RESUMO
Loop-mediated isothermal amplification (LAMP) is a cost-effective, rapid, and highly specific method of replicating nucleic acids. Adding multiple targets into a single LAMP assay to create a multiplex format is highly desirable for clinical applications but has been challenging due to a need to develop specific detection techniques and strict primer design criteria. This study describes the evaluation of a rapid triplex LAMP assay, MAST ISOPLEX®VTEC, for the simultaneous detection of Shiga toxin/verotoxin 1 and 2 (stx1/vt1 and stx2/vt2) genes in verotoxigenic Escherichia coli (E. coli) (VTEC) isolates with inhibition control (IC) synthetic DNA using a single fluorophore-oligonucleotide probe, MAST ISOPLEX®Probes, integrated into the primer set of each target. MAST ISOPLEX®Probes used in the MAST ISOPLEX®VTEC kit produce fluorescent signals as they integrate with reaction products specific to each target, allowing tracking of multiple amplifications in real time using a real-time analyzer. Initial validation on DNA extracts from fecal cultures and synthetic DNA sequences (gBlocks) showed that the MAST ISOPLEX®VTEC kit provides a method for sensitive simultaneous triplex detection in a single assay with a limit of detection (LOD) of less than 100 target copies/assay and 96% and 100% sensitivity and specificity, respectively.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Técnicas de Amplificação de Ácido Nucleico/métodos , Humanos , Sensibilidade e Especificidade , Toxina Shiga I/genética , Técnicas de Diagnóstico Molecular/métodos , Toxina Shiga II/genética , Limite de Detecção , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/diagnóstico , Kit de Reagentes para DiagnósticoRESUMO
Shiga toxin-producing Escherichia coli (STEC) is an important waterborne pathogen capable of causing serious gastrointestinal infections with potentially fatal complications, including haemolytic-uremic syndrome. All STEC serogroups harbour genes that encode at least one Shiga toxin (stx1 and/or stx2), which constitute the primary virulence factors of STEC. Loop-mediated isothermal amplification (LAMP) enables rapid real-time pathogen detection with a high degree of specificity and sensitivity. The aim of this study was to develop and validate an on-site portable diagnostics workstation employing LAMP technology to permit rapid real-time STEC detection in environmental water samples. Water samples (n=28) were collected from groundwater wells (n=13), rivers (n=12), a turlough (n=2) and an agricultural drain (n=1) from the Corrib catchment in Galway. Water samples (100 ml) were passed through a 0.22 µm filter, and buffer was added to elute captured cells. Following filtration, eluates were tested directly using LAMP assays targeting stx1, stx2 and E. coli phoA genes. The portable diagnostics workstation was used in field studies to demonstrate the on-site testing capabilities of the instrument. Real-time PCR assays targeting stx1 and stx2 genes were used to confirm the results. The limit of detection for stx1, stx2 and phoA LAMP assays were 2, 2 and 6 copies, respectively. Overall, stx1, stx2 and phoA genes were detected by LAMP in 15/28 (53.6â%), 9/28 (32.2â%) and 24/28 (85.7â%) samples, respectively. For confirmation, the LAMP results for stx1 and stx2 correlated perfectly (100â%) with those obtained using PCR. The portable diagnostics workstation exhibited high sensitivity throughout the on-site operation, and the average time from sample collection to final result was 40 min. We describe a simple, transferable and efficient diagnostic technology for on-site molecular analysis of various water sources. This method allows on-site testing of drinking water, enabling evidence-based decision-making by public health and water management authorities.
Assuntos
Técnicas de Amplificação de Ácido Nucleico , Escherichia coli Shiga Toxigênica , Microbiologia da Água , Técnicas de Amplificação de Ácido Nucleico/métodos , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Técnicas de Diagnóstico Molecular/métodos , Técnicas de Diagnóstico Molecular/instrumentação , Sensibilidade e Especificidade , Rios/microbiologia , Toxina Shiga I/genética , Água Subterrânea/microbiologiaRESUMO
Shiga toxigenic E. coli are important foodborne zoonotic pathogens. The present study was envisaged to standardize loop-mediated isothermal amplification assays targeting stx1 and stx2 genes for rapid and visual detection of STEC and compare its sensitivity with PCR. The study also assessed the effect of short enrichment on the detection limit of LAMP and PCR. The developed LAMP assays were found to be highly specific. Analytical sensitivity of LAMP was 94 fg/µLand 25.8 fg/µL for stx-1 and stx-2 while LOD of 5 CFU/g of carabeef was measured after 6-12 h enrichment. The study highlights the importance of short (6-12 h) enrichment for improving the sensitivity of LAMP. The entire detection protocol could be performed within 9 h yielding results on the same day. The developed LAMP assays proved to be a handy and cost-effective alternative for screening STEC contamination in meat.
Assuntos
Carne , Técnicas de Diagnóstico Molecular , Técnicas de Amplificação de Ácido Nucleico , Sensibilidade e Especificidade , Escherichia coli Shiga Toxigênica , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Técnicas de Amplificação de Ácido Nucleico/métodos , Animais , Técnicas de Diagnóstico Molecular/métodos , Carne/microbiologia , Microbiologia de Alimentos/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Contaminação de Alimentos/análiseRESUMO
Escherichia coli O157:H7 (E. coli O157:H7) and Campylobacter jejuni (C. jejuni) are pathogenic microorganisms that can cause severe clinical symptoms in humans and are associated with bovine meat consumption. Specific monitoring for E. coli O157: H7 or C. jejuni in meat is not mandatory under Chilean regulations. In this study, we analyzed 544 samples for the detection of both microorganisms, obtained from 272 bovine carcasses (280 kg average) at two slaughterhouses in the Bio-Bío District, Chile. Sampling was carried out at post-shower of carcasses and after channel passage through the cold chamber. Eleven samples were found to be positive for E. coli O157:H7 (4.0%) using microbiological and biochemical detection techniques and were subjected to a multiplex PCR to detect fliC and rfbE genes. Six samples (2.2%) were also found to be positive for the pathogenicity genes stx1, stx2, and eaeA. Twenty-two carcasses (8.0%) were found to be positive for C. jejuni using microbiological and biochemical detection techniques, but no sample with amplified mapA gene was found.
Assuntos
Matadouros , Campylobacter jejuni , Escherichia coli O157 , Proteínas de Escherichia coli , Microbiologia de Alimentos , Animais , Bovinos , Campylobacter jejuni/isolamento & purificação , Campylobacter jejuni/genética , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/genética , Chile , Proteínas de Escherichia coli/genética , Flagelina/genética , Carne/microbiologia , Contaminação de Alimentos/análise , Adesinas Bacterianas/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Reação em Cadeia da Polimerase Multiplex , Proteínas de Bactérias/genética , Transaminases , Carboidratos EpimerasesRESUMO
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) is a significant cause of foodborne illness causing various gastrointestinal diseases including hemolytic uremic syndrome (HUS), the most severe form, which can lead to kidney failure or even death. OBJECTIVE: Here, we report the development of recombinase aided amplification (RAA)-exo-probe assays targeting the stx1 and stx2 genes for the rapid detection of STEC in food samples. METHODS: Primers and exo-probes were designed and optimized for the detection of stx1 and stx2 using RAA technology. The optimal STEC RAA-exo-probe assays were then tested for specificity and sensitivity, and validated in both spiked and real food samples. RESULTS: These assays were found to be 100% specific to STEC strains and were also highly sensitive with a detection limit of 1.6 × 103 CFU/mL or 32 copies/reaction. Importantly, the assays were able to successfully detect STEC in spiked and real food samples (beef, mutton, and pork), with a detection limit as low as 0.35 CFU/25g in beef samples after an overnight enrichment step. CONCLUSIONS: Overall, the RAA assay reactions completed within â¼20 min and were less dependent on expensive equipment, suggesting they can be easily adopted for in-field testing requiring only a fluorescent reader. HIGHLIGHTS: As such, we have developed two rapid, sensitive, and specific assays that can be used for the routine monitoring of STEC contamination in food samples, particularly in the field or in poorly equipped labs.
Assuntos
Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica , Animais , Bovinos , Escherichia coli Shiga Toxigênica/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Recombinases , Microbiologia de AlimentosRESUMO
An important challenge relating to clinical diagnostics of the foodborne pathogen Shiga toxin-producing E. coli (STEC), is that PCR-detection of the shiga-toxin gene (stx) in DNA from stool samples can be accompanied by a failure to identify an STEC isolate in pure culture on agar. In this study, we have explored the use of MinION long-read sequencing of DNA from bacterial culture swipes to detect the presence of STEC, and bioinformatic tools to characterize the STEC virulence factors. The online workflow "What's in my pot" (WIMP) in the Epi2me cloud service, rapidly identified STEC also when it was present in culture swipes together with multiple other E. coli serovars, given sufficient abundance. These preliminary results provide useful information about the sensitivity of the method, which has potential to be used in clinical diagnostic of STEC, particularly in cases where a pure culture of the STEC isolate is not obtained due to the 'STEC lost Shiga toxin' phenomenon.
Assuntos
Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Humanos , Escherichia coli Shiga Toxigênica/genética , Sorogrupo , Infecções por Escherichia coli/diagnóstico , Infecções por Escherichia coli/microbiologia , Toxina Shiga I/genética , Toxina Shiga/genética , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Proteínas de Transporte/genéticaRESUMO
In this study, we reported Shiga toxin-producing Escherichia coli (STEC) in 847 samples, including those in coastal waters, sediments, and fish samples in the Southeast Coast of India. A total of 3742 E. coli strains were identified using conventional and molecular identification methods. Of these, 1518 isolates expressed virulent genes Stx1, Stx2, and Eae; effects on these genes on toxicity were examined. Furthermore, 2224 non-STEC isolates caused hemolytic uremic syndrome and played a key role in the persistence of STEC contamination. We conclude that toxin production is not adequate to cause disease, and the pathogenic mechanism of STEC remains poorly defined. Therefore, the present study indicates the status of pollution, highlighting the need for sanitation in public health.
Assuntos
Proteínas de Escherichia coli , Água do Mar/microbiologia , Escherichia coli Shiga Toxigênica , Animais , Proteínas de Escherichia coli/genética , Peixes/microbiologia , Sedimentos Geológicos/microbiologia , Índia , Toxina Shiga , Toxina Shiga I/genética , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoRESUMO
Enterohaemorrhagic Escherichia coli (EHEC) produces Shiga toxin 1 (Stx1) and Shiga toxin 2 (Stx2). Although stx1 and stx2 were found within the late operons of the Stx-encoding phages (Stx-phages), stx1 could mainly be transcribed from the stx1 promoter (PStx1), which represents the functional operator-binding site (Fur box) for the transcriptional regulator Fur (ferric uptake regulator), upstream of stx1. In this study, we found that the production of Stx1 by EHEC was affected by oxygen concentration. Increased Stx1 production in the presence of oxygen is dependent on Fur, which is an Fe2+-responsive transcription factor. The intracellular Fe2+ pool was lower under microaerobic conditions than under anaerobic conditions, suggesting that lower Fe2+ availability drove the formation of less Fe2+-Fur, less DNA binding to the PStx1 region, and an increase in Stx1 production.
Assuntos
Bacteriófagos , Escherichia coli Êntero-Hemorrágica , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/metabolismo , Oxigênio/metabolismo , Toxina Shiga I/genética , Toxina Shiga I/metabolismo , Toxina Shiga II/genética , Toxina Shiga II/metabolismoRESUMO
Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.
Assuntos
Bacteriófagos/genética , Escherichia coli Êntero-Hemorrágica/fisiologia , Escherichia coli Êntero-Hemorrágica/virologia , Infecções por Escherichia coli/microbiologia , Ribonucleases/metabolismo , Toxina Shiga/biossíntese , Toxina Shiga/genética , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica , Mitomicina/farmacologia , Plasmídeos/genética , Ribonucleases/genética , Toxina Shiga I/genética , Toxina Shiga II/genética , Ensaio de Placa Viral , Fatores de Virulência/genéticaRESUMO
Introduction. Shiga toxin-producing Escherichia coli (STEC) can cause severe disease and large outbreaks. In England, the incidence and clinical significance of STEC serogroups other than O157 (non-O157) is unknown due to a testing bias for detection of STEC O157. Since 2013, the implementation of PCR to detect all STEC serogroups by an increasing number of diagnostic laboratories has led to an increase in the detection of non-O157 STEC.Hypothesis/Gap statement. Due to a bias in testing methodologies to select for STEC serogroup O157 in frontline diagnostic laboratories in most countries, very little surveillance data have been previously generated on non-O157 STEC.Aim. Five years (2014-2018) of STEC national surveillance data were extracted and descriptive analysis undertaken to assess disease severity of non-O157 STEC strains.Methods. Data from 1 January 2014 to 31 December 2018 were extracted from the National Enhanced Surveillance System for STEC and analysed.Results. The implementation of Gastrointestinal Polymerase Chain Reaction (GI-PCR) has resulted in a four-fold increase in the detection of non-O157 STEC cases between 2014 and 2018. There were 2579 cases infected with 97 different non-O157 serogroups. The gender distribution was similar amongst STEC O157 and non-O157 STEC cases with 57 and 56â% of cases being female respectively, but a significantly higher proportion of cases (P <0.001) under 5 years of age was observed among STEC O157 (22â%) cases compared to non-O157 STEC (14â%). The most common non-O157 serogroups were O26 (16â%), O146 (11â%), O91 (10â%), O128 (7â%), O103 (5â%) and O117 (3â%). Overall, rates of bloody diarrhoea were highest in O26 (44â%) and O103 (48â%) cases and lowest in STEC O117 cases (17â%). Strains harbouring Shiga toxin stx1a caused the highest proportion of diarrhoea (93â%) and caused the same level of bloody diarrhoea as stx2a (39â%). However, stx2a caused the highest proportion of vomiting (46â%), hospitalisation (49â%) and considerably more HUS (29â%) than other stx profiles.Conclusion. The implementation of PCR targeting stx at diagnostic laboratories has shown that non-O157 STEC, most notably STEC O26, are an emerging risk to public health.
Assuntos
Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Adolescente , Adulto , Distribuição por Idade , Criança , Pré-Escolar , Inglaterra/epidemiologia , Feminino , Humanos , Masculino , Técnicas de Diagnóstico Molecular , Reação em Cadeia da Polimerase , Sorogrupo , Distribuição por Sexo , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Virulência/genética , Adulto JovemRESUMO
The performance of the eazyplex® EHEC complete (Amplex) for the detection of Shiga toxin genes in stool samples was evaluated. The assay performed well in distinguishing between stx1 and stx2 but suboptimal sensitivity may limit its use to complementary testing rather than primary diagnosis of Shiga toxin-producing Escherichia coli infections.
Assuntos
Infecções por Escherichia coli/diagnóstico , Fezes/microbiologia , Técnicas de Diagnóstico Molecular/métodos , Toxina Shiga I/genética , Toxina Shiga II/genética , Técnicas Bacteriológicas/métodos , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Toxina Shiga , Escherichia coli Shiga ToxigênicaRESUMO
Shiga toxin-producing Escherichia coli (STEC) is an important foodborne pathogens, known to cause enteric infections especially diarrhea, mainly attributed to Shiga toxins (Stxs). The use of certain antibiotics for treating this infection is controversial, owing to an increased risk for producing Stxs (Stx 1 and Stx 2). Increased antibiotic resistance is also thought to be involved in the pathogenesis of STEC diseases. The purpose of this study was to analyze the effects of antibiotics on induction of Stx 1 and Stx 2 in clinical STEC isolates and to investigate the relationships between increased resistance and Stx production. Fifteen clinical isolates were treated with sub minimum inhibitory concentrations (Sub MIC) of clinically used antibiotics (ciprofloxacin, fosfomycin, tigecycline, and meropenem), and the changes in expression levels of stx1 and stx2 genes were estimated using qRT-PCR. The expressions of Shiga toxins were found to be increased up to 6.5- and eightfold under ciprofloxacin and tigecycline Sub MIC, respectively. Fosfomycin had weak induction effect of up to twofold, whereas meropenem had the weakest influence on such expression. Resistant isolates were found to be more prone to increased expression of toxins.
Assuntos
Regulação Bacteriana da Expressão Gênica , Toxina Shiga I , Toxina Shiga II , Escherichia coli Shiga Toxigênica , Antibacterianos/farmacologia , Infecções por Escherichia coli/microbiologia , Perfilação da Expressão Gênica , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Humanos , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/genéticaRESUMO
BACKGROUND: Various primer and probe sets have been developed and standardized, but certain sets may have low efficiency or miss some stx-subtypes. OBJECTIVE: To compare the efficiency of the recommended stx screening primers and probe sets in four standardized methods and develop a new primers and probe system with an internal amplification control (IAC) for all known stx2 subtypes. METHOD: The inclusivity and specificity of recommended screening primers and probe sets in four standardized methods were compared. A new pan-stx2 primer and probe set was adapted from the International Organization for Standardization (ISO) method for all known stx2 subtypes. The robustness of the new method was assessed in seven laboratories and also assessed in ground beef and bean sprout samples. RESULTS: None of the recommended screening primers and probe sets in the four standardized methods could efficiently amplify all the stx2 subtypes because of various mismatches in the primers or the probe sequences. A new primers and probe system adapted from the ISO method, through introducing degenerate bases in primers and probe sequences with an IAC, showed high amplification efficiency and specificity for all known stx2 subtypes in ground beef and bean sprouts samples. The specificity of the new method was assessed in seven laboratories and showed robust and consistent results. CONCLUSIONS: This study provided evidence for Shiga-toxin producing Escherichia coli (STEC) screening method development, and the newly developed primers and probes system should be considered in the revision of the standardized methods. HIGHLIGHTS: None of the recommended screening primer and probe set in the four official methods could efficiently amplify all the stx2 subtypes. A new developed primer and probe set showed high amplification efficiency and specificity for all known stx2 subtypes in fresh ground beef and bean sprouts samples. The newly developed stx2 screening system showed robustness and consistency during interlaboratory study.
Assuntos
Toxina Shiga , Escherichia coli Shiga Toxigênica , Animais , Bovinos , Microbiologia de Alimentos , Reação em Cadeia da Polimerase em Tempo Real , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/genéticaRESUMO
We investigated the efficiency of the Verigene Enteric Pathogens Nucleic Acid Test (Verigene EP test), which is an automated microarray-based assay system that enables rapid and simultaneous genetic detection of gastrointestinal pathogens and toxins, including those in the Campylobacter Group, Salmonella species, Shigella species, the Vibrio Group, Yersinia enterocolitica, Shiga toxin 1 and 2, norovirus GI/GII, and rotavirus A. Three clinical laboratories evaluated the Verigene EP test, using 268 stool samples for bacterial and toxin genes and 167 samples for viral genes. Culture-based reference methods were used for the detection of bacteria and toxins, while a different molecular assay was used for viral detection. The overall concordance rate between the Verigene EP test and the reference methods for the 1940 assays was 99.0%. The overall sensitivity and specificity of the Verigene EP test were 97.0% and 99.3%, respectively. Of the 19 samples with discordant results, 13 samples were false positives and six were false negatives. The Verigene EP test simultaneously detected two targets in 11 samples; overall, the test demonstrated high efficiency in detecting crucial diarrheagenic pathogens, indicating its suitability for clinical practice.
Assuntos
Toxinas Bacterianas/isolamento & purificação , Diarreia/diagnóstico , Gastroenterite/microbiologia , Microbioma Gastrointestinal , Toxinas Bacterianas/genética , Diarreia/genética , Diarreia/microbiologia , Fezes/microbiologia , Gastroenterite/diagnóstico , Gastroenterite/genética , Humanos , Técnicas de Diagnóstico Molecular , Norovirus/genética , Norovirus/isolamento & purificação , Norovirus/patogenicidade , Técnicas de Amplificação de Ácido Nucleico/métodos , Toxina Shiga I/química , Toxina Shiga I/genética , Toxina Shiga I/isolamento & purificação , Shigella/genética , Shigella/isolamento & purificação , Shigella/patogenicidadeRESUMO
Enterohemorrhagic Escherichia coli (EHEC), an enteropathogen that colonizes in the intestine, causes severe diarrhea and hemorrhagic colitis in humans by the expression of the type III secretion system (T3SS) and Shiga-like toxins (Stxs). However, how EHEC can sense and respond to the changes in the alimentary tract and coordinate the expression of these virulence genes remains elusive. The T3SS-related genes are known to be regulated by the locus of enterocyte effacement (LEE)-encoded regulators, such as Ler, as well as non-LEE-encoded regulators in response to different environmental cues. Herein, we report that OmpR, which participates in the adaptation of E. coli to osmolarity and pH alterations, is required for EHEC infection in Caenorhabditis elegans. OmpR protein was able to directly bind to the promoters of ler and stx1 (Shiga-like toxin 1) and regulate the expression of T3SS and Stx1, respectively, at the transcriptional level. Moreover, we demonstrated that the expression of ler in EHEC is in response to the intestinal environment and is regulated by OmpR in C. elegans. Taken together, we reveal that OmpR is an important regulator of EHEC which coordinates the expression of virulence factors during gastrointestinal infection in vivo.
Assuntos
Proteínas de Bactérias/genética , Caenorhabditis elegans/microbiologia , Escherichia coli Êntero-Hemorrágica/patogenicidade , Toxina Shiga I/biossíntese , Transativadores/genética , Fatores de Virulência/biossíntese , Animais , Proteínas de Bactérias/metabolismo , Sistema Digestório/microbiologia , Escherichia coli Êntero-Hemorrágica/genética , Proteínas de Escherichia coli/biossíntese , Proteínas de Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica/genética , Regiões Promotoras Genéticas/genética , Toxina Shiga I/genética , Transativadores/biossíntese , Transativadores/metabolismo , Transcrição Gênica/genética , Ativação Transcricional/genética , Sistemas de Secreção Tipo III/biossíntese , Sistemas de Secreção Tipo III/genética , Fatores de Virulência/genéticaRESUMO
BACKGROUND: Acute diarrhea is a major public health problem, particularly in developing countries. Shigellosis is one of the substantial causative agents of microbial dysentery and still has a remarkable prevalence, particularly in areas with poor hygienic infrastructures. The probable existence of the deadly Shiga toxin (Stx) protein in some Shigella strains would manifest life-threatening clinical symptoms of the infection. METHODS: The aim of this study was to determine the presence of Shigella toxin 1 (Stx1) in isolated from patients with diarrhea. Totally, 227 Shigella species, including 60 S. flexneri, 157 S. sonnei, and 10 S. boydii were collected from diarrheal patients in the tropical infectious diseases research center of Ahvaz, Iran, during 2013-2015. The isolates were collected mostly from the intensive care unit, infectious disease, and surgery settings. The isolates were identified, and the polymerase chain reaction (PCR) was performed to detect the stx gene. RESULTS: The results indicated that none of them encode the stx1 gene. CONCLUSION: Isolates of this study were not capable of stx1 encoding. Future investigations should consider the relations between other Shigella species and Shigella toxin in Iran.
Assuntos
Disenteria Bacilar , Diarreia/epidemiologia , Disenteria Bacilar/epidemiologia , Humanos , Irã (Geográfico)/epidemiologia , Prevalência , Toxina Shiga I/genéticaRESUMO
The expression of synaptic vesicle exocytosis-regulator SNARE complex component genes (snap25, stx1 and vamp2) was examined in the olfactory nervous system during seaward and homeward migration by pink salmon (Oncorhynchus gorbuscha). The expression levels of snares in the olfactory organ were higher in seaward fry than in feeding and homeward adults, reflecting the development of the olfactory nervous system. The expression of snap25a, b and stx1a was upregulated or stable in the adult olfactory bulb and telencephalon. This upregulated expression suggested alterations in olfactory neuronal plasticity that may be related to the discrimination of natal rivers. The expression of stx1b was downregulated in the adult olfactory bulb, but remained stable in the adult telencephalon. The expression of vamp2 was initially strong in seaward fry, but was downregulated in adults in both the olfactory bulb and telencephalon. Pink salmon has the lowest diversity of maturation age, the largest population, and the most evolutional position in Pacific salmon (genus Oncorhynchus). The expression of snares in the olfactory center of pink salmon reflected the timing of sexual maturation and homeward migration. The present results and our previous studies indicate that snares show distinct expression patterns between two salmon species that depend on physiological and ecological features of migration.
Assuntos
Migração Animal/fisiologia , Encéfalo/fisiologia , Proteínas de Peixes/genética , Regulação da Expressão Gênica no Desenvolvimento , Salmão/genética , Olfato/genética , Animais , Toxina Shiga I/genética , Proteínas de Ligação a Fator Solúvel Sensível a N-Etilmaleimida/genética , Proteína 2 Associada à Membrana da Vesícula/genéticaRESUMO
BACKGROUND: Shigatoxin (Stx)-producing Escherichia coli (STEC) are the most common causes of hemolytic uremic syndrome (STEC-HUS). The aim of our study is to compare the risk of developing STEC-HUS in relation to the type of Stx genes (Stx1, Stx2, or both). METHODS: This is a prospective, observational, multicenter study involving 63 pediatric units in Northern Italy (ItalKid-HUS Network). STEC-infected children were identified within a screening program for bloody diarrhea during a 10-year period (2010-2019). Stx genes were detected by reverse dot blot or real-time PCR. After the identification of STEC infection, children were followed until diarrhea complete recovery for the possible development of STEC-HUS. RESULTS: Of the 214 Stx-positive patients, 34 (15.9%) developed STEC-HUS. The risk of HUS in STEC-infected children with Stx1 (n: 62; 29.0%) and Stx2 (n: 97; 45.3%) was respectively 0% and 23.7%, while in patients carrying both Stx1 and Stx2 (n: 55; 25.7%), the risk was 12.7% (p: 0.001). CONCLUSIONS: Our data confirm that Stx1 is a very rare cause of STEC-HUS and demonstrate that the risk of STEC-HUS halves in the case of Stx1+2-producing Escherichia coli infection compared with infections where Stx2 is present alone. This observation is helpful in assessing the risk of individual STEC-infected patients for the development of HUS and suggests that Stx1, in the presence of Stx2, might exert a protective role possibly by receptor competition.
Assuntos
Infecções por Escherichia coli/microbiologia , Síndrome Hemolítico-Urêmica/epidemiologia , Toxina Shiga I/toxicidade , Toxina Shiga II/toxicidade , Escherichia coli Shiga Toxigênica/genética , Criança , Pré-Escolar , Infecções por Escherichia coli/complicações , Feminino , Síndrome Hemolítico-Urêmica/microbiologia , Humanos , Lactente , Tipagem Molecular , Estudos Prospectivos , Fatores de Proteção , Medição de Risco , Toxina Shiga I/genética , Toxina Shiga I/isolamento & purificação , Toxina Shiga II/genética , Toxina Shiga II/isolamento & purificação , Escherichia coli Shiga Toxigênica/isolamento & purificaçãoRESUMO
Calf diarrhea is one of the considerable infectious diseases in calves, which results in tremendous economic losses globally. To determine the prevalence of Shiga-toxigenic E. coli (STEC) and Enterotoxigenic E. coli (ETEC) incriminated in calf diarrhea, with special reference to Shiga- toxins genes (stx1 and stx2) and enterotoxins genes (lt and sta) that govern their pathogenesis, as well as the virulence genes; eaeA (intimin) and f41(fimbrial adhesion), and the screening of their antibiogram and antimicrobial resistance genes; aadB, sul1, and bla-TEM, a total of 274 fecal samples were collected (April 2018-Feb 2019) from diarrheic calves at different farms in El-Sharqia Governorate, Egypt. The bacteriological examination revealed that the prevalence of E. coli in diarrheic calves was 28.8%. The serotyping of the isolated E. coli revealed 7 serogroups; O26, O128, O111, O125, O45, O119 and O91. Furthermore, the Congo red binding test was carried out, where 89.8% of the examined strains (n = 71) were positive. The antibiogram of the isolated strains was investigated; the majority of E. coli serotypes exhibit multidrug resistance (MDR) to four antimicrobial agents; neomycin, gentamycin, streptomycin, and amikacin. Polymerase chain reaction (PCR) was used to detect the prevalence of the virulence genes; stx1, stx2 lt, sta, f41 and eaeA, as well as the antibiotic resistance genes; aadB, sul1, and bla-TEM. The prevalence of STEC was 20.2% (n = 16), while the prevalence of ETEC was 30.4% (n = 24). Briefly, the Shiga toxins genes; stx1 and stx2, are the most prevalent virulence genes associated with STEC, which are responsible for the pathogenesis of the disease and helped by the intimin gene (eaeA). In addition, the lt gene is the most prevalent enterotoxin gene accompanied by the ETEC strains, either alone or in combination with sta and/or f41 genes. The majority of pathogenic E. coli incriminated in calf diarrhea possesses the aadB resistance gene, followed by the sul1 gene. Enrofloxacin, florfenicol, amoxicillin-clavulanic acid, and ampicillin-sulbactam, are the most effective antimicrobial agents against the isolated STEC and ETEC strains.
Assuntos
Proteínas de Bactérias/genética , Doenças dos Bovinos/microbiologia , Diarreia/veterinária , Farmacorresistência Bacteriana Múltipla/genética , Escherichia coli Enterotoxigênica/genética , Infecções por Escherichia coli/veterinária , Escherichia coli Shiga Toxigênica/genética , Fatores de Virulência/genética , Adesinas Bacterianas/genética , Animais , Antibacterianos/farmacologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/genética , Bovinos , Doenças dos Bovinos/tratamento farmacológico , Diarreia/tratamento farmacológico , Diarreia/microbiologia , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/patogenicidade , Enterotoxinas/genética , Infecções por Escherichia coli/tratamento farmacológico , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/genética , Fezes/microbiologia , Regulação Bacteriana da Expressão Gênica , Testes de Sensibilidade Microbiana , Toxina Shiga I/genética , Toxina Shiga II/genética , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/patogenicidade , Virulência , Fatores de Virulência/metabolismoRESUMO
Shiga toxin-producing Escherichia coli (STEC) strains are important food-borne pathogens that can be transmitted through the consumption of food products derived from pigs. Moreover, antimicrobial resistance in STEC has been a matter of increasing concern. The aim of this study was to investigate the prevalence and antimicrobial characteristics of STEC isolates from pork in Korea. We isolated 131 isolates of E. coli from 334 pork samples collected from slaughterhouses and retail markets from 2008 to 2009. Among the 131 isolates, 6 (4.58%) were confirmed to belong to 6 different serotypes of STEC. All six STEC isolates contained stx1 and eaeA virulence genes, and four of them additionally carried the hly gene. The minimum inhibitory concentration (MIC) of 15 antibiotics (amoxicillin/clavulanic acid, ampicillin, cephalothin, cefoxitin, ceftiofur, gentamicin, neomycin, streptomycin, nalidixic acid, ciprofloxacin, colistin, chloramphenicol, florfenicol, tetracycline and sulfamethoxazole/trimethoprim) toward the STEC isolates was determined. As a result, three strains were associated with high MICs for florfenicol and chloramphenicol (64 µg/mL). Furthermore, all three strains were found to contain the florfenicol-resistant gene (floR) but not the chloramphenicol-resistant gene (cat). Sequence alignment and BLAST analysis of the polymerase chain reaction products of the floR gene indicated that they contained sequences with homology to the floR gene of E. coli or Salmonella enterica serovar, Heidelberg. This is the first report on the detection of floR in STEC isolated from pork obtained from retail markets in Korea.