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Norovirus is the leading cause of viral gastroenteritis globally. While person-to-person transmission is most commonly reported route of infection, human norovirus is frequently associated with foodborne transmission, including through consumption of contaminated bivalve molluscan shellfish. Reverse transcription (RT)-qPCR is most commonly used method for detecting human norovirus detection in foods, but does not inform on its infectivity, posing challenges for assessing intervention strategies aimed at risk elimination. In this study, RT-qPCR was used in conjunction with a derivative of the photoreactive DNA binding dye propidium monoazide (PMAxx™) (PMAxx-RT-qPCR) to evaluate the viral capsid integrity of norovirus genogroup I and II (GI and GII) in shellfish following high pressure processing (HPP). Norovirus GI.3 and GII.4 bioaccumulated oysters were subjected to HPP at pressures of 300 and 450 MPa at 15 °C, and 300, 450 and 600 MPa at 20 °C. Samples were analysed using both RT-qPCR and PMAxx-RT-qPCR. For each sample, norovirus concentration (genome copies/g digestive tissue) determined by RT-qPCR was divided by the PMAxx-RT-qPCR concentration, giving the relative non-intact (RNI) ratio. The RNI ratio values relate to the amount of non-intact (non-infectious) viruses compared to fully intact (possible infectious) viruses. Our findings revealed an increasing RNI ratio value, indicating decreasing virus integrity, with increasing pressure and decreasing pressure. At 300 MPa, for norovirus GI, the median [95% confidence interval, CI] RNI ratio values were 2.6 [1.9, 3.0] at 15 °C compared to 1.1 [0.9, 1.8] at 20 °C. At 450 MPa, the RNI ratio values were 5.5 [2.9, 7.0] at 15 °C compared to 1.3 [1.0, 1.6] at 20 °C. At 600 MPa, the RNI ratio value was 5.1 [2.9, 13.4] at 20 °C. For norovirus GII, RT-qPCR and PMAxx-RT-qPCR detections were significantly reduced at 450 and 600 MPa at both 15 °C and 20 °C, with the median [95% CI] RNI ratio value at 300 MPa being 1.1 [0.8, 1.6]. Following HPP treatment, the use of PMAxx-RT-qPCR enables the selective detection of intact and potential infectious norovirus, enhancing our understanding of the inactivation profiles and supporting the development of more effective risk assessment strategies.
Assuntos
Manipulação de Alimentos , Norovirus , Ostreidae , Reação em Cadeia da Polimerase em Tempo Real , Frutos do Mar , Inativação de Vírus , Norovirus/genética , Norovirus/isolamento & purificação , Norovirus/fisiologia , Norovirus/classificação , Norovirus/crescimento & desenvolvimento , Animais , Ostreidae/virologia , Frutos do Mar/virologia , Manipulação de Alimentos/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Humanos , Contaminação de Alimentos/análise , Pressão Hidrostática , Propídio/química , Propídio/análogos & derivados , Azidas/química , Infecções por Caliciviridae/virologiaRESUMO
In this study, three generations of polymerase chain reaction (PCR) assays: (i) conventional PCR, (ii) qPCR and (iii) droplet digital PCR (ddPCR), were systematically tested for their abilities to detect non-pathogenic and pathogenic populations of Vibrio parahaemolyticus. The limit of detection (LOD) for the ddPCR was 1.1 pg/µL of purified DNA, followed by the qPCR (5.6 pg/µL) and the conventional PCR (8.8 pg/µL). Regarding the LOD for V. parahaemolyticus cells, the ddPCR assay was able to detect 29 cells, followed by the conventional PCR assay (58 cells) and the qPCR assay (115 cells). Regarding the sensitivities to detect this pathogen from PCR inhibition prone samples (naturally contaminated mussels), the ddPCR assay significantly outperformed the conventional PCR and qPCR. The ddPCR assay was able to consistently detect non-pathogenic and pathogenic populations of V. parahaemolyticus from naturally contaminated mussels, indicating its tolerance to various PCR inhibitors. This study also revealed the significant difference between conventional PCR and qPCR. The conventional PCR assay showed significantly greater sensitivity than that of the qPCR assay in detecting V. parahaemolyticus in crude samples, whereas the qPCR assay showed better sensitivity in detecting the presence of V. parahaemolyticus in purified DNA samples.
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Vibrio parahaemolyticus , Vibrio parahaemolyticus/genética , Sensibilidade e Especificidade , Reação em Cadeia da Polimerase , Alimentos Marinhos , DNARESUMO
The potential of using commercial peroxyacetic acid (PAA) for Vibrio parahaemolyticus sanitization was evaluated. Commercial PAA of 0.005 % (v/v, PAA: 2.24 mg/L, hydrogen peroxide: 11.79 mg/L) resulted in a planktonic cell reduction of >7.00 log10 CFU/mL when initial V. parahaemolyticus cells averaged 7.64 log10 CFU/mL. For cells on stainless steel coupons, treatment of 0.02 % PAA (v/v, PAA: 8.96 mg/L, hydrogen peroxide: 47.16 mg/L) achieved >5.00 log10 CFU/cm2 reductions in biofilm cells for eight strains but not for the two strongest biofilm formers. PAA of 0.05 % (v/v, PAA: 22.39 mg/L, hydrogen peroxide: 117.91 mg/L) was required to inactivate >5.00 log10 CFU/cm2 biofilm cells from mussel shell surfaces. The detection of PAA residues after biofilm treatment demonstrated that higher biofilm production resulted in higher PAA residues (p < 0.05), suggesting biofilm is acting as a barrier interfering with PAA diffusing into the matrices. Based on the comparative analysis of genomes, robust biofilm formation and metabolic heterogeneity within niches might have contributed to the variations in PAA resistance of V. parahaemolyticus biofilms.
Assuntos
Perna (Organismo) , Vibrio parahaemolyticus , Animais , Peróxido de Hidrogênio/farmacologia , Ácido Peracético/farmacologia , Aço Inoxidável , Biofilmes , PlânctonRESUMO
Vibrio parahaemolyticus has been identified as an emerging human pathogen worldwide with cases undergoing a global expansion over recent decades in phase with climate change. New Zealand had remained free of outbreaks until 2019, but different outbreaks have been reported consecutively since then. To provide new insights into the recent emergence of cases associated with outbreak clones over recent years, a comparative genomic study was carried out using a selection of clinical (mostly outbreak) and environmental isolates of V. parahaemolyticus obtained in New Zealand between 1973 and 2021. Among 151 isolates of clinical (n=60) and environmental (n=91) origin, 47 sequence types (STs) were identified, including 31 novel STs. The population of environmental isolates generated 30 novel STs, whereas only 1 novel ST (ST2658) was identified among the population of clinical isolates. The novel clinical ST was a single-locus variant of the pandemic ST36 strain, indicating further evolution of this pandemic strain. The environmental isolates exhibited a significant genetic heterogeneity compared to the clinical isolates. The whole-genome phylogeny separated the population of clinical isolates from their environmental counterparts, clearly indicating their distant genetic relatedness. In addition to differences in ancestral profiles and genetic relatedness, these two groups of isolates exhibited a profound difference in their virulence profiles. While the entire population of clinical isolates harboured the thermostable direct haemolysin (tdh) and/or the thermostable-related haemolysin (trh), only a few isolates of environmental origin possessed the same virulence genes. In contrast to tdh and trh, adhesin-encoding genes, vpadF and MSHA, showed a significantly (P<0.001) greater association with the environmental isolates compared to the clinical isolates. The effectors, VopQ, VPA0450 and VopS, which belong to T3SS1, were ubiquitous, being present in each isolate regardless of its origin. The effectors VopC and VopA, which belong to T3SS2, were rarely detected in any of the examined isolates. Our data indicate that the clinical and environmental isolates of V. parahaemolyticus from New Zealand differ in their population structures, ancestral profiles, genetic relatedness and virulence profiles. In addition, we identified numerous unique non-synonymous single-nucleotide polymorphisms (nsSNPs) in adhesins and effectors, exclusively associated with the clinical isolates tested, which may suggest a possible role of these mutations in the overall virulence of the clinical isolates.
Assuntos
Vibrio parahaemolyticus , Fatores de Virulência , Humanos , Fatores de Virulência/genética , Vibrio parahaemolyticus/genética , Nova Zelândia/epidemiologia , Virulência/genética , GenômicaRESUMO
Vibrio parahaemolyticus biofilms on the seafood processing plant surfaces are a potential source of seafood contamination and subsequent food poisoning. Strains differ in their ability to form biofilm, but little is known about the genetic characteristics responsible for biofilm development. In this study, pangenome and comparative genome analysis of V. parahaemolyticus strains reveals genetic attributes and gene repertoire that contribute to robust biofilm formation. The study identified 136 accessory genes that were exclusively present in strong biofilm forming strains and these were functionally assigned to the Gene Ontology (GO) pathways of cellulose biosynthesis, rhamnose metabolic and catabolic processes, UDP-glucose processes and O antigen biosynthesis (p < 0.05). Strategies of CRISPR-Cas defence and MSHA pilus-led attachment were implicated via Kyoto Encyclopedia of Genes and Genomes (KEGG) annotation. Higher levels of horizontal gene transfer (HGT) were inferred to confer more putatively novel properties on biofilm-forming V. parahaemolyticus. Furthermore, cellulose biosynthesis, a neglected potential virulence factor, was identified as being acquired from within the order Vibrionales. The cellulose synthase operons in V. parahaemolyticus were examined for their prevalence (22/138, 15.94 %) and were found to consist of the genes bcsG, bcsE, bcsQ, bcsA, bcsB, bcsZ, bcsC. This study provides insights into robust biofilm formation of V. parahaemolyticus at the genomic level and facilitates: identification of key attributes for robust biofilm formation, elucidation of biofilm formation mechanisms and development of potential targets for novel control strategies of persistent V. parahaemolyticus.
Assuntos
Vibrio parahaemolyticus , Vibrio parahaemolyticus/genética , Biofilmes , Genômica , Óperon , CeluloseRESUMO
Vibrio parahaemolyticus is a marine oriented pathogen; and biofilm formation enables its survival and persistence on seafood processing plant, complicating the hygienic practice. The objectives of this study are to assess the ability of V. parahaemolyticus isolated from seafood related environments to form biofilms, to determine the effective sodium hypochlorite concentrations required to inactivate planktonic and biofilm cells, and to evaluate the genetic diversity required for strong biofilm formation. Among nine isolates, PFR30J09 and PFR34B02 isolates were identified as strong biofilm forming strains, with biofilm cell counts of 7.20, 7.08 log10 CFU/cm2, respectively, on stainless steel coupons after incubation at 25 °C. Free available chlorine of 1176 mg/L and 4704 mg/L was required to eliminate biofilm cells of 1.74-2.28 log10 CFU/cm2 and > 7 log10 CFU/cm2, respectively, whereas 63 mg/L for planktonic cells, indicating the ineffectiveness of sodium hypochlorite in eliminating V. parahaemolyticus biofilm cells at recommended concentration in the food industry. These strong biofilm-forming isolates produced more polysaccharides and were less susceptible to sodium hypochlorite, implying a possible correlation between polysaccharide production and sodium hypochlorite susceptibility. Genetic diversity in mshA, mshC and mshD contributed to the observed variation in biofilm formation between isolates. This study identified strong biofilm-forming V. parahaemolyticus strains of new multilocus sequence typing (MLST) types, showed a relationship between polysaccharide production and sodium hypochlorite resistance.
Assuntos
Vibrio parahaemolyticus , Vibrio parahaemolyticus/genética , Hipoclorito de Sódio/farmacologia , Tipagem de Sequências Multilocus , Biofilmes , Variação GenéticaRESUMO
Listeria monocytogenes is a gram-positive foodborne pathogen that causes outbreaks of listeriosis associated with a diverse range of foods. L. monocytogenes forms biofilms as a strategy to enhance its survival in the environment. These biofilms then provide a source of contamination in processing plant environments. Cations like magnesium, calcium, and sodium are commonly found in the environment and are important to bacteria to maintain their homeostasis. It is, therefore, valuable to understand the relationship between these cations and biofilm formation. In this study, four isolates of L. monocytogenes from seafood processing environments were used to investigate the influence of magnesium, calcium, and sodium (1, 10, and 50 mM) on biofilms. The isolates selected were defined as being either a low biofilm former, a high biofilm former, an outbreak isolate, and a persistent isolate from the seafood industry. The study showed that the divalent cations magnesium and calcium increased biofilm formation compared with the monovalent cation, sodium. Fifty mM concentrations of the divalent cations significantly enhanced biofilm formation. The cations did not have a significant effect on the initial stages of biofilm formation but appeared to influence the later stages of biofilm development.
Assuntos
Listeria monocytogenes , Magnésio/farmacologia , Cálcio/farmacologia , Microbiologia de Alimentos , Biofilmes , Aderência Bacteriana , Sódio/farmacologia , Cátions Bivalentes/farmacologia , Contaminação de Alimentos/análiseRESUMO
Listeria monocytogenes, a causative agent of listeriosis, is a major foodborne pathogen. Among pathogens, L. monocytogenes stands out for its unique ecological and physiological characteristics. This distinct lifestyle of L. monocytogenes has a significant impact on food safety and public health, mainly through the ability of this pathogen to multiply at refrigeration temperature and to persist in the food processing environment. Due to a combination of these characteristics and emerging trends in consumer preference for ready-to-eat and minimally processed food, there is a need to develop effective and sustainable approaches to control contamination of food products with L. monocytogenes. Implementation of an efficient and reliable control strategy for L. monocytogenes must first address the problem of cross-contamination. Besides the preventive control strategies, cross-contamination may be addressed with the introduction of emerging post packaging non-thermal or thermal hurdles that can ensure delivery of a listericidal step in a packed product without interfering with the organoleptic characteristics of a food product. This review aims to present the most relevant findings underlying the distinct lifestyle of L. monocytogenes and its impact on food safety. We also discuss emerging food decontamination technologies that can be used to better control L. monocytogenes.
RESUMO
Inhibitors of cyclin-dependent kinases 4 and 6 (CDK4/6i) are standard first-line treatments for metastatic ER+ breast cancer. However, acquired resistance to CDK4/6i invariably develops, and the molecular phenotypes and exploitable vulnerabilities associated with resistance are not yet fully characterized. We developed a panel of CDK4/6i-resistant breast cancer cell lines and patient-derived organoids and demonstrate that a subset of resistant models accumulates mitotic segregation errors and micronuclei, displaying increased sensitivity to inhibitors of mitotic checkpoint regulators TTK and Aurora kinase A/B. RB1 loss, a well-recognized mechanism of CDK4/6i resistance, causes such mitotic defects and confers enhanced sensitivity to TTK inhibition. In these models, inhibition of TTK with CFI-402257 induces premature chromosome segregation, leading to excessive mitotic segregation errors, DNA damage, and cell death. These findings nominate the TTK inhibitor CFI-402257 as a therapeutic strategy for a defined subset of ER+ breast cancer patients who develop resistance to CDK4/6i.
Assuntos
Pontos de Checagem da Fase M do Ciclo Celular , Neoplasias , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/genéticaRESUMO
The formation of biofilms is a survival strategy employed by bacteria to help protect them from changing or unfavourable environments. In this research, 319 genes which govern biofilm formation in V. parahaemolyticus, as reported in 1,625 publications, were analysed using protein-protein-interaction (PPI) network analysis. CsrA was identified as a motility-sessility switch and biofilm formation regulator. Through robust rank aggregation (RRA) analysis of GSE65340, the generation of viable but non-culturable (VBNC) cells that may enhance cell tolerance to stress, was found to be associated with the TCA cycle and carbon metabolism biological pathways. The finding that CsrA is likely to play a role in the development of VBNC cells improves understanding of the molecular mechanisms of VBNC formation in V. parahaemolyticus and contributes to on-going efforts to reduce the hazard posed by this foodborne pathogen.
Assuntos
Vibrio parahaemolyticus , Bactérias , Biofilmes , Simulação por ComputadorRESUMO
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 ZelândiaRESUMO
Listeria monocytogenes is a ubiquitous foodborne pathogen that results in a high rate of mortality in sensitive and immunocompromised people. Contamination of food with L. monocytogenes is thought to occur during food processing, most often as a result of the pathogen producing a biofilm that persists in the environment and acting as the source for subsequent dispersal of cells onto food. A survey of seafood-processing plants in New Zealand identified the persistent strain 15G01, which has a high capacity to form biofilms. In this study, a transposon library of L. monocytogenes 15G01 was screened for mutants with altered biofilm formation, assessed by a crystal violet assay, to identify genes involved in biofilm formation. This screen identified 36 transposants that showed a significant change in biofilm formation compared to the wild type. The insertion sites were in 27 genes, 20 of which led to decreased biofilm formation and seven to an increase. Two insertions were in intergenic regions. Annotation of the genes suggested that they are involved in diverse cellular processes, including stress response, autolysis, transporter systems, and cell wall/membrane synthesis. Analysis of the biofilms produced by the transposants using scanning electron microscopy and fluorescence microscopy showed notable differences in the structure of the biofilms compared to the wild type. In particular, inactivation of uvrB and mltD produced coccoid-shaped cells and elongated cells in long chains, respectively, and the mgtB mutant produced a unique biofilm with a sandwich structure which was reversed to the wild-type level upon magnesium addition. The mltD transposant was successfully complemented with the wild-type gene, whereas the phenotypes were not or only partially restored for the remaining mutants.IMPORTANCE The major source of contamination of food with Listeria monocytogenes is thought to be due to biofilm formation and/or persistence in food-processing plants. By establishing as a biofilm, L. monocytogenes cells become harder to eradicate due to their increased resistance to environmental threats. Understanding the genes involved in biofilm formation and their influence on biofilm structure will help identify new ways to eliminate harmful biofilms in food processing environments. To date, multiple genes have been identified as being involved in biofilm formation by L. monocytogenes; however, the exact mechanism remains unclear. This study identified four genes associated with biofilm formation by a persistent strain. Extensive microscopic analysis illustrated the effect of the disruption of mgtB, clsA, uvrB, and mltD and the influence of magnesium on the biofilm structure. The results strongly suggest an involvement in biofilm formation for the four genes and provide a basis for further studies to analyze gene regulation to assess the specific role of these biofilm-associated genes.
Assuntos
Proteínas de Bactérias/genética , Biofilmes , Manipulação de Alimentos , Listeria monocytogenes/fisiologia , Alimentos Marinhos , Genes Bacterianos , Listeria monocytogenes/genética , Mutação , Nova ZelândiaRESUMO
Isolation of the pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis from foods typically rely on slow (10-21 day) "cold enrichment" protocols before confirmed results are obtained. We describe an approach that yields results in 39 h that combines an alternative enrichment method with culture on a non-selective medium, and subsequent identification of suspect colonies using elastic light scatter (ELS) analysis. A prototype database of ELS profiles from five Yersinia species and six other bacterial genera found in pork mince was established, and used to compare similar profiles of colonies obtained from enrichment cultures from pork mince samples seeded with representative strains of Y. enterocolitica and Y. pseudotuberculosis. The presumptive identification by ELS using computerised or visual analyses of 83/90 colonies in these experiments as the target species was confirmed by partial 16S rDNA sequencing. In addition to seeded cultures, our method recovered two naturally occurring Yersinia strains. Our results indicate that modified enrichment combined with ELS is a promising new approach for expedited detection of foodborne pathogenic yersiniae.
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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 ZelândiaRESUMO
Studies conducted in seawaters around New Zealand have shown the numbers of human pathogenic Vibrio spp. are usually low, but high numbers sometimes occur during warmer summer/autumn months (January - April). In this study, Pacific oysters (Crassostrea gigas) were grown at Kaipara Harbour and Mahurangi Harbour in New Zealand at different heights from the seafloor in different ways: fixed positons intertidally and subtidally, and as floating long lines over the 2013 and 2014 summer periods. Two geographically distinct commercial harvest areas: Coromandel Harbour (North Island) and Croisilles Harbour (South Island) in New Zealand were also compared in 2015 where oysters are grown under different methods. Detection and enumeration of Vibrio spp. was performed according to the Bacteriological Analytical Manual using the Most Probable Number approach and real-time polymerase chain reaction technique. The only significant growing method effect was observed in Mahurangi Harbour, where intertidal oysters at 1.5 m from the seafloor had higher numbers of trh + Vibrio parahaemolyticus than other intertidal samples from Kaipara Harbour and Coromandel Harbour. All other samples showed a relationship with surface seawater temperature, but not with distance from seafloor or farming method. Overall, there is no clear evidence that different oyster farming methods (floating, subtidal or intertidal at different depths) affect Vibrio spp. population sizes, which were dominated by seasonal changes and environmental parameters.
Assuntos
Crassostrea/microbiologia , Alimentos Marinhos/microbiologia , Vibrio parahaemolyticus/crescimento & desenvolvimento , Vibrio vulnificus/crescimento & desenvolvimento , Agricultura , Animais , Fazendas , Contaminação de Alimentos/análise , Humanos , Nova Zelândia , Reação em Cadeia da Polimerase em Tempo Real , Estações do Ano , Água do Mar/microbiologia , Temperatura , Vibrio parahaemolyticus/isolamento & purificação , Vibrio vulnificus/isolamento & purificaçãoRESUMO
A laboratory-based study testing 9 Listeria innocua strains independently and a cocktail of 11 Listeria monocytogenes strains was carried out. The aim was to identify suitable L. innocua strain(s) to model L. monocytogenes in inactivation experiments. Three separate inactivation procedures and a hurdle combination of the three were employed: thermal inactivation (55°C), UV-C irradiation (245 nm), and chemical sanitizer (TsunamiTM 100, a mixture of acetic acid, peroxyacetic acid, and hydrogen peroxide). The responses were strain dependent in the case of L. innocua with different strains responding differently to different regimes and L. innocua isolates generally responded differently to the L. monocytogenes cocktail. In the thermal inactivation treatment, inactivation of all strains including the L. monocytogenes cocktail plateaued after 120 min. In the case of chemical sanitizer, inactivation could be achieved at concentrations of 10 and 20 ppm with inactivation increasing with contact time up to 8 min, beyond which there was no significant benefit. All L. innocua strains except PFR16D08 were more sensitive than the L. monocytogenes cocktail to the hurdle treatment. PFR16D08 almost matched the resistance of the L. monocytogenes cocktail but was much more resistant to the individual treatments. A cocktail of two L. innocua strains (PFR 05A07 and PFR 05A10) had the closest responses to the hurdle treatment to those of the L. monocytogenes cocktail and is therefore recommended for hurdle experiments.
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Contamination of mussels with the human pathogen Listeria monocytogenes occurs during processing in the factory, possibly from bacteria persisting in the factory's indoor and outdoor areas. In this study, a selection of persistent (n=8) and sporadic (n=8) L. monocytogenes isolates associated with mussel-processing premises in New Zealand were investigated for their phenotypic and genomic characteristics. To identify traits that favour or contribute to bacterial persistence, biofilm formation, heat resistance, motility and recovery from dry surfaces were compared between persistent and sporadic isolates. All isolates exhibited low biofilm formation at 20°C, however, at 30°C persistent isolates showed significantly higher biofilm formation after 48h using cell enumeration and near significant difference using the crystal violet assay. All 16 isolates were motile at 20°C and 30°C and motility was fractionally higher for sporadic isolates, but no significant difference was observed. We found persistent isolates tend to exhibit greater recovery after incubation on dry surfaces compared to sporadic isolates. Two of the three most heat-resistant isolates were persistent, while four of five isolates lacking heat resistance were sporadic isolates. Comparison of genome sequences of persistent and sporadic isolates showed that there was no overall clustering of persistent or sporadic isolates, and that differences in prophages and plasmids were not associated with persistence. Our results suggest a link between persistence and biofilm formation, which is most likely multifactorial, combining subtle phenotypic and genotypic differences between isolates.
Assuntos
Biofilmes/crescimento & desenvolvimento , Bivalves/microbiologia , Manipulação de Alimentos , Listeria monocytogenes , Animais , Sequência de Bases , DNA Bacteriano/genética , Marcadores Genéticos/genética , Genoma Bacteriano/genética , Genótipo , Temperatura Alta , Humanos , Listeria monocytogenes/genética , Listeria monocytogenes/isolamento & purificação , Listeria monocytogenes/metabolismo , Tipagem de Sequências Multilocus , Nova Zelândia , Fenótipo , Prófagos/genética , Análise de Sequência de DNARESUMO
Loss of cell-cycle control is a hallmark of human cancer. Cell-cycle checkpoints are essential for maintaining genome integrity and balanced growth and division. They are specifically deregulated in cancer cells and contain regulators that represent potential therapeutic targets. Monopolar spindle 1 (Mps1; also known as TTK protein kinase) is a core component of the spindle assembly checkpoint (SAC), a genome-surveillance mechanism that is important for cell survival, and has emerged as a candidate target for anticancer therapy. Here, we report the cellular and antitumor effects of CFI-402257, a potent (Mps1 Ki = 0.09 ± 0.02 nM; cellular Mps1 EC50 = 6.5 ± 0.5 nM), highly selective, and orally active small-molecule inhibitor of Mps1 that was identified through a drug-discovery program. Human cancer cells treated with CFI-402257 exhibit effects consistent with Mps1 kinase inhibition, specifically SAC inactivation, leading to chromosome missegregation, aneuploidy, and ultimately cell death. Oral administration of CFI-402257 in monotherapy or in combination with an anti-programmed cell death 1 (PD-1) antibody in mouse models of human cancer results in inhibition of tumor growth at doses that are well-tolerated. Our findings provide a rationale for the clinical evaluation of CFI-402257 in patients with solid tumors.
Assuntos
Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Animais , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular Tumoral , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Terapia de Alvo Molecular , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Neoplasias/patologia , Inibidores de Proteínas Quinases/administração & dosagem , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/metabolismo , Pirazóis/administração & dosagem , Pirazóis/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/farmacocinética , Interferência de RNA , RNA Interferente Pequeno/genética , Carga Tumoral , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This work describes a scaffold hopping exercise that begins with known imidazo[1,2-a]pyrazines, briefly explores pyrazolo[1,5-a][1,3,5]triazines, and ultimately yields pyrazolo[1,5-a]pyrimidines as a novel class of potent TTK inhibitors. An X-ray structure of a representative compound is consistent with 1(1)/2 type inhibition and provides structural insight to aid subsequent optimization of in vitro activity and physicochemical and pharmacokinetic properties. Incorporation of polar moieties in the hydrophobic and solvent accessible regions modulates physicochemical properties while maintaining potency. Compounds with enhanced oral exposure were identified for xenograft studies. The work culminates in the identification of a potent (TTK K i = 0.1 nM), highly selective, orally bioavailable anticancer agent (CFI-402257) for IND enabling studies.