The potential role of Listeria monocytogenes in promoting colorectal adenocarcinoma tumorigenic process.

BACKGROUND: Listeria monocytogenes is a foodborne pathogen, which can cause a severe illness, especially in people with a weakened immune system or comorbidities. The interactions between host and pathogens and between pathogens and tumor cells have been debated in recent years. However, it is still unclear how bacteria can interact with tumor cells, and if this interaction can affect tumor progression and therapy. METHODS: In this study, we evaluated the involvement of L. monocytogenes in pre-neoplastic and colorectal cancer cell proliferation and tumorigenic potential. RESULTS: Our findings showed that the interaction between heat-killed L. monocytogenes and pre-neoplastic or colorectal cancer cells led to a proliferative induction; furthermore, by using a three-dimensional cell culture model, the obtained data indicated that L. monocytogenes was able to increase the tumorigenic potential of both pre-neoplastic and colorectal cancer cells. The observed effects were then confirmed as L. monocytogenes-specific, using Listeria innocua as negative control. Lastly, data suggested the Insulin Growth Factor 1 Receptor (IGF1R) cascade as one of the possible mechanisms involved in the effects induced by L. monocytogenes in the human colorectal adenocarcinoma cell line. CONCLUSIONS: These findings, although preliminary, suggest that the presence of pathogenic bacterial cells in the tumor niches may directly induce, increase, and stimulate tumor progression.

From farm to fork: Spread of a multidrug resistant Salmonella Infantis clone encoding blaCTX-M-1 on pESI-like plasmids in Central Italy.

Salmonella enterica subsp. enterica serovar Infantis (S. Infantis) is one of the "top five Salmonella serovars" of clinical significance in the European Union (EU). Antimicrobial resistant and extended spectrum ß-lactamase (ESBL) AmpC-producing S. Infantis have been described in food production systems and human clinical samples in Italy. Recently, an increase of MDR S. Infantis carrying blaCTX-M genes involved in 3rd generation cephalosporin resistance was noticed in the EU, including Italy, mainly due to the spread of S. Infantis harboring a pESI-like plasmid. The aim of this study was to investigate the occurrence of the S. Infantis pESI-like plasmid among antibiotic resistant S. Infantis strains isolated at different points of the food chain, and to provide a phylogenetic analysis to gain further insight on their transmission pathways from 'farm to fork'. MDR S. Infantis strains (n. 35) isolated from 2016 to 2021 at different stages of the food chain (animals, food, food-related environments, and humans) were investigated with in depth molecular characterization using real-time PCR, S1 nuclease pulsed-field gel electrophoresis (S1-PFGE) and whole genome sequencing (WGS). Our study reported the occurrence of S. Infantis strains harboring pESI-like plasmids, carrying blaCTX-M-1 genes, in Central Italy, at different sampling points along the food chain. Results confirmed the presence of a plasmid with a molecular size around 224-310 kb, thus consistent with the pESI-like, in 97 % of the 35 samples investigated. Two variants of S. Infantis pESI-like IncFIB(K)_1_Kpn3 were detected, one associated with the European clone carrying blaCTX-M-1 (21 isolates) and the other associated with U.S. isolates carrying blaCTX-M-65 (2 isolates, pESI-like U.S. variant). The majority was resistant to 3rd generation cephalosporins but none of the strains tested positive for the carbapenemase encoding genes. A total of 118 virulence genes were identified in isolates harboring the pESI-like plasmid. cgMLST and SNP-based analysis revealed the presence of one main cluster, composed by strains isolated from the environment, animals, food and humans. The results of this investigation underline the importance of phylogenetic studies to monitor and understand pathogen and AMR spread in a One Health approach.

Widespread Multidrug Resistance of Arcobacter butzleri Isolated from Clinical and Food Sources in Central Italy.

The Arcobacter genus comprises a group of bacteria widely distributed in different habitats that can be spread throughout the food chain. Fluoroquinolones and aminoglycosides represent the most common antimicrobial agents used for the treatment of Arcobacter infections. However, the increasing trend of the antimicrobial resistance of this pathogen leads to treatment failures. Moreover, the test implementation and interpretation are hindered by the lack of reference protocols and standard interpretive criteria. The purpose of our study was to assess the antibiotic resistance pattern of 17 A. butzleri strains isolated in Central Italy from fresh vegetables, sushi, chicken breast, and clinical human samples to provide new and updated information about the antimicrobial resistance epidemiology of this species. Antimicrobial susceptibility testing was carried out by the European Committee on Antimicrobial Susceptibility Testing (EUCAST)'s disc diffusion method. All the strains were multidrug resistant, with 100% resistance to tetracyclines and cefotaxime (third generation cephalosporins). Some differences were noticed among the strains, according to the isolation source (clinical isolates, food of animal origin, or fresh vegetables), with a higher sensitivity to streptomycin detected only in the strains isolated from fresh vegetables. Our data, together with other epidemiological information at the national or European Union (EU) level, may contribute to developing homogeneous breakpoints. However, the high prevalence of resistance to a wide range of antimicrobial classes makes this microorganism a threat to human health and suggests that its monitoring should be considered by authorities designated for food safety.

Listeria monocytogenes Strains Persisting in a Meat Processing Plant in Central Italy: Use of Whole Genome Sequencing and In Vitro Adhesion and Invasion Assays to Decipher Their Virulence Potential.

In this study, we used both a WGS and an in vitro approach to study the virulence potential of nine Listeria monocytogenes (Lm) strains belonging to genetic clusters persisting in a meat processing plant in Central Italy. The studied clusters belonged to CC1-ST1, CC9-ST9, and CC218-ST2801. All the CC1 and CC218 strains presented the same accessory virulence genes (LIPI-3, gltA, gltB, and aut_IVb). CC1 and CC9 strains presented a gene profile similarity of 22.6% as well as CC9 and CC218 isolates. CC1 and CC218 showed a similarity of 45.2% of the same virulence profile. The hypervirulent strains of lineage I (CC1 and CC218) presented a greater ability to adhere and invade Caco-2 cells than hypovirulent ones (CC9). CC1 strains were significantly more adhesive and invasive compared with CC9 and CC218 strains, although these last CCs presented the same accessory virulence genes. No statistically significant difference was found comparing CC218 with CC9 strains. This study provided for the first time data on the in vitro adhesiveness and invasiveness of CC218-ST2801 and added more data on the virulence characteristics of CC1 and CC9. What we observed confirmed that the ability of Lm to adhere to and invade human cells in vitro is not always decipherable from its virulence gene profile.

Evidence of Virulent Multi-Drug Resistant and Biofilm-Forming Listeria Species Isolated from Various Sources in South Africa.

Listeriosis is a foodborne disease caused by Listeria monocytogenes species and is known to cause severe complications, particularly in pregnant women, young children, the elderly, and immunocompromised individuals. The aim of this study was to investigate the presence of Listeria species in food and water using both biochemical and species-specific PCR analysis. L. monocytogenes isolates were further screened for the presence of various antibiotic resistance, virulence, and biofilm-forming determinants profiles using phenotypic and genotypic assays. A total of 207 samples (composed of meat, milk, vegetables, and water) were collected and analyzed for presence of L. monocytogenes using species specific PCR analysis. Out of 267 presumptive isolates, 53 (19.85%) were confirmed as the Listeria species, and these comprised 26 L. monocytogenes, 3 L. innocua, 2 L. welshimeri, and 1 L. thailandensis. The remaining 21 Listeria species were classified as uncultured Listeria, based on 16SrRNA sequence analysis results. A large proportion (76% to 100%) of the L. monocytogenes were resistant to erythromycin (76%), clindamycin (100%), gentamicin (100%), tetracycline (100%), novobiocin (100%), oxacillin (100%), nalidixic acid (100%), and kanamycin (100%). The isolates revealed various multi-drug resistant (MDR) phenotypes, with E-DA-GM-T-NO-OX-NA-K being the most predominant MDR phenotypes observed in the L. monocytogenes isolates. The virulence genes prfA, hlyA, actA, and plcB were detected in 100%, 68%, 56%, and 20% of the isolates, respectively. In addition, L. monocytogenes isolates were capable of forming strong biofilm at 4 °C (%) after 24 to 72 h incubation periods, moderate for 8% isolates at 48 h and 20% at 72 h (p < 0.05). Moreover, at 25 °C and 37 °C, small proportions of the isolates displayed moderate (8−20%) biofilm formation after 48 and 72 h incubation periods. Biofilm formation genes flaA and luxS were detected in 72% and 56% of the isolates, respectively. These findings suggest that proper hygiene measures must be enforced along the food chain to ensure food safety.

Prevalence, Antibiotic-Resistance, and Replicon-Typing of Salmonella Strains among Serovars Mainly Isolated from Food Chain in Marche Region, Italy.

Nontyphoidal salmonellosis (NTS) is the second most commonly reported gastrointestinal infection in humans and an important cause of food-borne outbreaks in Europe. The use of antimicrobial agents for animals, plants, and food production contributes to the development of antibiotic-resistant Salmonella strains that are transmissible to humans through food. The aim of this study was to investigate the presence and the potential dissemination of multidrug-resistant (MDR) Salmonella strains isolated in the Marche Region (Central Italy) via the food chain. Strains were isolated from different sources: food, human, food animal/livestock, and the food-processing environment. Among them, we selected MDR strains to perform their further characterization in terms of resistance to tetracycline agent, carriage of tet genes, and plasmid profiles. Tetracycline resistance genes were detected by PCR and plasmid replicons by PCR-based replicon typing (PBRT). A total of 102 MDR Salmonella strains were selected among the most prevalent serovars: S. Infantis (n = 36/102), S. Derby (n = 20/102), S. Typhimurium (n = 18/102), and a monophasic variant of S. Typhimurium (MVST, n = 28/102). Resistance to sulfisoxazole (86%) and tetracycline (81%) were the most common, followed by ampicillin (76%). FIIS was the most predominant replicon (17%), followed by FII (11%) and FIB (11%) belonging to the IncF incompatibility group. Concerning the characterization of tet genes, tetB was the most frequently detected (27/89), followed by tetA (10/89), tetG (5/89), and tetM (1/89). This study showed the potential risk associated with the MDR Salmonella strains circulating along the food chain. Hence, epidemiological surveillance supported by molecular typing could be a very useful tool to prevent transmission of resistant Salmonella from food to humans, in line with the One Health approach.

SARS-CoV-2 RNA Detection on Environmental Surfaces in a University Setting of Central Italy.

The transmission of SARS-CoV-2 occurs through direct contact (person to person) and indirect contact by means of objects and surfaces contaminated by secretions from individuals with COVID-19 or asymptomatic carriers. In this study, we evaluated the presence of SARS-CoV-2 RNA on surfaces made of different materials located in university environments frequented by students and staff involved in academy activity during the fourth pandemic wave (December 2021). A total of 189 environmental samples were collected from classrooms, the library, computer room, gym and common areas and subjected to real-time PCR assay to evaluate the presence of SARS-CoV-2 RNA by amplification of the RNA-dependent RNA polymerase (RdRp) gene. All samples gave a valid result for Internal Process Control and nine (4.8%) tested very low positive for SARS-CoV-2 RNA amplification with a median Ct value of 39.44 [IQR: 37.31-42.66] (≤1 copy of viral genome). Our results show that, despite the prevention measures implemented, the presence of infected subjects cannot be excluded, as evidenced by the recovery of SARS-CoV-2 RNA from surfaces. The monitoring of environmental SARS-CoV-2 RNA could support public health prevention strategies in the academic and school world.

Airborne Microorganism Inactivation by a UV-C LED and Ionizer-Based Continuous Sanitation Air (CSA) System in Train Environments.

Improving indoor air quality present in environments where people live is important to protect human health. This particularly applies to public transportation, where air quality may affect the health and safety of passengers, workers and staff. To provide better air quality, many buildings and transports are provided with heating, ventilation and air conditioning (HVAC) systems, which are always equipped with filters to retain the particulate present in the airflow, but they lack continuous air sanitization systems. In this study, a new UV-C LED and ionizer-based continuous sanitation air (CSA) system to be installed in a train HVAC was developed (international patent: N.PCT/IB2021/054194) and its sanitation efficacy against various microbial species (bacteria and fungi) was assessed. The device proved to be very effective at the microbial killing of aerodispersed microorganisms, both in its experimental configuration (ISO 15714:2019) and in a train setting. The installation of this CSA system on public transportation appears to be a promising solution to guarantee high microbiological air quality with a very low environmental impact due to its eco-friendly components.

Salmonella Abortusovis: An Epidemiologically Relevant Pathogen.

The ovine pathogen Salmonella enterica serovar Abortusovis (SAO), a pathogen strictly adapted to ovine hosts, is endemic in several European and Asian countries, where it causes significant economic losses due to the high rates of abortion in infected flocks. In some countries (i.e. Switzerland and Croatia), re-emergence of infection by SAO occurred after decades during which the disease has not been reported. The introduction of (SAO) epidemic strains in new areas is difficult to control due to the asymptomatic behaviors in infected adult lambs, rams, and nonpregnant ewes. Culture-based diagnosis may provide false-negative results. Moreover, the retrospective identification of Salmonella infection in ewes is challenging as excretion of the causative agent is transient and the serum antibodies fall to low titres soon after the abortion. Therefore, regular monitoring of pathogen exposure, mainly through seroconversion assessment, is advisable to prevent disease introduction and spread in SAO-free areas, especially in case of animal export, and to reduce abortion risk.

Assessment of hygienic conditions of recreational facility restrooms: an integrated approach.

INTRODUCTION: Microbiological quality of recreational environments included restrooms, is generally assessed by water and surface monitoring. In this study, an environmental monitoring, conducted in spring, of swimming pool restrooms of a recreation center located in the Marche region has been carried out. Seven water samples and seven surface swabs were collected. Moreover, six air samples have been included. The aim of this study was to evaluate if air microbiological monitoring, along with molecular detection in real-time PCR, could give additional useful information about the hygienic conditions of the facility. METHODS: Heterotrophic Plate Count (HPC) both at 22°C (psychrophilic) and 37°C (mesophilic) was determined by separate cultures in all samples. The presence of Legionella pneumophila and Pseudomonas aeruginosa was evaluated by both culture and real-time PCR. RESULTS: The analysis of shower water recorded a HPC load of mesophilic bacteria (37°C) more than 10-fold higher in men restroom, respect to women's one (> 100 vs < 10 CFU/ml), while in air samples was between < 100 and > 500. Concerning pathogen presence, both species Legionella pneumophila and Pseudomonas aeruginosa were detected only in men restroom, but in different sample types by using different methods (culture and real-time PCR). CONCLUSIONS: Air sampling may offer the advantage of giving more representative data about microbial presence in restrooms, including bacterial species transmitted through aerosol, like Legionella. Moreover, the concurrent use of molecular and microbiological detection in an integrated approach could offer the advantage of greater sensitivity.

Detection and Virulence Characterization of Listeria monocytogenes Strains in Ready-to-Eat Products.

The public health risk posed by Listeria monocytogenes in ready-to-eat (RTE) foods depends on the effectiveness of its control at every stage of the production process and the strain involved. Analytical methods currently in use are limited to the identification/quantification of L. monocytogenes at the species level, without distinguishing virulent from hypovirulent strains. In these products, according to EU Regulation 2073/2005, L. monocytogenes is a mandatory criterion irrespective of strain virulence level. Indeed, this species encompasses a diversity of strains with various pathogenic potential, reflecting genetic heterogeneity of the species itself. Thus, the detection of specific L. monocytogenes virulence genes can be considered an important target in laboratory food analysis to assign different risk levels to foods contaminated by strains carrying different genes. In 2015-2016, a severe invasive listeriosis outbreak occurred in central Italy, leading to the intensification of routine surveillance and strain characterization for virulence genetic markers. A new multiplex real-time polymerase chain reaction targeting main virulence genes has been developed and validated against the enzyme-linked fluorescent assay (ELFA) culture-based method. Results of the improved surveillance program are now reported in this study.

Hypo- and Hyper-Virulent Listeria monocytogenes Clones Persisting in Two Different Food Processing Plants of Central Italy.

A total of 66 Listeria monocytogenes (Lm) isolated from 2013 to 2018 in a small-scale meat processing plant and a dairy facility of Central Italy were studied. Whole Genome Sequencing and bioinformatics analysis were used to assess the genetic relationships between the strains and investigate persistence and virulence abilities. The biofilm forming-ability was assessed in vitro. Cluster analysis grouped the Lm from the meat plant into three main clusters: two of them, both belonging to CC9, persisted for years in the plant and one (CC121) was isolated in the last year of sampling. In the dairy facility, all the strains grouped in a CC2 four-year persistent cluster. All the studied strains carried multidrug efflux-pumps genetic determinants (sugE, mdrl, lde, norM, mepA). CC121 also harbored the Tn6188 specific for tolerance to Benzalkonium Chloride. Only CC9 and CC121 carried a Stress Survival Islet and presented high-level cadmium resistance genes (cadA1C1) carried by different plasmids. They showed a greater biofilm production when compared with CC2. All the CC2 carried a full-length inlA while CC9 and CC121 presented a Premature Stop Codon mutation correlated with less virulence. The hypo-virulent clones CC9 and CC121 appeared the most adapted to food-processing environments; however, even the hyper-virulent clone CC2 warningly persisted for a long time. The identification of the main mechanisms promoting Lm persistence in a specific food processing plant is important to provide recommendations to Food Business Operators (FBOs) in order to remove or reduce resident Lm.

Comparison of UV, Peracetic Acid and Sodium Hypochlorite Treatment in the Disinfection of Urban Wastewater.

One source of water contamination is the release of wastewater that has not undergone efficient treatment. The aim of this study was to evaluate the reduction obtained with sodium hypochlorite (NaClO), UV and peracetic acid disinfection treatment of Salmonella spp., pathogenic Campylobacter, STEC and bacterial indicators in three full-scale municipal wastewater plants. A general reduction in Salmonella was observed after disinfection, but these bacteria were detected in one UV-treated sample (culture method) and in 33%, 50% and 17% of samples collected after NaClO, UV and PAA disinfection treatments, respectively (PCR method). A better reduction was also observed under NaClO disinfection for the microbial indicators. Independent of the disinfection treatment, E. coli O157:H7 was not detected in the disinfected samples, whereas some samples treated with UV and PAA showed the presence of the stx1 gene. No reduction in the presence of stx2 genes was verified for any of the disinfection treatments. Campylobacter was not detected in any of the analysed samples. The overall results highlight a better reduction in microbiological parameters with a NaClO disinfection treatment in a full-scale municipal wastewater plant compared with UV and PAA. However, the results indicate that a complete and specific monitoring program is necessary to prevent a possible risk to public health.

Whole-Genome Sequencing Characterization of Virulence Profiles of Listeria monocytogenes Food and Human Isolates and In Vitro Adhesion/Invasion Assessment.

Listeria monocytogenes (Lm) is the causative agent of human listeriosis. Lm strains have different virulence potential. For this reason, we preliminarily characterised via Whole-Genome Sequencing (WGS) some Lm strains for their key genomic features and virulence-associated determinants, assigning the clonal complex (CC). Moreover, the ability of the same strains to adhere to and invade human colon carcinoma cell line Caco-2, evaluating the possible correspondence with their genetic virulence profile, was also assessed. The clinical strains typed belonged to clonal complex (CC)1, CC31, and CC101 and showed a very low invasiveness. The Lm strains isolated from food were assigned to CC1, CC7, CC9, and CC121. All CC1 carried the hypervirulence pathogenicity island LIPI-3 in addition to LIPI-1. Premature stop codons in the inlA gene were found only in Lm of food origin belonging to CC9 and CC121. The presence of LIPI2_inlII was observed in all the CCs except CC1. The CC7 strain, belonging to an epidemic cluster, also carried the internalin genes inlG and inlL and showed the highest level of invasion. In contrast, the human CC31 strain lacked the lapB and vip genes and presented the lowest level of invasiveness. In Lm, the genetic determinants of hypo- or hypervirulence are not necessarily predictive of a cell adhesion and/or invasion ability in vitro. Moreover, since listeriosis results from the interplay between host and virulence features of the pathogen, even hypovirulent clones are able to cause infection in immunocompromised people.

Physical activity as a tool for health promotion: the evolution of international strategies and interventions.

Physical activity (PA) has a great potential impact in the prevention and control of non-communicable diseases. However, epidemiologic data reporting a high percentage of inactive people, still indicate a scarce perception of PA benefits. Therefore, in the past decades, a number of documents has been produced by international organizations with the aim of changing policies and institutional actions towards the promotion of PA. Several actions have been put in place and an evolution process in international strategies for PA promotion is ongoing. Nevertheless, there is a need to continue updating these policies in light of new knowledge about evidence-based PA health effects. A stimulating discussion about effective PA promotion programs is useful for future planning of interventions. The aim of this work is to report the evolution of international strategies aimed to PA promotion, from early PA recommendations, to the recent WHO Global Action Plan on Physical Activity 2018-2030.

Redox homeostasis as a target for new antimycobacterial agents.

Despite early treatment with antimycobacterial combination therapy, drug resistance continues to emerge. Maintenance of redox homeostasis is essential for Mycobacterium avium (M. avium) survival and growth. The aim of the present study was to investigate the antimycobacterial activity of two pro-glutathione (pro-GSH) drugs that are able to induce redox stress in M. avium and to modulate cytokine production by macrophages. Hence, we investigated two molecules shown to possess antiviral and immunomodulatory properties: C4-GSH, an N-butanoyl GSH derivative; and I-152, a prodrug of N-acetyl-cysteine (NAC) and ß-mercaptoethylamine (MEA). Both molecules showed activity against replicating M. avium, both in the cell-free model and inside macrophages. Moreover, they were even more effective in reducing the viability of bacteria that had been kept in water for 7 days, proving to be active both against replicating and non-replicating bacteria. By regulating the macrophage redox state, I-152 modulated cytokine production. In particular, higher levels of interferon-gamma (IFN-γ), interleukin 1 beta (IL-1ß), IL-18 and IL-12, which are known to be crucial for the control of intracellular pathogens, were found after I-152 treatment. Our results show that C4-GSH and I-152, by inducing perturbation of redox equilibrium, exert bacteriostatic and bactericidal activity against M. avium. Moreover, I-152 can boost the host response by inducing the production of cytokines that serve as key regulators of the Th1 response.

Mediterranean diet adherence and weight status among Sicilian Middle school adolescents.

During the last decades, an important modification of dietary habits has been observed in the Mediterranean countries, especially among young people. The aim of this study was to evaluate adherence to the Mediterranean diet (MedDiet) and its relationship with weight status in a group of Italian middle school adolescents by using the KIDMED test. The evaluation of weight status revealed that 61.5, 26.8, and 11.7% were normal weight, overweight, and obese, respectively. MedDiet adherence was high in 13.3%, average in 27.1%, and low in 59.6% of the students with no differences by gender and age. MedDiet adherence was found significatively higher in normal weight and in played sport adolescents, in comparison to the overweight and obese ones (p < .001) who showed incorrect nutritional habits. This cross-sectional study shows a very low MedDiet adherence among adolescent living in the Mediterranean basin and highlights the role of Mediterranean dietary pattern in the fight against obesity.

Listeriosis Outbreak in South Africa: A Comparative Analysis with Previously Reported Cases Worldwide.

Listeria species are Gram-positive, rod-shaped, facultative anaerobic bacteria, which do not produce endospores. The genus, Listeria, currently comprises 17 characterised species of which only two (L. monocytogenes and L. ivanovii) are known to be pathogenic to humans. Food products and related processing environments are commonly contaminated with pathogenic species. Outbreaks and sporadic cases of human infections resulted in considerable economic loss. South Africa witnessed the world's largest listeriosis outbreak, characterised by a progressive increase in cases of the disease from January 2017 to July 2018. Of the 1060 laboratory-confirmed cases of listeriosis reported by the National Institute of Communicable Diseases (NICD), 216 deaths were recorded. Epidemiological investigations indicated that ready-to-eat processed meat products from a food production facility contaminated with L. monocytogenes was responsible for the outbreak. Multilocus sequence typing (MLST) revealed that a large proportion (91%) of the isolates from patients were sequence type 6 (ST6). Recent studies revealed a recurrent occurrence of small outbreaks of listeriosis with more severe side-effects in humans. This review provides a comparative analysis of a recently reported and most severe outbreak of listeriosis in South Africa, with those previously encountered in other countries worldwide. The review focuses on the transmission of the pathogen, clinical symptoms of the disease and its pathogenicity. The review also focuses on the major outbreaks of listeriosis reported in different parts of the world, sources of contamination, morbidity, and mortality rates as well as cost implications. Based on data generated during the outbreak of the disease in South Africa, listeriosis was added to the South African list of mandatory notifiable medical conditions. Surveillance systems were strengthened in the South African food chain in order to assist in preventing and facilitating early detection of both sporadic cases and outbreaks of infections caused by these pathogens in humans.

Evaluation of PCR-based methods for the identification of enteroaggregative hemorrhagic Escherichia coli in sprouts.

In this study real-time PCR assays were evaluated for the detection of enteroaggregative hemorrhagic Escherichia coli (EAHEC) O104:H4 in artificially contaminated mung bean and/alfalfa sprouts inoculated with 1, 10, and 100 CFU of EAHEC O104:H4 per 25 g sample (20, 10, and 2 replicates respectively). After selective culture enrichment the samples were tested using commercial real-time PCR kits detecting aggR/aaiC, stx/eae, and wzxO104. Using the commercial real-time PCR kits, the artificially contaminated samples were detected in the range of 75-80% positive results when contaminated with approximately 1 CFU, and 100% at 10 and 100 CFU. Microbiological detection employing O104-specific immunomagnetic capture and plating onto chromogenic media (modified Rainbow Agar and CHROMagar STEC) and confirmation by latex agglutination and PCR gave similar results (Cohen's kappa value between 0.61 and 1). In addition, the real-time PCR assay targeting the aggR and aaiC genes, indicative of enteroaggregative Escherichia coli (EAggEC), was tested against a panel of 60 bacterial strains and demonstrated 100% exclusivity (54 strains) and 100% inclusivity (6 strains). This study demonstrates the efficacy of the real-time PCR assays for the specific and sensitive detection of EAHEC from spouts.