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1.
BMC Infect Dis ; 21(1): 564, 2021 Jun 12.
Artigo em Inglês | MEDLINE | ID: mdl-34118865

RESUMO

BACKGROUND: Listeria monocytogenes (LM) has come to be a major public health issue of at-risk groups, causing high morbidity and mortality. Despite this data, studies are very limited in developing countries like Ethiopia. Thus, we aimed to isolate and characterize LM in terms of antibiogram and biofilm formation among pregnant women with fever, women with a history of spontaneous abortion, women with a history of fetal loss, and women with preterm delivery at Jimma University Medical Center (JUMC), southwest Ethiopia. METHODS: A cross-sectional study was done among 144 women from June to August 2019. Isolates were tested for antibiotic susceptibility and biofilm formation using disc diffusion and microtiter plate method, respectively. Data were collected using a structured questionnaire, entered into Epidata 3.1 and logistic regression was done by SPSS v25.0. RESULTS: LM was isolated in 8 (5.56%) of 144 screened women. The isolation rate of LM was relatively higher among women with a history of fetal loss (9.7%), followed by women with preterm delivery (6.25%). One of the six cord blood was positive for LM, indicating that the transplacental transmission rate at JUMC was 16.7%. More than 2% of women with an ongoing pregnancy were found to have LM septicemia, which could hurt their fetus. All of the isolates tested were susceptible to Ampicillin. However, all of the isolates were resistant to Penicillin and Meropenem and were biofilm producers. CONCLUSIONS: The high magnitude of pregnancy-related listeriosis in the current study setting appears that implementation of educational programs targeting risk reduction and more studies to identify sources of LM are warranted. The choice of antibiotics should be after susceptibility testing.


Assuntos
Antibacterianos/farmacologia , Listeria monocytogenes , Listeriose/microbiologia , Complicações Infecciosas na Gravidez/microbiologia , Centros Médicos Acadêmicos , Estudos Transversais , Etiópia/epidemiologia , Feminino , Humanos , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/isolamento & purificação , Listeriose/prevenção & controle , Testes de Sensibilidade Microbiana , Gravidez , Complicações Infecciosas na Gravidez/prevenção & controle
2.
Food Chem ; 359: 129974, 2021 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-33964662

RESUMO

The effect of curcumin-mediated blue light-emitting diode (LED) photodynamic inactivation (PDI) for preserving the quality of salmon contaminated with Listeria monocytogenes was investigated by microbiological, physical, chemical and histological methods during sample storage at 4 â„ƒ and 25 â„ƒ. The results showed that PDI decelerated the proliferation of L. monocytogenes on salmon during storage at 25 â„ƒ, with the maximum inhibition reaching 4.0 log10 CFU/g (99.99%), compared to the negative control. Moreover, PDI greatly retarded the increase in pH (P < 0.05) and the production of TVB-N, retarded the accumulation of free fatty acids, and decelerated the degradation of proteins, ultimately preserving the high nutritional value of the salmon. In addition, PDI effectively prevented a change in colour and retarded the loss of water from the salmon, thereby conserving its texture and sensory properties. Therefore, PDI is a promising and valid non-thermal technology to use for fish preservation.


Assuntos
Curcumina/farmacologia , Listeria monocytogenes/isolamento & purificação , Fotoquimioterapia , Salmão/microbiologia , Animais , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Conservação de Alimentos/métodos
3.
J Int Med Res ; 49(4): 300060521998568, 2021 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-33866842

RESUMO

Listeria monocytogenes is a Gram-positive facultative intracellular bacterium that causes central nervous system infection. We report a case of rhombencephalitis caused by L. monocytogenes infection, which mimicked Bickerstaff's brainstem encephalitis, and GQ1b antibody positivity and multiple intracranial foci were observed. A 68-year-old male patient presented with a nonspecific prodrome of faintness, forehead tightness, and walking instability. This was followed by progressive cranial nerve palsies, limb weakness, cerebellar signs, hyperpyrexia, and impaired consciousness. Brain imaging showed multiple abnormal brainstem and cerebellar signals that were accompanied by blood infiltration without any lesion enhancement. Serum GQ1b antibody positivity led to an initial diagnosis of Bickerstaff's brainstem encephalitis, which was treated with immunosuppressive therapy with limited efficacy. A pathogen examination helped confirm L. monocytogenes infection. A combination of meropenem and trimethoprim-sulfamethoxazole therapy was applied and the patient recovered without sequelae. The symptoms and imaging of Listeria rhombencephalitis are nonspecific. Accurate diagnosis and prompt treatment of this condition are essential. Whether Listeria infection triggers an autoimmune response remains unclear.


Assuntos
Hemorragias Intracranianas/etiologia , Listeria monocytogenes/isolamento & purificação , Listeriose/diagnóstico , Rombencéfalo/diagnóstico por imagem , Idoso , Infecções do Sistema Nervoso Central , Encefalite/diagnóstico por imagem , Encefalite/tratamento farmacológico , Sequenciamento de Nucleotídeos em Larga Escala , Humanos , Listeria monocytogenes/genética , Listeriose/tratamento farmacológico , Masculino , Neuroimagem
4.
Int J Food Microbiol ; 348: 109204, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-33930838

RESUMO

The survival kinetics of Listeria monocytogenes (9 log CFU/mL) as a post-fermentation contaminant in probiotic fermented milk (Lactobacillus acidophilus La-5, 8-9 log CFU/mL) processed with milk subjected to ohmic heating (0, 4, 6, and 8 V/cm; CONV, OH4, OH6, OH8, 90-95 °C/5 min) was investigated using Weibull predictive model. Additionally, the presence of bioactive compounds (antioxidant activity, inhibition of the enzymes α-glucosidase, α-amylase, and angiotensin-converting) and sensory analysis (consumer test) of probiotic fermented milks were evaluated. Overall, OH provided a decrease in the viability of Listeria monocytogenes, suitable Lactobacillus acidophilus counts, and satisfactory results in the gastrointestinal tract survival. The Weibull model presented an excellent fit to the data of all conditions. Furthermore, lower δ values (217-298 against 665 h, CONV), and increased R2 values (0.99 against 0.98, CONV) were obtained for the OH-treated samples, emphasizing the best performance of OH data. In addition, OH improved the generation of bioactive compounds as well as the sensory acceptance. Indeed, considering functional and safety purposes, OH presented as an interesting technology to be used in milk for manufacturing probiotic fermented milk.


Assuntos
Produtos Fermentados do Leite/microbiologia , Lactobacillus acidophilus/metabolismo , Listeria monocytogenes/isolamento & purificação , Leite/microbiologia , Probióticos/metabolismo , Inibidores da Enzima Conversora de Angiotensina/análise , Animais , Antioxidantes/análise , Reatores Biológicos/microbiologia , Produtos Fermentados do Leite/análise , Fermentação , Inibidores de Glicosídeo Hidrolases/análise , Calefação , Cinética , Percepção Gustatória , alfa-Amilases/antagonistas & inibidores , alfa-Glucosidases/química
5.
Int J Food Microbiol ; 347: 109166, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-33838478

RESUMO

Listeria monocytogenes is a foodborne human pathogen that causes systemic infection, fetal-placental infection in pregnant women causing abortion and stillbirth and meningoencephalitis in elderly and immunocompromised individuals. This study aimed to analyse L. monocytogenes from different sources from New Zealand (NZ) and to compare them with international strains. We used pulsed-field gel electrophoresis (PFGE), multilocus sequence typing (MLST) and whole-genome single nucleotide polymorphisms (SNP) to study the population structure of the NZ L. monocytogenes isolates and their relationship with the international strains. The NZ isolates formed unique clusters in PFGE, MLST and whole-genome SNP comparisons compared to the international isolates for which data were available. PFGE identified 31 AscI and 29 ApaI PFGE patterns with indistinguishable pulsotypes being present in seafood, horticultural products and environmental samples. Apart from the Asc0002:Apa0002 pulsotype which was distributed across different sources, other pulsotypes were site or factory associated. Whole-genome analysis of 200 randomly selected L. monocytogenes isolates revealed that lineage II dominated the NZ L. monocytogenes populations. MLST comparison of international and NZ isolates with lineage II accounted for 89% (177 of 200) of the total L. monocytogenes population, while the international representation was 45.3% (1674 of 3473). Rarefaction analysis showed that sequence type richness was greater in NZ isolates compared to international trend, however, it should be noted that NZ isolates predominantly came from seafood, horticulture and their respective processing environments or factories, unlike international isolates where there was a good mixture of clinical, food and environmental isolates.


Assuntos
Microbiologia Ambiental , Genoma Bacteriano/genética , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Alimentos Marinhos/microbiologia , Manipulação de Alimentos , Microbiologia de Alimentos , Variação Genética , Horticultura , Humanos , Listeria monocytogenes/classificação , Nova Zelândia
6.
Int J Food Microbiol ; 347: 109198, 2021 Jun 02.
Artigo em Inglês | MEDLINE | ID: mdl-33894462

RESUMO

The U.S. FDA Food Safety Modernization Act Preventive Controls for Human Food Rule underlines the importance of an effective environmental monitoring (EM) program. EM is used to determine harborage sites of microorganisms on processing equipment, assess effectiveness of sanitation programs, and prevent transmission of foodborne pathogens. This study characterizes commercially-available polyurethane foam (PUF) and cellulose (CELL) EM tools for their efficacy in the release of foodborne pathogens from their sponge matrices. Specifically, the objectives of this study were to 1) compare the ability of EM tools to release microorganisms into a recovery eluent, 2) characterize EM tool performance at decreasing inoculum concentrations, and 3) assess the impact of various operators during the processing of EM samples. Two bacteria (Listeria monocytogenes, Salmonella Typhimurium) and one human norovirus surrogate (Tulane virus [TV]) were compared at decreasing inoculum levels utilizing two elution techniques (mechanical stomacher, manually by operator), and across six operators. Data indicated that EM tool material composition impacted the release of microorganisms (p = 0.0001), where the PUF EM tool released TV more readily than the CELL EM tool. Conversely, the decreasing inoculum levels did not statistically differ in the release of microorganisms from the EM tool matrices. In addition, no significant difference was found between the machine stomacher and manual elution by human operator or between operators. Overall, the study provides a detailed characterization of two commercially-available EM tools, and the differences identified in this study can be used to improve the effectiveness of EM programs.


Assuntos
Celulose/farmacologia , Monitoramento Ambiental/métodos , Listeria monocytogenes/isolamento & purificação , Norovirus/isolamento & purificação , Poliuretanos/farmacologia , Salmonella typhimurium/isolamento & purificação , Carga Bacteriana/métodos , Contagem de Colônia Microbiana , Microbiologia de Alimentos , Inocuidade dos Alimentos , Humanos , Listeria monocytogenes/genética , Norovirus/genética , Salmonella typhimurium/genética , Carga Viral/métodos
7.
Food Microbiol ; 98: 103756, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875198

RESUMO

Phenotypic and genotypic resistance to benzalkonium chloride (BC), cadmium and arsenic was tested (by susceptibility assays and molecular methods) in 287 Listeria monocytogenes strains isolated from fish and fish products, and food-producing factories in Poland. Overall, 40% of the isolates were resistant to BC, 56% to cadmium and 41% to arsenic (57% displayed resistance to more than one of the tested compounds). Among BC-resistant isolates, the most commonly detected resistance determinant was the qacH gene (83%). Three distinct types of cadA gene determining resistance to cadmium were detected, with the cadA1 variant predominant (88%), while most arsenic-resistant isolates (86%) harbored the arsA gene associated with a Tn554-like transposon (one strain harbored two copies of arsA in different arsenic resistance cassettes). 53% of all tested isolates contained plasmids (from 4 kb to > 90 kb in size), which were classified into 11 groups (p1-p11) based on their restriction patterns. Interestingly, 12 isolates harbored the small mobilizable pLMST6-like plasmid pLIS3 encoding multidrug efflux pump EmrC. Clustering analysis of PFGE patterns revealed that these isolates represent several diverse bacterial populations, which strongly suggests mobility of the pLMST6-like plasmids among L. monocytogenes strains and their role in dissemination of BC resistance.


Assuntos
Antibacterianos/farmacologia , Arsênio/farmacologia , Compostos de Benzalcônio/farmacologia , Farmacorresistência Bacteriana , Produtos Pesqueiros/microbiologia , Peixes/microbiologia , Listeria monocytogenes/efeitos dos fármacos , Listeria monocytogenes/isolamento & purificação , Animais , Peixes/classificação , Contaminação de Alimentos/análise , Listeria monocytogenes/classificação , Listeria monocytogenes/genética , Polônia
8.
Food Microbiol ; 98: 103770, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-33875206

RESUMO

Food business operators are responsible for food safety and assessment of shelf lives for their ready-to-eat products. For assisting them, a customized software based on predictive models, ListWare, is being developed. The aim of this study was to develop and validate a predictive model for the growth of Listeria monocytogenes in sliced roast beef. A challenge study was performed comprising 51 different combinations of variables. The growth curves followed the Baranyi and Roberts model with no clear lag phase and specific growth rates in the range <0.005-0.110 hr-1. A linear regression model was developed based on 528 observations and had an adjusted R-square of 0.80. The significant predictors were storage temperature, sodium lactate, interactions between sodium acetate and temperature, and MAP packaging and temperature. The model was validated in four laboratories in three countries. For conditions where the model predicted up to + log 2 cfu/g Listeria concentration, the observed concentrations were true or below the predicted concentration in 90% of the cases. For the remaining 10%, the roast beef was coated with spices and therefore different from the others. The model will be implemented in ListWare web-application for calculation of "Listeria shelf life".


Assuntos
Fast Foods/microbiologia , Contaminação de Alimentos/estatística & dados numéricos , Listeria monocytogenes/crescimento & desenvolvimento , Produtos da Carne/microbiologia , Animais , Bovinos , Contaminação de Alimentos/análise , Inocuidade dos Alimentos , Armazenamento de Alimentos , Cinética , Listeria monocytogenes/química , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Produtos da Carne/análise , Modelos Biológicos , Análise de Regressão , Temperatura
9.
Int J Food Microbiol ; 340: 109043, 2021 Feb 16.
Artigo em Inglês | MEDLINE | ID: mdl-33454520

RESUMO

The food processing environments of a newly opened meat processing facility were sampled in ten visits carried out during its first 1.5 years of activity and analyzed for the presence of Listeria monocytogenes. A total of 18 L. monocytogenes isolates were obtained from 229 samples, and their genomes were sequenced to perform comparative genomic analyses. An increase in the frequency of isolation of L. monocytogenes and in the diversity of sequence types (STs) detected was observed along time. Although the strains isolated belonged to six different STs (ST8, ST9, ST14, ST37, ST121 and ST155), ST9 was the most abundant (8 out of 18 strains). Low (0 and 2) single nucleotide polymorphism (SNP) distances were found between two pairs of ST9 strains isolated in both cases 3 months apart from the same processing room (Lm-1267 and Lm-1705, with a 2 SNPs distance in the core genome; Lm-1265 and Lm-1706, with a 0 SNPs distance), which suggests that these strains may be persistent L. monocytogenes strains in the food processing environment. Most strains showed an in silico attenuated virulence potential either through the truncation of InlA (in 67% of the isolates) or the absence of other virulence factors involved in cell adhesion or invasion. Twelve of the eighteen L. monocytogenes isolates contained a plasmid, which ranged in size from 4 to 87 Kb and harbored stress survival, in addition to heavy metals and biocides resistance determinants. Identical or highly similar plasmids were identified for various sets of L. monocytogenes ST9 isolates, which suggests the clonal expansion and persistence of plasmid-containing ST9 strains in the processing environments of the meat facility. Finally, the analysis of the L. monocytogenes genomes available in the NCBI database, and their associated metadata, evidenced that strains from ST9 are more frequently reported in Europe, linked to foods, particularly to meat and pork products, and less represented among clinical isolates than other L. monocytogenes STs. It also showed that the ST9 strains here isolated were more closely related to the European isolates, which clustered together and separated from ST9 North American isolates.


Assuntos
Contaminação de Equipamentos , Manipulação de Alimentos , Variação Genética , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Instalações Industriais e de Manufatura , Carne , Animais , Desinfetantes , Europa (Continente) , Pisos e Cobertura de Pisos , Microbiologia de Alimentos , Genes Bacterianos , Listeria monocytogenes/classificação , Listeria monocytogenes/patogenicidade , Plasmídeos , Suínos , Virulência/genética , Fatores de Virulência/genética , Sequenciamento Completo do Genoma
10.
Int J Food Microbiol ; 341: 109069, 2021 Mar 02.
Artigo em Inglês | MEDLINE | ID: mdl-33508582

RESUMO

Listeria monocytogenes has caused outbreaks of foodborne illness from apples in the USA, and is also a major issue for regulatory compliance worldwide. Due to apple's significance as an important export product from New Zealand, we aimed to determine the effect of long-term, low-temperature sea-freight from New Zealand to the USA (July) and Europe (March-April), two key New Zealand markets, on the survival and/or growth of L. monocytogenes on fresh apples. Temperature and humidity values were recorded during a shipment to each market (USA and Europe), then the observed variations around the 0.5 °C target temperature were simulated in laboratory trials using open ('Scired') and closed ('Royal Gala' for the USA and 'Cripps Pink' for Europe) calyx cultivars of apples inoculated with a cocktail of 107-108 cells of seven strains of L. monocytogenes. Samples were analysed for L. monocytogenes quantification at various intervals during the simulation and on each occasion, an extra set was analysed after a subsequent 8 days at 20 °C. When both the sea-freight simulations concluded, L. monocytogenes showed 5 log reductions on the equatorial surface of skin of apples, but only about 2.5 log reduction for USA and about 3.3 log reduction for Europe in the calyx. Cultivar type had no significant effect on the survival of L. monocytogenes for both sea-freight simulations, either in the calyx or on the skin (P > 0.05). Most of the reduction in the culturable cells on the skin occurred during the initial 2 weeks of the long-term storage simulations. There was also no significant difference in the reduction of L. monocytogenes at 0.5 or 20 °C. No correlation was observed between firmness or total soluble solids and survival of L. monocytogenes. Because the inoculated bacterial log reduction was lower in the calyx than on the skin, it is speculated that the risk of causing illness is higher if contaminated apple cores are eaten. The result suggested that the international sea-freight transportation does not result in the growth of L. monocytogenes irrespective of time and temperature. The results of this study provide useful insights into the survival of L. monocytogenes on different apple cultivars that can be used to develop effective risk mitigation strategies for fresh apples during long-term, low-temperature international sea-freight transportation.


Assuntos
Manipulação de Alimentos/métodos , Listeria monocytogenes/isolamento & purificação , Malus/microbiologia , Refrigeração/métodos , Carga Bacteriana , Temperatura Baixa , Contagem de Colônia Microbiana , Europa (Continente) , Microbiologia de Alimentos/métodos , Doenças Transmitidas por Alimentos/microbiologia , Doenças Transmitidas por Alimentos/prevenção & controle , Nova Zelândia
11.
Int J Infect Dis ; 104: 718-724, 2021 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-33515776

RESUMO

OBJECTIVE: To determine serogroups, multilocus sequence typing (MLST) of Listeria monocytogenes isolates and analyze clinical characteristics of these clones focusing on non-perinatal cases. METHODS: From 2000 to 2015, we analyzed 123 human listeriosis cases at a medical center in northern Taiwan using PCR serogrouping, MLST, and clinical presentations. RESULTS: The annual incidence of listeriosis increased since 2005 with a peak in 2008 (0.2 per 1000 admission) and decreased thereafter. Of the 115 non-perinatal listeriosis cases, we found a male predominance (60%) with an average age of 63.9 years old (standard deviation: 15.3 years), and almost all patients had underlying conditions including malignancies (61.7%), steroid usage (39.1%), diabetes mellitus (31.3%), renal insufficiency (27.8%), and liver cirrhosis (17.4%). Clinical presentations included bacteremia (74.8%), neurolisteriosis (20.0%), and spontaneous bacterial peritonitis (5.2%). The most frequently identified serogroup-sequence types (ST) were IIB-ST87 (30.9%), followed by IIA-ST378 (16.3%) and IIA-ST155 (14.6%). The 30-day all-cause mortality of non-perinatal listeriosis was 25.2% and was associated with age (Hazard ratio: 1.04, 95% C.I. = 1.01-1.07, p = 0.021), steroid usage (Hazard ratio: 2.54, 95% C.I. = 1.06-6.11, p = 0.038) and respiratory distress at presentation (Hazard ratio: 2.59, 95% C.I. = 1.05-6.39, p = 0.038); while no association was found with serogroups (IIA, IIB, and IVB) or three major ST types by multivariable analysis. All 8 mothers of perinatal listeriosis patients survived and three neonates died (mortality, 37.5%), and IIB-ST87 was the major type (62.5%). CONCLUSION: Predominant strains in Taiwan could cause significant morbidity and mortality. Further disease monitoring and source surveillance are warranted despite a declining trend of human listeriosis in Taiwan.


Assuntos
Listeria monocytogenes/genética , Listeriose/epidemiologia , Listeriose/microbiologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Bacteriemia/diagnóstico , Bacteriemia/epidemiologia , Bacteriemia/microbiologia , Bacteriemia/mortalidade , Feminino , Humanos , Incidência , Listeria monocytogenes/isolamento & purificação , Listeriose/diagnóstico , Listeriose/mortalidade , Masculino , Pessoa de Meia-Idade , Epidemiologia Molecular , Tipagem de Sequências Multilocus , Reação em Cadeia da Polimerase , Taiwan/epidemiologia
12.
Methods Mol Biol ; 2220: 3-16, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975762

RESUMO

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


Assuntos
Listeria monocytogenes/isolamento & purificação , Contagem de Colônia Microbiana/métodos , Meios de Cultura/química , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Humanos , Listeriose/microbiologia
13.
Methods Mol Biol ; 2220: 17-29, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975763

RESUMO

Listeria monocytogenes is a major food-borne pathogen and causative agent of a fatal disease, listeriosis. Stringent regulatory guidelines and zero tolerance policy toward this bacterium necessitate rapid, accurate, and reliable methods of identification and subtyping. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF MS) has recently become a method of choice for routine identification of pathogens in clinical settings and has largely replaced biochemical assays. Identification relies on well-curated databases such as SARAMIS. Extensive use of SARAMIS to generate consensus mass spectra, in conjunction with statistical analysis, such as partial least square-discriminant analysis and hierarchical cluster analysis, is useful in subtyping bacteria. While MALDI-ToF MS has been extensively used for pathogen detection, its application in bacterial subtyping has been limited. The protocol describes a MALDI-ToF MS workflow as a single tool for simultaneous identification and subtyping of L. monocytogenes directly from solid culture medium.


Assuntos
Listeria monocytogenes/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Técnicas de Tipagem Bacteriana/economia , Técnicas de Tipagem Bacteriana/métodos , Humanos , Listeria monocytogenes/química , Listeria monocytogenes/classificação , Listeriose/microbiologia , Software , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/economia , Fatores de Tempo , Fluxo de Trabalho
14.
Methods Mol Biol ; 2220: 31-40, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975764

RESUMO

Quantitative PCR, if performed properly, is a highly sensitive and robust tool. Nevertheless, its application to the particular case of pathogen detection from foodstuffs necessitates special requirements for reliable results. Firstly, a robust analytical chain, involving sample preparation and DNA isolation with purification, is necessary to ensure optimal performance. Secondly, for reliable quantification of Listeria monocytogenes from food, reproducible controls for all steps of the analytical chain are needed, which can give quantitative information about the performance of each individual step of the detection chain. Ideally, each individual sample should include a so-called internal sample process control (ISPC) which passes through all steps of the analytical chain and is phenotypically similar to the target organism (in this case L. monocytogenes).This chapter describes the modular and rapid (3 h) sample preparation method "matrix lysis" for the quantification of L. monocytogenes from food and gives detailed information regarding the application of an ISPC based on the example of the L. monocytogenes Δ-prfA/+IAC strain.


Assuntos
Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Animais , DNA Bacteriano/genética , DNA Bacteriano/isolamento & purificação , Humanos , Listeria monocytogenes/genética , Listeriose/microbiologia , Carne/microbiologia , Reação em Cadeia da Polimerase em Tempo Real/métodos , Manejo de Espécimes/métodos
15.
Methods Mol Biol ; 2220: 41-53, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975765

RESUMO

Quantitative real-time polymerase chain reaction (qPCR) is one of the most used molecular methods. There are numerous qPCR assays on the market, some of them for pathogen detection, and the development of new assays still continues. However, what methods are suitable for assay performance validation and which information do they provide? For conclusions based on qPCR data, it is essential to know which capacities and limitations an assay has. This chapter gives an overview of methods for qPCR assay performance validation and the respective insights and how to combine them. Most of those validation methods have been published in connection with the prfA assay, which specifically detects Listeria monocytogenes. Thereby, it could be demonstrated that this assay reliably quantifies even a single copy of the prfA gene and is thus suitable for detection of Listeria monocytogenes.


Assuntos
Proteínas de Bactérias/genética , Listeria monocytogenes/genética , Fatores de Terminação de Peptídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , DNA Bacteriano/genética , Microbiologia de Alimentos , Humanos , Limite de Detecção , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia
16.
Methods Mol Biol ; 2220: 57-78, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975766

RESUMO

For assessing isolates of Listeria monocytogenes, serotype designation is the first subtyping method used. Methodologies used to assign serotype are currently evolving and will eventually be replaced with whole genome sequencing. Traditionally, serotyping has been done with agglutination reactions; however, alternative methods utilizing enzyme linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR) are common. Described here are the three non-genomic methods and the advantages and disadvantages of each.


Assuntos
Testes de Aglutinação/métodos , Ensaio de Imunoadsorção Enzimática/métodos , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase/métodos , Sorotipagem/métodos , Humanos , Listeria monocytogenes/classificação , Listeriose/diagnóstico
17.
Methods Mol Biol ; 2220: 79-88, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975767

RESUMO

PFGE is a valuable tool for assessing L. monocytogenes strain interrelatedness. It is based on the study of total bacterial DNA restriction patterns. Cells are embedded in agarose plugs before being lysed. The released DNA is then digested into large fragments by restriction enzymes. As DNA fragments are too large to be separated by traditional electrophoresis in an agarose gel, changes in the direction of the electrical current are periodically applied in order to allow the proper migration of large DNA fragments. Strains are characterized by the obtained DNA fragment patterns or pulsotypes which vary depending on the number and size of bands.


Assuntos
DNA Bacteriano/análise , Eletroforese em Gel de Campo Pulsado/métodos , Listeria monocytogenes/isolamento & purificação , Enzimas de Restrição do DNA/química , DNA Bacteriano/genética , Microbiologia de Alimentos , Humanos , Listeria monocytogenes/genética , Listeriose/microbiologia , Sefarose/química
18.
Methods Mol Biol ; 2220: 201-215, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975777

RESUMO

The pathogen Listeria monocytogenes is a facultative intracellular bacterium, which targets a large range of cell types. Following entry, bacteria disrupt the invasion vacuole and reach the cytoplasm where they replicate and use the actin cytoskeleton to propel themselves from cell to cell. Mammalian epithelial cells grown in vitro can be used to study the different steps of the intracellular life of Listeria. However, rapid multiplication and dissemination of bacteria can induce important cell death and detachment, resulting in the formation of lytic plaques. Thus, in vitro infections with L. monocytogenes are usually restricted to short time courses, from a few minutes to one day. Here, we present a method to study long-term L. monocytogenes infections in epithelial cells using epifluorescence microscopy. This protocol enables the observation of actin-based motility, intercellular dissemination foci, and entrapment of L. monocytogenes within vacuoles of persistence termed "Listeria-Containing Vacuoles" (LisCVs). We also describe a protocol to study the recruitment of cytoskeletal proteins at Listeria actin comet tails, as well as a method to assess the membrane integrity of intracellular bacteria using a LIVE/DEAD viability assay.


Assuntos
Células Epiteliais/microbiologia , Listeria monocytogenes/fisiologia , Listeriose/patologia , Microscopia de Fluorescência/métodos , Linhagem Celular , Proteínas do Citoesqueleto/análise , Células Epiteliais/patologia , Imunofluorescência/métodos , Interações Hospedeiro-Patógeno , Humanos , Listeria monocytogenes/isolamento & purificação , Listeriose/microbiologia
19.
Methods Mol Biol ; 2220: 219-231, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975778

RESUMO

Biofilm-forming ability may vary significantly among different Listeria (L.) monocytogenes strains. This interstrain variation is also observed in L. monocytogenes biofilm resistance to antimicrobial compounds commonly used in the food-processing environment. The screening of a large set of L. monocytogenes strains with specific characteristics, such as serotype, MLST type, and other genetic characteristics under various environmental conditions, may lead to a better understanding of the mechanisms underlying the establishment of the pathogen on food contact surfaces. In this chapter, traditional methods for L. monocytogenes strains characterization with regard to biofilm formation and novel biofilm control methods will be described.


Assuntos
Biofilmes , Manipulação de Alimentos , Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/fisiologia , Carga Bacteriana/métodos , Desinfecção/métodos , Manipulação de Alimentos/métodos , Humanos , Listeriose/microbiologia , Microscopia de Fluorescência/métodos , Propriedades de Superfície
20.
Methods Mol Biol ; 2220: 233-242, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-32975779

RESUMO

The Listeria monitoring program for Austrian dairies and cheese factories was established in 1988. The aim was to control the entrance of L. monocytogenes into the food-processing environment (FPE), preventing the contamination of food under processing. The Austrian Listeria monitoring program comprises four levels of investigation, dealing with routine monitoring of samples and consequences of finding a positive sample. Preventive quality control concepts attempt to detect a foodborne hazard along the food-processing chain, prior to food delivery, retailing, and consumption. The implementation of a preventive food safety concept provokes a deepened insight by the manufacturers into problems concerning food safety. The development of preventive quality assurance strategies contributes to the national food safety status and protects public health.


Assuntos
Contaminação de Alimentos/análise , Manipulação de Alimentos , Microbiologia de Alimentos , Listeria monocytogenes/isolamento & purificação , Manipulação de Alimentos/métodos , Qualidade dos Alimentos , Humanos , Listeriose/microbiologia
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