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1.
Nat Commun ; 11(1): 267, 2020 01 14.
Artículo en Inglés | MEDLINE | ID: mdl-31937772

RESUMEN

The ability to detect low numbers of microbial cells in food and clinical samples is highly valuable but remains a challenge. Here we present a detection system (called 'APC-Cas') that can detect very low numbers of a bacterial pathogen without isolation, using a three-stage amplification to generate powerful fluorescence signals. APC-Cas involves a combination of nucleic acid-based allosteric probes and CRISPR-Cas13a components. It can selectively and sensitively quantify Salmonella Enteritidis cells (from 1 to 105 CFU) in various types of samples such as milk, showing similar or higher sensitivity and accuracy compared with conventional real-time PCR. Furthermore, APC-Cas can identify low numbers of S. Enteritidis cells in mouse serum, distinguishing mice with early- and late-stage infection from uninfected mice. Our method may have potential clinical applications for early diagnosis of pathogens.


Asunto(s)
Bacterias/aislamiento & purificación , Técnicas Microbiológicas/métodos , Regulación Alostérica , Animales , Aptámeros de Nucleótidos , Sistemas CRISPR-Cas , Catálisis , ADN Bacteriano/genética , Fluorescencia , Microbiología de Alimentos , Ratones , Leche/microbiología , Reacción en Cadena en Tiempo Real de la Polimerasa , Infecciones por Salmonella/sangre , Infecciones por Salmonella/diagnóstico , Salmonella enteritidis/aislamiento & purificación
2.
Talanta ; 206: 120204, 2020 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-31514833

RESUMEN

As a rapid and facile means for foodborne bacteria detection in situ, lateral flow immunoassay (LFA) still has intrinsic limitations in the construction of the existing sandwich LFA format, e.g. screening difficulties of paired antibodies (Abs), poor stability of Ab probe, etc. Here, combined the strong affinity of antibiotic with the superior specificity of antibody molecules, a novel and robust LFA based on a dual recognition strategy and magnetic separation was designed to achieve specific and sensitive determination of Salmonella enteritidis (S. enteritidis). In this work, ampicillin (Amp), a broad-spectrum antibiotic against bacteria, was employed as an ideal Ab replacer to anchor cells of target bacteria. By coating Amp on magnetite nanoparticles (MNPs), the Amp-MNPs showed remarkable binding, separation and enrichment capacities toward bacteria even under complex sample matrices. To ensure the selectivity of this protocol, anti-S. enteritidis monoclonal antibody was then adopted as the second anchoring agent to form a sandwich complex with Amp-MNPs. Based on these facts, S. enteritidis, as low as 102-103 CFU/mL, could be detected by naked eyes in food samples. Therefore, this creative antibiotic-bacteria-antibody LFA sandwich pattern shows great application potential in the monitoring of food contamination and infectious diseases caused by pathogenic bacteria. Compared to the common paired Abs based sandwich method, the proposed approach was cost-effective, non-labor intensive, stable, sensitive and efficient.


Asunto(s)
Contaminación de Alimentos/análisis , Inmunoensayo/métodos , Separación Inmunomagnética/métodos , Salmonella enteritidis/aislamiento & purificación , Ampicilina/química , Animales , Anticuerpos Monoclonales/inmunología , Citrullus/microbiología , Jugos de Frutas y Vegetales/análisis , Jugos de Frutas y Vegetales/microbiología , Límite de Detección , Nanopartículas de Magnetita/química , Leche/microbiología , Carne de Cerdo/análisis , Carne de Cerdo/microbiología , Salmonella enteritidis/inmunología , Porcinos/microbiología
3.
Anal Bioanal Chem ; 411(29): 7899-7906, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31745614

RESUMEN

The presence of Salmonella in natural freshwater and drinking water is a leading cause of intestinal illness all over the world; thus, the detection of Salmonella in water is of great importance to public health. The objective of this study is to develop a rapid screening method for the detection of Salmonella enterica serovar Enteritidis in water involving surface-enhanced Raman spectroscopy (SERS), aptamers, and filtration. SERS offers a great alternative to traditional methods of pathogen detection, with a simplified detection assay and shortened detection time. The specific capturing and labeling of Salmonella Enteritidis are realized by a specific single-stranded DNA aptamer, which is modified with an additional chain of adenine and fluorescein (FAM) and used as presence/absence indicator of Salmonella Enteritidis. By incorporating a vacuum filtration system, bacterial cells recognized by the specific aptamer are concentrated onto a membrane. With additional filtration of gold nanoparticles, the aptamer signals were captured and used to construct a SERS mapping indicating the presence and absence of target bacterial strains with potential quantitative capability. The specificity of the method was validated by using other strains of bacteria such as Escherichia coli and Listeria monocytogenes. The sensitivity of the method goes down to 103 CFU/mL for 1 mL of sample with a total detection and analyzing time within 3 h. This study demonstrates the capability of the filtration-based SERS platform for detecting Salmonella Enteritidis in various aqueous matrices such as distilled water and rinsing water from fresh produce with high selectivity and sensitivity. Graphical abstract.


Asunto(s)
Filtración/métodos , Salmonella enteritidis/aislamiento & purificación , Espectrometría Raman/métodos , Escherichia coli/aislamiento & purificación , Límite de Detección , Listeria monocytogenes/aislamiento & purificación , Reproducibilidad de los Resultados , Microbiología del Agua
4.
PLoS Negl Trop Dis ; 13(10): e0007782, 2019 10.
Artículo en Inglés | MEDLINE | ID: mdl-31609964

RESUMEN

BACKGROUND: Salmonella Typhimurium and Enteritidis are major causes of bloodstream infection in children in sub-Saharan Africa. This study assessed evidence for their zoonotic versus human reservoir. METHODS: Index patients were children with blood culture confirmed Salmonella infection recruited during a microbiological surveillance study in Nanoro, rural Burkina between May 2013 and August 2014. After consent, their households were visited. Stool from household members and livestock (pooled samples per species) as well as drinking water were cultured for Salmonella. Isolates with identical serotype obtained from index patient and any household sample were defined as "paired isolates" and assessed for genetic relatedness by multilocus variable number tandem-repeat analysis (MLVA) and whole-genome sequencing (WGS). RESULTS: Twenty-nine households were visited for 32/42 (76.2%) eligible index patients: two households comprised two index patients each, and in a third household the index patient had a recurrent infection. Among the 32 index patients, serotypes were Salmonella Typhimurium (n = 26), Salmonella Enteritidis (n = 5) and Salmonella Freetown (n = 1). All Typhimurium isolates were sequence type (ST)313. Median delay between blood culture sampling and household visits was 13 days (range 6-26). Salmonella was obtained from 16/186 (8.6%) livestock samples (13 serotypes) and 18/290 (6.2%) household members (9 serotypes). None of the water samples yielded Salmonella. Paired Salmonella Typhimurium isolates were obtained from three households representing four index patients. MLVA types were identical in two pairs and similar in the third (consisting of two index patients and one household member). WGS showed a strong genetic relatedness with 0 to 2 core genome SNPs difference between pairs on a household level. Livestock samples did not yield any Salmonella Typhimurium or Salmonella Enteritidis, and the latter was exclusively obtained from blood culture. Other serotypes shared by human and/or livestock carriers in the same household were Salmonella Derby, Drac, Tennessee and Muenster. CONCLUSIONS/SIGNIFICANCE: The current study provides further evidence of a human reservoir for invasive non-Typhoidal Salmonella (iNTS) in sub-Saharan Africa.


Asunto(s)
Reservorios de Enfermedades/microbiología , Composición Familiar , Infecciones por Salmonella/microbiología , Salmonella/clasificación , Salmonella/aislamiento & purificación , Adolescente , Animales , Burkina Faso/epidemiología , Niño , Preescolar , Monitoreo del Ambiente , Heces/microbiología , Femenino , Humanos , Lactante , Ganado , Masculino , Tipificación de Secuencias Multilocus , Filogenia , Salmonella/genética , Infecciones por Salmonella/epidemiología , Salmonella enterica/genética , Salmonella enterica/aislamiento & purificación , Salmonella enteritidis/genética , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/genética , Salmonella typhimurium/aislamiento & purificación , Serogrupo , Microbiología del Agua , Secuenciación Completa del Genoma
5.
BMC Infect Dis ; 19(1): 806, 2019 Sep 14.
Artículo en Inglés | MEDLINE | ID: mdl-31521113

RESUMEN

BACKGROUND: Non-typhoidal salmonellae (NTS) have been associated with invasive disease, notably meningitis, in immunocompromised individuals. Infections of this nature carry high rates of morbidity and mortality. Colistin resistance in salmonellae is a rare finding, more so in an invasive isolate such as cerebrospinal fluid (CSF). Colistin resistance has important infection control implications and failure to manage this phenomenon may lead to the loss of our last line of defence against multi-drug resistant Gram-negative organisms. To our knowledge, this is the first reported clinical case of colistin-resistant Salmonella Enteritidis meningitis in South Africa. CASE PRESENTATION: We report a case of a young male patient with advanced human immunodeficiency virus (HIV) infection who presented to hospital with symptoms of meningitis. Cerebrospinal fluid (CSF) cultured a Salmonella Enteritidis strain. Antimicrobial susceptibility testing (AST) of the isolate, revealed the strain to be colistin resistant. Despite early and aggressive antimicrobial therapy, the patient succumbed to the illness after a short stay in hospital. Subsequent genomic analysis of the isolate showed no presence of the mcr genes or resistance-conferring mutations in phoPQ, pmrAB, pmrHFIJKLME/arnBCADTEF, mgrB, and acrAB genes, suggesting the presence of a novel colistin resistance mechanism. CONCLUSION: Invasive non-typhoidal salmonellae infection should be suspected in patients with advanced immunosuppression who present with clinical features of meningitis. Despite early and appropriate empiric therapy, these infections are commonly associated with adverse outcomes to the patient. Combination therapy with two active anti-Salmonella agents may be a consideration in the future to overcome the high mortality associated with NTS meningitis. Colistin resistance in clinical Salmonella isolates, although a rare finding at present, has significant public health and infection control implications. The causative mechanism of resistance should be sought in all cases.


Asunto(s)
Antibacterianos/uso terapéutico , Colistina/uso terapéutico , Farmacorresistencia Bacteriana , Seropositividad para VIH/complicaciones , Meningitis Bacterianas/complicaciones , Meningitis Bacterianas/tratamiento farmacológico , Infecciones por Salmonella/complicaciones , Infecciones por Salmonella/tratamiento farmacológico , Adulto , Antibacterianos/efectos adversos , Colistina/efectos adversos , Servicio de Urgencia en Hospital , Resultado Fatal , Escala de Coma de Glasgow , Humanos , Masculino , Salmonella enteritidis/aislamiento & purificación , Sudáfrica , Centros de Atención Terciaria
6.
Future Microbiol ; 14: 885-898, 2019 07.
Artículo en Inglés | MEDLINE | ID: mdl-31368788

RESUMEN

Aim: The aim of this study is to formulate a new single nonselective pre-enrichment medium (ELSS) that can support the concurrent growth of four major foodborne pathogens containing E. coli O157: H7, L. monocytogenes, S. aureus and S. enterica serovar Entertidis to develop a multiplex TaqMan Real-time PCR (mRT-PCR). Methods: The mRT-PCR with a new pre-enrichment was carried out for simultaneous detection and quantification of these foodborne bacteria. Results: By using mRT-PCR after 16 h pre-enrichment in ELSS, the detection limit of each pathogen was 1 CFU/25 ml contaminated milk, as well as inclusivity and exclusivity reached 100%. Conclusion: The mRT-PCR assay with pre-enrichment step is a fast and reliable technique for detecting single or multiple pathogens in food products.


Asunto(s)
Bacterias/genética , Técnicas Bacteriológicas/métodos , Microbiología de Alimentos/métodos , Enfermedades Transmitidas por los Alimentos/microbiología , Leche/microbiología , Animales , Bacterias/crecimiento & desarrollo , Bacterias/aislamiento & purificación , Medios de Cultivo , ADN Bacteriano/genética , Escherichia coli O157/genética , Escherichia coli O157/crecimiento & desarrollo , Escherichia coli O157/aislamiento & purificación , Listeria monocytogenes/genética , Listeria monocytogenes/crecimiento & desarrollo , Listeria monocytogenes/aislamiento & purificación , Reacción en Cadena de la Polimerasa , Salmonella enteritidis/genética , Salmonella enteritidis/crecimiento & desarrollo , Salmonella enteritidis/aislamiento & purificación , Sensibilidad y Especificidad , Staphylococcus aureus/genética , Staphylococcus aureus/crecimiento & desarrollo , Staphylococcus aureus/aislamiento & purificación
7.
Artículo en Inglés | MEDLINE | ID: mdl-31214517

RESUMEN

Non-typhoidal Salmonella (NTS) strains are Gram negative bacterial pathogens that are associated with foodborne illness worldwide. During the process of infection, Salmonella uses two molecular injectisomes known as Type 3 Secretion Systems (T3SS) to secrete virulence factors that are encoded by Salmonella Pathogenicity Island-1 (SPI-1) and SPI-2 into host cells. These secretion systems play a major role in virulence, as shown in various animal models, but little is known about their role in human infections. In Saudi Arabia, NTS strains frequently cause human infections but data regarding these pathogenic strains is fairly limited. The aim of this study was to characterize Salmonella human clinical isolates in Riyadh, Saudi Arabia, by determining their serotype, testing for the presence of SPI-1 and SPI-2 genes and to determine the antibiotic resistance profiles of these strains. Using the rapid Check and Trace Salmonella ™ (CTS) system our results demonstrate that S. Enteritidis and S. Typhimurium were the predominant serovars, followed by S. Livingstone, S. Kentucky and S. Poona among a list of 36 serovars reported for the first time in the country. In addition, SPI-1 genes were detected in 99% of the isolates, while the sifA gene (SPI-2) was not detected in 13.5% of the isolates. These results suggest that both the SPI-1 and SPI-2 virulence determinants are important for human infection. Moreover, we report the presence of a Multi-Drug (MDR) carbapenem resistant S. Kentucky isolate harboring the bla OXA-48 gene not reported previously in Saudi Arabia.


Asunto(s)
Proteínas Bacterianas/aislamiento & purificación , Proteínas de la Membrana/aislamiento & purificación , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/aislamiento & purificación , Serogrupo , Fiebre Tifoidea/microbiología , Factores de Virulencia/genética , Animales , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Carbapenémicos/farmacología , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple , Genoma Bacteriano , Genotipo , Humanos , Proteínas de la Membrana/genética , Salmonella enteritidis/clasificación , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/genética , Salmonella typhimurium/clasificación , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Arabia Saudita/epidemiología , Serotipificación , Sistemas de Secreción Tipo III/genética , Sistemas de Secreción Tipo III/aislamiento & purificación , Virulencia , beta-Lactamasas/genética
8.
PLoS Negl Trop Dis ; 13(6): e0007485, 2019 06.
Artículo en Inglés | MEDLINE | ID: mdl-31220112

RESUMEN

BACKGROUND: Salmonella enterica serovar Enteritidis is a cause of both poultry- and egg-associated enterocolitis globally and bloodstream-invasive nontyphoidal Salmonella (iNTS) disease in sub-Saharan Africa (sSA). Distinct, multi-drug resistant genotypes associated with iNTS disease in sSA have recently been described, often requiring treatment with fluoroquinolone antibiotics. In industrialised countries, antimicrobial use in poultry production has led to frequent fluoroquinolone resistance amongst globally prevalent enterocolitis-associated lineages. METHODOLOGY/PRINCIPAL FINDINGS: Twenty seven S. Enteritidis isolates from patients with iNTS disease and two poultry isolates, collected between 2007 and 2015 in the Ashanti region of Ghana, were whole-genome sequenced. These isolates, notable for a high rate of diminished ciprofloxacin susceptibility (DCS), were placed in the phyletic context of 1,067 sequences from the Public Health England (PHE) S. Enteritidis genome database to understand whether DCS was associated with African or globally-circulating clades of S. Enteritidis. Analysis showed four of the major S. Enteritidis clades were represented, two global and two African. All thirteen DCS isolates, containing a single gyrA mutation at codon 87, belonged to a global PT4-like clade responsible for epidemics of poultry-associated enterocolitis. Apart from two DCS isolates, which clustered with PHE isolates associated with travel to Spain and Brazil, the remaining DCS isolates, including one poultry isolate, belonged to two monophyletic clusters in which gyrA 87 mutations appear to have developed within the region. CONCLUSIONS/SIGNIFICANCE: Extensive phylogenetic diversity is evident amongst iNTS disease-associated S. Enteritidis in Ghana. Antimicrobial resistance profiles differed by clade, highlighting the challenges of devising empirical sepsis guidelines. The detection of fluoroquinolone resistance in phyletically-related poultry and human isolates is of major concern and surveillance and control measures within the region's burgeoning poultry industry are required to protect a human population at high risk of iNTS disease.


Asunto(s)
Antibacterianos/farmacología , Enfermedades Transmisibles Emergentes/epidemiología , Fluoroquinolonas/farmacología , Salmonelosis Animal/epidemiología , Infecciones por Salmonella/epidemiología , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/aislamiento & purificación , Adolescente , Animales , Niño , Preescolar , Enfermedades Transmisibles Emergentes/microbiología , Enfermedades Transmisibles Emergentes/veterinaria , Enterocolitis/epidemiología , Enterocolitis/microbiología , Enterocolitis/veterinaria , Femenino , Variación Genética , Genotipo , Ghana/epidemiología , Humanos , Lactante , Masculino , Pruebas de Sensibilidad Microbiana , Epidemiología Molecular , Filogenia , Aves de Corral , Infecciones por Salmonella/microbiología , Salmonelosis Animal/microbiología , Salmonella enteritidis/clasificación , Salmonella enteritidis/genética , Secuenciación Completa del Genoma
9.
BMJ Case Rep ; 12(5)2019 May 19.
Artículo en Inglés | MEDLINE | ID: mdl-31110064

RESUMEN

Non-typhoidal Salmonella osteomyelitis in healthy children is a very rare condition. A previously healthy 7-year-old boy presented with foot pain following a small injury. Local inflammatory signs of the foot were observed, with a normal radiograph. Two weeks before, he had had gastroenteritis. Assuming cellulitis, flucloxacillin was began. Early during admission, surgical subcutaneous abscess drainage was performed and Salmonella enteritidis was identified. According to bacterial susceptibility, antibiotherapy was changed to ceftriaxone. A foot radiograph and an MRI scan, performed on the 9th and the 12th days, revealed findings suggestive of osteomyelitis of the metaphysis of the fifth metatarsal. Clinical worsening persisted and surgical extensive debridement was needed. A 6-week antibiotic treatment was completed, with a good outcome. Osteomyelitis with a poor evolution in children with risk factors (prior gastrointestinal illness, warm weather and previous exposure to antibiotics) can raise the possibility of a non-typhoidal Salmonella infection.


Asunto(s)
Huesos Metatarsianos/microbiología , Infecciones por Salmonella/diagnóstico , Administración Intravenosa , Antibacterianos/administración & dosificación , Ceftriaxona/administración & dosificación , Niño , Desbridamiento , Humanos , Imagen por Resonancia Magnética , Masculino , Huesos Metatarsianos/diagnóstico por imagen , Huesos Metatarsianos/cirugía , Infecciones por Salmonella/tratamiento farmacológico , Salmonella enteritidis/aislamiento & purificación
10.
Talanta ; 201: 245-252, 2019 Aug 15.
Artículo en Inglés | MEDLINE | ID: mdl-31122419

RESUMEN

In this study, the coupling of magnetic enrichment of bacteria from real samples with rapid surface enhanced Raman spectroscopy (SERS) detection was reported. The selective isolation and enrichment for the model bacteria Escherichia coli (E. coli) was performed using E. coli (primary) antibody bound-magnetic gold (Fe3O4@Au) nanoparticles. Following isolation and enrichment, the rennet enzyme was used to cleave of casein modified Fe3O4/Au-PEI nanoparticles from primary antibody-bound bacteria to prevent the nanoparticle aggregation and provide the movement of bacteria on nitrocellulose membrane. In the first part of the study, optimization studies were carried out namely; the amounts of gold nanoparticles (AuNPs), polyethyleneimine coated magnetic gold (Fe3O4/Au-PEI) nanoparticles, casein and rennet enzyme. The SERS signals of DTNB (5,5'-Dithiobis(2-nitrobenzoic acid)) molecule were collected on the test line and a calibration curve was plotted by using signal intensities. The correlation between the concentration of E. coli and SERS signal was found to be linear within the range of 101-107 cfu/mL (R2 = 0.984, LOD = 0.52 cfu/mL and LOQ = 1.57 cfu/mL). The selectivity of the paper-based lateral flow immunoassay (LFIA) was examined with Bacillus subtilis (B. subtilis), Micrococcus luteus (M. luteus), Salmonella enteritidis (S. enteritidis) which did not produce any significant response compared with E. coli measurement. Finally, the developed paper-based LFIA was tested with urine and milk samples. The obtained SERS results were compared with a plate counting method results which were in a good accordance. The developed method was found as rapid and sensitive to E. coli with a total analysis time of less than 60 min.


Asunto(s)
Bacterias/aislamiento & purificación , Técnicas de Tipificación Bacteriana/métodos , Nanopartículas de Magnetita/química , Leche/microbiología , Papel , Orina/microbiología , Animales , Anticuerpos/inmunología , Bacillus subtilis/aislamiento & purificación , Caseínas/inmunología , Quimosina/química , Escherichia coli/aislamiento & purificación , Oro/química , Límite de Detección , Micrococcus luteus/aislamiento & purificación , Salmonella enteritidis/aislamiento & purificación
11.
Acta Biomed ; 90(2): 333-338, 2019 05 23.
Artículo en Inglés | MEDLINE | ID: mdl-31125015

RESUMEN

BACKGROUND: Non-typhoid Salmonella (NTS) is an important cause of bacterial meningitis in newborn and infants in developing countries, but rarely in industrialized ones. We describe an unusual presentation of bacterial meningitis in an infant, focusing on his diagnostic and therapeutic management. CASE REPORT: An Italian two-month old male presented high fever and diarrhea with blood, associated with irritability. Inflammatory markers were high, cerebrospinal fluid analysis was compatible with bacterial meningitides but microbiological investigations were negative. Salmonella enteritidis was isolated from blood. Cerebral ultrasound and MRI showed periencephalic collection of purulent material. Specific antibiotic therapy with cefotaxime was initiated with improvement of clinical conditions and blood tests. Brain MRI follow up improved progressively. CONCLUSIONS: Most of pediatric patients with NTS infection develop self-limited gastroenteritis, but in 3-8% of the cases complications such as bacteremia and meningitis may occur, especially in weak patients. Cerebral imaging can be useful to identify neurological findings. Although there is no standardized treatment for this condition, specific antibiotic therapy for at least four weeks is recommended. Neuroimaging follow up is required due to high risk of relapse.


Asunto(s)
Cefotaxima/uso terapéutico , Meningitis Bacterianas/tratamiento farmacológico , Meningitis Bacterianas/microbiología , Salmonella enteritidis/aislamiento & purificación , Estudios de Seguimiento , Humanos , Lactante , Italia , Imagen por Resonancia Magnética/métodos , Masculino , Meningitis Bacterianas/diagnóstico , Enfermedades Raras , Medición de Riesgo , Infecciones por Salmonella/diagnóstico , Infecciones por Salmonella/tratamiento farmacológico , Tomografía Computarizada por Rayos X/métodos , Resultado del Tratamiento
12.
Br Poult Sci ; 60(4): 388-394, 2019 Aug.
Artículo en Inglés | MEDLINE | ID: mdl-31066296

RESUMEN

1. Salmonella is one of the most important pathogens in public health and it is usually associated with food-borne diseases. Salmonella serovars Enteritidis and Typhimurium are widespread in the world with outbreaks frequently associated with consumption of poultry products; furthermore, there is an increasing public health concern with the wide dissemination of the serovar Heidelberg in poultry flocks. 2. The aim of the experiment was to develop and to validate rapid methods to detect Salmonella serovars Enteritidis, Typhimurium, and Heidelberg by real-time PCRs and test isolates from pre-enriched poultry samples. 3. Three real-time PCRs were developed and used in combination to detect the serovars Enteritidis, Typhimurium and Heidelberg. These assays were validated by the analysis of 126 Salmonella isolates, eight other enteric bacterial species and 34 naturally contaminated poultry samples after pre-enrichment with buffered peptone water (BPW). 4. Real-time PCRs detected the isolates of the most important poultry serovars (Enteritidis, Typhimurium and Heidelberg) with 100% inclusivity and exclusivity in each assay. The PCR identified monophasic variants of the serovars Typhimurium and Heidelberg. All PCRs were validated in detecting these specific serovars directly from pre-enriched poultry samples. The whole analytical procedure was performed in less than 24 h in a veterinary diagnostic laboratory.


Asunto(s)
Técnicas Bacteriológicas/métodos , Pollos , Enfermedades de las Aves de Corral/tratamiento farmacológico , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Salmonelosis Animal/tratamiento farmacológico , Salmonella enterica/aislamiento & purificación , Pavos , Animales , Técnicas Bacteriológicas/instrumentación , Enfermedades de las Aves de Corral/microbiología , Salmonelosis Animal/microbiología , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/aislamiento & purificación
13.
J Agric Food Chem ; 67(23): 6642-6649, 2019 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-31117488

RESUMEN

The rapid monitoring of foodborne pathogens by monoclonal antibody (McAb)-based immunochromatographic tests (ICTs) is desirable but highly challenging as a result of the screening obstacle for a superior performance probe, which will greatly determine the capture efficiency of targets and the sensitivity of the immunoassay. In this work, on the basis of two-dimensional (2D) nanosheets (including MoS2 and graphene) as the extraordinary capture probe and signal indicator, we fabricated a label-free ICT method for Salmonella enteritidis detection. Especially, without the customarily labeled antibody probe, these 2D versatile probes presented strong capture ability toward bacteria by directly assembling onto the surface of bacteria. An ideal analytical performance with high sensitivity and specificity was achieved by virtue of the novel nanosheet-bacteria-McAb sandwich format. On the basis of MoS2 2D nanosheets as a fabulous probe element, the developed ICT exhibited a lowest detectable concentration of 103 colony-forming units/mL for S. enteritidis and could be well-applied in drinking water and watermelon juice samples. By the smart design, this work removes a series of conditionality issues of traditional double antibody sandwich-based ICTs and can give a new application direction for 2D nanosheet materials in the rapid detection field.


Asunto(s)
Agua Potable/microbiología , Jugos de Frutas y Vegetales/microbiología , Inmunoensayo/métodos , Salmonella enteritidis/aislamiento & purificación , Citrullus/química , Citrullus/microbiología , Grafito/química , Inmunoensayo/instrumentación , Nanoestructuras/química , Salmonella enteritidis/química , Sensibilidad y Especificidad
14.
BMC Infect Dis ; 19(1): 330, 2019 Apr 18.
Artículo en Inglés | MEDLINE | ID: mdl-30999894

RESUMEN

BACKGROUND: Invasive Salmonella infections result in significant morbidity and mortality in developing countries. In Asia, typhoid and paratyphoid fever are reported to be the major invasive Salmonella infections, while invasive non-typhoidal Salmonella (iNTS) infections are believed to be uncommon. Data from Sarawak, in Malaysian Borneo, are limited. METHODS: A retrospective study identifying all children aged < 15 years with invasive Salmonella infections from 2011 to 2016 was conducted in Bintulu Hospital in Sarawak. Population incidences, clinical and bacterial characteristics were examined. RESULTS: Forty-four patients were identified during the 6-year study period: 43 had iNTS infection and 1 had typhoid fever. The average annual iNTS incidence was 32.4 per 100,000 children aged < 5 years. None of the children had malaria or HIV infection, and only 7% were severely malnourished. Salmonella Enteritidis and Salmonella Java were the commonest NTS serovars identified. Pneumonia was the most common manifestation of iNTS disease, present in 20 (47%) children. Other manifestations included gastroenteritis, fever without a source, septic arthritis and meningitis. Salmonella Enteritidis was identified in 76% of those with pneumonia, significantly more frequently than in children with other manifestations. Over 25% of children with iNTS developed severe disease and nearly 10% suffered long term morbidity or mortality. While 78% of Salmonella Java isolates were multi-drug resistant, nearly all other isolates were susceptible to most antimicrobials, including ampicillin. CONCLUSIONS: Bintulu Division in Sarawak observed a very high incidence of childhood iNTS infections. Enteric fever was uncommon. The epidemiology of invasive Salmonella infections in Malaysian Borneo differs considerably from that of neighbouring countries in Asia.


Asunto(s)
Infecciones por Salmonella/diagnóstico , Adolescente , Antiinfecciosos/farmacología , Borneo/epidemiología , Niño , Preescolar , Farmacorresistencia Bacteriana Múltiple , Femenino , Humanos , Lactante , Malasia/epidemiología , Masculino , Pruebas de Sensibilidad Microbiana , Neumonía/diagnóstico , Neumonía/epidemiología , Estudios Retrospectivos , Salmonella/efectos de los fármacos , Salmonella/aislamiento & purificación , Infecciones por Salmonella/epidemiología , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/aislamiento & purificación , Serogrupo , Fiebre Tifoidea/diagnóstico , Fiebre Tifoidea/epidemiología
15.
J Appl Microbiol ; 127(1): 262-273, 2019 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-31002451

RESUMEN

AIMS: The aim of this study was to develop a multiplex touchdown PCR (multiplex TD-PCR) for rapid and simultaneous detection of four major foodborne pathogens to avoid mispriming and unwanted production during gene amplification. Touchdown PCR is the modified form of standard PCR, which enhances specificity, sensitivity. METHODS AND RESULTS: For this reason, a multiplex TD-PCR assay with a pre-enrichment step was developed to detect four foodborne pathogens namely Escherichia coli O157:H7, Listeria monocytogenes, Staphylococcus aureus, and Salmonella enterica serovar Enteritidis in pure culture and raw milk samples. The results showed that this protocol can eliminate the unwanted band or reduce significantly. The detection sensitivity of the single and multiplex TD-PCR was one cell per ml in pure culture. Furthermore, the detection limit of multiplex TD-PCR was one cell per 25 ml for artificially contaminated raw milk. We obtained similar results for detection of aforementioned pathogens in raw milk, after comparing the multiplex TD-PCR method with the traditional culture, except in one or two samples. CONCLUSIONS: Hence, the proposed multiplex TD-PCR method could be confirmed as an effective way for rapid optimization of PCR reactions to increase specificity, sensitivity during gene amplification. SIGNIFICANCE AND IMPACT OF THE STUDY: Hence, due to its simplicity, cost-effectiveness and being time-saving, it seems that this method is reasonable and economical for rapid optimization of PCR reactions.


Asunto(s)
Bacterias/aislamiento & purificación , Microbiología de Alimentos/métodos , Leche/microbiología , Reacción en Cadena de la Polimerasa Multiplex , Animales , Bacterias/genética , Escherichia coli O157/genética , Escherichia coli O157/aislamiento & purificación , Listeria monocytogenes/genética , Listeria monocytogenes/aislamiento & purificación , Salmonella enteritidis/genética , Salmonella enteritidis/aislamiento & purificación , Sensibilidad y Especificidad , Staphylococcus aureus/genética , Staphylococcus aureus/aislamiento & purificación
16.
BMJ Case Rep ; 12(3)2019 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-30898965

RESUMEN

Few data reported non-typhoidal Salmonella peritonitis in immunocompromised patients. We reported the case of a man without immunosuppression or predisposing factor, who developed Salmonella enterica serovar Enteritidis peritonitis with spontaneous intestinal perforation. After emergent surgery, the patient was transferred to intensive care unit (ICU) because of respiratory, renal and haemodynamic failures. When S. enterica serovar Enteritidis was identified, antibiotics were de-escalated for ceftriaxone and metronidazole for 5 days. No immunosuppression was found. Evolution was favourable, and the patient has been discharged from the ICU on day 8. The originality of this case arises from a perforation peritonitis secondary to S. enterica without any immunosuppression. In absence of non-Typhi Salmonella data, we treated this patient as a typhoid perforation: surgical treatment, antibiotic association and supportive care.


Asunto(s)
Perforación Intestinal/etiología , Peritonitis/complicaciones , Infecciones por Salmonella/complicaciones , Antibacterianos/uso terapéutico , Ceftriaxona/uso terapéutico , Humanos , Perforación Intestinal/diagnóstico por imagen , Perforación Intestinal/cirugía , Masculino , Metronidazol/uso terapéutico , Persona de Mediana Edad , Peritonitis/tratamiento farmacológico , Infecciones por Salmonella/sangre , Infecciones por Salmonella/tratamiento farmacológico , Salmonella enteritidis/aislamiento & purificación
17.
Biosens Bioelectron ; 132: 360-367, 2019 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-30897543

RESUMEN

Lateral flow immunoassay (LFIA) is a class and widespread applied point-of-care biosensor in the rapid monitoring field. To address the matched antibodies and antibody labeling dependence in the conventional LFIAs, in this work, an innovative label-free LFIA was proposed for the sensitive detection of Salmonella enteritidis (S. enteritidis) by introducing a new nanoparticles-bacteria-antibody sandwich strategy in the sensor. Surface positively charged nitrogen-rich carbon (pNC) nanoparticles, synthesized via calcination and etching reactions, were used as adsorbent to capture bacteria as well as for generating signals. In the presence of target pathogens, bacterial cells could combine with pNC through electrostatic interaction and hydrogen bonding, then the complex would be captured specifically by the anti-bacteria monoclonal antibody (McAb) coated on the test line (T-line). With the accumulation of nanoparticles-bacteria, the color on T-line would be gradually deepened from nearly colorless to deep black. Importantly, the pNC-based immunoassay could exhibit high sensitivity for target pathogens detection with a linear range of 102-108 cfu mL-1 and a low detection limit of 102 cfu mL-1. Furthermore, this system was validated preliminarily to screen S. enteritidis in different food samples with recoveries ranging from 85% to 110%. Taking advantages of simplicity, label-free, convenience, and sensitivity, the pNC-based LFIA has the application potential for pathogenic microorganisms monitoring in food safety and early clinical diagnosis fields.


Asunto(s)
Anticuerpos Inmovilizados/química , Técnicas Biosensibles/instrumentación , Carbono/química , Análisis de los Alimentos/instrumentación , Nanopartículas/química , Nitrógeno/química , Salmonella enteritidis/aislamiento & purificación , Microbiología de Alimentos , Humanos , Inmunoensayo/instrumentación , Límite de Detección , Modelos Moleculares , Tiras Reactivas/análisis , Infecciones por Salmonella/microbiología
18.
Microb Pathog ; 129: 118-124, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30738177

RESUMEN

For detection and isolation of Salmonella enterica, 650 meat and tissue samples were processed using Rappaport-Vassiliadis Enrichment broth and Salmonella Chromogenic agar followed by confirmation through specific antisera and polymerase chain reaction (PCR) targeting their Specific Serovar Genomic Regions (SSGRS). Isolates were tested for 15 antibiotics (CRO, AMX, GEN, STR, TET, CHL, CLR, LVX, OFX, GAT, CIP, SXT, AMP, LIN and AZM) according to the disc diffusion method and antimicrobial resistant genes (tet(A), tet(B), tet(C), strA/strB, aadA, aac(3)IV), aadB, sul1, sul2 and sul3, blaCMY-2, blaTEM and blaSHV) using PCR. The overall prevalence of Salmonella enterica was 12%, being higher in markets (15%) as compared to poultry farms (37.2%). The MPN of all positive meat and tissue samples was found 3.6 MPN/gram (0.17-18). A total of 234 isolates were obtained, serovar Typimurium (139) and Enteridits (95) were the most prevalent. Antimicrobial resistance patterns were different in different serovars according to origin of Salmonella isolates. The overall isolates were highly resistant for LIN (93.1%, 218/234) followed by AMX (80%, 187/234), AMP (74.3%, 174/234), TET (64.5%, 151/234) and STR (64.5%, 151/234). Overall, the most common ARG was blaTEM (76%, 178/234), followed by blaSHV (71.7%, 168/234), tet(A) (64%, 151/234) and tet(B) (64%, 150/234), while the least ARG was aadB (7.2%, 17/234). Both Typimurium and Enteridits were tested in the Balb/C mice for pathogenicity. Both Typimurium and Enteridits were found to cause successful colonization, 100% morbidity but Enteriditis were found to cause 33% mortality.


Asunto(s)
Farmacorresistencia Bacteriana , Enfermedades de las Aves de Corral/microbiología , Productos Avícolas/microbiología , Salmonelosis Animal/microbiología , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/aislamiento & purificación , Animales , Antibacterianos/farmacología , Técnicas Bacteriológicas , Femenino , Genes Bacterianos , Ratones Endogámicos BALB C , Modelos Animales , Reacción en Cadena de la Polimerasa , Aves de Corral , Prevalencia , Salmonelosis Animal/epidemiología , Salmonella enteritidis/efectos de los fármacos , Salmonella enteritidis/genética , Salmonella typhimurium/efectos de los fármacos , Salmonella typhimurium/genética , Análisis de Supervivencia
19.
J Microbiol ; 57(2): 170-179, 2019 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-30706346

RESUMEN

Since Salmonella Enteritidis is one of the major foodborne pathogens, on-site applicable rapid detection methods have been required for its control. The purpose of this study was to isolate and purify S. Enteritidis-specific phage (KFS-SE2 phage) from an eel farm and to investigate its feasibility as a novel, efficient, and reliable bio-receptor for its employment. KFS-SE2 phage was successfully isolated at a high concentration of (2.31 ± 0.43) × 1011 PFU/ml, and consisted of an icosahedral head of 65.44 ± 10.08 nm with a non-contractile tail of 135.21 ± 12.41 nm. The morphological and phylogenetic analysis confirmed that it belongs to the Pis4avirus genus in the family of Siphoviridae. KFS-SE2 genome consisted of 48,608 bp with 45.7% of GC content. Genome analysis represented KFS-SE2 to have distinctive characteristics as a novel phage. Comparative analysis of KFS-SE2 phage with closely related strains confirmed its novelty by the presence of unique proteins. KFS-SE2 phage exhibited excellent specificity to S. Enteritidis and was stable under the temperature range of 4 to 50°C and pH of 3 to 11 (P < 0.05). The latent time was determined to be 20 min. Overall, a new lytic KFS-SE2 phage was successfully isolated from the environment at a high concentration and the excellent feasibility of KFS-SE2 phage was demonstrated as a new bio-receptor for S. Enteritidis detection.


Asunto(s)
Bacteriófagos/clasificación , Bacteriófagos/aislamiento & purificación , Filogenia , Salmonella enteritidis/aislamiento & purificación , Salmonella enteritidis/virología , Bacteriófagos/genética , Bacteriófagos/ultraestructura , Composición de Base , Técnicas Biosensibles , ADN Viral/genética , Genes Virales , Genoma Viral , Especificidad del Huésped , Análisis de Secuencia de ADN , Siphoviridae/clasificación , Siphoviridae/genética , Proteínas Virales/genética
20.
Braz J Microbiol ; 50(2): 445-448, 2019 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-30796714

RESUMEN

This study investigated the serotypes and antimicrobial resistance of Salmonella isolates in urine cultures from 38 hospitalized patients. Nine serotypes were detected, and a large proportion was Typhimurium and Enteritidis. The strains presented resistance to 11 different antibiotics. Thirteen isolates (11 from serotype Typhimurium) exhibited multidrug resistance.


Asunto(s)
Antibacterianos/farmacología , Farmacorresistencia Bacteriana Múltiple/fisiología , Infecciones por Salmonella/microbiología , Salmonella enteritidis/efectos de los fármacos , Salmonella typhimurium/efectos de los fármacos , Orina/microbiología , Adolescente , Adulto , Niño , Preescolar , Femenino , Humanos , Lactante , Recién Nacido , Masculino , Pruebas de Sensibilidad Microbiana , Persona de Mediana Edad , Infecciones por Salmonella/orina , Salmonella enteritidis/aislamiento & purificación , Salmonella typhimurium/aislamiento & purificación , Serotipificación , Adulto Joven
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