Gonçalo Almeida

Incidence of Listeria monocytogenes in different food products commercialized in Portugal

Several types of food products on sale in Portugal, were examined for the presence of Listeria monocytogenes. Secondary enrichments, in Fraser broth, were analysed by the mini-Vidas LMO, enzyme-linked fluorescent immunoassay technique. Positive samples were confirmed by isolation on Oxford and PALCAM selective agars followed by biochemical characterization. Of 1035 samples, 72 (7.0%) were positive for L. monocytogenes, the majority being from raw products (milk, meat, fish, flour) although some heat-processed or fermented foods (ready-to-eat) were also positive. In Portugal, a predilection for fresh cheese was indicated as a potential risk for consumers.

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.

Listeriosis in Portugal: an existing but under reported infection

BackgroundListeriosis is a rare disease caused by the bacterium Listeria monocytogenes, the normal vehicle of which is food. The disease, which is largely confined to its risk groups of pregnant women, the elderly and immunocompromised individuals, has increased in incidence in recent years.In Portugal, listeriosis is not a notifiable infection and available data are scarce.The objective of this work was to collate the available information concerning listeriosis in Portugal by compiling a retrospective study of cases recorded over a decade.MethodsRequests for case data on clinically confirmed listeriosis, recorded over the previous decade, were replied to by 23 hospitals and a National Institute of Health delegation.Results35 cases of listeriosis were identified for the period between 1994 and 2003 inclusive, the mortality rate being greater than 17%. According to the data collected in this study for the year 2003, the incidence of this disease in Portugal was at least 1.4 cases per million inhabitants in that year.ConclusionThe study demonstrates, for the first time in the widely available literature, that despite their being no cases of listeriosis in Portugal recorded in official reports, the threat of L. monocytogenes to public health is of a similar dimension to that in other countries.

Microbiological characterization of randomly selected Portuguese raw milk cheeses with reference to food safety

Seventy raw milk cheeses made in different regions of Portugal, both hard and soft varieties, made with cows, ewes, or goats milk or combinations of these, were sampled within their quoted shelf lives for microbiological safety. On the basis of the presence or numbers of Escherichia coli, E. coli O157, Staphylococcus aureus, Salmonella, and Listeria monocytogenes, cheeses were categorized as satisfactory, acceptable, unsatisfactory, or unacceptable and potentially hazardous. Twenty-two of the 70 cheeses were classified as satisfactory or acceptable. Thirty-seven of the cheeses were considered unsatisfactory because of the presence of E. coli, S. aureus, or both, while 11 of the cheeses were graded as unacceptable and potentially hazardous because of the presence of excessive numbers of S. aureus, E. coli, or L. monocytogenes and the presence of Salmonella in three of these. All cheeses graded as unacceptable and potentially hazardous were soft or semisoft cheeses made with ewes and goats milk, with the exception of two hard cheeses made with cows milk. E. coli O157 was not detected in any of the cheeses. According to the present results, it seems that the presence or counts of pathogenic or indicator organisms in raw milk cheeses cannot be related to the processing conditions, milk type, or region of production.

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.

Comparison of Oxford Agar, PALCAM and Listeria monocytogenes Blood Agar for the recovery of L. monocytogenes from foods and environmental samples

Abstract This work had as the main objective a comparison between Listeria monocytogenes Blood Agar (LMBA) and the conventional selective agar media, Oxford and PALCAM, relative to its efficacy in the detection of L. monocytogenes in naturally contaminated food and environmental samples. 173 environmental samples and 272 samples of foods were analysed. A higher sensitivity for detection of L. monocytogenes was verified for LMBA than for PALCAM and Oxford. In LMBA L. monocytogenes could be distinguished from other Listeria spp. by detection of hemolysis. In Oxford and PALCAM this distinction was not possible. The higher growth rate of L. innocua cf. L. monocytogenes in selective liquid media could result in a high number of false negatives (non-detection of the target organism on plates, although its presence was observed by other tests, eg. mini-VIDAS LMO). The need for specific media for the detection of L. monocytogenes in food was confirmed. LMBA could be an alternative medium to use together with PALCAM or Oxford.

Survival of Clinical and Food Isolates of Listeria monocytogenes Through Simulated Gastrointestinal Tract Conditions

Twenty-seven strains of Listeria monocytogenes previously isolated from food (n = 16) and human patients of listeriosis (n = 11) were characterized and compared based on their ability to survive through the simulated gastrointestinal tract conditions. Cells were exposed (60 or 120 min) to low pH in the presence of pepsin, to simulate the digestion in the stomach, and subsequently to bile salts to simulate the digestion in the small intestine (60 or 120 min). Their survival was shown to be origin- (food and clinical) and strain dependent (p < 0.001) and also significantly dependent on the imposed simulated gastric conditions (long vs. quick exposure) (p < 0.001). In comparison to the food isolates, the clinical strains were in general more resistant and survived better to the two challenges imposed. Some of the tested strains, after the exposure to low pH in the presence of pepsin, became injured and subsequently more susceptible to the bile salts challenge. It was demonstrated that one of the most important natural barriers against foodborne pathogens might not be effective since it was shown that L. monocytogenes isolates that survived through the pH challenge were also able to survive the subsequent challenge to bile salts.

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.

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.