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1.
J Agric Food Chem ; 68(4): 1118-1125, 2020 Jan 29.
Artigo em Inglês | MEDLINE | ID: mdl-31895982

RESUMO

The conventional colloidal gold immunochromatographic assay (AuNP-ICA) cannot meet the requirements for the rapid and sensitive detection of Escherichia coli (E. coli) O157:H7 because of its poor sensitivity. Herein, a novel two-step cascade signal amplification strategy that integrates in situ gold growth and nanozyme-mediated catalytic deposition was proposed to enhance the detection sensitivity of conventional AuNP-ICA dramatically. The enhanced strip displayed ultrahigh sensitivity in E. coli O157:H7 detection and had a detection limit of 1.25 × 101 CFU/mL, which is approximately 400-fold lower than that of traditional AuNP-ICA (5 × 103 CFU/mL). The amplified strip had no background signal in the T-line zone in the absence of E. coli O157:H7 even after one round of cascade signal amplification. The enhanced strip demonstrated excellent selectivity against E. coli O157:H7 with a negligible cross-reaction to nine other common pathogens. Intra-assays and interassays showed that the improved strip has acceptable accuracy and precision for determining E. coli O157:H7. The average recoveries in a real milk sample ranged from 87.33 to 112.15%, and the coefficients of variation were less than 10%. The enhanced strip also showed great potential in detecting a single E. coli O157:H7 cell in a 75 µL solution.


Assuntos
Escherichia coli O157/isolamento & purificação , Imunoensaio/métodos , Leite/microbiologia , Animais , Bovinos , Escherichia coli O157/genética , Limite de Detecção
2.
Food Microbiol ; 86: 103303, 2020 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-31703885

RESUMO

Escherichia coli O157:H7 and Salmonella enterica are foodborne pathogens with major public health concern in the U.S. These pathogens utilize several virulence factors to initiate infections in humans. The antimicrobial effect of seven glucosinolate hydrolysis compounds against Salmonella and E. coli O157:H7 was investigated by the disc diffusion assay. Among the tested compounds, benzyl isothiocyanate (BIT), which exerted the highest antimicrobial activity, was evaluated for its anti-virulence properties against these pathogens. The effect of BIT on motility of Salmonella and E. coli O157:H7 and Shiga toxin production by E. coli O157:H7 was determined by the motility assay and ELISA procedure, respectively. Confocal and transmission electron microscopy (TEM) procedures were used to determine bacterial damage at the cellular level. Results revealed that sub-inhibitory concentrations (SICs) of BIT significantly inhibited the motility of both bacteria (P < 0.05). Shiga toxin production by E. coli O157:H7 was decreased by ~32% in the presence of BIT at SICs. TEM results showed the disruption of outer membrane, release of cytoplasmic contents, and cell lysis following BIT treatment. Results suggest that BIT could be potentially used to attenuate Salmonella and E. coli O157:H7 infections by reducing the virulence factors including bacterial motility and Shiga toxin production.


Assuntos
Antibacterianos/farmacologia , Escherichia coli O157/efeitos dos fármacos , Isotiocianatos/farmacologia , Salmonella enterica/efeitos dos fármacos , Fatores de Virulência/metabolismo , Escherichia coli O157/citologia , Escherichia coli O157/genética , Escherichia coli O157/metabolismo , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Salmonella enterica/citologia , Salmonella enterica/genética , Salmonella enterica/metabolismo , Toxina Shiga/metabolismo , Fatores de Virulência/antagonistas & inibidores , Fatores de Virulência/genética
3.
J Appl Microbiol ; 128(1): 301-309, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31541508

RESUMO

AIMS: The aim of this study was to evaluate the microbiological quality of commercially prepared ready-to-eat (RTE) sushi by enumerating aerobic mesophilic bacteria (AMB) and thermotolerant coliforms (TC) and detecting Escherichia coli and Salmonella ssp. An isolate was identified as E. coli O157:H7 which was evaluated for its virulence and antimicrobial resistance profiling as well as its ability to form biofilms on stainless steel. METHODS AND RESULTS: There were four sampling events in seven establishments, totalling 28 pools of sushi samples. Mean AMB counts ranged between 5·2 and 7·7 log CFU per gram. The enumeration of TC varied between 2·1 and 2·7 log MPN per gram. Salmonella ssp. were not detected, and one sample was positive for E. coli and was identified as E. coli O157:H7. To the best of our knowledge, this is the first report of E. coli O157:H7 in sushi samples in the world literature. This isolate presented virulence genes stx1, stx2, eae and hlyA. It was also susceptible to 14 antimicrobials tested and had the ability to form biofilms on stainless steel. CONCLUSIONS: There is a need to improve the good hygiene practices adopted in establishments selling sushi in the city of Pelotas, Brazil. In addition, the isolated E. coli O157:H7 carries a range of important virulence genes being a potential risk to consumer health, as sushi is a RTE food. This isolate also presents biofilm formation ability, therefore, may trigger a constant source of contamination in the production line of this food. SIGNIFICANCE AND IMPACT OF THE STUDY: The increase in the consumption of sushi worldwide attracts attention regarding the microbiological point of view, since it is a ready-to-eat food. To our knowledge, this was the first time that E. coli O157:H7 was identified in sushi samples.


Assuntos
Escherichia coli O157/isolamento & purificação , Microbiologia de Alimentos , Alimentos Marinhos/microbiologia , Animais , Antibacterianos/farmacologia , Biofilmes/crescimento & desenvolvimento , Brasil , Contagem de Colônia Microbiana , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Testes de Sensibilidade Microbiana , Aço Inoxidável , Termotolerância , Virulência/genética
4.
Sensors (Basel) ; 19(24)2019 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-31842267

RESUMO

The detection of bacterial deoxyribonucleic acid (DNA) is of great significance in the quality control of food and water. In this study, a light-addressable potentiometric sensor (LAPS) deposited with highly oriented ZnO nanorod arrays (NRAs) was used for the label-free detection of single-stranded bacterial DNA (ssDNA). A functional, sensitive surface for the detection of Escherichia coli (E. coli) O157:H7 DNA was prepared by the covalent immobilization of the specific probe single-stranded DNA (ssDNA) on the LAPS surface. The functional surface was exposed to solutions containing the target E. coli ssDNA molecules, which allowed for the hybridization of the target ssDNA with the probe ssDNA. The surface charge changes induced by the hybridization of the probe ssDNA with the target E. coli ssDNA were monitored using LAPS measurements in a label-free manner. The results indicate that distinct signal changes can be registered and recorded to detect the target E. coli ssDNA. The lower detection limit of the target ssDNA corresponded to 1.0 × 102 colony forming units (CFUs)/mL of E. coli O157:H7 cells. All the results demonstrate that this DNA biosensor, based on the electrostatic detection of ssDNA, provides a novel approach for the sensitive and effective detection of bacterial DNA, which has promising prospects and potential applications in the quality control of food and water.


Assuntos
Técnicas Biossensoriais , DNA Bacteriano/isolamento & purificação , Infecções por Escherichia coli/diagnóstico , Escherichia coli O157/isolamento & purificação , DNA Bacteriano/genética , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Humanos , Luz , Nanotubos/química , Potenciometria/métodos
5.
Res Vet Sci ; 127: 27-32, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31670090

RESUMO

Bovines are the primary reservoir of enterohemorrhagic Escherichia coli (EHEC) O157:H7 and the main source of its transmission to humans. Here, we present a one-year longitudinal study of fecal shedding of E. coli O157. E. coli O157 obtained from recto-anal mucosal samples were characterized by multiplex PCR. The E. coli O157 prevalence ranged from 0.84% in July to 15.25% in November. The confinement within pens resulted in prevalence of 11%. Most animals (61.86%; 75/118) shed E. coli O157 at least in one sampling occasion. Of the positive animals, 82.19%, 16.44%, and 1.37% were stx positive on one, two and three sampling occasions, respectively. All the E. coli O157 isolated strains carried the genes eae and rfbO157, whereas 11%, 33% and 56% contained stx1, stx2 and stx1/stx2, respectively. The stx1/stx2 and stx2 types were significantly higher during the grazing and finishing periods, respectively, in comparison with the rearing and grazing periods. The presence of stx2a subtype was evident in four isolates, whereas stx2c was present in at least seven. However, both subtypes were present simultaneously in two isolates. The stx1/stx2c, stx1/stx2d and stx1/stx2NT genotypes occurred in 24, 2 and 15 isolates, respectively. The simultaneous occurrence of stx1 and stx2c significantly increased during grazing. Some cases of within-pen and between-pen transmission occurred throughout the study. Contagion levels during in-field grazing were higher than during permanent confinement in the pens. Thus, the individual patterns of shedding varied depending on the proportion of animals shedding the bacteria within pens and the time of shedding.


Assuntos
Derrame de Bactérias , Doenças dos Bovinos/microbiologia , Infecções por Escherichia coli/veterinária , Escherichia coli O157/fisiologia , Adesinas Bacterianas/análise , Animais , Argentina , Bovinos , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/análise , Fezes/microbiologia , Genótipo , Estudos Longitudinais , Masculino , Toxina Shiga/análise , Virulência
6.
J Food Sci ; 84(11): 3241-3245, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31604365

RESUMO

Escherichia coli O157:H7 is an important foodborne pathogen and has been implicated in numerous food poisoning outbreaks worldwide. Although several microbiological and molecular methods have been developed to detect E. coli O157:H7, the difficulty to rapidly detect low levels of the foodborne bacteria persists. Here, the optimization of a filtration technique to concentrate and rapidly detect E. coli O157:H7 was conducted. Using homogenates prepared from freshly cut lettuce and cabbage samples, the E. coli O157:H7 concentration efficiencies of seven membrane filters were compared. Mixed cellulose ester (MCE) and polyvinylidene difluoride (PVDF) filters demonstrated the highest bacterial recoveries. In addition, the optimal E. coli O157:H7 detachment method from MCE filters after filtration was investigated. Tapping for 80 s was demonstrated to be the most effective method for detaching bacteria from the filters. Further, the possibility of the rapid detection of low levels of E. coli O157:H7 in lettuce and cabbage was evaluated using real-time polymerase chain reaction after bacterial concentration using MCE and PVDF filters. The use of MCE filters enabled the detection of 10° CFU/g (5 CFU/g) of E. coli O157:H7 within 2 hr without microbial enrichment culture. Therefore, concentration by filtration can be used for the rapid detection of low levels of foodborne pathogens. PRACTICAL APPLICATION: The modified method, which has been verified in this study, has been optimized to reduce the analysis time and to detect very low concentrations of E. coli O157:H7 within 2 hr. All these detection systems have a direct economic impact on the food analysis of producers, health authorities, or third-party laboratories.


Assuntos
Escherichia coli O157/isolamento & purificação , Filtração/métodos , Reação em Cadeia da Polimerase em Tempo Real/métodos , Brassica/microbiologia , Contagem de Colônia Microbiana , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Contaminação de Alimentos/análise , Microbiologia de Alimentos , Alface/microbiologia
7.
J Food Sci ; 84(10): 2736-2744, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31573690

RESUMO

Controlling the free chlorine (FC) availability in wash water during sanitization of fresh produce enhances our ability to reduce microbial levels and prevent cross-contamination. However, maintaining an ideal concentration of FC that could prevent the risk of contamination within the wash system is still a technical challenge in the industry, indicating the need to better understand wash water chemistry dynamics. Using bench-scale experiments and modeling approaches, we developed a comprehensive mathematical model to predict the FC concentration during fresh-cut produce wash processes for different lettuce types (romaine, iceberg, green leaf, and red leaf), carrots, and green cabbage as well as Escherichia coli O157:H7 cross-contamination during fresh-cut iceberg lettuce washing. Fresh-cut produce exudates, as measured by chemical oxygen demand (COD) levels, appear to be the primary source of consumption of FC in wash water, with an apparent reaction rate ranging from 4.74 × 10 - 4 to 7.42 × 10 - 4 L/mg·min for all produce types tested, at stable pH levels (6.5 to 7.0) in the wash water. COD levels increased over time as more produce was washed and the lettuce type impacted the rate of increase in organic load. The model parameters from our experimental data were compared to those obtained from a pilot-plant scale study for lettuce, and similar reaction rate constant (5.38 × 10-4 L/mg·min) was noted, supporting our hypothesis that rise in COD is the main cause of consumption of FC levels in the wash water. We also identified that the bacterial transfer mechanism described by our model is robust relative to experimental scale and pathogen levels in the wash water. Finally, we proposed functions that quantify an upper bound on pathogen levels in the water and on cross-contaminated lettuce, indicating the maximum potential of water-mediated cross-contamination. Our model results could help indicate the limits of FC control to prevent cross-contamination during lettuce washing.


Assuntos
Cloro/metabolismo , Escherichia coli O157/isolamento & purificação , Manipulação de Alimentos , Cloro/análise , Contagem de Colônia Microbiana , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Contaminação de Alimentos/análise , Manipulação de Alimentos/métodos , Microbiologia de Alimentos , Humanos , Alface/química , Alface/microbiologia , Modelos Biológicos , Folhas de Planta/química , Folhas de Planta/microbiologia , Verduras/química , Verduras/microbiologia
8.
J Food Sci ; 84(10): 2916-2924, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31502678

RESUMO

A method combining surface-enhanced Raman scattering (SERS) with a lateral flow strip (LFS) was developed for the quantitative and sensitive analysis of Escherichia coli O157:H7. AuMBA @Ag nanoparticles were prepared as SERS probes, and 4-methylthiobenzoic acid (MBA) as a Raman reporter was inserted into the interior gap of the Au@Ag core-shell nanoparticles, which replaced the Au nanoparticles that serve as SERS nanotags in traditional LFS. Using this developed SERS-LFS, the presence of the target bacteria could be tested through the appearance of a red band on the test line. Furthermore, quantitative analysis of E. coli O157:H7 was achieved by measuring the specific Raman intensity of MBA on the test line. The sensitivity of this SERS-LFS biosensor is 5 × 104 CFU/mL of E. coli O157:H7, which is 10-fold higher than that of a naked eye-based colorimetric LFS. This quantitative detection of E. coli O157:H7 ( Y = 1993.86 X - 6812.17, R2 = 0.9947) was obtained with a wide linear range (5 × 104 to 5 × 108 ) due to the signal enhancement of the SERS nanotags. In addition, the SERS-LFS could differentiate E. coli O157:H7 from closely related bacterial species or nontarget contaminants, suggesting high specificity of this assay. The applicability of SERS-LFS to the analysis of E. coli O157:H7 in milk, chicken breast, and beef was also validated, indicating that the sensitivity was not disturbed by the food matrix. In summary, the SERS-LFS developed in this study could be a powerful tool for the quantitative and sensitive screening of E. coli O157:H7 in a food matrix. PRACTICAL APPLICATION: This study demonstrates that a surface-enhanced Raman scattering (SERS)-based lateral flow strip (LFS) could be used as a rapid and sensitive method for Escherichia coli O157:H7 detection. Furthermore, this SERS-based LFS could achieve quantitative detection of the target, eliminating the defect of the traditional colloidal gold LFS, which is not quantifiable.


Assuntos
Escherichia coli O157/isolamento & purificação , Nanopartículas Metálicas/química , Prata/química , Análise Espectral Raman/métodos , Animais , Bovinos , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Leite/microbiologia , Sensibilidade e Especificidade , Análise Espectral Raman/instrumentação
9.
J Appl Genet ; 60(3-4): 417-426, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31485951

RESUMO

Escherichia coli O157:H7 bacterium as a zoonotic pathogen is one of the most important causative agents of foodborne illnesses worldwide. Due to the serious concerns in public health and enormous economic losses in agriculture and food industry, it is very necessary to develop novel technology-based methods for sensitive and rapid detection of this bacterium in contaminated resources. In this study, a sensitive and selective fluorescence DNA nanosensing platform based on graphene oxide (GO) and the 3' end of eae gene as specific sequence was developed for the detection of E. coli O157:H7. In this platform, fluorescence resonance energy transfer (FRET) process between GO- and FAM-labeled eae gene probe was used for the diagnosis of E. coli O157:H7. Following the immobilization of the eae gene probe on GO, fluorescence emission of FAM was quenched. In hybridization reaction, by adding the complementary DNA, fluorescence emission of FAM was significantly increased and recovered to 93%. The performance of sensor for detection of E. coli O157:H7 genomic DNA was determined 10 pg genomic DNA per 1 ml Tris-HCl hybridization buffer which was significantly more sensitive than PCR method. In conclusion, the results indicated that GO eae gene-based nanosensor has potential to be developed as a rapid and sensitive diagnostic device besides PCR methods for the detection of E. coli O157:H7 bacteria.


Assuntos
Técnicas Biossensoriais , DNA Bacteriano/isolamento & purificação , Escherichia coli O157/isolamento & purificação , Microbiologia de Alimentos , Primers do DNA , DNA Bacteriano/genética , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Grafite/química , Humanos , Nanocompostos/química , Reação em Cadeia da Polimerase/métodos
10.
Future Microbiol ; 14: 885-898, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31368788

RESUMO

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.


Assuntos
Bactérias/genética , Técnicas Bacteriológicas/métodos , Microbiologia de Alimentos/métodos , Doenças Transmitidas por Alimentos/microbiologia , Leite/microbiologia , Animais , Bactérias/crescimento & desenvolvimento , Bactérias/isolamento & purificação , Meios de Cultura , DNA Bacteriano/genética , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/isolamento & purificação , Listeria monocytogenes/genética , Listeria monocytogenes/crescimento & desenvolvimento , Listeria monocytogenes/isolamento & purificação , Reação em Cadeia da Polimerase , Salmonella enteritidis/genética , Salmonella enteritidis/crescimento & desenvolvimento , Salmonella enteritidis/isolamento & purificação , Sensibilidade e Especificidade , Staphylococcus aureus/genética , Staphylococcus aureus/crescimento & desenvolvimento , Staphylococcus aureus/isolamento & purificação
11.
PLoS Pathog ; 15(8): e1007652, 2019 08.
Artigo em Inglês | MEDLINE | ID: mdl-31404118

RESUMO

Enterohemorrhagic Escherichia coli O157:H7 (EHEC) is an important food-borne pathogen that colonizes the colon. Transposon-insertion sequencing (TIS) was used to identify genes required for EHEC and E. coli K-12 growth in vitro and for EHEC growth in vivo in the infant rabbit colon. Surprisingly, many conserved loci contribute to EHEC's but not to K-12's growth in vitro. There was a restrictive bottleneck for EHEC colonization of the rabbit colon, which complicated identification of EHEC genes facilitating growth in vivo. Both a refined version of an existing analytic framework as well as PCA-based analysis were used to compensate for the effects of the infection bottleneck. These analyses confirmed that the EHEC LEE-encoded type III secretion apparatus is required for growth in vivo and revealed that only a few effectors are critical for in vivo fitness. Over 200 mutants not previously associated with EHEC survival/growth in vivo also appeared attenuated in vivo, and a subset of these putative in vivo fitness factors were validated. Some were found to contribute to efficient type-three secretion while others, including tatABC, oxyR, envC, acrAB, and cvpA, promote EHEC resistance to host-derived stresses. cvpA is also required for intestinal growth of several other enteric pathogens, and proved to be required for EHEC, Vibrio cholerae and Vibrio parahaemolyticus resistance to the bile salt deoxycholate, highlighting the important role of this previously uncharacterized protein in pathogen survival. Collectively, our findings provide a comprehensive framework for understanding EHEC growth in the intestine.


Assuntos
Elementos de DNA Transponíveis , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/crescimento & desenvolvimento , Proteínas de Escherichia coli/metabolismo , Intestinos/microbiologia , Fatores de Virulência/metabolismo , Animais , Infecções por Escherichia coli/genética , Infecções por Escherichia coli/metabolismo , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Coelhos , Análise de Sequência de DNA , Fatores de Virulência/genética
12.
Mem. Inst. Invest. Cienc. Salud (Impr.) ; 17(2): 71-76, ago. 2019. tab, ilus
Artigo em Espanhol | LILACS, BDNPAR | ID: biblio-1008486

RESUMO

Los serogrupos O26, O45, O103, O104, O111, O121, O145 y O157 de STEC se relacionan con un elevado número de casos de SUH a nivel mundial, por lo que están incluidos dentro de las categorías de mayor riesgo para los humanos, según los criterios de autoridades alimentarias de Estados Unidos y Europa. El método convencional de identificación de antígenos O y H se realiza por aglutinación con antisueros de conejo. Este método además de ser muy costoso y laborioso, no se encuentra disponible en el país para empleo masivo. En este contexto, el objetivo de este estudio observacional descriptivo de corte transverso ha sido la estandarización de una técnica de PCR múltiple para la detección de estos 8 serogrupos, a fin de contar con un sistema de detección eficiente, sensible y con potencial de aplicación en la industria alimentaria. Se estandarizaron reacciones de PCR empleando como controles positivos cepas E. coli de referencia correspondientes a la totalidad de los serogrupos citados. Se obtuvieron productos de tamaños esperados para cada serogrupo, no se observaron amplificaciones cruzadas o falsos positivos. Esta técnica estandarizada podría representar una herramienta rápida y menos costosa que la técnica serológica, con la capacidad de ser aplicada a diferentes matrices, permitiendo la detección de estos serogrupos en aislados STEC de ganado en pie, fuentes de agua de consumo, alimentos e incluso en aislamientos clínicos asociados a enfermedades humanas(AU)


STEC serogroups O26, O45, O103, O104, O111, O121, O145, and O157, are related to a high number of cases of HUS worldwide, so they are included in the categories of greatest risk for humans, according to the food administration criteria of the United States and Europe. The conventional method of identifying antigens O and H is carried out by agglutination with rabbit antisera. This method is very expensive and laborious and is not available in the country for massive-scale use. In this context, the objective of this cross-sectional descriptive observational study has been the standardization of a multiplex PCR technique for the detection of these 8 serogroups, in order to have an efficient and sensitive detection system with the potential for application in the food industry. PCR reactions were standardized using as positive controls reference E. coli strains to correspond to all the mentioned serogroups. Products of expected sizes were obtained for each serogroup; no cross-amplification or false positives were observed. This standardized technique could represent a quick and less expensive tool than the serological technique, with the possibility to be applied to different kind of samples, allowing the detection of these serogroups in STEC isolates of live cattle, sources of drinking water, food and even in clinical isolates associated with human diseases(AU)


Assuntos
Escherichia coli Shiga Toxigênica/isolamento & purificação , Reação em Cadeia da Polimerase Multiplex , Estudos Transversais , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/genética , Escherichia coli Shiga Toxigênica/genética , Escherichia coli O104/isolamento & purificação , Escherichia coli O104/genética
13.
Nucleic Acids Res ; 47(18): e103, 2019 10 10.
Artigo em Inglês | MEDLINE | ID: mdl-31269198

RESUMO

Targeted PCR amplification and high-throughput sequencing (amplicon sequencing) of 16S rRNA gene fragments is widely used to profile microbial communities. New long-read sequencing technologies can sequence the entire 16S rRNA gene, but higher error rates have limited their attractiveness when accuracy is important. Here we present a high-throughput amplicon sequencing methodology based on PacBio circular consensus sequencing and the DADA2 sample inference method that measures the full-length 16S rRNA gene with single-nucleotide resolution and a near-zero error rate. In two artificial communities of known composition, our method recovered the full complement of full-length 16S sequence variants from expected community members without residual errors. The measured abundances of intra-genomic sequence variants were in the integral ratios expected from the genuine allelic variants within a genome. The full-length 16S gene sequences recovered by our approach allowed Escherichia coli strains to be correctly classified to the O157:H7 and K12 sub-species clades. In human fecal samples, our method showed strong technical replication and was able to recover the full complement of 16S rRNA alleles in several E. coli strains. There are likely many applications beyond microbial profiling for which high-throughput amplicon sequencing of complete genes with single-nucleotide resolution will be of use.


Assuntos
Sequenciamento de Nucleotídeos em Larga Escala , Microbiota/genética , Filogenia , RNA Ribossômico 16S/genética , Escherichia coli K12/classificação , Escherichia coli K12/genética , Escherichia coli O157/classificação , Escherichia coli O157/genética , Fezes/microbiologia , Humanos , Reação em Cadeia da Polimerase/métodos , Polimorfismo de Nucleotídeo Único/genética , Análise de Sequência de DNA
14.
Biomed Microdevices ; 21(3): 72, 2019 07 08.
Artigo em Inglês | MEDLINE | ID: mdl-31286242

RESUMO

In this study, we integrated sample purification and genetic amplification in a seamless polycarbonate microdevice to facilitate foodborne pathogen detection. The sample purification process was realized based on the increased affinity of the boronic acid-modified surface toward the cis-diol group present on the bacterial outer membrane. The modification procedure was conducted at room temperature using disposable syringe. The visible color and fluorescence signals of alizarin red sodium were used to confirm the success of the surface modification process. Escherichia coli O157:H7 containing green fluorescence protein (GFP) and Staphylococcus aureus were chosen as the microbial models to demonstrate the nonspecific immobilization using the microdevice. Bacterial solutions of various concentrations were injected into the microdevice at three flow rates to optimize the operation conditions. This microdevice successfully amplified the 384-bp fragment of the eaeA gene of the captured E. coli O157:H7 within 1 h. Its detection limit for E. coli O157:H7 was determined to be 1 × 103 colony-forming units per milliliter (CFU mL-1). The proposed microdevice serves as a monolithic platform for facile and on-site identification of major foodborne pathogens.


Assuntos
Métodos Analíticos de Preparação de Amostras/instrumentação , Ácidos Borônicos/química , Microbiologia de Alimentos , Dispositivos Lab-On-A-Chip , Cimento de Policarboxilato/química , Reação em Cadeia da Polimerase/instrumentação , Escherichia coli O157/genética , Escherichia coli O157/isolamento & purificação , Limite de Detecção , Staphylococcus aureus/genética , Staphylococcus aureus/isolamento & purificação , Propriedades de Superfície , Temperatura
15.
Sci Total Environ ; 692: 297-304, 2019 Nov 20.
Artigo em Inglês | MEDLINE | ID: mdl-31351277

RESUMO

Enterohaemorrhagic Escherichia coli (EHEC) O157:H7 is one of the major foodborne and waterborne pathogens causing severe diseases and outbreaks worldwide. There is scarcity of EHEC O157:H7 data in South Africa. This study was carried out to determine the molecular characteristics and genotypic diversity of EHEC O157:H7 isolates in the Gauteng region, South Africa. Samples were cultured on selective chromogenic media. Antibiotic susceptibility profile of isolates was determined using the VITEK®-2 automated system. Isolates were characterised using multiplex PCR assays and the genetic diversity was determined using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST). A total of 520 samples of which 270 environmental water samples and 250 stool specimens were collected and analysed. Overall, EHEC O157:H7 was recovered from 2.3% (12/520) of samples collected. Environmental water samples and clinical stool specimens showed a prevalence of 4.07% (11/270) and 0.4% (1/250) respectively. Antibiotic susceptibility profile varied from isolates with full susceptibility to isolates with resistance to multiple antibiotics. Most resistance was detected to the penicillins, specifically ampicillin (7/12), amoxicillin (3/12) and piperacillin/Tazobactam (3/12) followed by one of the folate inhibitors, trimethoprim (3/12) and the carbapenems, imipenem and meropenem (2/12) each. Three isolates harboured a combination of Shiga-toxins (Stx)-2, intimin (eae) and enterohaemolysin (hlyA) genes, while two isolates harboured the Stx-1, Stx-2 and hlyA genes. The PFGE performed showed that EHEC O157:H7 isolates were genetically diverse, with two minor pulsotypes and eight singletons. The MLST analysis identified three sequence types (STs) (ST10, ST11 and ST1204) that have been previously reported associated with outbreaks. The STs identified in this study pose a potential public health risk to consumers of untreated environmental water and closed human contacts. There is necessity to enhance surveillance in reducing the propagation of this bacterium which is a public health problem.


Assuntos
Escherichia coli O157/genética , Fezes/microbiologia , Água Doce/microbiologia , Variação Genética , Genótipo , Esgotos/microbiologia , Águas Residuárias/microbiologia , Eletroforese em Gel de Campo Pulsado , Escherichia coli O157/isolamento & purificação , Tipagem de Sequências Multilocus , África do Sul
16.
Appl Microbiol Biotechnol ; 103(18): 7317-7324, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31359104

RESUMO

Long polar fimbria (LPF) is one of the few fimbrial adhesins of enterohemorrhagic Escherichia coli (E. coli) O157:H7 associated with colonization on host intestine, and both two types of LPF (including LPF1 and LPF2) play essential roles during the bacterial infection process. Though the fimbriae had been well studied in intestinal pathogenic E. coli strains, new evidences from our research revealed that it might be the key virulence for bovine mastitis pathogenic E. coli (MPEC) as well. This article summarizes the current knowledge on the LPF in E. coli, focusing on its genetic characteristics, prevalence, expression regulation, and adherence mechanism in different pathotypes of E. coli strains.


Assuntos
Aderência Bacteriana , Escherichia coli O157/genética , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/fisiologia , Proteínas de Fímbrias/fisiologia , Fímbrias Bacterianas/fisiologia , Animais , Bovinos , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Feminino , Proteínas de Fímbrias/genética , Fímbrias Bacterianas/genética , Humanos , Intestinos/microbiologia , Mastite Bovina/microbiologia , Virulência
17.
APMIS ; 127(10): 671-680, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31344276

RESUMO

Regardless of the communal impact of Shiga toxins, till today neither a specific treatment nor licensed vaccine is available. Lactococcus lactis (L. lactis), generally regarded as safe organism, is well known to provide a valuable approach regarding the oral delivery of vaccines. This study was undertaken to evaluate the protective efficacy of Stx2a1 expressed in nisin-inducible L. lactis, against Shiga toxins (Stx1, Stx2) in mouse model. Oral immunization of BALB/c mice with LL-Stx2a1 elicited significant serum antibody titer with elevated fecal and serum IgA, along with minimized intestinal and kidney damage resulting in survival of immunized animals at 84% and 100% when challenged with 10 × LD50 of Escherichia coli O157 and Shigella dysenteriae toxins, respectively. HeLa cells incubated with immune sera and toxin mixture revealed high neutralizing capacity with 90% cell survivability against both the toxins. Mice immunized passively with both toxins and antibody mixture survived the observation period of 15 days, and the controls administered with sham sera and toxins were succumbed to death within 3 days. Our results revealed protective efficacy and toxin neutralization ability of LL-Stx2a1, proposing it as an oral vaccine candidate against Shiga toxicity mediated by E. coli O157 and S. dysenteriae.


Assuntos
Vacinas Bacterianas/administração & dosagem , Vacinas Bacterianas/imunologia , Escherichia coli O157/imunologia , Envenenamento/prevenção & controle , Toxina Shiga/imunologia , Toxina Shiga/toxicidade , Shigella dysenteriae/imunologia , Administração Oral , Animais , Anticorpos Antibacterianos/administração & dosagem , Anticorpos Antibacterianos/sangue , Anticorpos Neutralizantes/administração & dosagem , Anticorpos Neutralizantes/sangue , Antitoxinas/administração & dosagem , Antitoxinas/sangue , Vacinas Bacterianas/genética , Sobrevivência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Portadores de Fármacos/administração & dosagem , Escherichia coli O157/genética , Vetores Genéticos/administração & dosagem , Células HeLa , Humanos , Lactococcus lactis/genética , Camundongos , Camundongos Endogâmicos BALB C , Toxina Shiga/genética , Shigella dysenteriae/genética , Análise de Sobrevida , Resultado do Tratamento , Vacinas Sintéticas/administração & dosagem , Vacinas Sintéticas/genética , Vacinas Sintéticas/imunologia
18.
Biosens Bioelectron ; 141: 111415, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31202189

RESUMO

We focused on the development of a hand-held pathogen-detection device using smartphone-embedded electronic elements combined with functionalized magnetic particles (MPs) and sepharose. To perform affinity chromatography for evaluating DNA amplicons, avidin-conjugated MPs and succinimide-linked sepharose were used with biotin-primers. To mimic the centrifugal-based affinity ligand chromatography, a smartphone-mountable low-power fan was plugged into the charging port of a smartphone. The charging port stably emitted electric current at 3.0 V, and the fan blades were modified for use as a portable rotor. Based on the binding variation of MPs with DNA amplicons, the position of MPs in sepharose changed significantly during centrifugation. The change in distance was optically analyzed using the illumination sensor of the smartphone with respect to the altered transmittance due to the MPs. Amplified genes from Escherichia. coli O157:H7 samples ranging from 1.0 × 101 to 1.0 × 106 colony-forming units could be rapidly and immediately detected by the naked eye using a simple smartphone-based optical device. The results indicated that this novel biosensing technique is suitable for use as a point-of-care testing device in both industrial and clinical fields.


Assuntos
Técnicas Biossensoriais/instrumentação , Infecções por Escherichia coli/microbiologia , Escherichia coli O157/isolamento & purificação , Reação em Cadeia da Polimerase/instrumentação , Smartphone/instrumentação , Animais , Técnicas Biossensoriais/economia , DNA Bacteriano/análise , DNA Bacteriano/genética , Escherichia coli O157/genética , Análise de Alimentos/economia , Análise de Alimentos/instrumentação , Humanos , Leite/microbiologia , Sistemas Automatizados de Assistência Junto ao Leito/economia , Reação em Cadeia da Polimerase/economia , Smartphone/economia , Fatores de Tempo
19.
Artif Cells Nanomed Biotechnol ; 47(1): 2593-2604, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-31240960

RESUMO

Escherichia coli O157:H7 is considered as emerging foodborne pathogens that occur globally. Three major virulence protein factors; EspA(E), intimin(I), Tir(T) and Stx2 toxin have been found to be highly associated with bloody diarrhoea or, Haemolytic Uremic Syndrome. In this study, a trivalent recombinant EIT in combination with the binding domain of STX toxin were encapsulated with chitosan nanoparticles as a combination vaccine candidate. Mice were immunized either subcutaneously or orally with these antigens and challenged with E. coli O157:H7. Results of the binding inhibition assay with caco2 cell monolayer show a significant reduction in the adhesion percentage of pre-treated E. coli O157:H7 with immunized mice sera. Evaluation of neutralizing abilities of immune sera pre-incubated with CD50 dose of STX2 by Vero cells cytotoxicity neutralization assay shows less morphological reforms in comparison with the control groups. Results of mice mortality challenge with STX2 demonstrate around 66% of survived in immunized mice. In a challenge experiment with E. coli O157:H7, all the immunized mice showed a significant decrease in bacterial colonization and shedding. The results indicate that the use of multiple recombinant proteins in combination with natural nanostructure effectively evocated strong humoral and mucosal response, increasing the protection capacity of the synthetic antigen.


Assuntos
Antígenos de Bactérias/química , Antígenos de Bactérias/imunologia , Quitosana/química , Portadores de Fármacos/química , Escherichia coli O157/imunologia , Imunização , Nanopartículas/química , Animais , Anticorpos Antibacterianos/imunologia , Células 3T3 BALB , Aderência Bacteriana , DNA Recombinante/genética , Escherichia coli O157/genética , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/fisiologia , Feminino , Camundongos , Células Vero
20.
Food Microbiol ; 82: 378-387, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027797

RESUMO

The majority of foodborne illnesses associated with E. coli O157 are attributed to the consumption of foods of bovine origin. In this study, RNA-Seq experiments were undertaken with E. coli O157 to identify genes that may be associated with growth and survival on meat and the beef carcass at low temperature. In addition, the response of an E. coli O157 isolate representative of the general genetic 'type' found in Australia (E. coli O157:H- strain EC2422) was compared to that of a pathogenic clinical isolate (E. coli O157:H7 strain Sakai) not typically found in Australia. Both strains up-regulated genes involved in the acid stress response, cold shock response, quorum sensing, biofilm formation and Shiga toxin production. Differences were also observed, with E. coli O157:H7 Sakai up-regulating genes playing a critical role in the barrier function of the outer membrane, lipopolysaccharide biosynthesis, extracellular polysaccharide synthesis and curli production. In contrast, E. coli O157:H- EC2422 down-regulated genes involved in peptidoglycan biosynthesis and of the primary envelope stress response Cpx system. The unique gene expression profiles of the strains, indicate that these genotypes may differ in their ability to persist in the meat production environment and therefore also in their ability to cause disease.


Assuntos
Escherichia coli O157/genética , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos/microbiologia , Carne Vermelha/microbiologia , Transcriptoma , Animais , Austrália , Bovinos , Temperatura Baixa , Contagem de Colônia Microbiana , Escherichia coli O157/crescimento & desenvolvimento , Escherichia coli O157/isolamento & purificação , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Genoma Bacteriano/genética , Polissacarídeos Bacterianos/genética , Especificidade da Espécie , Estresse Fisiológico/genética , Fatores de Virulência/genética
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