains of Escherichia coli were detected in 9 salad (7.9%) and 25 herb samples (33.3%). Significant differences in bacterial counts were found between conventionally and organically-grown products: in herbs the counts of moulds were significantly higher in organically-grown products, while E. coli was only detected in conventionally-grown products. In conventionally-grown salad samples, yeast counts were significantly higher. Salmonella Enteritidis was only detected in two conventionally- and in one organically-produced salad samples (2.6%). No coagulase-positive staphylococci were detected in fresh salads as well as in herbs. High levels of B. cereus sensu lato (≥3 log CFU/g) were detected in 19 vegetable salads (16.7%) and even in 55 samples of fresh herbs (73.3%). Listeria monocytogenes could not be detected in fresh herbs; however, three L. monocytogenes strains were isolated from two conventionally-produced salad samples and belonged to PCR serogroup IIa. Although our results indicate a high microbial load in fresh salads and herbs in Southwest Germany in 2015, the incidences of human pathogenic bacteria, that is, L. monocytogenes, Salmonella spp., and coagulase-positive staphylococci strains, were low.