RESUMEN
Food processing and food handling environments may contain residual levels of sanitizers or cleaners which may trigger oxidative stress adaptation in Listeria monocytogenes. The aim of this study was to determine the induction and stability of oxidative stress adaptation in L. monocytogenes EGD (Bug600) (serotype 1/2a) and F1057 (serotype 4b) at different concentrations and times of sublethal oxidative stress induced by H2O2 or sublethal alkali stress induced by NaOH at 37°C. Both L. monocytogenes Bug600 and F1057 strains showed significantly higher survival in lethal oxidative stress (1000ppm H2O2) after pre-exposure to 50ppm H2O2 for 30min compared to control cells (no pre-exposure to H2O2). When the cells were pre-exposed to sublethal alkali stress by NaOH, the oxidative stress adaptation was induced within 5min in L. monocytogenes. The survival of both L. monocytogenes strains was increased by 2 to 4.5 logs in lethal oxidative stress when the cells were pre-exposed to sublethal alkali stress at pH9 from 5 to 120min by NaOH compared to control cells (no pre-exposure to sublethal alkali pH). Two other alkali reagents tested (KOH and NH4OH) also induced oxidative stress adaptation in L. monocytogenes. For both L. monocytogenes strains, the oxidative stress adaptation induced by sublethal H2O2 was reversible in 30min and that induced by sublethal alkali stress was reversible within 60min at 37°C in the absence of such sublethal stress. These findings show that sublethal oxidative or alkali stress conditions can induce oxidative stress adaptation that may increase the risk of survival of L. monocytogenes cells in lethal oxidative stress.
Asunto(s)
Amoníaco/farmacología , Peróxido de Hidrógeno/farmacología , Hidróxidos/farmacología , Listeria monocytogenes/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Compuestos de Potasio/farmacología , Hidróxido de Sodio/farmacología , Adaptación Fisiológica/efectos de los fármacos , Manipulación de Alimentos , Concentración de Iones de HidrógenoRESUMEN
The stability of acid stress adaptation in Listeria monocytogenes and its induced cross protection effect against GRAS (generally recognized as safe) antimicrobial compounds has never been investigated before. In the present study, the acid stress adaptation in L. monocytogenes was initially induced in pH 5.0 tryptic soy broth supplemented with 0.6% yeast extract (TSB-YE) at 37 °C. Subsequently, the stability of acid stress adaptation, which was defined as the capacity to maintain its acquired acid adaptation after induction in the absence of sublethal acid stress, was determined at 37 °C, 22 °C or 4 °C in broth and in different food substrates. Then, the acid stress adaptation induced cross protection against lauric arginate (LAE) and its stability was investigated in TSB-YE, milk and carrot juice. Our findings show that the acid stress adaptation was stable at 4 °C up to 24h but was reversed at 37 °C or 22 °C within 2h. In the cross protection assay with LAE, the acid stress adapted cells had approximately 2 log CFU/ml greater survival than non-adapted cells in broth at 22 °C or in milk and carrot juice at 4 °C. The acid adaptation induced cross protection against LAE in L. monocytogenes was reversible within 1h at 4 °C in the absence of sublethal acid stress. Our findings suggest that the stability of acid adaptation in L. monocytogenes under cold conditions should be taken into account when the risk analysis is performed during food processing.
Asunto(s)
Ácidos/farmacología , Arginina/análogos & derivados , Microbiología de Alimentos , Listeria monocytogenes/efectos de los fármacos , Adaptación Fisiológica/efectos de los fármacos , Animales , Antiinfecciosos/farmacología , Arginina/farmacología , Bebidas/microbiología , Daucus carota/microbiología , Manipulación de Alimentos/normas , Listeria monocytogenes/fisiología , Viabilidad Microbiana , Leche/microbiología , Estrés Fisiológico , TemperaturaRESUMEN
A group of 37 strains representing all 13 serotypes of Listeria monocytogenes with an initial cell density of 10(7) CFU/ml were analyzed for their heat tolerance at 60°C for 10 min. These L. monocytogenes strains were categorized into three heat tolerance groups: low (<2 log CFU/ml survival), medium (2 to 4 log CFU/ml survival), and high (4 to 6 log CFU/ml survival). Serotype 1/2a strains had relatively low heat tolerance; seven of the eight tested strains were classified as low heat tolerant. Of the two serotype 1/2b strains tested, one was very heat sensitive (not detectable) and the other was very heat resistant (5.4 log CFU/ml survival). Among the 16 serotype 4b strains, survival ranged from not detectable to 4 log CFU/ml. When one L. monocytogenes strain from each heat tolerance group was subjected to sublethal heat stress at 48°C for 30 or 60 min, the survival of heat-stressed cells at 60°C for 10 min increased by 5 log CFU/ml (D60°C-values nearly doubled) compared with the nonstressed control cells. Sublethal heat stress at 48°C for 60 or 90 min increased the lag phase of L. monocytogenes in tryptic soy broth supplemented with 0.6% yeast extract at room temperature by 3 to 5 h compared with nonstressed control cells. The heat stress adaptation in L. monocytogenes was reversed after 2 h at room temperature but was maintained for up to 24 h at 4°C. Our results indicate a high diversity in heat tolerance among strains of L. monocytogenes, and once acquired this heat stress adaptation persists after cooling, which should be taken into account while conducting risk analyses for this pathogen.
Asunto(s)
Listeria monocytogenes/química , Listeria monocytogenes/crecimiento & desarrollo , Adaptación Fisiológica , Recuento de Colonia Microbiana , Calor , Humanos , Listeria monocytogenes/fisiología , Viabilidad MicrobianaRESUMEN
Our findings show that temperature plays a significant role in the induction of acid-stress adaptation in Listeria monocytogenes, and two distinct patterns were observed: (1) Presence of sublethal acid at 37°C or 22°C significantly induced acid-stress adaptation; and (2) Presence of sublethal acid at 4°C did not induce any acid-stress adaptation. Both patterns were confirmed by two experimental models: (1) L. monocytogenes cells were first grown at 37°C and then exposed to sublethal acid at 37°C, 22°C, and 4°C prior to lethal acid challenge; (2) Alternatively, L. monocytogenes cells were first grown at 4°C for 20 days before pre-exposure to sublethal acid and then challenged with lethal acid. Regardless of whether L. monocytogenes cells were simultaneously exposed with both cold stress and sublethal acid stress, or subjected to cold growth first before exposure to sublethal acid, no acid-stress adaptation was induced at 4°C. We also found that acid-stress adaptation in L. monocytogenes did not occur in acidic whey at 4°C. Bead beating treatment prior to mild acid pre-exposure at 4°C partially induced acid adaptation in L. monocytogenes. Our findings suggest that cold temperature can prevent the risk of acid-stress adaptation in L. monocytogenes.
Asunto(s)
Ácidos/metabolismo , Adaptación Fisiológica , Frío , Listeria monocytogenes/crecimiento & desarrollo , Estrés Fisiológico , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Manipulación de Alimentos , Microbiología de Alimentos , Concentración de Iones de Hidrógeno , Cloruro de Sodio/metabolismoRESUMEN
In the present study, low concentrations of carvacrol (0.025 to 0.2%) and lauric arginate (LAE; 25 to 200 ppm) were tested at 4, 22, and 45°C in a broth model, and higher concentrations of carvacrol (0.1 to 5%) and LAE (200 to 5,000 ppm) were tested individually and in combination at 4°C in 3 different ground turkey samples (with 15, 7, and 1% fat content) for their effectiveness against a 3-strain mixture of Salmonella. A low concentration of 25 ppm of LAE or 0.025% carvacrol had no effect on Salmonella in a broth model, but their mixture showed a synergistic action by reducing 6 log cfu/mL Salmonella counts to a nondetectable level within 30 min of exposure. The US Food and Drug Administration-recommended 200 ppm of LAE was not sufficient for Salmonella reductions in ground turkey when applied internally. High concentrations of 2,000 to 5,000 ppm of LAE or 1 to 2% carvacrol were needed to reduce Salmonella counts by 2 to 5 log cfu/g in ground turkey by internal application. No specific relationship existed between fat content and LAE or carvacrol concentrations for Salmonella reductions. For example, 2,000 ppm of LAE could reduce Salmonella counts by 4 log cfu/g in 1% fat-containing turkey samples but very similar ~1.5 log cfu/g reductions in both 7 and 15% fat-containing ground turkey samples. For the total microbial load, about 2,000 ppm of LAE or 2% of carvacrol treatments were needed to achieve 2 to 3 log (P ≤ 0.05) cfu/g reductions in different turkey samples. A mixture of 1% carvacrol and 2,000 ppm of LAE exhibited a synergistic action in ground turkey containing 7% fat by reducing the Salmonella counts by 4 log cfu/g, which was not found with individual antimicrobial treatments.
Asunto(s)
Antibacterianos/farmacología , Arginina/análogos & derivados , Microbiología de Alimentos , Conservantes de Alimentos/farmacología , Carne/microbiología , Monoterpenos/farmacología , Aceites Volátiles/farmacología , Salmonella/efectos de los fármacos , Tejido Adiposo/química , Animales , Arginina/farmacología , Carga Bacteriana/efectos de los fármacos , Cimenos , Relación Dosis-Respuesta a Droga , Sinergismo Farmacológico , Almacenamiento de Alimentos , Temperatura , PavosRESUMEN
Persistence of Salmonella biofilms within food processing environments is an important source of Salmonella contamination in the food chain. In this study, essential oils of thyme and oregano and their antimicrobial phenolic constituent carvacrol were evaluated for their ability to inhibit biofilm formation and inactivate preformed Salmonella biofilms. A crystal violet staining assay and CFU measurements were utilized to quantify biofilm cell mass, with evaluating factors such as strain variation, essential oil type, their concentrations, exposure time, as well as biofilm formation surface. Of the three Salmonella strains, Salmonella Typhimurium ATCC 23564 and Salmonella Typhimurium ATCC 19585 produced stronger biofilms than Salmonella Typhimurium ATCC 14028. Biofilm formation by different Salmonella strains was 1.5- to 2-fold higher at 22°C than at 30 or 37°C. The presence of nonbiocidal concentrations of thyme oil, oregano oil, and phenolic carvacrol at 0.006 to 0.012% suppressed Salmonella spp. biofilm formation 2- to 4-fold, but could not completely eliminate biofilm formation. There was high correlation in terms of biofilm inactivation, as determined by the crystal violet-stained optical density (at a 562-nm wavelength) readings and the viable CFU counts. Reduction of biofilm cell mass was dependent on antimicrobial concentration. A minimum concentration of 0.05 to 0.1% of these antimicrobial agents was needed to reduce a 7-log CFU biofilm mass to a nondetectable level on both polystyrene and stainless steel surfaces within 1 h of exposure time.
Asunto(s)
Adhesión Bacteriana/fisiología , Biopelículas/crecimiento & desarrollo , Poliestirenos , Salmonella typhimurium/fisiología , Acero Inoxidable , Cimenos , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Manipulación de Alimentos/métodos , Monoterpenos/farmacología , Aceites Volátiles/farmacologíaRESUMEN
Plant-derived essential oils were tested for their ability to eliminate biofilms of Listeria monocytogenes on polystyrene and stainless steel surfaces. Various concentrations of essential oils were tested with different contact times on biofilms of various ages. Preliminarily screening of nine essential oils and related phenolic compounds in a disk diffusion assay revealed that thyme oil, oregano oil, and carvacrol had the highest antimicrobial activity. Further screening of these three compounds against 21 L. monocytogenes strains representing all 13 serotypes indicated some strain-specific variations in antimicrobial activity. For 1-day-old biofilms of mixed L. monocytogenes strains produced at 22°C on polystyrene microtiter plates, only 0.1% concentrations of thyme oil, oregano oil, and carvacrol were needed to eliminate 7 log CFU per well. On the stainless steel coupons, a 0.5% concentration of these compounds was adequate to completely eliminate 4-day-old biofilms at 7 log CFU per coupon. Our findings indicate that these compounds are potential candidates for elimination of L. monocytogenes biofilms on stainless steel and polystyrene surfaces.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/fisiología , Aceites Volátiles/farmacología , Poliestirenos , Acero Inoxidable , Antibacterianos/farmacología , Biopelículas/efectos de los fármacos , Recuento de Colonia Microbiana , Desinfección/métodos , Contaminación de Equipos/prevención & control , Contaminación de Alimentos/prevención & control , HumanosRESUMEN
The antimicrobial activity of various essential oils and carvacrol was determined on fresh raw catfish fillets against a 4-strain Listeria monocytogenes mixture representing serotypes 1/2b, 3b, 4b, and 4c that were predominantly isolated from catfish processing environments. Thyme oil, oregano oil and carvacrol exhibited concentration and time dependent responses in broth against L. monocytogenes; for example 0.5% concentrations resulted in 4 log CFU/mL reduction within 30 min whereas 0.1% concentrations required more than 24 h for the same level of reduction. Lemon, orange, and tangerine oils, at 0.5% showed listeriostatic effect in which 4 log CFU/mL of the initial L. monocytogenes load was unchanged at 4 °C in 10 d whereas 1% concentrations were listericidal in a time dependent manner. Apart from carvacrol, efficacy of tested essential oils in reducing L. monocytogenes and total microbial load from catfish fillet was very limited. Dipping treatment of catfish fillets in 2% carvacrol solution for 30 min at 4 °C reduced L. monocytogenes to an undetectable level from their initial load of 5 log CFU/g and reduced total microbial load from catfish fillets by approximately 5 log CFU/g. In sensory analysis trained panelist preferred control samples over 2% carvacrol treated samples implying potential limitation in applicability of carvacrol for fillet treatments.
Asunto(s)
Antibacterianos/farmacología , Microbiología de Alimentos , Conservación de Alimentos/métodos , Listeria monocytogenes/aislamiento & purificación , Monoterpenos/farmacología , Aceites Volátiles/farmacología , Animales , Carga Bacteriana , Bagres , Cimenos , Contaminación de Alimentos/análisis , Manipulación de Alimentos , Listeria monocytogenes/efectos de los fármacos , Fenoles/farmacología , Alimentos MarinosRESUMEN
Single and combined effects of three GRAS (generally recognized as safe) antimicrobials including, bacteriophage P100 (phage P100), lauric arginate (LAE), and potassium lactate-sodium diacetate mixture (PL-SD) were evaluated against Listeria monocytogenes cold growth in queso fresco cheese (QFC). The fate of phage P100 when exposed to LAE (200 ppm) or PL-SD (2.8% PL and 0.2% SD) was determined at 4°C and 30°C in a broth model. Phage P100 was found to be stable in the presence of these antimicrobial agents as plaque forming units (PFU) did not vary between control, LAE or PL-SD treatments. When 9 log CFU/ml of stationary phase cells of L. monocytogenes was exposed to these antimicrobials in tryptic soy broth, there was a 3 to 5 log CFU/ml reduction with phage P100 and a complete 9 log CFU/ml reduction with LAE but no measurable reduction with PL-SD after 24h at 4°C or 30°C. In QFC, the L. monocytogenes populations increased from the initial 3.5 log CFU/cm(2) to 7.7 log CFU/cm(2) in 28 days at 4°C. Treatment with 7.8 log PFU/cm(2) of phage P100 or 200 ppm of LAE showed strong listericidal effect initially by reducing L. monocytogenes counts by 2 to 3.5-4 log CFU/cm(2) while there was a subsequent regrowth of L. monocytogenes at 4°C. Treatment with PL-SD showed strong listeriostatic effect without decreasing L. monocytogenes counts but growth was prevented for 28 days at 4°C. Only the combined treatment of listericidal phage P100 or LAE with listeriostatic PL-SD reduced the initial L. monocytogenes counts by 2-4 log CFU/cm(2) and also kept the L. monocytogenes counts at that reduced level in QFC for 28 days at 4°C.
Asunto(s)
Antibacterianos/farmacología , Queso/microbiología , Manipulación de Alimentos , Microbiología de Alimentos/métodos , Listeria monocytogenes/efectos de los fármacos , Listeria monocytogenes/fisiología , Acetatos/farmacología , Arginina/análogos & derivados , Arginina/farmacología , Bacteriófagos/efectos de los fármacos , Bacteriófagos/fisiología , Frío , Recuento de Colonia Microbiana , Humanos , Lactatos/farmacología , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/virologíaRESUMEN
The foodborne transmission of Listeria monocytogenes requires physiological adaptation to various conditions, including the cold, osmotic, heat, acid, alkaline, and oxidative stresses, associated with food hygiene, processing, and preservation measures. We review the current knowledge on the molecular stress adaptation responses in L. monocytogenes cells as revealed through transcriptome, proteome, genetic, and physiological analysis. The adaptation of L. monocytogenes to stress exposure is achieved through global expression changes in a large number of cellular components. In addition, the cross-protection of L. monocytogenes exposed to different stress environments might be conferred through various cellular machineries that seem to be commonly activated by the different stresses. To assist in designing L. monocytogenes mitigation strategies for ready-to-eat food products, further experiments are warranted to specifically evaluate the effects of food composition, additives, preservatives, and processing technologies on the modulation of L. monocytogenes cellular components in response to specific stresses.
Asunto(s)
Perfilación de la Expresión Génica , Listeria monocytogenes/fisiología , Proteómica , Estrés Fisiológico/genética , Estrés Fisiológico/fisiología , Adaptación Fisiológica , Plásmidos de Bacteriocinas , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/prevención & control , Calor , Concentración de Iones de Hidrógeno , Listeria monocytogenes/genética , Listeriosis , Concentración Osmolar , Estrés OxidativoRESUMEN
Listeria monocytogenes is an important foodborne pathogen with a persistent ability to form biofilm matrices in the food processing environments. In this study, we have determined the ability of bacteriophage P100 to reduce L. monocytogenes cell populations under biofilm conditions by using 21 L. monocytogenes strains representing 13 different serotypes. There were considerable differences in the ability of various strains of L. monocytogenes to form biofilms, with strains of serotype 1/2a showing maximum biofilm formation. Irrespective of the serotype, growth conditions, or biofilm levels, the phage P100 treatment significantly reduced L. monocytogenes cell populations under biofilm conditions. On the stainless steel coupon surface, there was a 3.5- to 5.4-log/cm2 reduction in L. monocytogenes cells by phage treatment. These findings illustrate that phage P100 is active against a wide range of L. monocytogenes strains in biofilm conditions.
Asunto(s)
Bacteriófagos/fisiología , Biopelículas/crecimiento & desarrollo , Contaminación de Equipos/prevención & control , Industria de Procesamiento de Alimentos/normas , Listeria monocytogenes/fisiología , Acero Inoxidable , Bacteriólisis , Recuento de Colonia Microbiana , Microbiología de Alimentos , Humanos , Listeria monocytogenes/clasificación , SerotipificaciónRESUMEN
We have demonstrated the antilisterial activity of generally recognized as safe (GRAS) bacteriophage LISTEX P100 (phage P100) on the surface of raw salmon fillet tissue against Listeria monocytogenes serotypes 1/2a and 4b. In a broth model system, phage P100 completely inhibited L. monocytogenes growth at 4 degrees Celsius for 12 days, at 10 degrees Celsius for 8 days, and at 30 degrees Celsius for 4 days, at all three phage concentrations of 10(4), 10(6), and 10(8) PFU/ml. On raw salmon fillet tissue, a higher phage concentration of 10(8) PFU/g was required to yield 1.8-, 2.5-, and 3.5-log CFU/g reductions of L. monocytogenes from its initial loads of 2, 3, and 4.5 log CFU/g at 4 or 22 degrees Celsius. Over the 10 days of storage at 4 degrees Celsius, L. monocytogenes growth was inhibited by phage P100 on the raw salmon fillet tissue to as low as 0.3 log CFU/g versus normal growth of 2.6 log CFU/g in the absence of phage. Phage P100 remained stable on the raw salmon fillet tissue over a 10-day storage period, with only a marginal loss of 0.6 log PFU/g from an initial phage treatment of 8 log PFU/g. These findings illustrate that the GRAS bacteriophage LISTEX P100 is listericidal on raw salmon fillets and is useful in quantitatively reducing L. monocytogenes.
Asunto(s)
Bacteriólisis , Bacteriófagos/fisiología , Seguridad de Productos para el Consumidor , Listeria monocytogenes/crecimiento & desarrollo , Salmón/microbiología , Animales , Recuento de Colonia Microbiana , Contaminación de Alimentos/análisis , Contaminación de Alimentos/prevención & control , Manipulación de Alimentos/métodos , Conservación de Alimentos/métodos , Humanos , Alimentos Marinos/microbiología , Alimentos Marinos/normas , Temperatura , Factores de TiempoRESUMEN
The enteric pathogen Salmonella enterica serovar Typhimurium uses autoinducer-2 (AI-2) as a signaling molecule. AI-2 requires the luxS gene for its synthesis. The regulation of global gene expression in Salmonella Typhimurium by luxS/AI-2 is currently not known; therefore, the focus of this study was to elucidate the global gene expression patterns in Salmonella Typhimurium as regulated by luxS/AI-2. The genes controlled by luxS/AI-2 were identified using microarrays with RNA samples from wild-type (WT) Salmonella Typhimurium and its isogenic DeltaluxS mutant, in two growth conditions (presence and absence of glucose) at mid-log and early stationary phases. The results indicate that luxS/AI-2 has very different effects in Salmonella Typhimurium depending on the stage of cell growth and the levels of glucose. Genes with p < or = 0.05 were considered to be significantly expressed differentially between WT and DeltaluxS mutant. In the mid-log phase of growth, AI-2 activity was higher (1500-fold) in the presence of glucose than in its absence (450-fold). There was differential gene expression of 13 genes between the WT and its isogenic DeltaluxS mutant in the presence of glucose and 547 genes in its absence. In early stationary phase, AI-2 activity was higher (650-fold) in the presence of glucose than in its absence (1.5-fold). In the presence of glucose, 16 genes were differentially expressed, and in its absence, 60 genes were differentially expressed. Our microarray study indicates that both luxS and AI-2 could play a vital role in several cellular processes including metabolism, biofilm formation, transcription, translation, transport, and binding proteins, signal transduction, and regulatory functions in addition to previously identified functions. Phenotypic analysis of DeltaluxS mutant confirmed the microarray results and revealed that luxS did not influence growth but played a role in the biofilm formation and motility.
Asunto(s)
Proteínas Bacterianas/fisiología , Liasas de Carbono-Azufre/fisiología , Perfilación de la Expresión Génica , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Homoserina/análogos & derivados , Salmonella typhimurium/genética , Proteínas Bacterianas/genética , Biopelículas , Bioensayo , Transporte Biológico/genética , Liasas de Carbono-Azufre/genética , Ciclo Celular , Quimiotaxis/genética , Fimbrias Bacterianas/genética , Flagelos/genética , Glucosa/administración & dosificación , Homoserina/análisis , Homoserina/fisiología , Lactonas/análisis , Análisis de Secuencia por Matrices de Oligonucleótidos , Fenotipo , Biosíntesis de Proteínas , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Salmonella typhimurium/crecimiento & desarrollo , Salmonella typhimurium/fisiología , Transducción de Señal/genética , Transcripción GenéticaRESUMEN
Bacteriophage Listex P100 (phage P100) was approved by the U.S. Food and Drug Administration and U.S. Department of Agriculture's Food Safety and Inspection Service for Listeria monocytogenes control on both raw and ready-to-eat food products. In this article, we present the proof of concept on the influence of phage dose, phage contact time, and storage temperature on the listericidal activity of phage P100 in reducing the L. monocytogenes loads on the surface of fresh channel catfish fillet. The fresh catfish fillet samples were surface inoculated with approximately 4.3 log(10) colony forming units (CFU)/g of a two serotype mix (1/2a and 4b) of L. monocytogenes cells and then surface treated with phage P100. L. monocytogenes reduction was influenced by phage contact time and phage dose regardless of higher or lower temperature regimes tested on catfish fillet. The reduction in L. monocytogenes loads (p < 0.05) with the phage P100 dose of 2 x 10(7) plaque forming units (PFU)/g (7.3 log(10) PFU/g) was 1.4-2.0 log(10) CFU/g at 4 degrees C, 1.7-2.1 log(10) CFU/g at 10 degrees C, and 1.6-2.3 log(10) CFU/g at room temperature (22 degrees C) on raw catfish fillet. The phage contact time of 30 min was adequate to yield greater than 1 log(10) CFU/g reduction in L. monocytogenes, whereas 15 min contact time with phage yielded less than 1 log(10) CFU/g reduction in L. monocytogenes loads on catfish fillet. Phage P100 titer was stable on catfish fillet samples, and overall reductions in L. monocytogenes counts were still maintained over a 10-day shelf life at 4 degrees C or 10 degrees C by phage P100 treatment. These findings illustrate the effectiveness of an alternative generally recognized as safe antimicrobial such as bacteriophage Listex P100 in quantitatively reducing L. monocytogenes from fresh catfish fillet surfaces.
Asunto(s)
Productos Pesqueros/microbiología , Microbiología de Alimentos , Ictaluridae/microbiología , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/virología , Myoviridae/fisiología , Animales , Bacteriólisis , Recuento de Colonia Microbiana , Manipulación de Alimentos/métodos , Myoviridae/crecimiento & desarrollo , Propiedades de Superficie , Temperatura , Factores de Tiempo , Ensayo de Placa ViralRESUMEN
Deliberate or accidental contamination of foods such as milk, soft drinks, and drinking water with infectious agents or toxins is a major concern to health authorities. There is a critical need to develop technologies that can rapidly and efficiently separate and concentrate biothreat agents from food matrices. A key limitation of current centrifugation and filtration technologies is that they are batch processes with extensive hands-on involvement and processing times. The objective of our studies was to evaluate the continuous flow centrifugation (CFC) technique for the rapid separation and concentration of bacterial spores from large volumes of milk. We determined the effectiveness of the CFC technology for concentrating approximately 10(3) bacterial spores in 3.7 liters (1 gal) of whole milk and skim milk, using Bacillus subtilis, Bacillus atrophaeus, and Clostridium sporogenes spores as surrogates for biothreat agents. The spores in the concentrated samples were enumerated by using standard plating techniques. Three independent experiments were performed at 10,000 rpm and 0.7 liters/min flow rate. The mean B. subtilis spore recoveries were 71.3 and 56.5% in skim and whole milk, respectively, and those for B. atrophaeus were 55 and 59.3% in skim and whole milk, respectively. In contrast, mean C. sporogenes spore recoveries were 88.2 and 78.6% in skim and whole milk, respectively. The successful use of CFC to concentrate these bacterial spores from 3.7 liters of milk in 10 min shows promise for rapidly concentrating other spores from large volumes of milk.
Asunto(s)
Bacillus/aislamiento & purificación , Centrifugación/métodos , Clostridium/aislamiento & purificación , Contaminación de Alimentos/análisis , Leche/microbiología , Esporas Bacterianas/aislamiento & purificación , Animales , Recuento de Colonia Microbiana , Seguridad de Productos para el Consumidor , Análisis de Inyección de Flujo , HumanosRESUMEN
Cell-to-cell communications in bacteria mediated by small diffusible molecules termed as autoinducers (AI) are known to influence gene expression and pathogenicity. Oligopeptides and N-acylhomoserine lactones (AHL) are major AI molecules involved in intra-specific communication in gram-positive and gram-negative bacteria respectively, whereas boronated-diester molecules (AI-2) are involved in inter-specific communication among both gram-positive and gram-negative bacteria. Naturally occurring furocoumarins from grapefruit showed >95% inhibition of AI-1 and AI-2 activities based on the Vibrio harveyi based autoinducer bioassay. Grapefruit juice and furocoumarins also inhibited biofilm formation by Escherichia coli O157:H7, Salmonella typhimurium and Pseudomonas aeruginosa. These results suggest that grape fruit juice and furocoumarins could serve as a source to develop bacterial intervention strategies targeting microbial cell signaling processes.
Asunto(s)
Biopelículas/crecimiento & desarrollo , Contaminación de Alimentos/prevención & control , Furocumarinas/farmacología , Bacterias Gramnegativas/fisiología , Bacterias Grampositivas/fisiología , Análisis de Varianza , Bebidas , Biomasa , Citrus paradisi , Recuento de Colonia Microbiana , Escherichia coli O157/efectos de los fármacos , Escherichia coli O157/fisiología , Microbiología de Alimentos , Furocumarinas/aislamiento & purificación , Bacterias Gramnegativas/efectos de los fármacos , Bacterias Grampositivas/efectos de los fármacos , Pseudomonas aeruginosa/efectos de los fármacos , Pseudomonas aeruginosa/fisiología , Percepción de Quorum , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/fisiología , Transducción de SeñalRESUMEN
Salmonella Typhimurium is known to exhibit LuxS/AI-2-mediated cell signaling. We investigated the role of LuxS/AI-2 system on Salmonella Typhimurium protein expression using a proteomics approach based on two-dimensional gel electrophoresis (2DGE)-MALDI-MS. The global protein expression profiles of the wild-type, a luxS mutant, and a luxS mutant strain supplemented with AI-2 were compared. Seven proteins were differentially expressed when comparing the wild-type and luxS mutant strains, whereas 13 proteins were differentially expressed when comparisons were made between luxS mutant strains with and without AI-2 supplementation. The seven proteins that were differentially expressed between the wild-type and the luxS mutant strain were also differentially expressed in the luxS mutant strain supplemented with AI-2. The level of PhoP, a virulence determinant, was higher in the presence of AI-2. Proteins associated with the carbohydrate metabolism (pfkA, gpmI, and talB) and ATP synthesis (Pta gene product) were up-regulated by the presence of AI-2 molecules. These results provide experimental evidence that AI-2 molecules regulate a variety of cellular processes in Salmonella Typhimurium.
Asunto(s)
Proteínas Bacterianas/análisis , Liasas de Carbono-Azufre/fisiología , Homoserina/análogos & derivados , Proteómica , Salmonella typhimurium/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Bioensayo , Metabolismo de los Hidratos de Carbono , Liasas de Carbono-Azufre/genética , Electroforesis en Gel Bidimensional , Regulación Bacteriana de la Expresión Génica , Homoserina/biosíntesis , Homoserina/genética , Homoserina/fisiología , Lactonas , Mutación , Salmonella typhimurium/genética , Salmonella typhimurium/patogenicidad , Transducción de Señal , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Transcripción Genética , Regulación hacia Arriba , Virulencia/genéticaRESUMEN
Autoinducer-2 (AI-2) molecules are used by several microorganisms to modulate various processes, including bioluminescence, biofilm formation, and virulence expression. Certain food matrices, including ground beef extracts, possess compounds capable of inhibiting AI-2 activity. In the present study, we identified and characterized these AI-2 inhibitors from ground beef extract using hexane solvent extraction and gas chromatography. Gas chromatographic analysis revealed the presence of several fatty acids such as palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:omega9), and linoleic acid (C18:omega6) that were capable of inhibiting AI-2 activity. These fatty acids were tested (using Vibrio harveyi BB170 and MM32 reporter strains) at different concentrations (1, 5, and 10 mM) to identify differences in the level of AI-2 activity inhibition. AI-2 inhibition ranged from 25 to 90%. A mixture of these fatty acids (prepared at concentrations equivalent to those present in the ground beef extract) produced 52 to 65% inhibition of AI-2 activity. The fatty acid mixture also negatively influenced Escherichia coli K-12 biofilm formation. These results demonstrate that both medium- and long-chain fatty acids in ground beef have the ability to interfere with AI-2-based cell signaling.
Asunto(s)
Ácidos Grasos/farmacología , Regulación Bacteriana de la Expresión Génica , Homoserina/análogos & derivados , Lactonas/antagonistas & inhibidores , Productos de la Carne/microbiología , Transducción de Señal , Animales , Biopelículas , Bovinos , Escherichia coli K12/efectos de los fármacos , Escherichia coli K12/crecimiento & desarrollo , Escherichia coli K12/patogenicidad , Homoserina/antagonistas & inhibidores , Humanos , Productos de la Carne/análisis , Percepción de Quorum , Vibrio/efectos de los fármacos , Vibrio/crecimiento & desarrollo , Vibrio/patogenicidadRESUMEN
The zeta potentials of E. coli, GFP (green fluorescence protein)-labeled E. coli, Salmonella Newport, and Pseudomonas sp. in different states (nutrient-starved and dead) and grown in rich and minimal media were measured. Capillary electrophoresis experiments were conducted to measure the zeta potential of the different cells suspended in a drinking water sample. Salmonella Newport strain showed a lower zeta potential compared to E. coli, GFP-labeled E. coli, and Pseudomonas sp. Starved E. coli cells had a lower zeta potential compared to E. coli cells grown under rich media conditions. Salmonella Newport cells grown in minimal media also had a lower zeta potential compared to rich, starved, and dead cells. The different bacterial cell types exhibited differences in size as well. These results suggest that when bacterial cells are present in drinking water they can exhibit significant heterogeneity in the size and zeta potential, depending on their physiological state.
Asunto(s)
Escherichia coli/fisiología , Agua Dulce/microbiología , Pseudomonas/fisiología , Salmonella/fisiología , Electricidad Estática , Electroforesis CapilarRESUMEN
A microfluidic device based on electrophoretic transport and electrostatic trapping of charged particles has been developed for continuous capture and concentration of microorganisms from water. Reclaimed and bottled water samples at pH values ranging from 5.2-6.5 were seeded with bacteria (E. coli, Salmonella, and Pseudomonas) and viruses (MS-2 and Echovirus). Negative control and capture experiments were performed simultaneously using two identical devices. Culture based methods were utilized to characterize the capture efficiency as a function of the species type, time, flow rate, and applied electric field. Based on differences between the capture and negative control data, capture efficiencies of 90% to 99% are reported for E. coli, Salmonella, Pseudomonas, and MS-2, while the capture efficiency for Echovirus was between 70% and 80%. Overall, the device exhibits a 16.67 fold sample volume reduction within an hour at 6 mL h(-1) flow rate, resulting in a concentration factor of 14.2 at 85.2% capture efficiency. The device can function either as a filter or a sample concentrator without using any chemical additives. It can function as an integral component of a continuous, microbial capture and concentration system from large volumes of potable water.