Rui Magalhães

Foci of contamination of Listeria monocytogenes in different cheese processing plants.

Listeria monocytogenes is a ubiquitous bacterium widely distributed in the environment that can cause a severe disease in humans when contaminated foods are ingested. Cheese has been implicated in sporadic cases and in outbreaks of listeriosis worldwide. Environmental contamination, in several occasions by persistent strains, has been considered an important source of finished product contamination. The objectives of this research were to (i) evaluate the presence of L. monocytogenes within the factory environments and cheeses of three processing plants, artisanal producer of raw ewes milk cheeses (APC), small-scale industrial cheese producer (SSI) and industrial cheese producer (ICP) each producing a distinct style of cheese, all with history of contamination by L. monocytogenes (ii) and identify possible sources of contamination using different typing methods (arsenic and cadmium susceptibility, geno-serotyping, PFGE). The presence of markers specific for 3 epidemic clones (ECI-ECIII) of L. monocytogenes was also investigated. Samples were collected from raw milk (n = 179), whey (n = 3), cheese brining solution (n = 7), cheese brine sludge (n = 505), finished product (n = 3016), and environment (n = 2560) during, at least, a four-year period. Listeria monocytogenes was detected in environmental, raw milk and cheese samples, respectively, at 15.4%, 1.1% and 13.6% in APC; at 8.9%, 2.9% and 3.4% in SSI; and at 0%, 21.1% and 0.2% in ICP. Typing of isolates revealed that raw ewes milk and the dairy plant environment are important sources of contamination, and that some strains persisted for at least four years in the environment. Although cheeses produced in the three plants investigated were never associated with any case or outbreak of listeriosis, some L. monocytogenes belonging to specific PFGE types that caused disease (including putative epidemic clone strains isolated from final products) were found in this study.

Cheese-related listeriosis outbreak, Portugal, March 2009 to February 2012

In Portugal, listeriosis has been notifiable since April 2014, but there is no active surveillance programme for the disease. A retrospective study involving 25 national hospitals led to the detection of an outbreak that occurred between March 2009 and February 2012. The amount of time between the start of the outbreak and its detection was 16 months. Of the 30 cases of listeriosis reported, 27 were in the Lisbon and Vale do Tejo region. Two cases were maternal/neonatal infections and one resulted in fetal loss. The mean age of the non-maternal/neonatal cases was 59 years (standard deviation: 17); 13 cases were more than 65 years old. The case fatality rate was 36.7%. All cases were caused by molecular serogroup IVb isolates indistinguishable by pulsed-field gel electrophoresis and ribotype profiles. Collaborative investigations with the national health and food safety authorities identified cheese as the probable source of infection, traced to a processing plant. The magnitude of this outbreak, the first reported food-borne listeriosis outbreak in Portugal, highlights the importance of having an effective listeriosis surveillance system in place for early detection and resolution of outbreaks, as well as the need for a process for the prompt submission of Listeria monocytogenes isolates for routine laboratory typing.

Evaluation of Antibiotic Resistance Patterns of Food and Clinical Listeria monocytogenes Isolates in Portugal

The aim of this study was to characterize a broad collection of isolates of Listeria monocytogenes, of different serotypes, recovered in Portugal between 2003 and 2007 from foods (n=353) and from clinical cases of human listeriosis (n=95), in terms of antimicrobial susceptibility. All the isolates were susceptible to ampicillin, the preferred agent to treat listeriosis. Resistances to nitrofurantoin (n=99), to ciprofloxacin (n=18), to erythromycin (n=10), to tetracycline (n=2), to gentamicin (n=1) and to rifampicin (n=1) were observed. One hundred (28.3%) and 20 (21.0%) food and clinical isolates, respectively, were resistant to at least one antibiotic. Eight isolates (1.8%) were resistant to two or more antimicrobials of different classes, and all were collected from foods. Serogroup IVb included the highest percentage of isolates resistant to erythromycin. The highest percentages of isolates resistant to nitrofurantoin were of serogroup IVb and IIc. It was demonstrated that the incidence of antibiotic-resistant isolates of L. monocytogenes, during the period 2003 to 2007, was low in Portugal but still higher than that observed in other countries. Given the increasing population at greater risk of listeriosis, namely, the elderly, the high mortality rate of the infection and the detection of resistant isolates, monitoring for antibiotic resistance in strains of L. monocytogenes on a large scale, and assessing the risk of infection by these strains, is highly recommended.

Bacteria: Listeria monocytogenes

Listeria monocytogenes is a ubiquitous microorganism responsible for listeriosis, a rare but severe disease in humans, who can become infected by ingesting contaminated food products, namely dairy, meat, fish, and vegetables. Although it can occur in healthy humans, listeriosis mainly affects the elderly, immunocompromised, persons pregnant women, and newborns, with a high case-fatality rate (20–30%). This article will summarize what is currently known about L. monocytogenes , listeriosis, methods of detection and enumeration, subtyping, persistence, virulence traits, and regulatory control.

Survival of Listeria monocytogenes with different antibiotic resistance patterns to food-associated stresses

The ongoing rise of antibiotic resistant microbial pathogens has become one of the major public health threats worldwide. Despite all the effort and actions taken so far, a proliferation of antibiotic resistant (AR) and multi-antibiotic resistant (MAR) strains is still observed, including in foodborne pathogens. This trend has been also noted recently for isolates of Listeria monocytogenes, a species that, although remaining largely sensitive to clinically relevant antimicrobials, has been reported to develop increased tolerance to antibiotics, particularly in isolates recovered from the food-chain. In this study we compared the ability of MAR (n=8), AR (n=18) and antibiotic susceptible (AS, n=11) L. monocytogenes strains from food and clinical origin to survive to different environmental stress conditions, including temperature (58°C), acidic stress (1% v/v lactic acid, pH3.5), and osmotic stress (37% w/v NaCl). The presence of antibiotic active efflux among MAR and AR strains, and its role on L. monocytogenes tolerance to different antimicrobial compounds was also investigated, namely; hydrogen peroxide; organic acids (acetic, citric and lactic); nisin; benzalkonium chloride (BC); and, sodium nitrite. While no significant differences were observed in the survival of the 37 strains exposed to high temperature (58°C), overall the mean logarithmic reduction of clinical strains was statistically lower after acid and salt exposure than that observed for strains of food origin; but both food and clinical strains resistant to two or three antibiotics were significantly less susceptible to acid (lactic acid 1% v/v) and osmotic stresses (37% w/v NaCl) when compared to AS strains. Using the EtBr-agar Cartwheel method, it was possible to detect efflux pumps in three of the 26 MAR and AR isolates, including one control strain; the active efflux in theses isolates was proven to be associated with fluoroquinolone resistance, and possible extrusion of BC and hydrogen peroxide. The mechanisms responsible for the possible correlation between resistance to antibiotics and to acid or salt stress in L. monocytogenes have yet to be understood.

Genetic and phenotypic characterization of Listeria monocytogenes from human clinical cases that occurred in Portugal between 2008 and 2012.

Listeria monocytogenes infection (listeriosis) is an uncommon but severe foodborne illness that affects mainly individuals with recognized underlying conditions: the elderly, immunocompromised individuals, and pregnant women and their fetuses. The aim of this study was to obtain epidemiological data on cases of listeriosis occurring in Portugal from 2008 through 2012, collected in hospitals on a voluntary basis. L. monocytogenes isolates were characterized by genoserotyping by multiplex polymerase chain reaction, DNA macrorestriction pulsed-field gel electrophoresis (PFGE), and determination of minimal inhibitory concentration (MIC, μg/mL) for 12 antibiotics. During this period, 203 cases of listeriosis were detected. The annual incidence rate observed ranged from 0.2 to 0.7 cases per 100,000 inhabitants. Nineteen cases (9.5%) corresponded to maternal/neonatal (MN) infections. The mean age of the nonmaternal/neonatal (non-MN) cases with documented age was 59 years, and 46.4% occurred in patients aged over 65 years. The majority of listeriosis cases were caused by genoserogroup IVb isolates, and PFGE analysis revealed a high molecular diversity, suggesting that most were sporadic. Nevertheless, several clusters of isolates presenting different geographic and time distributions were detected. The incidence of antibiotic-resistant isolates of L. monocytogenes was low but significantly higher than in previous years (2003-2007). The implementation of a national surveillance system monitoring the incidence of listeriosis and antimicrobial resistance of strains would be most valuable, allowing identification of sporadic and outbreak cases, to detect general trends in antibiotic susceptibilities, and potentially identify food sources of clinical strains.

Biofilm Formation among Clinical and Food Isolates of Listeria monocytogenes

Objective. A total of 725 Listeria monocytogenes isolates, 607 from various foods and 118 from clinical cases of listeriosis, were investigated concerning their ability to form biofilms, at 4°C during 5 days and at 37°C during 24 h. Methods. Biofilm production was carried out on polystyrene tissue culture plates. Five L. monocytogenes isolates were tested for biofilm formation after being exposed to acidic and osmotic stress conditions. Results. Significant differences (P < 0.01) between clinical and food isolates were observed. At 37°C for 24 h, most food isolates were classified as weak or moderate biofilm formers whereas all the clinical isolates were biofilm producers, although the majority were weak. At 4°C during 5 days, 65 and 59% isolates, from food and clinical cases, respectively, were classified as weak. After both sublethal stresses, at 37°C just one of the five isolates tested was shown to be more sensitive to subsequent acidic exposure. However, at 4°C both stresses did not confer either sensitivity or resistance. Conclusions. Significant differences between isolates origin, temperature, and sublethal acidic stress were observed concerning the ability to form biofilms. Strain, origin, and environmental conditions can determine the level of biofilm production by L. monocytogenes isolates.

Biofilm formation by persistent and non-persistent Listeria monocytogenes strains on abiotic surfaces

Contaminated food with Listeria monocytogenes is the predominant route of transmission of listeriosis to humans, a severe illness with a high mortality rate. Food processing environments can be colonized by persistent strains, repeatedly isolated for months or years. This study aimed to investigate the biofilm formation capacity of six strains of L. monocytogenes that persisted for long periods in two cheese processing plants in comparison to seven strains isolated sporadically from the same plants. Persistent strains produced more biofilm than non-persistent strains on stainless steel and silicon rubber surfaces; no significant differences were observed on polyvinyl chloride (PVC). In a polystyrene microtiter plate assay with crystal violet staining, no evidence was found that persistent strains have higher ability to form biofilm than non-persistent strains, and no correlation was identified between biofilm formation in the microtiter plate and on the three other surfaces tested.

Traditional Methods for Isolation of Listeria monocytogenes

Conventional methods for the detection of Listeria monocytogenes in foods and environmental samples relies on selective pre-enrichment, enrichment, and plating. This is followed by confirmation of suspected colonies by testing a limited number of biochemical markers.

Biocontrol strategies for Mediterranean-style fermented sausages

Naturally fermented meat sausages have a long tradition in Mediterranean countries and are one of the most important groups of traditional foods consumed throughout Europe. Despite all the advances in food science and technology and increased regulatory requirements and concerns for safety and quality during the last decades, the challenge to control important foodborne pathogens in this type of meat products still persists. Simultaneously, growing consumer interest in safe, high quality and minimal processed products, with less additives/preservatives have driven the food industry and scientists in a crusade for innovative technologies to maintain the safety of these products by natural means. Biological control (biocontrol) fits well within this tendency. This review summarizes the latest achievements on biocontrol strategies applied to Mediterranean-style fermented sausages, namely: (i) bioprotective cultures; (ii) bacteriocins; and, (iii) essential oils (EOs).