A high level of antibiotic resistance in Klebsiella and Aeromonas isolates from street water sold in Mozambique, associated with the prevalence of extended-spectrum and AmpC ß-lactamases.

This study aims to evaluate the resistance profile and the prevalence of antibiotic resistance genes in 30 isolates of Klebsiella spp. and Aeromonas spp. recovered from water sold in the streets of Maputo. Susceptibility profiles to 15 antibiotics were performed according to Clinical Laboratory Standard Institute guidelines with antibiotic disks on Mueller-Hinton agar plates. Multiplex PCRs were performed targeting 10 ß-lactamase genes, five ESBL (blaTEM-variants, blaOXA-variants, BlaSHV-variants, MCTX-M Group 1 and Group 9 variants) and five AmpC (ACC variants, FOX variants, MOX variants, CIT variants and DHA variants). The results showed a high prevalence of Klebsiella resistance to ß-lactam antibiotics, such as amoxicillin/clavulanic acid (62.5%), amoxicillin (56.3%), ampicillin (50%), cefoxitin (43.8%), and cefotaxime (43.8%). Aeromonas showed resistance to cefoxitin and ampicillin (71.4%), amoxicillin/clavulanic acid (57.1%) and imipenem (42.9%). ESBL blaOXA-variants, blaSVH-variants, MCTX-M Group 1 variants, and MCTX-M Group 9 variants were the most prevalent b-lactam genes, followed by the b-lactams AmpC, ACC variants and FOX variants. It is extremely important to improve waterborne disease control strategies, especially in terms of public awareness of the potential health implications of multidrug-resistant strains of Klebsiella and Aeromonas, which are often neglected.

Pineapple (Ananas comosus L.) By-Products Valorization: Novel Bio Ingredients for Functional Foods.

Pineapple is consumed on a large scale around the world due to its appreciated sensorial characteristics. The industry of minimally processed pineapple produces enormous quantities of by-products (30-50%) which are generally undervalued. The end-of-life of pineapple by-products (PBP) can be replaced by reuse and renewal flows in an integrated process to promote economic growth by reducing consumption of natural resources and diminishing food waste. In our study, pineapple shell (PS) and pineapple core (PC), vacuum-packed separately, were subjected to moderate hydrostatic pressure (225 MPa, 8.5 min) (MHP) as abiotic stress to increase bromelain activity and antioxidant capacity. Pressurized and raw PBP were lyophilized to produce a stable powder. The dehydrated samples were characterized by the following methodologies: chemical and physical characterization, total phenolic compounds (TPC), antioxidant capacity, bromelain activity, microbiology, and mycotoxins. Results demonstrated that PBP are naturally rich in carbohydrates (66-88%), insoluble (16-28%) and soluble (2-4%) fiber, and minerals (4-5%). MHP was demonstrated to be beneficial in improving TPC (2-4%), antioxidant activity (2-6%), and bromelain activity (6-32%) without affecting the nutritional value. Furthermore, microbial and mycotoxical analysis demonstrated that powdered PC is a safe by-product. PS application is possible but requires previous decontamination to reduce the microbiological load.

Odontogenic cutaneous sinus tracts due to infection with nontuberculous mycobacteria: a report of three cases.

BACKGROUND: Soft tissue or skin infections due to nontuberculous mycobacteria (NTM) have been reported frequently and are mostly associated with trauma or cosmetic interventions like plastic surgery. However, infection with NTM as a result of a dental procedure have rarely been described and the lack of clinical suspicion and a clear clinical manifestation makes diagnosis challenging. CASE PRESENTATION: We report on three patients with a facial cutaneous sinus tract of dental origin, due to an infection with respectively Mycobacterium fortuitum, M. abscessus and M. peregrinum. The infection source was the dental unit waterlines (DUWLs), which were colonized with NTM. CONCLUSIONS: Water of the DUWL can pose a health risk. This report emphasizes the need for quality control and certification of water flowing through DUWLs, including the absence of NTM. Our report also shows the need for a rapid recognition of NTM infections and accurate laboratory diagnosis in order to avoid long-term ineffective antibiotic treatment.

Biofilm Formation and Disinfectant Susceptibility of Persistent and Nonpersistent Listeria monocytogenes Isolates from Gorgonzola Cheese Processing Plants.

The aim of this study was to investigate whether the biofilm-forming ability and/or the disinfectant susceptibility accounted for the persistence of Listeria monocytogenes in Gorgonzola cheese processing plants. For this purpose, a set of 16 L. monocytogenes isolates collected in the 2004-2007 period was analyzed, including 11 persistent isolates collected in different years, within the collection period, and displaying identical or highly correlated pulsotypes. The evaluation of biofilm-forming ability was assessed using crystal violet (CV) staining and the enumeration of viable cells on stainless steel coupons (SSC). Absorbance values obtained with CV staining for persistent and nonpersistent isolates were not significantly different (rm-ANOVA p > 0.05) and the cell counts from nonpersistent isolates showed to be higher compared with persistent isolates (rm-ANOVA p < 0.05). A simulation of disinfectant treatments was performed on spot inoculated coupons in clean and dirty conditions, according to EN 13697, and on biofilms on SSC, grown in nutrient-rich (dirty) and limiting (clean) conditions using acid acetic-hydrogen peroxide (P3) and acid citric-hydrogen peroxide (MS) commercial disinfectants. The treatment was considered effective when a 4 Log reduction in viable cell count was observed. The Log reductions of persistent and nonpersistent isolates, obtained with both the assays in clean and dirty conditions, were compared and no significant differences were detected (rm-ANOVA p > 0.05). A greater influence of organic matter on MS could explain why P3 was efficient in reducing to effective levels the majority of the isolates at the lowest concentration suggested by the manufacturer (0.2% [v/v]), while the same purpose required a higher concentration (1% [v/v]) of MS. In conclusion, our results demonstrate that the persistence of these isolates in Gorgonzola cheese processing plants was linked neither to the biofilm-forming ability nor to their susceptibility to hydrogen peroxide-based disinfectants; therefore, other factors should contribute to the persistent colonization of the dairies.

Differences in the Expression of Cold Stress-Related Genes and in the Swarming Motility Among Persistent and Sporadic Strains of Listeria monocytogenes.

The persistence of certain Listeria monocytogenes strains in food-related environments suggests niche adaptation of these strains and therefore constitutes a major risk to consumer health and results in economic losses for the food producer. In this study, a set of 23 L. monocytogenes isolates, including a group of persistent and a group of sporadic strains, was evaluated regarding their swarming motility at 11°C. In each group, significant (p<0.05) differences in motility were observed. The transcript levels of nine cold stress-related genes were analyzed by real-time quantitative PCR in two representatives of persistent (CBISA3077) and sporadic (CBISA3049) strains isolated from the dairy environment, and significant (p<0.05) differences between the two strains were observed. The persistent strain showed significantly higher transcript levels of dtpT and sigB genes, and significantly lower levels of flaA, oppA, lmo1722, and lmo0866 genes. In the persistent strain, the upregulation of sigB, involved in the tolerance to low temperature and to osmotic stress, could account for the persistence of this strain in its original dairy environment. In a similar way, the downregulation of two helicase-encoding genes lmo1722 and lmo0866, in this strain, may be an evolutionary trait that could facilitate cold stress adaptation. Even though this analysis should be extended to more sporadic and more persistent strains, the results presented here strongly suggest gene expression networks differently adjusted, in the two strains, to the low-temperature environment from where they were collected. Moreover, our findings suggest that bacterial motility per se should not be considered a key feature for the persistence of L. monocytogenes in the food environment.

Bisphenol A disrupts transcription and decreases viability in aging vascular endothelial cells.

Bisphenol A (BPA) is a widely utilized endocrine disruptor capable of mimicking endogenous hormones, employed in the manufacture of numerous consumer products, thereby interfering with physiological cellular functions. Recent research has shown that BPA alters epigenetic cellular mechanisms in mammals and may be correlated to enhanced cellular senescence. Here, the effects of BPA at 10 ng/mL and 1 µg/mL, concentrations found in human samples, were analyzed on HT29 human colon adenocarcinona cell line and Human Umbilical Vein Endothelial Cells (HUVEC). Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR) transcriptional analysis of the Long Interspersed Element-1 (LINE-1) retroelement showed that BPA induces global transcription deregulation in both cell lines, although with more pronounced effects in HUVEC cells. Whereas there was an increase in global transcription in HT29 exclusively after 24 h of exposure, this chemical had prolonged effects on HUVEC. Immunoblotting revealed that this was not accompanied by alterations in the overall content of H3K9me2 and H3K4me3 epigenetic marks. Importantly, cell viability assays and transcriptional analysis indicated that prolonged BPA exposure affects aging processes in senescent HUVEC. To our knowledge this is the first report that BPA interferes with senescence in primary vascular endothelial cells, therefore, suggesting its association to the etiology of age-related human pathologies, such as atherosclerosis.

Metagenomic analysis of the bacterial microbiome, resistome and virulome distinguishes Portuguese Serra da Estrela PDO cheeses from similar non-PDO cheeses: An exploratory approach.

This study aimed to evaluate the microbiome, resistome and virulome of two types of Portuguese cheese using high throughput sequencing (HTS). Culture-dependent chromogenic methods were also used for certain groups/microorganisms. Eight samples of raw ewe's milk cheese were obtained from four producers: two producers with cheeses with a PDO (Protected Designation of Origin) label and the other two producers with cheeses without a PDO label. Agar-based culture methods were used to quantify total mesophiles, Enterobacteriaceae, Escherichia coli, Staphylococcus, Enterococcus and lactic acid bacteria. The presence of Listeria monocytogenes and Salmonella was also investigated. The selected isolates were identified by 16S rRNA gene sequencing and evaluated to determine antibiotic resistance and the presence of virulence genes. The eight cheese samples analyzed broadly complied with EC regulations in terms of the microbiological safety criteria. The HTS results demonstrated that Leuconostoc mesenteroides, Lactococcus lactis, Lactobacillus plantarum, Lacticaseibacillus rhamnosus, Enterococcus durans and Lactobacillus coryniformis were the most prevalent bacterial species in cheeses. The composition of the bacterial community varied, not only between PDO and non-PDO cheeses, but also between producers, particularly between the two non-PDO cheeses. Alpha-diversity analyses showed that PDO cheeses had greater bacterial diversity than non-PDO cheeses, demonstrating that the diversity of spontaneously fermented foods is significantly higher in cheeses produced without the addition of food preservatives and dairy ferments. Despite complying with microbiological regulations, both PDO and non-PDO cheeses harbored potential virulence genes as well as antibiotic resistance genes. However, PDO cheeses exhibited fewer of these virulence and antibiotic resistance genes compared to non-PDO cheeses. Therefore, the combination of conventional microbiological methods and the metagenomic approach could contribute to improving the attribution of the PDO label to this type of cheese.

Comparison of Listeria monocytogenes Exoproteomes from biofilm and planktonic state: Lmo2504, a protein associated with biofilms.

The food-borne pathogen Listeria monocytogenes is the causative agent of the severe human and animal disease listeriosis. The persistence of this bacterium in food processing environments is mainly attributed to its ability to form biofilms. The search for proteins associated with biofilm formation is an issue of great interest, with most studies targeting the whole bacterial proteome. Nevertheless, exoproteins constitute an important class of molecules participating in various physiological processes, such as cell signaling, pathogenesis, and matrix remodeling. The aim of this work was to quantify differences in protein abundance between exoproteomes from a biofilm and from the planktonic state. For this, two field strains previously evaluated to be good biofilm producers (3119 and J311) were used, and a procedure for the recovery of biofilm exoproteins was optimized. Proteins were resolved by two-dimensional difference gel electrophoresis and identified by electrospray ionization-tandem mass spectrometry. One of the proteins identified in higher abundance in the biofilm exoproteomes of both strains was the putative cell wall binding protein Lmo2504. A mutant strain with deletion of the gene for Lmo2504 was produced (3119Δlmo2504), and its biofilm-forming ability was compared to that of the wild type using the crystal violet and the ruthenium red assays as well as scanning electron microscopy. The results confirmed the involvement of Lmo2504 in biofilm formation, as strain 3119Δlmo2504 showed a significantly (P < 0.05) lower biofilm-forming ability than the wild type. The identification of additional exoproteins associated with biofilm formation may lead to new strategies for controlling this pathogen in food processing facilities.

Comparative analysis of the exoproteomes of Listeria monocytogenes strains grown at low temperatures.

This article presents the major differences in the exoproteomes of Listeria monocytogenes strains grown at 11°C and 20°C, and their comparison to 37°C, the optimal temperature of growth of this foodborne pathogenic bacteria. A set of four strains previously characterized and representing the genetic diversity of the species was used. Two were virulent, of which one was persistent, and two were low virulent strains. The proteins secreted by the strains grown in minimal medium were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and identified by liquid chromatography tandem mass spectrometry. The heterogeneity among the four strains concerning the 15 major proteins detected was noticed. No clear association of exoproteome with virulence or genotype was found. Cluster analysis of the protein patterns of the strains suggests an increasing differentiation of strain response with low temperatures, highlighting the importance of the study of the exoproteomes. The main finding was the lack of some proteins in the exoproteome of the persistent strain, namely, flagellin (FlaA) and of OppA/oligopeptide ABC transporter, when compared to the other strains. In fact, these two proteins differ in abundance between strains grown at low temperature. Moreover, FlaA was the only glycoprotein identified in the exoproteomes. An attempt is made here to assess the relevance of the major exoproteins differentially detected. The investigation of the exoproteomes of other persistent and sporadic strains will allow identification of proteins involved in adaptation of particular L. monocytogenes strains to low temperatures in use throughout the food chain.

The Socioeconomic Factors of Street Food Vending in Developing Countries and Its Implications for Public Health: A Systematic Review.

The sale of ready-to-eat (RTE) street food represents an important source of income in many developing countries. However, these foods are frequently implicated in outbreaks of gastrointestinal diseases. Street food vendors face several constraints that hamper improvement in the microbiological quality of their products. The aim of this review was to update knowledge about the main causes of foodborne illnesses in developing countries, including the growing concern with the microbial transmission of antibiotic resistance. Following PRISMA guidelines, this systematic review was conducted on original articles published from January 2010 to July 2023. The search was carried out using Scopus, PubMed, Web of Science, Food Science and Technology Abstracts (FSTA), the International Information System for Agricultural Sciences and Technology (AGRIS), as well as isolated searches of relevant articles from Google Scholar. The initial search identified 915 articles, 50 of which were included in this systematic review. The results indicate that, in the majority of the 15 countries examined, women constitute the predominant segment of street food vendors, representing more than 55% of the total number of these vendors. In 11 countries, street food vendors under the age of 18 were identified. Most vendors had a low level of education and, consequently, were unaware of good hygiene practices when handling food. The combination of factors such as poor hygiene practices on the part of food handlers and the lack of facilities, namely, the absence of available potable water, were frequently listed as the main causes of food contamination. Enterobacteriaceae such as Escherichia coli (61.9%), Salmonella (30.1%), and Shigella spp. (9.5%), as well as Staphylococcus aureus (30.1%) and Listeria monocytogenes (14.3%), were the most common pathogens found in RTE street foods. In 22 studies from 13 developing countries, 59% (13/22) reported high multidrug resistance in Enterobacteriaceae (40% to 86.4% in E. coli, 16.7 to 70% in Salmonella, and 31 to 76.4% in S. aureus). To address the challenges faced by street vendors and improve their economic activities, it is necessary for government entities, consumers, and vendors to work together collaboratively.

Multidrug Resistance in Enterococci Isolated from Cheese and Capable of Producing Benzalkonium Chloride-Resistant Biofilms.

This study aimed to investigate enterococci recovered from eight Portuguese cheeses made with raw ewe's milk, regarding antibiotic resistance, virulence genes, minimum inhibitory concentration (MIC) of benzalkonium chloride (BAC), biofilm formation capacity, and biofilm eradication (MBEC) by BAC. Antimicrobial resistance against seven antibiotics of five groups was evaluated using the disk diffusion method. The presence of the genes that encode resistance to the antibiotics penicillin (blaZ), erythromycin (ermA, ermB, and ermC), vancomycin (vanA and vanB), aminoglycoside (aac(6')-Ie-aph(2″)-Ia), and ß-lactam (pbp5) and the genes that encode virulence factors, frsB, cylA, gelE, esp, and agg, were investigated via multiplex PCR. The susceptibility of planktonic cells to BAC was evaluated by the MIC and MBC values of the isolates, using the broth microdilution method. To assess the biofilm-forming ability and resistance of biofilms to BAC, biofilms were produced on stainless steel coupons, followed by exposure to BAC. The results showed a high resistance to the antibiotics vancomycin (87.5%), erythromycin (75%), tetracycline (50%), and penicillin (37.5%). Multidrug resistance was observed in 68.8% of the isolates. Genes encoding the virulence factors FrsB (frsB) and gelatinase E (gelE) were detected in all isolates. The esp and cylA genes were found in 56.3% and 37.5% of the isolates, respectively. All isolates exhibited a biofilm-forming ability, regardless of incubation time and temperature tested. However, after 72 h at 37 °C, E. faecium and E. faecalis biofilms showed significant differences (p ≤ 0.05). Although most isolates (62.5%) were susceptible to BAC (MIC ≤ 10 mg/L), biofilms of the same isolates were, generally, resistant to the higher concentration of BAC (80 mg/mL) tested. This study using Enterococcus isolates from a ready-to-eat food, such as cheese, reveals the high percentages of vancomycin resistance and multidrug resistance, associated with the presence of virulence genes, in isolates also capable of producing biofilms resistant to BAC, an important active ingredient of many disinfectants. These results emphasize the need for effective control measures to ensure the safety and quality of dairy products.

A rare cause of acute abdomen after a Good Friday.

Small bowel perforation caused by an ingested fish bone is rare but can involve the appendix or Meckel's diverticulum. We report the case of a 25-year-old man who presented to the emergency department with acute abdomen caused by perforation of a Meckel's diverticulum with a fish bone ingested in a Good Friday.

Storage Stability and In Vitro Bioaccessibility of Microencapsulated Tomato (Solanum Lycopersicum L.) Pomace Extract.

Tomato pomace is rich in carotenoids (mainly lycopene), which are related to important bioactive properties. In general, carotenoids are known to react easily under environmental conditions, which may create a barrier in producing stable functional components for food. This work intended to evaluate the storage stability and in vitro release of lycopene from encapsulated tomato pomace extract, and its bioaccessibility when encapsulates were incorporated in yogurt. Microencapsulation assays were carried out with tomato pomace extract as the core material and arabic gum or inulin (10 and 20 wt%) as wall materials by spray drying (160 and 200 °C). The storage stability results indicate that lycopene degradation was highly influenced by the presence of oxygen and light, even when encapsulated. In vitro release studies revealed that 63% of encapsulated lycopene was released from the arabic gum particles in simulated gastric fluid, whereas for the inulin particles, the release was only around 13%. The feed composition with 20% inulin showed the best protective ability and the one that enabled releasing the bioactives preferentially in the intestine. The bioaccessibility of the microencapsulated lycopene added to yogurt increased during simulated gastrointestinal digestion as compared to the microencapsulated lycopene alone. We anticipate a high potential for the inulin microparticles containing lycopene to be used in functional food formulations.

High Fecal Contamination and High Levels of Antibiotic-Resistant Enterobacteriaceae in Water Consumed in the City of Maputo, Mozambique.

In the city of Maputo, Mozambique, food and water are often sold on the streets. Street water is packaged, distributed, and sold not paying attention to good hygienic practices, and its consumption is often associated with the occurrence of diarrheal diseases. Coincidentally, the increase of diarrheal diseases promotes the inappropriate use of antibiotics that might cause the emergence of antibiotic-resistant bacterial strains. In this context, the present study aimed to assess the microbiological quality of water sold on the streets of Maputo, as well as the antibiotic resistance profile of selected Enterobacteriaceae isolates. The 118 water samples analyzed were from street home-bottled water (n = 81), municipal water distribution systems (tap water) (n = 25), and selected supply wells in several neighborhoods (n = 12). The samples were analyzed for total mesophilic microorganisms, fecal enterococci, fecal coliforms, Escherichia coli, and Vibrio spp. The results showed a high level of fecal contamination in all types of water samples. In home-bottled water, fecal coliforms were found in 88% of the samples, and E. coli in 66% of the samples. In tap water, fecal coliforms were found in 64%, and E. coli in 28% of the samples. In water from supply wells, fecal coliforms and E. coli were found in 83% of the samples. From 33 presumptive Vibrio spp. colonies, only three were identified as V. fluvialis. The remaining isolates belonged to Aeromonas spp. (n = 14) and Klebsiella spp. (n = 16). Of 44 selected Enterobacteriaceae isolates from water samples (28 isolates of E. coli and 16 isolates of Klebsiella spp.), 45.5% were not susceptible to the beta-lactams ampicillin and imipenem, 43.2% to amoxicillin, and 31.8% to amoxicillin/clavulanic acid. Regarding non-beta-lactam antibiotics, there was a high percentage of isolates with tolerance to tetracycline (52.3%) and azithromycin (31.8%). In conclusion, water in Maputo represents a risk for human health due to its high fecal contamination. This situation is made more serious by the fact that a relatively high percentage of isolates with multidrug resistance (40%) were found among Enterobacteriaceae. The dissemination of these results can raise awareness of the urgent need to reduce water contamination in Maputo and other cities in Mozambique.

Lactobacillus plantarum in Dual-Species Biofilms With Listeria monocytogenes Enhanced the Anti-Listeria Activity of a Commercial Disinfectant Based on Hydrogen Peroxide and Peracetic Acid.

The aim of this work was to investigate the effect of dual-species biofilms of Listeria monocytogenes with Lactobacillus plantarum on the anti-Listeria activity of a hydrogen peroxide/peracetic acid based commercial disinfectant (P3, Oxonia) when using conditions approaching the food industry environment. Nine strains of L. monocytogenes, including eight persistent strains collected from the meat industry and one laboratory control strain, were used in mono and in dual-species biofilms with a strain of L. plantarum. Biofilms were produced on stainless steel coupons (SSCs), at 11°C (low temperature) or at 25°C (control temperature), in TSB-YE (control rich medium) or in 1/10 diluted TSB-YE (mimicking the situation of biofilm formation after a deficient industrial cleaning procedure). The biofilm forming ability of the strains was evaluated by enumeration of viable cells, and the antibiofilm activity of P3 was assessed by the log reduction of viable cells on SSC. In both nutrient conditions and at low temperature, there was no significant difference (p > 0.05) between L. monocytogenes biofilm forming ability in mono- and in dual-species biofilms. In dual-species biofilms, L. monocytogenes was the dominant species. However, it was generally more susceptible to the lower concentration of P3 0.5% (v/v) than in pure culture biofilms. The presence of L. plantarum, although without significant interference in the number of viable cells of L. monocytogenes, enhanced the efficacy of the anti-Listeria activity of P3, since dual-species biofilms were easier to control. The results presented here reinforce the importance of the investigation into co-culture biofilms produced in food industry conditions, namely at low temperatures, when susceptibility to sanitizers is being assessed.

Resistance to ß-lactams in bacteria isolated from different types of Portuguese cheese.

The purpose of this study was to investigate the presence of beta-lactam-resistant bacteria in six different types of Portuguese cheese. The numbers of ampicillin resistant (AMP(r)) bacteria varied from 4.7 x 10(2) to 1.5 x 10(7) CFU/g. Within 172 randomly selected beta-lactam-resistant bacteria, 44 resistant phenotypes were found and 31.4% were multidrug resistant. The majority (85%) of the isolates identified belonged to the Enterobacteriaceae family. The presence of the bla(TEM) gene was detected in 80.9% of the tested isolates. The results suggest that without thermal processing of the milk and good hygienic practices, cheese may act as a vehicle of transfer of beta-lactam-resistant bacteria to the gastrointestinal tract of consumers.

Virulence of Listeria monocytogenes isolated from the cheese dairy environment, other foods and clinical cases.

The virulence potential of 51 Listeria monocytogenes isolates, including strains from cheese, cheese production environments and from human cases of listeriosis, was evaluated in this study. The isolates were used to infect HT-29 cell monolayers in an in vitro test of virulence, based on a plaque-forming assay (PFA). Fifteen selected isolates were used for subcutaneous footpad inoculation in mice and subsequent recovery of the bacterium from the spleen 3 days after inoculation. In the PFA, two isolates from milk (serovar 1/2a) were not significantly different (P<0.05) from the low-virulence strain (442) used as reference. Thirty-three isolates were not significantly different (P<0.05) from the virulent strain (EGDe) used as reference. Nine isolates were significantly more virulent (highly virulent) than the EGDe strain and seven isolates were significantly less virulent. The nine highly virulent isolates were either from humans (four), from cheese dairy environments (two isolates of a strain were found persistently in two dairies), from cheese (one), from milk (one) and the reference strain for serovar 1/2b (CECT 936). The two milk isolates with low virulence in the PFA were found to be virulent in mice. In conclusion, all the isolates from food and food-related environments were potentially virulent or highly virulent. These results stress the risk of listeriosis associated with the consumption of cheese contaminated with L. monocytogenes, and once more emphasize the importance of good manufacturing practices (GMPs) together with sanitation standard operating procedures (SSOPs) throughout the food chain.

Pulsed-field gel electrophoresis (PFGE) analysis of Listeria monocytogenes isolates from different sources and geographical origins and representative of the twelve serovars.

Multiplex-PCR (MPCR) serogrouping and pulsed-field gel electrophoresis (PFGE) subtyping analysis are currently used by several public and private laboratories for the characterization of Listeria monocytogenes. In this study a set of 80 L. monocytogenes isolates belonging to the twelve serovars was used to investigate (i) the typeability of the rare serovars, (ii) the ability of PFGE analysis with ApaI and AscI to differentiate serovars within MPCR serogroups and (iii) the association of molecular types with the specific source or geographical origin of the isolates. With the exception of three isolates (rare serovars 4a and 4c) that were not amenable to restriction with ApaI, all the other analyzed isolates were subtyped by both enzymes. PFGE discriminated the 80 isolates into 62 combined ApaI and AscI PFGE patterns (pulsotypes), but could not differentiate serovars within MPCR serogroups, in which isolates from different serovars displaying the same pulsotype were found. Clustering analysis suggests that for some pulsotypes grouping according to Portuguese origin or source can be suggested. On the other hand, some L. monocytogenes clones are widely distributed. Two pulsotypes from Portuguese human isolates were identical to the ones displayed by human outbreak clones in the UK and in the USA and Switzerland, respectively, although they were not temporally matched. Computer-assisted data analysis of large and diverse PFGE type databases will improve the correct interpretation of subtyping data in epidemiological studies and in tracing routes and sources of contamination in the food industry.

Listeria monocytogenes cells under nutrient deprivation showed reduced ability to infect the human intestinal cell line HT-29.

PURPOSE: This study aimed to evaluate the effect of two types of stress, cold and nutritional, on the viability and the in vitro virulence of the foodborne pathogenic bacteria Listeria monocytogenes. METHODOLOGY: Ten diverse isolates were kept in phosphate-buffered saline (PBS) at optimal (37 °C) or at refrigeration temperature (7 °C), for 1 and 7 days. The viability of the cells [log colony-forming units (c.f.u.)/ml] and their in vitro virulence, before and after storage in these conditions, were investigated. In vitro virulence (log PFA) was evaluated using the human intestinal epithelial cell line HT-29 in plaque-forming assays (PFAs).Results/Key findings. In general, when compared with the conditions at 37 °C, the exposure at 7 °C for 7 days seemed to increase the resistance of the isolates to nutritional stress. Nutritional stress per se acted significantly to decrease the in vitro virulence of the isolates. After 7 days of nutrient deprivation, whether at optimal or at refrigeration temperature, the majority of the isolates assumed a low-virulence phenotype. CONCLUSION: Our results suggest that when L. monocytogenes are in refrigerated post-processing environments that are unable to support their growth they may increase their resistance to nutritional stress and may decrease their virulence. This should be considered when performing risk assessments for refrigerated ready-to-eat (RTE) foods.

Comparative characterization of Listeria monocytogenes isolated from Portuguese farmhouse ewe's cheese and from humans.

In order to investigate the possible relationships between Listeria monocytogenes strains isolated from farmhouse ewe's cheese and clinical strains collected, in partially overlapping dates, from the same geographical area in Portugal, a total of 109 isolates from seven ewe's cheese manufactures (n=94) and from humans (n=15) were characterized by serotyping, RAPD, PFGE and allelic analysis of the virulent actA gene. Serotyping indicated the presence of four different serovars: 1/2a, 1/2b, 1/2c and 4b. The 15 clinical isolates were either serovar 4b (86.7%) or serovar 1/2b (13.3%). Among the 94 isolates from cheese and related environments the serovars prevalence was 1/2a (1.1%), 1/2b (17.0%), 1/2c (12.8%) and, unexpectedly, 4b (69.1%). Based on results obtained with PFGE typing of the strains, 25 genotypes were identified, 10 from farmhouses and 15 from human cases. Isolates from serovars 1/2a and 1/2c were assigned to single genotypes, respectively. Within serovars 1/2b and 4b three and 20 genotypes were established, respectively. RAPD typing of the isolates rendered 18 types indicating the lack of accuracy of the primers used in strain differentiation within serovar 4b. The actA gene typing of the strains showed a prevalence of actA gene type I (90.4%) compared with the rest of the strains that were all actA gene type II (9.6%). In spite of the fact that all the farmhouses were completely independent, the distribution of L. monocytogenes genotypes, intra and inter cheese manufactures, was relatively homogeneous, suggesting the existence of resident strains. In contrast, among human isolates there was a great genetic diversity. There was no common genotype between L. monocytogenes implicated in the cases of listeriosis and these cheese-related isolates, suggesting the absence of a causal relationship.