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- The risk of salt reduction in dry-cured sausage assessed by the influence on water activity and the survival of salmonellaPublication . Patarata, Luis; Fernandes, Liliana; Silva, José António; Fraqueza, M. J.Water activity (aw) is the main hurdle for microbial control in dry-cured sausages. The aw can be influenced by drying or adding electrolytes or humectants. Dry-cured meat products are partially dried, which, together with added salt, results in safe aw values. Currently, there is a trend to reduce salt in meat products, which can compromise the preservation process. The present work aims to evaluate the influences of added salt levels (1% or 3%) and the use or omission of phosphates and wine on the aw of a dry-cured sausage, and to evaluate the possibility of estimating the aw from the moisture loss and the behavior of Salmonella during dry-cured sausage (chouriço) processing. There was a strong relationship between moisture and aw, regardless of the salt level and the presence of phosphates or wine. Predicting aw from moisture loss is possible using the Boltzmann sigmoid function. The salt level strongly influences Salmonella behavior, mainly through aw reduction. An increase in aw by 0.01 units reduced the odds of achieving a 5-log reduction in Salmonella counts to half. Increasing added salt from 1% to 3% increased the odds of achieving a 5-log Salmonella reduction 7.5-fold. The current trend to reduce salt in foods must be carefully approached if applied to cured meat products, as it has substantial consequences on aw evolution and Salmonella survival
- Editorial: natural compounds in food safety and preservationPublication . Ferreira, Susana; Sanchez, Gloria; Alves, Maria J.; Fraqueza, M. J.
- Staphylococcus saprophyticus causing infections in humans is associated with high resistance to heavy metalsPublication . Lawal, Opeyemi U.; Fraqueza, M. J.; Worning, Peder; Bouchami, OnsStaphylococcus saprophyticus is a common pathogen of the urinary tract, a heavy metal-rich environment, but information regarding its heavy metal resistance is unknown. We investigated 422 S. saprophyticus isolates from human infection and colonization/contamination, animals, and environmental sources for resistance to copper, zinc, arsenic, and cadmium using the agar dilution method. To identify the genes associated with metal resistance and assess possible links to pathogenicity, we accessed the wholegenome sequence of all isolates and used in silico and pangenome-wide association approaches. The MIC values for copper and zinc were uniformly high (1,600mg/liter). Genes encoding copper efflux pumps (copA, copB, copZ, mco, and csoR) and zinc transporters (zinT, czrAB, znuBC, and zur) were abundant in the population (20 to 100%). Arsenic and cadmium showed various susceptibility levels. Genes encoding the ars operon (arsRDABC), an ABC transporter and a two-component permease, were linked to resistance to arsenic (MICs$ 1,600mg/liter; 14% [58/422]; P, 0.05). At least three cad genes (cadA or cadC and cadD-cadX or czrC) and genes encoding multidrug efflux pumps and hyperosmoregulation in acidified conditions were associated with resistance to cadmium (MICs$ 200mg/liter; 20% [85/422]; P, 0.05). These resistance genes were frequently carried by mobile genetic elements. Resistance to arsenic and cadmium were linked to human infection and a clonal lineage originating in animals (P, 0.05). Altogether, S. saprophyticus was highly resistant to heavy metals and accumulated multiple metal resistance determinants. The highest arsenic and cadmium resistance levels were associated with infection, suggesting resistance to these metals is relevant for S. saprophyticus pathogenicity
- Bacteriocin-producing strain lactiplantibacillus plantarum LP17L/1 isolated from traditional stored Ewe’s milk cheese and its beneficial potentialPublication . Lauková, Andrea; Tomáška, Martin; Fraqueza, M. J.; Szabóová, Renáta; Bino, Eva; Šcerbová, Jana; Simonová, Monika Pogány; Dvorožnáková, EmíliaStored ewe’s milk lump cheese is a local product that can be a source of autochthonous beneficial microbiota, especially lactic acid bacteria. The aim of this study was to show the antimicrobial potential of Lactiplantibacillus plantarum LP17L/1 isolated from stored ewe’s milk lump cheese. Lpb. plantarum LP17L/1 is a non-hemolytic, non-biofilm-forming strain, susceptible to antibiotics. It contains genes for 10 bacteriocins—plantaricins and exerted active bacteriocin with in vitro antistaphylococcal and anti-listerial effect. It does not produce damaging enzymes, but it produces β-galactosidase. It also sufficiently survives in Balb/c mice without side effects which indicate its safety. Moreover, a reduction in coliforms in mice jejunum was noted. LP17L/1 is supposed to be a promising additive for Slovak local dairy products.
- Effect of high hydrostatic pressure challenge on biogenic amines, microbiota, and sensory profile in traditional poultry- and pork-based semidried fermented sausagePublication . Borges, Ana F.; Cozar, Almudena; Patarata, Luis; Gama, Luis; Alfaia, Cristina; Fernandes, Maria J.; Fernandes, Maria H.; Perez Vergara, Herminia; Fraqueza, M. J.The processing of traditional poultry- and pork-based semidried fermented smoked sausages needs to bemodernized to improve product quality and further extend its shelf life. The aim of the present study was to applydifferent combinations of high pressure (300 to 600 MPa) and time (154 to 1,800 s) on the sausages using an experimentaldesign based on response surface methodology. The chemical, microbial, and sensory characteristics of sausages treatedwith high-pressure processing (HPP) were investigated. HPP application to semidried fermented sausages resulted incolor changes, which could be dependent on the ingredients, formulation, and smoking conditions used. Nevertheless,none of the HPP treatments applied resulted in detectable changes in sensory properties, as tested in a triangle test andconfirmed by the analysis of focus groups assessment. Significant differences were detected for lactic acid bacteria (LAB)counts from 344 MPa and 1,530 s onward, with a marked decrease for the combination of 600 MPa and 960 s (P<0.05).Coagulase-negativestaphylococcishowed higher tolerance to the increase in pressure than LAB. HPP induced a microbialreduction onEnterobacteriaceae, molds, and yeasts, minimizing the production of the main biogenic amines. However,the polyamines (spermine and spermidine) increased since their metabolic use by microorganisms did not occur. Giventhe reduction of the main spoilage microbial indicators with no detectable sensory changes observed with the binomialcondition of 600 MPa and 960 s, this was chosen as the optimal combination to be further applied.
- Evidence for the dissemination to humans of Methicillin-Resistant Staphylococcus aureus ST398 through the pork production chain : a study in a portuguese slaughterhousePublication . Bouchami, Ons; Fraqueza, M. J.; Faria, Nuno Alexandre; Alves, Valquíria; Lawal, Opeyemi Uwangbaoje; Lencastre, Herminia de; Miragaia, MariaLivestock-associated methicillin-resistant Staphylococcus aureus (LA-MRSA) ST398 was recovered from infections in humans exposed to animals, raising public health concerns. However, contact with food producing chain as a means of transmission of LA-MRSA to humans remains poorly understood. We aimed to assess if pork production chain is a source of MRSA ST398 for human colonization and infection. MRSA from live pigs, meat, the environment, and slaughterhouse workers were analyzed by Pulsed-Field Gel Electrophoresis (PFGE), spa, MLST typing, SNPs and for antibiotic resistance and virulence gene profiles. We compared core and accessory genomes of MRSA ST398 isolated from slaughterhouse and hospital. We detected MRSA ST398 (t011, t108, t1451) along the entire pork production chain (live pigs: 60%; equipment: 38%; meat: 23%) and in workers (40%). All MRSA ST398 were multidrug resistant, and the majority carried genes encoding biocide resistance and enterotoxins. We found 23 cross-transmission events between live pigs, meat, and workers (6–55 SNPs). MRSA ST398 from infection and slaughterhouse environment belonged to the same clonal type (ST398, t011, SCCmec V), but differed in 321–378 SNPs. Pork production chain can be a source of MRSA ST398 for colonization of human slaughterhouse workers, which can represent a risk of subsequent meat contamination and human infection.
- Co-inoculation with Staphylococcus equorum and Lactobacillus sakei reduces vasoactive biogenic amines in traditional dry-cured sausagesPublication . Dias, Igor; Laranjo, Marta; Potes, Maria Eduarda; Santos, Ana Cristina Agulheiro; Ricardo Rodrigues, Sara; Fialho, Ana Rita; Véstia, Joana; Fraqueza, M. J.; Oliveira, Margarida; Elias, Miguel: Dry-cured sausages are traditional in Mediterranean countries, and Paio do Alentejo (PA) is one of the most popular in South Portugal. The aim of the present work was to evaluate the effect of combined starters on the safety and quality of PA preserving its sensory quality. Physicochemical parameters, namely pH and water activity (aW), microbiological parameters, biogenic amines, color, texture, and sensory attributes were assessed. Three starter cultures were used, namely Staphylococcus equorum S2M7 and Lactobacillus sakei CV3C2, both separate and combined with the 2RB4 yeast strain at a concentration of 106 cfu/g. Dextrose 0.25% was added to the meat batter. Starters had a significant effect on the reduction of aW values (0.845 to 0.823). The treatment with L. sakei as well as the co-inoculation of L. sakei with S. equorum effectively reduced the L. monocytogenes counts to undetectable levels. Sausages co-inoculated with S. equorum S2M7/L. sakei CV3C2 showed a significant reduction in the content of vasoactive amines, namely tryptamine (26.21 to 15.70) and β-phenylethylamine (4.80 to 3.69). Regarding texture, control PA showed higher hardness values, and the starters promoted the cohesiveness of the batter while reducing chewiness. The studied starters did not compromise the sensory characteristics of PA.
- Genomic and phenotypic characterization of Campylobacter fetus subsp. venerealis strainsPublication . Silva, Marta Filipa Serra da; Pereira, Ana L.; Fraqueza, M. J.; Pereira, Gonçalo; Mateus, Luisa; Lopes-da-Costa, Luís; Silva, EThe pathogenesis mechanisms of Campylobacter fetus subsp. venerealis (Cfv), the etiologic agent of Bovine Genital Campylobacteriosis remain elusive. This study evaluated the virulence potential and biovar characteristics of Cfv isolates (n = 13) by PCR screening of putative virulencefactor (VF) genes, Multilocus Sequence Typing (MLST) analysis, antimicrobial susceptibility to tetracycline, penicillin, enrofloxacin and streptomycin testing and whole-genome sequencing (WGS; n = 5), also comparing the latter with 26 other whole-genome sequences of Cfv strains. The putative VF genes encoding type IV secretion system of Cfv (virB2-virB11/virD4) were absent in 92% of isolates, including isolates from aborted foetuses, evidencing that these VF genes are not essential for Cfv pathogenicity. The parA gene, used as a Cfv diagnostic molecular target, was detected in only 3 of 13 isolates, invalidating its use for diagnosis purposes. Three novel sequence types were identified by MLST. Although no in vitro antimicrobial resistance was detected, WGS identified antimicrobial resistance-related genes, including those encoding the multidrug efflux pumps CmeABC and YkkCD, indicating that their presence is not enough to provide antimicrobial resistance. The SNP and accessory protein families analysis segregated the Cfv and Cfv biovar intermedius (Cfvi) strains into different clusters. In conclusion, this study evidenced virulence potential and biovar characteristics of Cfv and Cfvi, which are of relevance for the control of Bovine Genital Campylobacteriosis.
- Assessment of biofilm formation by Campylobacter spp. isolates mimicking poultry slaughterhouse conditionsPublication . Araújo, P. M.; Batista, E.; Fernandes, M. H.; Fernandes, M. J.; Gama, Luis; Fraqueza, M. J.This research aimed to assess the biofilm formation ability of Campylobacter strains under temperature and oxygen stress conditions, similar to those found in the industrial environment, to explain the persistence of this pathogen on the poultry slaughter line. A collection of C. jejuni and C. coli isolates (n = 143) obtained from poultry samples (cecal content and neck skin), collected at slaughterhouse level, from diverse flocks, on different working days, was genotyped by flaA-restriction fragment length polymorphism (RFLP) typing method. A clustering analysis resulted in the assignment of 10 main clusters, from which 15 strains with different flaA-RFLP genotypes were selected for the assessment of biofilm formation ability and antimicrobial susceptibility. Biofilm assays, performed by crystal violet staining method, were conducted with the goal of mimicking some conditions present at the slaughterhouse environment, based on temperature, atmosphere, and contamination levels. Results indicated that many C. jejuni strains with similar flaA-RFLP profiles were present at the slaughterhouse on different processing days. All the strains tested (n = 15) were multidrugresistant except for one. Biofilm formation ability was strain-dependent, and it appeared to have been affected by inoculum concentration, temperature, and tolerance to oxygen levels. At 10 degrees C, adherence levels were significantly lower than at 42 degrees C. Under microaerobic and aerobic atmospheres, at 42 degrees C, 3 strains (C. jejuni 46E, C. jejuni 61C, and C. coli 65B) stood out, exhibiting significant levels of biofilm formation. C. jejuni strains 46E and 61C were inserted in clusters with evidence of persistence at the slaughterhouse for a long period of time. This study demonstrated that Campylobacter strains from broilers are capable of forming biofilms under conditions resembling the slaughterhouse environment. These results should be seen as a cue to improve the programs of hygiene implemented, particularly in those zones that can promote biofilm formation.
- Nitrite-free implications on consumer acceptance and the behavior of pathogens in cured pork loinsPublication . Patarata, Luis; Carvalho, Filipa; Fraqueza, M. J.Cured pork loins are valued products due to their particular sensory characteristics. These products are usually prepared with nitrite to guarantee adequate color and pathogen control. The use of nitrite in meat products has been criticized due to its potential contribution to carcinogenic N-nitroso-compound formation. The present work aimed to evaluate the effect of eliminating nitrite from the manufacturing of cured loins made with wine- and water-based marinades on the color evaluation of consumers and on the behavior of Clostridium sporogenes, Listeria monocytogenes, and Salmonella. The use of nitrite in processing cured loins resulted in a color considered adequate by more than 50% of the consumers. When nitrite was not used, the color was described mainly as weak. The hedonic evaluation of cured loins did not reflect the color evaluation. The samples with a weak and an adequate color had similar hedonic evaluations. The present work did not allow us to infer the potential interest in injecting S. xylosus into meat to prepare cured loins. The use of nitrite did not affect the survival of Cl. sporogenes, L. monocytogenes, or Salmonella. The reduction in the aw was the primary determinant influencing pathogen survival. The production of nitrite-free cured loins seems possible once the control of pathogens can be achieved. However, the product will have a weaker color. Consumers appreciate sensory aspects other than color, which, combined with the positive impact of the “additive-free” claim, can support the possibility of producing cured loins without nitrite