RESUMEN
The use of direct nucleic acid amplification of pathogens from food matrices has the potential to reduce time to results over DNA extraction-based approaches as well as traditional culture-based approaches. Here we describe protocols for assay design and experiments for direct amplification of foodborne pathogens in food sample matrices using loop-mediated isothermal amplification (LAMP) and polymerase chain reaction (PCR). The examples provided include the detection of Escherichia coli in milk samples and Salmonella in pork meat samples. This protocol includes relevant reagents and methods including obtaining target sequences, assay design, sample processing, and amplification. These methods, though used for specific example matrices, could be applied to many other foodborne pathogens and sample types.
Asunto(s)
ADN Bacteriano , Microbiología de Alimentos , Leche , Técnicas de Amplificación de Ácido Nucleico , Reacción en Cadena de la Polimerasa , Salmonella , Técnicas de Amplificación de Ácido Nucleico/métodos , Microbiología de Alimentos/métodos , Animales , Leche/microbiología , Salmonella/genética , Salmonella/aislamiento & purificación , ADN Bacteriano/genética , ADN Bacteriano/aislamiento & purificación , Reacción en Cadena de la Polimerasa/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Técnicas de Diagnóstico Molecular/métodos , PorcinosRESUMEN
Foodborne pathogens continue to be a major health concern worldwide. Culture-dependent methodologies are still considered the gold standard to perform pathogen detection and quantification. These methods present several drawbacks, such as being time-consuming and labor intensive. The implementation of real-time PCR has allowed to overcome these limitations, and even reduce the cost associated with the analyses, due to the possibility of simultaneously and accurately detecting several pathogens in one single assay, with results comparable to those obtained by classical approaches. In this chapter, a protocol for the simultaneous detection of two of the most important foodborne pathogens, Salmonella spp. and Listeria monocytogenes, is described.
Asunto(s)
Microbiología de Alimentos , Enfermedades Transmitidas por los Alimentos , Listeria monocytogenes , Reacción en Cadena de la Polimerasa Multiplex , Salmonella , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Microbiología de Alimentos/métodos , Salmonella/genética , Salmonella/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/diagnóstico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Humanos , ADN Bacteriano/genética , ADN Bacteriano/análisisRESUMEN
BACKGROUND: Foodborne diseases are a growing public health concern worldwide and households are a common setting. This study aimed to explore the epidemiological characteristics of household foodborne disease outbreaks in Zhejiang Province and propose targeted prevention and control measures. METHODS: Descriptive statistical methods were used to analyze household foodborne disease outbreak data collected from the Foodborne Disease Outbreaks Surveillance System in Zhejiang Province from 2010 to 2022. RESULTS: Household foodborne disease outbreaks showed an upward trend during the study period (Cox-Staurt trend test, p = 0.01563 < 0.05). These outbreaks mainly occurred from June to September, with 62.08% (352/567) of all reported outbreaks. The number of reported outbreaks varied in 11 prefectures, with a maximum of 100 and a minimum of only 7. Household foodborne disease outbreaks had a wide spectrum of etiologic factors. Mushroom toxins accounted for the largest proportion of all etiologies (43.39 %) and caused the highest proportion of hospitalization (54.18%) and death (78.26%). Such outbreaks are caused by accidently eating wild poisonous mushrooms. Bacterial infection (16.23%) was the second most common etiology, with Salmonella spp. and Vibrio parahaemolyticus being the primary pathogens. These outbreaks were caused by improper storage, improper processing or a combination of factors, and the foods involved were mainly aquatic animals, eggs and cooked meat. Other identified etiologies included plant toxins (9.52%), chemicals (7.23%), animal toxins (3.70%), and viruses (1.76%). Among the above-mentioned etiologies, mushroom toxins, bacteria, and animal toxins had seasonal characteristics. Analysis of regions and etiologies revealed that the proportion of various etiologies was different in 11 prefectures. Wild mushrooms (43.39%), aquatic animals (9.88%), and toxic plants (8.47%) were the top three foods involved in these outbreaks. The most common factors contributing to household foodborne disease outbreaks were inedibility and misuse (59.08%), followed by multiple factors (7.58%), improper storage (7.41%), and improper processing (7.41%). CONCLUSIONS: Household foodborne disease outbreaks were closely related to the lack of knowledge regarding foodborne disease prevention. Therefore, public health agencies should strengthen residents' surveillance and health education to improve food safety awareness and effectively reduce foodborne diseases in households. In addition, timely publicity and early warning by relevant government departments, the introduction of standards to control the contamination of pathogenic bacteria in raw materials, and strengthened supervision of the sale of substances that may cause health hazards, such as poisonous mushrooms and nitrites, will also help reduce such outbreaks.
Asunto(s)
Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos , China/epidemiología , Humanos , Enfermedades Transmitidas por los Alimentos/epidemiología , Enfermedades Transmitidas por los Alimentos/microbiología , Composición Familiar , Contaminación de Alimentos/análisis , Contaminación de Alimentos/estadística & datos numéricos , Vibrio parahaemolyticus/aislamiento & purificación , Salmonella/aislamiento & purificación , AnimalesRESUMEN
Salmonella is a major bacterial concern for public health globally. Although there are limited documentation on the prevalence of Salmonella species in Cambodia's food chain, some reports indicate that salmonellosis is a severe gastrointestinal infection in its population and especially in children. To investigate the presence of Salmonella spp., 285 food samples (75 meat, 50 seafood, and 160 leafy green vegetable samples) were randomly collected from various local markets in Phnom Penh capital and nearby farms in Cambodia. Concurrently, field observations were conducted to collect data on food hygiene and practices among the relevant actors. All food samples were analyzed using bacterial culture and plate counts, and the findings were confirmed serially with biochemical, serological, and PCR tests. The observational data on food hygiene and practices from farm to market revealed that the spread of Salmonella in the food-value chain from farm to market could pose health risks to consumers. The overall prevalence of Salmonella spp. was 48.4% (138/285), while the prevalence in meat, seafood, and vegetables was 71% (53/75), 64% (32/50), and 33% (53/160), respectively. Mean Salmonella plate count ranged from 1.2 to 7.40 log10 CFU/g, and there was no significant difference in bacterial counts between meat, seafood, and vegetable samples (p > 0.05). The most common serogroups among the isolated Salmonella spp. were B and C. These results suggest that a large proportion of meat, seafood, and vegetable products sold at local markets in Phnom Penh are contaminated with Salmonella spp. This is likely linked to inadequate hygiene and sanitation practices, including handling, storage, and preservation conditions. Observations on farms suggested that the prevalence of Salmonella in vegetables sold at the market could be linked to contamination relating to agricultural practices. Thus, controlling the spread of foodborne salmonellosis through the food-value chain from farms and retailers to consumers is warranted to enhance food safety in Cambodia.
Asunto(s)
Granjas , Contaminación de Alimentos , Carne , Salmonella , Alimentos Marinos , Verduras , Cambodia/epidemiología , Verduras/microbiología , Salmonella/aislamiento & purificación , Salmonella/clasificación , Contaminación de Alimentos/análisis , Contaminación de Alimentos/estadística & datos numéricos , Prevalencia , Alimentos Marinos/microbiología , Carne/microbiología , Animales , Microbiología de Alimentos , Humanos , HigieneRESUMEN
Objective: To investigate the cause of a foodborne outbreak that occurred in Dong Nai province, Viet Nam, in 2024, and implement control measures. Methods: An initial investigation was conducted to confirm the outbreak, which was followed by epidemiological and environmental investigations to find the plausible causative food item. Clinical specimens and food samples were tested to identify the pathogen. Results: A total of 547 symptomatic cases were recorded, of whom two were in severe condition requiring extracorporeal membrane oxygenation and ventilation, one of whom died. Among 99 interviewed cases, the mean incubation time was 9 hours (range 2-24 hours), with the main symptoms being fever, abdominal pain, diarrhoea and vomiting. All patients had eaten banh mi from a local bakery. Salmonella spp. were identified in food samples and clinical specimens. The bakery halted production, and the outbreak ended after 1 week. Discussion: All the patients were exposed to only one food in common, which facilitated the investigation process. This outbreak is a reminder to small retailers and take-away shops of the importance of food safety management in preventing similar future outbreaks. All food handlers must comply with food hygiene principles, especially in hot temperatures, which boosts bacterial growth.
Asunto(s)
Brotes de Enfermedades , Intoxicación Alimentaria por Salmonella , Humanos , Vietnam/epidemiología , Masculino , Adulto , Femenino , Intoxicación Alimentaria por Salmonella/epidemiología , Intoxicación Alimentaria por Salmonella/microbiología , Persona de Mediana Edad , Preescolar , Niño , Adolescente , Lactante , Salmonella/aislamiento & purificación , Adulto Joven , Enfermedades Transmitidas por los Alimentos/epidemiología , Enfermedades Transmitidas por los Alimentos/microbiología , Microbiología de Alimentos , AncianoRESUMEN
Large-scale poultry production in low- and middle-income countries may be a source of adulterated products (e.g., Salmonella contamination, antibiotic residues) that can be disseminated over wide areas. We employed a cross-sectional survey of 199 randomly selected poultry farms in Lagos State, Nigeria, to estimate the prevalence of non-typhoidal Salmonella (NTS), and biosecurity and antibiotic use practices. Pooled fecal samples were collected from laying chickens and from poultry handlers. Selective culture, biochemical assays, and PCR (invA) were used to isolate and confirm NTS isolates. NTS was detected at 14% of farms (28/199) and from 10% of farm workers (6/60). Multivariate logistic regression analysis indicated that antiseptic foot dips reduced the odds ratio (OR) for detecting NTS in chicken feces [OR: 0.55; 95% confidence interval (CI) 0.07-0.58]. Most farms (94.5%, 188/199) used antibiotics for treatment and prophylaxis, but no farms (0/199) exercised withdrawal before sale of products. Most farms (86.4%, 172/199) reported using antibiotic cocktails that included medically important colistin, ciprofloxacin, chloramphenicol, and gentamicin. Egg production in Lagos State relies heavily on antibiotics and antibiotic residues are likely passed to consumers through poultry products, but there is evidence that low-cost biosecurity controls are effective for limiting the presence of NTS on farms.
Asunto(s)
Antibacterianos , Pollos , Enfermedades de las Aves de Corral , Salmonella , Animales , Nigeria/epidemiología , Antibacterianos/farmacología , Salmonella/aislamiento & purificación , Salmonella/efectos de los fármacos , Pollos/microbiología , Enfermedades de las Aves de Corral/microbiología , Enfermedades de las Aves de Corral/prevención & control , Estudios Transversales , Salmonelosis Animal/prevención & control , Salmonelosis Animal/epidemiología , Heces/microbiología , Aves de Corral/microbiología , Crianza de Animales Domésticos/métodos , Humanos , Granjas , PrevalenciaRESUMEN
BACKGROUND: Foodborne diseases (FBDs) represent a significant risk to public health, with nearly one in ten people falling ill every year globally. The large incidence of foodborne diseases in African low- and middle-income countries (LMIC) shows the immediate need for action, but there is still far to a robust and efficient outbreak detection system. The detection of outbreak heavily relies on clinical diagnosis, which are often delayed or ignored due to resource limitations and inadequate surveillance systems. METHODS: In total, 68 samples of non-typhoidal Salmonella isolates from human, animal and environmental sources collected between November 2021 and January 2023 were analyzed using sequencing methods to infer phylogenetic relationships between the samples. A source attribution model using a machine-learning logit-boost that predicted the likely source of infection for 20 cases of human salmonellosis was also run and compared with the results of the cluster detection. RESULTS: Three clusters of samples with close relation (SNP difference < 30) were identified as non-typhoidal Salmonella in Harar town and Kersa district, Ethiopia. These three clusters were comprised of isolates from different sources, including at least two human isolates. The isolates within each cluster showed identical serovar and sequence type (ST), with few exceptions in cluster 3. The close proximity of the samples suggested the occurrence of three potential outbreaks of non-typhoidal Salmonella in the region. The results of the source attribution model found that human cases of salmonellosis could primarily be attributed to bovine meat, which the results of the phylogenetic analysis corroborated. CONCLUSIONS: The findings of this study suggested the occurrence of three possible outbreaks of non-typhoidal Salmonella in eastern Ethiopia, emphasizing the importance of targeted intervention of food safety protocols in LMICs. It also highlighted the potential of integrated surveillance for detecting outbreak and identifying the most probable source. Source attribution models in combination with other epidemiological methods is recommended as part of a more robust and integrated surveillance system for foodborne diseases.
Asunto(s)
Brotes de Enfermedades , Enfermedades Transmitidas por los Alimentos , Filogenia , Infecciones por Salmonella , Salmonella , Humanos , Etiopía/epidemiología , Salmonella/genética , Salmonella/aislamiento & purificación , Salmonella/clasificación , Enfermedades Transmitidas por los Alimentos/microbiología , Enfermedades Transmitidas por los Alimentos/epidemiología , Animales , Infecciones por Salmonella/epidemiología , Infecciones por Salmonella/microbiologíaRESUMEN
Salmonella-related foodborne illness is a significant public health concern, with the primary source of human infection being animal-based food products, particularly chicken meat. Lebanon is currently experiencing a dual crisis: the COVID-19 pandemic and an unprecedented economic crisis, which has resulted in substantial challenges to the public health system and food safety. This study aims to assess the prevalence and antibiotic resistance profile of Salmonella in raw poultry meat sold in North Lebanon during this dual crisis. A cross-sectional study was carried out between May 2021 and April 2022 across six different districts in North Lebanon. A total of 288 whole, unprocessed chickens were examined. The isolation and identification of Salmonella isolates were done based on cultural and biochemical properties. All isolates were subjected to antimicrobial susceptibility testing and phenotypic assays for Extended-Spectrum Beta-lactamase (ESBL) detection. The prevalence of Salmonella in raw poultry meat purchased in North Lebanon reached 18.05â¯% (52/288). The dry season and chilled chicken were significantly associated with an increased risk of Salmonella contamination (P < 0.05). Additionally, 34.61â¯% of the isolates were potential ESBL producers, and 57.69â¯% exhibited multidrug resistance (MDR). This study highlights the existence of MDR in chicken meat in North Lebanon, posing a potential health risk if undercooked chicken meat is consumed. This emphasizes the importance of the implementation of preventive strategies and hygienic procedures throughout the food chain to reduce the risk of Salmonella spp. contamination in chicken meats and its potential transmission to humans.
Asunto(s)
COVID-19 , Pollos , Salmonella , Animales , Líbano/epidemiología , Salmonella/efectos de los fármacos , Salmonella/aislamiento & purificación , Estudios Transversales , Prevalencia , COVID-19/epidemiología , COVID-19/prevención & control , Carne/microbiología , Recesión Económica , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , SARS-CoV-2 , Microbiología de Alimentos , Enfermedades de las Aves de Corral/epidemiología , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/epidemiología , Salmonelosis Animal/microbiologíaRESUMEN
Concerns are rising about the contamination of recreational waters from human and animal waste, along with associated risks to public health. However, existing guidelines for managing pathogens in these environments have not yet fully integrated risk-based pathogen-specific criteria, which, along with recent advancements in indicators and markers, are essential to improve the protection of public health. This study aimed to establish risk-based critical concentration benchmarks for significant enteric pathogens, i.e., norovirus, rotavirus, adenovirus, Cryptosporidium spp., Giardia lamblia, Campylobacter jejuni, Salmonella spp., and Escherichia coli O157:H7. Applying a 0.036 risk benchmark to both marine and freshwater environments, the study identified the lowest critical concentrations for children, who are the most susceptible group. Norovirus, C. jejuni, and Cryptosporidium presented lowest median critical concentrations for virus, bacteria, and protozoa, respectively: 0.74 GC, 1.73 CFU, and 0.39 viable oocysts per 100 mL in freshwater for children. These values were then used to determine minimum sample volumes corresponding to different recovery rates for culture method, digital polymerase chain reaction and quantitative PCR methods. The results indicate that for children, norovirus required the largest sample volumes of freshwater and marine water (52.08 to 178.57 L, based on the 5th percentile with a 10 % recovery rate), reflecting its low critical concentration and high potential for causing illness. In contrast, adenovirus and rotavirus required significantly smaller volumes (approximately 0.24 to 1.33 L). C. jejuni and Cryptosporidium, which required the highest sampling volumes for bacteria and protozoa, needed 1.72 to 11.09 L and 4.17 to 25.51 L, respectively. Additionally, the presented risk-based framework could provide a model for establishing pathogen thresholds, potentially guiding the creation of extensive risk-based criteria for various pathogens in recreational waters, thus aiding public health authorities in decision-making, strengthening pathogen monitoring, and improving water quality testing accuracy for enhanced health protection.
Asunto(s)
Cryptosporidium , Monitoreo del Ambiente , Microbiología del Agua , Monitoreo del Ambiente/métodos , Humanos , Cryptosporidium/aislamiento & purificación , Norovirus/aislamiento & purificación , Agua Dulce/virología , Medición de Riesgo/métodos , Giardia lamblia/aislamiento & purificación , Recreación , Agua de Mar/virología , Campylobacter jejuni/aislamiento & purificación , Rotavirus/aislamiento & purificación , Salmonella/aislamiento & purificaciónRESUMEN
Nontyphoidal Salmonella (NTS) is the main etiological agent of human nontyphoidal salmonellosis. The aim of this study was to analyze the epidemiological characteristics and horizontal transfer mechanisms of antimicrobial resistance (AMR) genes from eight strains of NTS detected in Zhenjiang City, Jiangsu Province, China. Fecal samples from outpatients with food-borne diarrhea were collected in 2022. The NTS isolates were identified, and their susceptibility was tested with the Vitek 2 Compact system. The genomes of the NTS isolates were sequenced with the Illumina NovaSeq platform and Oxford Nanopore Technologies platform. The AMR genes and mobile genetic elements (MGEs) were predicted with the relevant open access resources. Eight strains of NTS were isolated from 153 specimens, and Salmonella Typhimurium ST19 was the most prevalent serotype. The AMR gene with the highest detection rate was AAC(6')-Iaa (10.5%) followed by TEM-1 (7.9%), sul2 (6.6%), and tet(A) (5.3%). Eleven MGEs carrying 34 AMR genes were identified on the chromosomes of 3 of the 8 NTS, including 3 resistance islands, 6 composite transposons (Tns), and 2 integrons. Eighteen plasmids carrying 40 AMR genes were detected in the 8 NTS strains, including 6 mobilizable plasmids, 3 conjugative plasmids, and 9 nontransferable plasmids, 7 of which carried 10 composite Tns and 3 integrons. This study provided a theoretical basis, from a genetic perspective, for the prevention and control of NTS resistance in Zhenjiang City. IMPORTANCE: Human nontyphoidal salmonellosis is one of the common causes of bacterial food-borne illnesses, with significant social and economic impacts, especially those caused by invasive multidrug-resistant nontyphoidal Salmonella, which entails high morbidity and mortality. Antimicrobial resistance is mainly mediated by drug resistance genes, and mobile genetic elements play key roles in the capture, accumulation, and dissemination of antimicrobial resistance genes. Therefore, it is necessary to study the epidemiological characteristics and horizontal transfer mechanisms of antimicrobial resistance genes of nontyphoidal Salmonella to prevent the spread of multidrug-resistant nontyphoidal Salmonella.
Asunto(s)
Secuencias Repetitivas Esparcidas , Infecciones por Salmonella , Salmonella , Humanos , Salmonella/genética , Salmonella/efectos de los fármacos , Salmonella/aislamiento & purificación , Secuencias Repetitivas Esparcidas/genética , Infecciones por Salmonella/microbiología , Infecciones por Salmonella/epidemiología , Antibacterianos/farmacología , China/epidemiología , Farmacorresistencia Bacteriana/genética , Pruebas de Sensibilidad Microbiana , Transferencia de Gen Horizontal , Genoma Bacteriano/genética , Plásmidos/genética , Genómica , Heces/microbiologíaRESUMEN
Salmonella spp. infections in animals are a concern due to their zoonotic nature, welfare effects and economic impact on the livestock industry. To enable targeted surveillance, it is important to identify risk factors for the introduction of Salmonella spp. in a herd. Since 2009, Dutch dairy processors require herds delivering milk to their plants to participate in a Salmonella programme. In this programme, bulk milk is tested three times a year (i.e. test rounds) by ELISA on presence of antibodies against Salmonella spp. serogroups B and D. Based on these bulk milk results we identified newly infected herds, and aimed to identify associated risk factors. Effects of putative risk factors for becoming newly infected were studied using a multivariable population average logistic regression (PA-GEE) model with binomial distribution. Per test round in 2019-2021, 0.85-4.10â¯% of the Dutch dairy herds at risk became newly infected, with large regional differences. Several risk factors for becoming newly infected in the context of the low herd-level prevalence were identified. The most evident risk factors that were identified were having at least one infected or recently recovered dairy herd within 500â¯m (OR = 2.67), on-farm presence of pigs (OR = 1.63), introduction of more than 2 cattle from other herds in the previous 12 months (OR = 1.17), being in an area with a relative soil moisture of >0.54â¯% (OR = 1.31), being located in an area with a high water surface area (>2â¯%; OR = 1.14) and a larger herd size (OR = 1.65). These results indicate that, in addition to introduction of cattle, local transmission plays an important role in the between-herd transmission of Salmonella spp. Information on risk factors for becoming newly infected based on regularly collected data, can be used to improve surveillance and to implement targeted control measures against salmonellosis.
Asunto(s)
Enfermedades de los Bovinos , Industria Lechera , Salmonelosis Animal , Salmonella , Serogrupo , Animales , Bovinos , Salmonelosis Animal/epidemiología , Salmonelosis Animal/microbiología , Factores de Riesgo , Países Bajos/epidemiología , Salmonella/aislamiento & purificación , Salmonella/clasificación , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/microbiología , Prevalencia , Femenino , Leche/microbiología , Ensayo de Inmunoadsorción Enzimática/veterinariaRESUMEN
A novel bacteriophage-targeted electrochemical biosensor designed for accurate and quantitative detection of live Salmonella in food samples is presented. The biosensor is simply constructed by electrostatic immobilizing bacteriophages on MXene-nanostructured electrodes. MXene, renowned for its high surface area, biocompatibility, and conductivity, serves as an ideal platform for bacteriophage immobilization. This allows for a high-density immobilization of bacteriophage particles, achieving approximately 71 pcs µm-2. Remarkably, the bacteriophages immobilized MXene nanostructured electrodes still maintain their viability and functionality, ensuring their effectiveness in pathogen detection. Therefore, the proposed biosensor exhibited enhanced sensitivity with a low limit of detection (LOD) of 5 CFU mL-1. Notably, the biosensor shows excellent specificity in the presence of other bacteria that commonly contaminate food and can distinguish live Salmonella from a mixed population. Furthermore, it is applicable in detecting live Salmonella in food samples, which highlights its potential in food safety monitoring. This biosensor offers simplicity, convenience, and suitability for resource-limited environments, making it a promising tool for on-site monitoring of foodborne pathogenic bacteria.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Microbiología de Alimentos , Límite de Detección , Azul de Metileno , Salmonella , Electricidad Estática , Técnicas Biosensibles/métodos , Técnicas Electroquímicas/métodos , Salmonella/aislamiento & purificación , Salmonella/virología , Microbiología de Alimentos/métodos , Azul de Metileno/química , Bacteriófagos/química , Electrodos , Contaminación de Alimentos/análisis , Nanoestructuras/químicaRESUMEN
Point-of-care testing of pathogens is becoming more and more important for the prevention and control of food poisoning. Herein, a power-free colorimetric biosensor was presented for rapid detection of Salmonella using a microfluidic SlipChip for fluidic control and Au@PtPd nanocatalysts for signal amplification. All the procedures, including solution mixing, immune reaction, magnetic separation, residual washing, mimicking catalysis and colorimetric detection, were integrated on this SlipChip. First, the mixture of the bacterial sample, immune magnetic nanobeads (IMBs) and immune Au@PtPd nanocatalysts (INCs), washing buffer and H2O2-TMB chromogenic substrate were preloaded into the sample, washing and catalysis chambers, respectively. After the top layer of this SlipChip was slid to connect the sample chamber with the separation chamber, the mixture was moved back and forth through the asymmetrical split-and-recombine micromixer by using a disposable syringe to form the IMB-Salmonella-INC sandwich conjugates. Then, the conjugates were captured in the separation chamber using a magnetic field, and the top layer was slid to connect the washing chamber with the separation chamber for washing away excessive INCs. Finally, the top layer was slid to connect the catalysis chamber with the separation chamber, and the colorless substrate was catalyzed by the INCs with peroxidase-mimic activity to generate color change, followed by using a smartphone app to collect and analyze the image to determine the bacterial concentration. This all-in-one microfluidic biosensor enabled simple detection of Salmonella as low as 101.2 CFU mL-1 within 30 min and was featured with low cost, straightforward operation, and compact design.
Asunto(s)
Técnicas Biosensibles , Oro , Dispositivos Laboratorio en un Chip , Salmonella , Técnicas Biosensibles/instrumentación , Salmonella/aislamiento & purificación , Oro/química , Colorimetría/instrumentación , Técnicas Analíticas Microfluídicas/instrumentación , Platino (Metal)/química , Paladio/química , Límite de Detección , Diseño de Equipo , Peróxido de Hidrógeno/químicaRESUMEN
Food contaminated by pathogenic bacteria poses a serious threat to human health. Consequently, we used Salmonella as a model and developed an electrochemical immunosensor based on a polydopamine/CoFe-MOFs@Nafion nanocomposite for the detection of Salmonella in milk. The CoFe-MOFs exhibit good stability, large specific surface area, and high porosity. However, after modification on the electrode surface, they were prone to detachment. This issue was effectively mitigated by incorporating Nafion into the nanocomposite. A polydopamine (PDA) film was deposited onto the surface of CoFe-MOFs@Nafion through cyclic voltammetry (CV), accompanied by an investigation into the polymerization mechanism of the PDA film. PDA contains a substantial number of quinone functional groups, which can covalently bind to amino or sulfhydryl groups via Michael addition reaction or Schiff base reaction, thereby immobilizing anti-Salmonella antibodies onto the modified electrode surface. Under the optimal experimental conditions, the Salmonella concentration exhibited a good linear relationship within the range of 1.38 × 102 to 1.38 × 108 CFU mL-1, with a detection limit of 1.38 × 102 CFU mL-1. Furthermore, the constructed immunosensor demonstrated good specificity, stability, and reproducibility, offering a novel approach for the rapid detection of foodborne pathogens.
Asunto(s)
Técnicas Biosensibles , Técnicas Electroquímicas , Electrodos , Polímeros de Fluorocarbono , Oro , Indoles , Leche , Polímeros , Salmonella , Polímeros/química , Salmonella/aislamiento & purificación , Leche/microbiología , Polímeros de Fluorocarbono/química , Animales , Oro/química , Técnicas Electroquímicas/métodos , Técnicas Biosensibles/métodos , Contaminación de Alimentos/análisis , Límite de Detección , Microbiología de Alimentos , Estructuras Metalorgánicas/química , Inmunoensayo/métodos , Reproducibilidad de los Resultados , Nanocompuestos/químicaRESUMEN
The resistance of foodborne pathogens to antimicrobial agents is a potential danger to human health. Hence, establishing the status of good agricultural practices (GAPs) and the antimicrobial susceptibility of major foodborne pathogens has a significant programmatic implication in planning interventions. The objective of this study was to assess the gap in attaining GAP and estimate the prevalence and antimicrobial susceptibility profile of Salmonella in vegetable farms fertilized with animal manure in Addis Ababa, Ethiopia. A total of 81 vegetable farms from four sub-cities in Addis Ababa were visited, and 1119 samples were collected: soil (n = 271), manure (n = 375), vegetables (n = 398), and dairy cattle feces (n = 75). Additional data were collected using a structured questionnaire. Isolation of Salmonella was done using standard microbiology techniques and antimicrobial susceptibility testing was conducted using disk diffusion assays. Carriage for antimicrobial resistance genes was tested using polymerase chain reaction (PCR). Among the 81 vegetable farms visited, 24.7% used animal manure without any treatment, 27.2% used properly stored animal manure and 80.2% were easily accessible to animals. The prevalence of Salmonella was 2.3% at the sample level, 17.3% at the vegetable farm level, and 2.5% in vegetables. The highest rate of resistance was recorded for streptomycin, 80.7% (21 of 26), followed by kanamycin, 65.4% (17 of 26), and gentamicin, 61.5% (16 of 26). Multidrug resistance was detected in 61.5% of the Salmonella isolates. Vegetable farms have a gap in attaining GAPs, which could contribute to increased contamination and the transfer of antimicrobial resistance to the vegetables. The application of GAPs, including proper preparation of compost and the appropriate use of antimicrobials in veterinary practices, are recommended to reduce the emergence and spread of antimicrobial resistance.
Asunto(s)
Antibacterianos , Granjas , Estiércol , Salmonella , Verduras , Etiopía/epidemiología , Animales , Salmonella/aislamiento & purificación , Salmonella/efectos de los fármacos , Salmonella/genética , Verduras/microbiología , Estiércol/microbiología , Prevalencia , Bovinos , Antibacterianos/farmacología , Pruebas de Sensibilidad Microbiana , Fertilizantes , Microbiología del Suelo , Farmacorresistencia Bacteriana , Humanos , Heces/microbiología , AgriculturaRESUMEN
BACKGROUND: Salmonella, a foodborne pathogen poses significant threats to food safety and human health. Immunochromatographic (ICTS) sensors have gained popularity in the field of food safety due to their convenience, speed, and cost-effectiveness. However, most existing ICTS sensors rely on antibody sandwich structures which are limited by their dependence on high-quality paired antibodies and restricted sensitivity. For the first time, we combined multi-line ICTS strips with fluorescent bacterial probes to develop a label-free multi-line immunochromatographic sensor capable of detecting broad-spectrum Salmonella. Salmonella was labeled with the aggregation-induced luminescence material TCBPE, resulting in its transformation into a green fluorescent probe. RESULTS: Using this sensor, we successfully detected Salmonella typhimurium within the concentration range of 104-108 CFU/mL with a visual detection limit of 6.0 × 104 CFU/mL. Compared to single-line sensors, our multi-line sensor exhibited significantly improved fluorescence intensity resulting in enhanced detection sensitivity by 50 %. Furthermore, our developed multi-line ICTS sensor demonstrated successful detection of 18 different strains of Salmonella without any cross-reaction observed with 5 common foodborne pathogens tested. The applicability and reliability were validated using milk samples, cabbage juice samples as well and drinking water samples suggesting its potential for rapid and accurate detection of Salmonella in real-world scenarios across both the food industry and clinical settings. SIGNIFICANCE: In this experiment, we developed a TCBPE-based multiline immunochromatographic sensor. Specifically, Salmonella was labeled with the aggregation-induced luminescence material TCBPE, resulting in its transformation into a green fluorescent probe. Through the multi-line analysis system, the detection sensitivity and accuracy of the sensor are improved. In brief, the sensor does not require complex antibody labeling and paired antibodies, and only one antibody is needed to complete the detection process.
Asunto(s)
Cromatografía de Afinidad , Cromatografía de Afinidad/métodos , Cromatografía de Afinidad/instrumentación , Leche/microbiología , Leche/química , Microbiología de Alimentos , Animales , Colorantes Fluorescentes/química , Salmonella/aislamiento & purificación , Salmonella/inmunología , Contaminación de Alimentos/análisis , Límite de Detección , Salmonella typhimurium/aislamiento & purificación , Salmonella typhimurium/inmunología , Brassica/química , Brassica/microbiologíaRESUMEN
Salmonella screening is essential to avoid food poisoning. A simple, fast and sensitive colorimetric biosensor was elaborately developed for Salmonella detection on a microfluidic chip through limiting air chambers for precise air control, switching rotary valves for accurate fluid selection, a convergence-and-divergence passive micromixer and an extrusion-and-suction active micromixer for efficient fluid mixing, and immune gold@platinum palladium nanocatalysts for effective signal amplification. The mixture of bacteria, immune magnetic nanobeads and nanocatalysts was first rapidly mixed to form nanobead-bacteria-nanocatalyst conjugates and magnetically separated for enrichment. After washing with water, the conjugates were used to catalyze colorless substrate and blue product was finally analyzed using ImageJ for quantifying bacterial concentration. The finger-actuated microfluidic chip enabled designated control of designated fluids in designated places towards designated directions by simple press-release operations on designated air chambers without any external power. Under optimal conditions, this sensor could detect Salmonella at 45 CFU/mL in 25 min.
Asunto(s)
Técnicas Biosensibles , Colorimetría , Salmonella , Técnicas Biosensibles/instrumentación , Colorimetría/instrumentación , Salmonella/aislamiento & purificación , Oro/química , Dispositivos Laboratorio en un Chip , Paladio/química , Técnicas Analíticas Microfluídicas/instrumentación , Platino (Metal)/química , Nanopartículas del Metal/químicaRESUMEN
Due to its increasing occurrence in cattle farms in various countries, leading to significant economic losses in affected livestock, Salmonella enterica subspecies enterica serovar Dublin (S. Dublin) has become a highly investigated pathogen in cattle production. In Austria, there have been occasional human cases of S. Dublin as well as an increase in laboratory-confirmed cases in cattle, indicating the need for a screening programme to determine the current status in Austria. The aims of this study were, firstly, to determine the seroprevalence of S. Dublin in dairy herds through bulk milk screenings in two federal states (Salzburg, Tyrol) of Austria. Secondly, the study aimed to identify the infection status of the herds through individual animal and herd level detection, comparing microbiological, molecular and serological detection methods. The results of the study will allow the development of a sampling strategy for a surveillance programme in Austria. A total of 6973 dairy farms were tested through serological bulk milk screening. The seroprevalence for the federal state of Tyrol was 14.8â¯% and for Salzburg it was 18.2â¯%, resulting in an average seroprevalence of 16.5â¯%. At an individual animal level, 205 (11.3â¯%) animals tested positive for shedding of S. Dublin in the faeces through microbiological detection, and 268 (17.0â¯%) animals had positive values (ct value ≤ 38) by qPCR. The association between microbiological and molecular detection was statistically significant (p < 0.001), with a calculated kappa value of 0.65 ± 0.27 (p ≤ 0.001), assuming a substantial level of agreement. In 17 herds, where an individual animal tested positive for shedding of S. Dublin, environmental sampling and testing were carried out. At a herd level 16 (94.1â¯%) out of the 17 participating herds, tested positive for S. Dublin either microbiologically or by molecular assay in boot swab samples. Bulk milk samples from 14 out of the 17 participating herds were analysed for antibodies to S. Dublin and 12 samples (85.7â¯%) were positive. In total 111 (18.9â¯%) out of 587 blood samples tested positive for S. Dublin antibodies, demonstrating a statistically significant correlation (p < 0.001) both with microbiological (κ = 0.32 ± 0.49; p ≤ 0.001) and molecular (κ=0.23 ± 0.06; p ≤ 0.001) findings. It was possible to identify S. Dublin by culture from boot swabs in 14 (82.4â¯%) out of 17 herds and by molecular assay using qPCR in 15 (88.2â¯%) out of 17 herds, indicating a suitable sample type for screening on a herd level-basis for acute infections, but not for identifying chronic infections or asymptomatic carriers. Other environmental samples, such as sponge-sticks, are only suitable to a limited extent for the detection of S. Dublin. The results of this study demonstrate a moderate S. Dublin prevalence in dairy herds in the selected Austrian regions, signalling further screening and management programmes for the future.
Asunto(s)
Enfermedades de los Bovinos , Industria Lechera , Leche , Salmonelosis Animal , Animales , Bovinos , Austria/epidemiología , Salmonelosis Animal/epidemiología , Salmonelosis Animal/microbiología , Salmonelosis Animal/diagnóstico , Enfermedades de los Bovinos/epidemiología , Enfermedades de los Bovinos/microbiología , Enfermedades de los Bovinos/diagnóstico , Estudios Seroepidemiológicos , Femenino , Leche/microbiología , Salmonella/aislamiento & purificación , Heces/microbiología , Prevalencia , Monitoreo Epidemiológico/veterinariaRESUMEN
This study aimed to understand the epidemiological characteristics of Salmonella in Tibetan pigs. We isolated, identified, and examined via antimicrobial susceptibility testing on Salmonella from Tibetan pigs breeder farms and slaughterhouses in Tibet, China. A genetic evolutionary tree was constructed on the basis of whole genome sequencing (WGS). A total of 81 Salmonella isolates were isolated from 987 samples. The main serovars were Salmonella Typhimurium and Salmonella London in Tibetan pigs. The isolated Salmonella Typhimurium isolates subjected to antimicrobial susceptibility testing showed varying degrees of resistance to ß-lactams, aminoglycosides, fluoroquinolones, sulfonamides, tetracyclines, and amphenicols. WGS analysis was performed on 20 Salmonella Typhimurium isolates in Tibet (n = 10), Jiangsu (n = 10), and 205 genome sequences downloaded from the Enterobase database to reveal their epidemiological and genetic characteristics. They were divided into two clusters based on core genome single-nucleotide polymorphisms: Cluster A with 112 isolates from Tibet and other regions in China and Cluster B with 113 isolates from Jiangsu and other regions. The isolates in Cluster A were further divided into two subclusters: A-1 with 40 isolates including Tibet and A-2 with 72 isolates from other regions. Virulence factors analysis revealed that all isolates from Tibet carried adeG, but this observation was not as common in Salmonella isolates from Jiangsu and other regions of China. Antibiotic resistance genes (ARGs) analysis showed that all isolates from Tibet carried blaTEM-55 and rmtB, which were absent in Salmonella isolates from Jiangsu and other regions of China. Genetic characteristic analysis and biofilm determination indicated that the biofilm formation capabilities of the isolates from Tibet were stronger than those of the isolates from Jiangsu and other regions of China. Our research revealed the epidemic patterns and genomic characteristics of Salmonella in Tibetan pigs and provided theoretical guidance for the prevention and control of local salmonellosis.
Asunto(s)
Antibacterianos , Pruebas de Sensibilidad Microbiana , Salmonelosis Animal , Salmonella , Enfermedades de los Porcinos , Animales , Porcinos , Tibet/epidemiología , Salmonelosis Animal/epidemiología , Salmonelosis Animal/microbiología , Enfermedades de los Porcinos/microbiología , Enfermedades de los Porcinos/epidemiología , Prevalencia , Salmonella/genética , Salmonella/aislamiento & purificación , Salmonella/efectos de los fármacos , Salmonella/clasificación , Antibacterianos/farmacología , Secuenciación Completa del Genoma , Factores de Virulencia/genética , Filogenia , Salmonella typhimurium/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/aislamiento & purificación , Farmacorresistencia Bacteriana Múltiple/genética , Polimorfismo de Nucleótido Simple , Genoma BacterianoRESUMEN
Timely screening for harmful pathogens is a great challenge in emergencies where traditional culture methods suffer from long assay time and alternative methods are limited by poor accuracy and low robustness. Herein, we present a dCas9-mediated colorimetric and surface-enhanced Raman scattering (SERS) dual-signal platform (dCas9-CSD) to address this challenge. Strategically, the platform used dCas9 to accurately recognize the repetitive sequences in amplicons produced by loop-mediated isothermal amplification (LAMP), forming nucleic acid frameworks that assemble numerous bifunctional gold-platinum (Au@Pt) nanozymes into chains on the surface of streptavidin-magnetic beads (SA-MB). The collected Au@Pt converted colorless 3,3',5,5'-tetramethylbenzidine (TMB) to blue oxidized TMB (oxTMB) via its Pt shell and then enhanced the Raman signal of oxTMB by its Au core. Therefore, the presence of Salmonella could be dexterously converted into cross-validated colorimetric and SERS signals, providing more reliable conclusions. Notably, dCas9-mediated secondary recognition of amplicons reduced background signal caused by nontarget amplification, and two-round signal amplification consisting of LAMP reaction and Au@Pt catalysis greatly improved the sensitivity. With this design, Salmonella as low as 1 CFU/mL could be detected within 50 min by colorimetric and SERS modes. The robustness of dCas9-CSD was further confirmed by various real samples such as lake water, cabbage, milk, orange juice, beer, and eggs. This work provides a promising point-of-need tool for pathogen detection.