Microbiological Quality of Ready-to-Eat Salad Products Collected from Retail and Catering Settings in England during 2020 to 2021.

ABSTRACT: Salad and other fresh produce were collected in England from retail and catering businesses during 2020 to 2021 and were tested for Salmonella, Shiga toxin-producing Escherichia coli (STEC), Listeria, Bacillus cereus, and E. coli. Of the 604 samples collected, 57% were from retail settings and 43% were from catering settings; 61% were either salad leaves or salad leaves mixed with other products. Equal numbers of samples were prepacked or loose, and 50% were refrigerated at the time of sampling. Combining results for all microbiological parameters, 84% were interpreted as satisfactory, 12% were interpreted as borderline, and 4% were interpreted as unsatisfactory. One sample (prepacked leaves, cucumber, and tomato from a caterer) was categorized as unacceptable and potentially injurious because of detection of STEC O76; no STEC from human infections in the United Kingdom matched this isolate. No Salmonella enterica was detected, but Listeria monocytogenes was recovered from 11 samples: 1 at 20 CFU/g and the remainder at <20 CFU/g. B. cereus was detected at borderline levels (103 to ≤105 CFU/g) in 9% of samples and at an unsatisfactory level (>105 CFU/g) in one sample. E. coli was detected in 3% of samples at borderline levels (20 to ≤102 CFU/g) and in 4% at unsatisfactory levels (>102 CFU/g). There was a significant association between detection of L. monocytogenes and borderline or unsatisfactory levels of E. coli. There were no specific risk profiles associated with products with the higher levels of B. cereus, STEC, or Listeria, but elevated levels of E. coli were predominantly confined to loose products from the United Kingdom collected from caterers in summer or autumn 2021 and may have resulted from relaxation of COVID-19 restrictions. Among the L. monocytogenes isolates, only one matched those from human cases and was recovered from a prepacked mixed salad from a catering business in 2021. This isolate was the same strain as that responsible for a multicountry outbreak (2015 to 2018) associated with Hungarian-produced frozen sweet corn; no link to the outbreak food chain was established.

Maternal sepsis caused by Listeria monocytogenes with a fatal fetal outcome.

Improving maternal and child health is a global priority. Although infection with Listeria monocytogenes (LM), a small facultative anaerobic, gram-positive motile bacillus is rare, when it infects the maternal-fetoplacental unit, it can result in adverse fetal sequelae such as chorioamnionitis, preterm labour, neonatal sepsis, meningitis and neonatal death. Pregnancy-associated listeriosis may present with a plethora of diverse, non-specific symptoms such as fever, influenza-like or gastrointestinal symptoms, premature contractions and preterm labour. It has a predilection for the second and third trimester of pregnancy, occurring sporadically or as part of an outbreak, most of which have involved unpasteurised dairy products, long shelf life products, contaminated ready-to-eat food, deli meats and soft cheeses. Strains belonging to the clonal complexes 1, 4 and 6 are hypervigilant and are commonly associated with maternal-neonatal infections. Maternal listeriosis occurs as a direct consequence of LM-specific placental tropism, which is mediated by the conjugated action of internalin A and internalin B at the placental barrier. The diagnosis is established from placental culture. Penicillin, ampicillin and amoxicillin are the antimicrobials of choice. It has a high fetal morbidity of up to 30%. The authors present the case of a multiparous woman in her early 20s presenting with sepsis and preterm premature rupture of her membranes at 21 weeks gestation. A live baby was delivered spontaneously and died shortly after birth. Placental cultures and postmortem examination were consistent with the diagnosis of disseminated Listeria infection. Due to the increased susceptibility of pregnant women for LM, a high index of clinical suspicion is required to establish the diagnosis and initiate appropriate antimicrobial therapy to reduce adverse fetal outcomes.

Evaluation of the Thermo ScientificTM SureTectTMListeria monocytogenes PCR Assay in a Broad Range of Foods and Selected Environmental Surfaces: Pre-Collaborative and Collaborative Study, First Action 2021.05.

BACKGROUND: The Thermo Scientific™ SureTect™ Listeria monocytogenes PCR Assay uses Solaris reagents for performing PCR for the rapid and specific detection of Listeria monocytogenes in a broad range of foods and selected environmental surfaces. OBJECTIVE: To demonstrate reproducibility of the SureTect Listeria monocytogenes PCR Assay in a collaborative study using a challenging matrix, full-fat cottage cheese (25 g). To extend the scope of the method. METHOD: In the collaborative study, the candidate method was compared to the United States Food and Drug Administration/Bacteriological Analytical Manual (FDA/BAM) Chapter 10 Listeria reference method. The candidate method used two PCR thermocyclers, the Applied Biosystems™ QuantStudio™ 5 Real-Time PCR instrument (QS5) and the Applied Biosystems 7500 Fast Real-Time PCR instrument (7500 Fast). Eighteen participants from 10 laboratories located within the United States and Europe were solicited for the collaborative study, with 12 participants submitting valid data. Three levels of contamination were evaluated for each matrix. Statistical analysis was conducted according to the probability of detection (POD) statistical model. In addition, to extend the scope, six matrix studies were performed comparing the candidate method to the FDA/BAM reference method. One of these matrixes was also compared to the ISO 11290-1:2017 Microbiology of the Food Chain-Horizontal Method for the Detection and Enumeration of Listeria monocytogenes and of Listeria spp.-Part 1: Detection Method Reference Method. RESULTS: In the collaborative study, the difference in laboratory results indicates equivalence between the candidate method and reference method for the matrix evaluated, and the method demonstrated acceptable inter-laboratory reproducibility as determined in the collaborative evaluation. The two PCR instruments used in the study performed equivalently. All presumptive positives were confirmed via the alternative confirmation procedure. In the pre-collaborative studies, the results showed comparable performances between the candidate method and the reference method for all matrixes tested. CONCLUSIONS: Based on the data generated, the method demonstrated acceptable inter-laboratory reproducibility data and statistical analysis. HIGHLIGHTS: Due to the COVID-19 pandemic, some participants had to be trained remotely. Additionally, 25 g full-fat cottage cheese is known to be a challenging matrix to test. No unusual cross-contamination or false positive/negative data were reported, highlighting the ease of use, reproducibility, and robustness of the method.

Microbial Contamination and Survival Rate on Different Types of Banknotes.

In the COVID-19 pandemic context, numerous concerns have been raised regarding the hygienic status of certain objects we interact with on a daily basis, and especially cash money and their potential to harbor and transmit pathogenic bacteria. Therefore, in the present study, we analyzed different currency bills represented by British pounds (5 £, 10 £ and 20 £), Romanian lei (1 leu, 5 lei and 10 lei), U.S. dollars (1 $, 5 $ and 10 $) and Euros (5 €, 10 € and 20 €) in order to evaluate the bacterial survival rate and bacterial adherence. We used five reference microorganisms by American Type Culture Collection (ATCC, Manassas, VA, USA): Staphylococcus aureus ATCC 6538, Escherichia coli ATCC 8739, Enterococcus sp. ATCC 19952, Salmonella enterica subsp. enterica serovar Typhi ATCC 6539, and Listeria monocytogenes ATCC 7644. Microorganisms were selected in accordance with the criteria of prevalence, pathogenicity, opportunism, and incidence. However, Maldi-TOF analysis from samples taken from the banknotes revealed only a few of the common pathogens that are traditionally thought to be found on banknotes. Some of the most important factors for the survival of pathogenic agents on surfaces are the presence of organic matter, temperature and humidity. Our data showed that Salmonella enterica survived 72 h on every banknote tested, while L. monocytogenes tended to improve persistence in humid conditions. Survival rate is also influenced by the substrate composition, being lower for polymer-based banknotes especially for Salmonella enterica, Listeria monocytogenes and Enterococcus sp. The adherence of bacterial strains was lower for polymer-based banknotes British pounds and Romanian Leu, in contrast to the cotton-based U.S dollars and Euro banknotes. The risk of bacterial contamination from the banknote bills is high as indicated by both a strong survival capacity and low adherence of tested bacteria with differences between the two types of materials used for the tested banknotes.

Phage Display-Derived Monoclonal Antibodies Against Internalins A and B Allow Specific Detection of Listeria monocytogenes.

Listeria monocytogenes is the causative agent of listeriosis, a highly lethal disease initiated after the ingestion of Listeria-contaminated food. This species comprises different serovars, from which 4b, 1/2a, and 1/2b cause most of the infections. Among the different proteins involved in pathogenesis, the internalins A (InlA) and B (InlB) are the best characterized, since they play a major role in the enterocyte entry of Listeria cells during early infection. Due to their covalent attachment to the cell wall and location on the bacterial surface, along with their exclusive presence in the pathogenic L. monocytogenes, these proteins are also used as detection targets for this species. Even though huge advancements were achieved in the enrichment steps for subsequent Listeria detection, few studies have focused on the improvement of the antibodies for immunodetection. In the present study, recombinant InlA and InlB produced in Escherichia coli were used as targets to generate antibodies via phage display using the human naïve antibody libraries HAL9 and HAL10. A set of five recombinant antibodies (four against InlA, and one against InlB) were produced in scFv-Fc format and tested in indirect ELISA against a panel of 19 Listeria strains (17 species; including the three main serovars of L. monocytogenes) and 16 non-Listeria species. All five antibodies were able to recognize L. monocytogenes with 100% sensitivity (CI 29.24-100.0) and specificity (CI 88.78-100.0) in all three analyzed antibody concentrations. These findings show that phage display-derived antibodies can improve the biological tools to develop better immunodiagnostics for L. monocytogenes.

Listeria toxin promotes phosphorylation of the inflammasome adaptor ASC through Lyn and Syk to exacerbate pathogen expansion.

Inflammasome activation exacerbates infectious disease caused by pathogens such as Listeria monocytogenes, Staphylococcus aureus, and severe acute respiratory syndrome coronavirus 2. Although these pathogens activate host inflammasomes to regulate pathogen expansion, the mechanisms by which pathogen toxins contribute to inflammasome activation remain poorly understood. Here we show that activation of inflammasomes by Listeria infection is promoted by amino acid residue T223 of listeriolysin O (LLO) independently of its pore-forming activity. LLO T223 is critical for phosphorylation of the inflammasome adaptor ASC at amino acid residue Y144 through Lyn-Syk signaling, which is essential for ASC oligomerization. Notably, a Listeria mutant expressing LLO T223A is impaired in inducing ASC phosphorylation and inflammasome activation. Furthermore, the virulence of LLO T223A mutant is markedly attenuated in vivo due to impaired ability to activate the inflammasome. Our results reveal a function of a pathogen toxin that exacerbates infection by promoting phosphorylation of ASC.

Hyper-Virulent Listeria monocytogenes Strains Associated With Respiratory Infections in Central Italy.

Listeria monocytogenes (Lm) is a foodborne pathogen causing listeriosis. Invasive forms of the disease mainly manifest as septicaemia, meningitis and maternal-neonatal infections. Lm-associated respiratory infections are very rare and little known. We reported two Lm respiratory infection cases occurred in Central Italy during the summer of 2020, in the midst of the SARS-CoV2 pandemic. In addition to collect the epidemiological and clinical characteristics of the patients, we used Whole Genome Sequencing to study the genomes of the Lm isolates investigating their virulence and antimicrobial profiles and the presence of genetic mobile elements. Both the strains belonged to hypervirulent MLST clonal complexes (CC). In addition to the Listeria Pathogenicity Island 1 (LIPI-1), the CC1 strain also carried LIPI-3 and the CC4 both LIPI-3 and LIPI-4. Genetic determinants for antimicrobial and disinfectants resistance were found. The CC1 genome presented prophage sequences but they did not interrupt the comK gene, involved in the phagosomal escape of Lm. None of the strains carried plasmids. Lm is an important, although rare, opportunistic pathogen for respiratory tract and lung infections. To avoid dangerous diagnostic delays of these severe clinical forms, it is important to sensitize hospital laboratories to this rare manifestation of listeriosis considering Lm in the differential diagnosis of respiratory infections.

Prevalence of Listeria monocytogenes and Salmonella spp. in Different Ready to Eat Foods from Large Retailers and Canteens over a 2-Year Period in Northern Italy.

This study aims to give an overview of the prevalence of Listeria monocytogenes and Salmonella spp. in 9727 samples (2996 for L. monocytogenes and 6731 for Salmonella spp.) from different categories of ready-to-eat (RTE) foods, collected over 2 years from 28 large retailers and 148 canteens in the regions of northern Italy. The RTE samples were classified into two groups according to the preparation methods: (i) multi-ingredient preparations consisting of fully cooked food ready for immediate consumption, or with minimal further handling before consumption (Group A), and (ii) multi-ingredient preparations consisting of cooked and uncooked food, or preparations consisting of only raw ingredients (Group B). L. monocytogenes and Salmonella spp. were investigated in both of these categories. The overall prevalence of L. monocytogenes and Salmonella spp. was 0.13% and 0.07%, respectively. More specifically, L. monocytogenes was found in 0.04% of 2442 analysed RTE food samples belonging to group A and in 0.54% of 554 samples belonging to group B. Furthermore, 0.03% of 5367 RTE food samples from group A and 0.21% of 1364 samples from group B tested positive for Salmonella spp. In conclusion, the results obtained in this study can provide a significant contribution to L. monocytogenes and Salmonella spp. risk analysis in RTE foods.

An implementation science approach to evaluating pathogen whole genome sequencing in public health.

BACKGROUND: Pathogen whole genome sequencing (WGS) is being incorporated into public health surveillance and disease control systems worldwide and has the potential to make significant contributions to infectious disease surveillance, outbreak investigation and infection prevention and control. However, to date, there are limited data regarding (i) the optimal models for integration of genomic data into epidemiological investigations and (ii) how to quantify and evaluate public health impacts resulting from genomic epidemiological investigations. METHODS: We developed the Pathogen Genomics in Public HeAlth Surveillance Evaluation (PG-PHASE) Framework to guide examination of the use of WGS in public health surveillance and disease control. We illustrate the use of this framework with three pathogens as case studies: Listeria monocytogenes, Mycobacterium tuberculosis and SARS-CoV-2. RESULTS: The framework utilises an adaptable whole-of-system approach towards understanding how interconnected elements in the public health application of pathogen genomics contribute to public health processes and outcomes. The three phases of the PG-PHASE Framework are designed to support understanding of WGS laboratory processes, analysis, reporting and data sharing, and how genomic data are utilised in public health practice across all stages, from the decision to send an isolate or sample for sequencing to the use of sequence data in public health surveillance, investigation and decision-making. Importantly, the phases can be used separately or in conjunction, depending on the need of the evaluator. Subsequent to conducting evaluation underpinned by the framework, avenues may be developed for strategic investment or interventions to improve utilisation of whole genome sequencing. CONCLUSIONS: Comprehensive evaluation is critical to support health departments, public health laboratories and other stakeholders to successfully incorporate microbial genomics into public health practice. The PG-PHASE Framework aims to assist public health laboratories, health departments and authorities who are either considering transitioning to whole genome sequencing or intending to assess the integration of WGS in public health practice, including the capacity to detect and respond to outbreaks and associated costs, challenges and facilitators in the utilisation of microbial genomics and public health impacts.

Pulmonary insults exacerbate susceptibility to oral Listeria monocytogenes infection through the production of IL-10 by NK cells.

Most individuals who consume foods contaminated with the bacterial pathogen Listeria monocytogenes (Lm) develop mild symptoms, while others are susceptible to life-threatening systemic infections (listeriosis). Although it is known that the risk of severe disease is increased in certain human populations, including the elderly, it remains unclear why others who consume contaminated food develop listeriosis. Here, we used a murine model to discover that pulmonary coinfections can impair the host's ability to adequately control and eradicate systemic Lm that cross from the intestines to the bloodstream. We found that the resistance of mice to oral Lm infection was dramatically reduced by coinfection with Streptococcus pneumoniae (Spn), a bacterium that colonizes the respiratory tract and can also cause severe infections in the elderly. Exposure to Spn or microbial products, including a recombinant Lm protein (L1S) and lipopolysaccharide (LPS), rendered otherwise resistant hosts susceptible to severe systemic Lm infection. In addition, we show that this increase in susceptibility was dependent on an increase in the production of interleukin-10 (IL-10) from Ncr1+ cells, including natural killer (NK) cells. Lastly, the ability of Ncr1+ cell derived IL-10 to increase disease susceptibility correlated with a dampening of both myeloid cell accumulation and myeloid cell phagocytic capacity in infected tissues. These data suggest that efforts to minimize inflammation in response to an insult at the respiratory mucosa render the host more susceptible to infections by Lm and possibly other pathogens that access the oral mucosa.

COVID-19 Impairs Immune Response to Candida albicans.

Infection with SARS-CoV-2 can lead to Coronavirus disease-2019 (COVID-19) and result in severe acute respiratory distress syndrome (ARDS). Recent reports indicate an increased rate of fungal coinfections during COVID-19. With incomplete understanding of the pathogenesis and without any causative therapy available, secondary infections may be detrimental to the prognosis. We monitored 11 COVID-19 patients with ARDS for their immune phenotype, plasma cytokines, and clinical parameters on the day of ICU admission and on day 4 and day 7 of their ICU stay. Whole blood stimulation assays with lipopolysaccharide (LPS), heat-killed Listeria monocytogenes (HKLM), Aspergillus fumigatus, and Candida albicans were used to mimic secondary infections, and changes in immune phenotype and cytokine release were assessed. COVID-19 patients displayed an immune phenotype characterized by increased HLA-DR+CD38+ and PD-1+ CD4+ and CD8+ T cells, and elevated CD8+CD244+ lymphocytes, compared to healthy controls. Monocyte activation markers and cytokines IL-6, IL-8, TNF, IL-10, and sIL2Rα were elevated, corresponding to monocyte activation syndrome, while IL-1ß levels were low. LPS, HKLM and Aspergillus fumigatus antigen stimulation provoked an immune response that did not differ between COVID-19 patients and healthy controls, while COVID-19 patients showed an attenuated monocyte CD80 upregulation and abrogated release of IL-6, TNF, IL-1α, and IL-1ß toward Candida albicans. This study adds further detail to the characterization of the immune response in critically ill COVID-19 patients and hints at an increased susceptibility for Candida albicans infection.

CD71+ Erythroid Cells in Human Neonates Exhibit Immunosuppressive Properties and Compromise Immune Response Against Systemic Infection in Neonatal Mice.

Newborns are highly susceptible to infectious diseases. The underlying mechanism of neonatal infection susceptibility has generally been related to their under-developed immune system. Nevertheless, this notion has recently been challenged by the discovery of the physiological abundance of immunosuppressive erythroid precursors CD71+erythroid cells (CECs) in newborn mice and human cord blood. Here, as proof of concept, we show that these cells are also abundant in the peripheral blood of human newborns. Although their frequency appears to be more variable compared to their counterparts in mice, they rapidly decline by 4 weeks of age. However, their proportion remains significantly higher in infants up to six months of age compared to older infants. We found CD45 expressing CECs, as erythroid progenitors, were the prominent source of reactive oxygen species (ROS) production in both humans and mice. Interestingly, a higher proportion of CD45+CECs was observed in the spleen versus bone marrow of neonatal mice, which was associated with a higher ROS production by splenic CECs compared to their siblings in the bone marrow. CECs from human newborns suppressed cytokine production by CD14 monocytes and T cells, which was partially abrogated by apocynin in vitro. Moreover, the depletion of CECs in neonatal mice increased the number of activated effector immune cells in their spleen and liver, which rendered them more resistant to Listeria monocytogenes infection. This was evident by a significant reduction in the bacteria load in the spleen, liver and brain of treated-mice compared to the control group, which enhanced their survival rate. Our finding highlights the immunoregulatory processes mediated by CECs in newborns. Thus, such tightly regulated immune system in newborns/infants may explain one potential mechanism for the asymptomatic or mild COVID-19 infection in this population.