RESUMO
Shiga-toxin producing Escherichia coli (STEC) O157 is a food-borne pathogen which causes gastrointestinal illness in humans. Ruminants are considered the main reservoir of infection, and STEC exceedance has been associated with heavy rainfall. In September 2022, a large outbreak of STEC O157:H7 was identified in the United Kingdom (UK). A national-level investigation was undertaken to identify the source of the outbreak and inform risk mitigation strategies. Whole genome sequencing (WGS) was used to identify outbreak cases. Overall, 259 cases with illness onset dates between 5 August and 12 October 2022, were confirmed across the UK. Epidemiological investigations supported a UK grown, nationally distributed, short shelf-life food item as the source of the outbreak. Analytical epidemiology and food chain analysis suggested lettuce as the likely vehicle of infection. Food supply chain tracing identified Grower X as the likely implicated producer. Independent of the food chain investigations, a novel geospatial analysis triangulating meteorological, flood risk, animal density and land use data was developed, also identifying Grower X as the likely source. Novel geospatial analysis and One Health approaches are potential tools for upstream data analysis to predict and prevent contamination events before they occur and to support evidence generation in outbreak investigations.
Assuntos
Mudança Climática , Surtos de Doenças , Infecções por Escherichia coli , Escherichia coli O157 , Microbiologia de Alimentos , Doenças Transmitidas por Alimentos , Lactuca , Lactuca/microbiologia , Humanos , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/transmissão , Reino Unido/epidemiologia , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/genética , Doenças Transmitidas por Alimentos/epidemiologia , Doenças Transmitidas por Alimentos/microbiologia , Sequenciamento Completo do Genoma , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/genética , Adulto , Pessoa de Meia-Idade , Feminino , Masculino , Contaminação de Alimentos/análise , Idoso , Animais , Adolescente , CriançaRESUMO
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
BACKGROUND: Due to the diversity of Shiga toxin-producing Escherichia coli (STEC) isolates, detecting highly pathogenic strains in foodstuffs is challenging. Currently, reference protocols for STEC rely on the molecular detection of eae and the stx1 and/or stx2 genes, followed by the detection of serogroup-specific wzx or wzy genes related to the top 7 serogroups. However, these screening methods do not distinguish between samples in which a STEC possessing both determinants are present and those containing two or more organisms, each containing one of these genes. This study aimed to evaluate ecf1, Z2098, Z2099, and nleA genes as single markers and their combinations (ecf1/Z2098, ecf1/Z2099, ecf1/nleA, Z2098/Z2099, Z2098/nleA, and Z2099/nleA) as genetic markers to detect potentially pathogenic STEC by the polymerase chain reaction (PCR) in 96 animal samples, as well as in 52 whole genome sequences of human samples via in silico PCR analyses. RESULTS: In animal isolates, Z2098 and Z2098/Z2099 showed a strong association with the detected top 7 isolates, with 100% and 69.2% of them testing positive, respectively. In human isolates, Z2099 was detected in 95% of the top 7 HUS isolates, while Z2098/Z2099 and ecf1/Z2099 were detected in 87.5% of the top 7 HUS isolates. CONCLUSIONS: Overall, using a single gene marker, Z2098, Z2099, and ecf1 are sensitive targets for screening the top 7 STEC isolates, and the combination of Z2098/Z2099 offers a more targeted initial screening method to detect the top 7 STEC isolates. Detecting non-top 7 STEC in both animal and human samples proved challenging due to inconsistent characteristics associated with the genetic markers studied.
Assuntos
Escherichia coli Êntero-Hemorrágica , Infecções por Escherichia coli , Escherichia coli Shiga Toxigênica , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Marcadores Genéticos , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/microbiologia , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Humanos , Plasmídeos/genética , Simulação por Computador , Bovinos , Reação em Cadeia da Polimerase/veterinária , Ovinos , Ilhas Genômicas/genéticaRESUMO
This study investigated the virulence potential and antibiotic susceptibility analysis of non-O157 Shiga toxin-producing Escherichia coli (STEC) serogroups, which are significant cause of food borne diseases. A study collected 800 samples of dairy bovine raw milk through various sources, 500 from milk shops, 200 from dairy farms, 26 from milk collection centers, and 74 from street vendors. Using a standard method, E. coli was detected in 321 out of the 800 samples collected. Out of the 321 E. coli-positive samples isolated, 148 were identified as STEC using selective media, specifically Cefixime Tellurite Sorbitol MacConkey's Agar (CT-SMA). Out of the 148 positive samples, 40 were confirmed as STEC non-O157 strains using multiplex PCR, indicating a prevalence of 5% (40 out of 800 samples). STEC isolates were subjected to antimicrobial susceptibility testing, and all isolates were resistant to at least one or more antimicrobials tested through the disk diffusion method, revealed high resistance to Amoxicillin 100%, Ceftriaxone 50%, and Penicillin 44.5%, and notably 44% of the strains exhibited Streptomycin resistance, while Enrofloxacin 55%, Florfenicol 50% and Norfloxacin 44%, demonstrated the highest susceptibility. Out of 40 STEC non-O157, twelve were subjected to Multi Locus Sequence Typing (MLST) sequencing through Illumina Inc. MiSeq platform's next-generation sequencing technology, United States. The genome investigation evidenced the persistence of twelve serotypes H4:O82, H30:O9a, H4:O82, H16:O187, H9:O9, H16:O113, H30:O9, H32:O, H32:O, H32, H32, and H38:O187, linked to the potential infections in humans. Conclusion: STEC isolates showed resistance to multiple antimicrobials, raising concerns for both animal and public health due to widespread use of these drugs in treatment and prevention. The study contributes new insights into monitoring STEC in raw milk, emphasizing the critical role of whole genome sequencing (WGS) for genotyping and sequencing diverse isolates. Still a deficiency in understanding STEC pathogenesis mechanisms, ongoing surveillance is crucial for safeguarding human health and enhancing understanding of STEC genetic characteristics.
Assuntos
Antibacterianos , Testes de Sensibilidade Microbiana , Leite , Escherichia coli Shiga Toxigênica , Animais , Bovinos , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Leite/microbiologia , Antibacterianos/farmacologia , Paquistão/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/epidemiologia , Farmacorresistência Bacteriana/genética , Sequenciamento Completo do Genoma , SorogrupoRESUMO
Subtilase exhibits strong cytotoxicity that was first described in O113:H21 strain in Australia as a plasmid- encoded cytotoxin (subAB1). Subsequently, chromosomal variants including subAB2-1, subAB2-2, and subAB2-3 were described. We aimed to investigate the presence of subAB genes in a collection of Shiga toxin-producing Escherichia coli (STEC) strains (n=101) isolated from different sources in Iran. A collection of 101 archived STEC strains isolated from cattle (n=50), goats (n=25), sheep (n=15), wild captive animals (n=8: persian fallow deer, n=3; caspian pony, n=1; Macaca mulatta, n=4), and humans (n=3) during 2007-2016 were analyzed for the detection of different genes encoding the Subtilase variants, plasmidic and chromosomal virulence genes, phylogroups and serogroups. Overall, 57 isolates (56.4%) carried at least one variant of subAB. Most strains from small ruminants including 93% of sheep and 96% of caprine isolates carried at least one chromosomally encoded variant (subAB-2-1 and/or subAb2-2). In contrast, 12 cattle isolates (24%) only harbored the plasmid encoded variant (subAB1). STEC strains from other sources, including deer, pony and humans were positive for subAB-2-1 and/or subAb2-2. Our results reveal the presence of potentially pathogenic genotypes among locus of enterocyte effacement (LEE)-negative isolates, and some host specificity related to Subtilase variants and other virulence markers that may aid in source tracking of STEC during outbreak investigations.
Assuntos
Proteínas de Escherichia coli , Escherichia coli Shiga Toxigênica , Subtilisinas , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Animais , Irã (Geográfico)/epidemiologia , Proteínas de Escherichia coli/genética , Subtilisinas/genética , Ovinos/microbiologia , Humanos , BovinosRESUMO
Shiga toxin-producing and Enteropathogenic Escherichia coli are foodborne pathogens commonly associated with diarrheal disease in humans. This study investigated the presence of STEC and EPEC in 771 dairy cattle fecal samples which were collected from 5 abattoirs and 9 dairy farms in South Africa. STEC and EPEC were detected, isolated and identified using culture and PCR. Furthermore, 339 STEC and 136 EPEC isolates were characterized by serotype and major virulence genes including stx1, stx2, eaeA and hlyA and the presence of eaeA and bfpA in EPEC. PCR screening of bacterial sweeps which were grown from fecal samples revealed that 42.2% and 23.3% were STEC and EPEC positive, respectively. PCR serotyping of 339 STEC and 136 EPEC isolates revealed 53 different STEC and 19 EPEC serotypes, respectively. The three most frequent STEC serotypes were O82:H8, OgX18:H2, and O157:H7. Only 10% of the isolates were classified as "Top 7" STEC serotypes: O26:H2, 0.3%; O26:H11, 3.2%; O103:H8, 0.6%; and O157:H7, 5.9%. The three most frequent EPEC serotypes were O10:H2, OgN9:H28, and O26:H11. The distribution of major virulence genes among the 339 STEC isolates was as follows: stx1, 72.9%; stx2, 85.7%; eaeA, 13.6% and hlyA, 69.9%. All the 136 EPEC isolates were eaeA-positive but bfpA-negative, while 46.5% carried hlyA. This study revealed that dairy cattle are a major reservoir of STEC and EPEC in South Africa. Further comparative studies of cattle and human STEC and EPEC isolates will be needed to determine the role played by dairy cattle STEC and EPEC in the occurrence of foodborne disease in humans.Please kindly check and confirm the country and city name in affiliation [6].This affiliation is correct.Please kindly check and confirm the affiliationsConfirmed. All Affiliations are accurate.
Assuntos
Escherichia coli Enteropatogênica , Infecções por Escherichia coli , Proteínas de Escherichia coli , Fezes , Sorogrupo , Escherichia coli Shiga Toxigênica , Fatores de Virulência , Animais , Bovinos , África do Sul , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/isolamento & purificação , Escherichia coli Enteropatogênica/classificação , Escherichia coli Enteropatogênica/patogenicidade , Fezes/microbiologia , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/classificação , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Fatores de Virulência/genética , Virulência/genética , Proteínas de Escherichia coli/genética , Sorotipagem , Doenças dos Bovinos/microbiologia , Indústria de Laticínios , Matadouros , Reação em Cadeia da PolimeraseRESUMO
BACKGROUND: Shiga toxin-producing Escherichia coli (STEC) infections are a significant public health concern as they can cause serious illness and outbreaks. In England, STEC incidence is highest among children and guidance recommends that children under six diagnosed with STEC are excluded from childcare until two consecutive stool cultures are negative. We aimed to describe the barriers and facilitators to implementing exclusion and the impact of exclusion policies on young children and their families. METHODS: Individual level data was obtained from a wider study focusing on shedding duration among STEC cases aged < 6 years between March 2018 - March 2022. Data was extracted from England's public health case management system. The case management system includes notes on telephone conversations, email correspondence and meeting minutes relating to the case. Collected data consisted of free text in three forms: (1) quotes from parents, either direct or indirect, (2) direct quotes from the case record by health protection practitioners or environmental health officers, and (3) summaries by the data collector after reviewing the entire case record. We analysed free text comments linked to 136 cases using thematic analysis with a framework approach. RESULTS: The median age of included cases was 3 years (IQR 1.5-5), with males accounting for 49%. Nine key themes were identified. Five themes focused on barriers to managing exclusion, including (i) financial losses, (ii) challenges with communication, engagement and collaboration, (iii) issues with sampling, processing, and results, (iv) adverse impact on children and their families and (v) conflicting exclusion advice. Four themes related to facilitators to exclusion, including (i) good communication with parents and childcare settings, (ii) support with childcare, (iii) improvements to sampling, testing, and reporting of results, and (iv) provision of supervised control measures. CONCLUSIONS: Qualitative analysis of public health case records can provide evidence-based insights around complex health protection issues to inform public health guidelines. Our analysis highlights the importance of considering wider social and economic consequences of exclusion when developing policies and practices for the management of STEC in children.
Assuntos
Infecções por Escherichia coli , Pesquisa Qualitativa , Escherichia coli Shiga Toxigênica , Humanos , Escherichia coli Shiga Toxigênica/isolamento & purificação , Masculino , Pré-Escolar , Feminino , Inglaterra , Lactente , Administração de Caso/organização & administração , Saúde Pública , CriançaRESUMO
The current study aimed to detect virulence, hetero-pathogenicity, and hybridization genes in Escherichia coli strains, previously isolated from cloacal swabs in commercial breeding psittacines and zoological collections, via multiplex PCR. A total of 68 strains of E. coli, previously isolated from psittacines in zoos and commercial breeding facilities in Ceará, Brazil, were assessed for the presence of the following genes and/or probes: eae, bfpA (EPEC - Enteropathogenic E. coli), CVD432 (EAEC - Enteroaggregative E. coli); LT gene and ST gene (ETEC - Enterotoxigenic E. coli); ipaH (EIEC - Enteroinvasive E. coli); stx1 and stx2 (STEC - Shiga toxin-producing E. coli); iroN, ompT, hlyF, iss, and iutA (APEC - Avian pathogenic E. coli). Of the 68 E. coli strains analyzed, 61 (98.7â¯%) were positive for the following genes and/or probes: Stx1 (61/98.7â¯%), ST gene (54/79.4â¯%), CVD432 (49/72â¯%), bfpA (44/64.7â¯%), eae (42/61.8â¯%), Stx2 (41/60.3â¯%), ipaH (34/50â¯%), LT gene (33/48.5â¯%), iroN (21/30.9â¯%), hlyF (11/6.2â¯%), iss (06/8.8â¯%) and iutA (06/8.8â¯%). The following diarrheagenic pathotypes were identified: 66 (97â¯%) from STEC, 49 (72â¯%) from EAEC, 35 (52â¯%) from EIEC, 25 (37â¯%) from ETEC, and one (1.5â¯%) from EPEC. Regarding hetero-pathogenicity, 50 (74â¯%) heterogeneous strains were identified. Positivity for APEC was seen in four (6â¯%) strains, all characterized as pathogenic hybrids. This study describes significant associations of virulence factors in E. coli strains DEC/DEC and DEC/APEC, which were isolated from psittacines and may be potentially harmful to One Health.
Assuntos
Animais de Zoológico , Doenças das Aves , Infecções por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli , Fatores de Virulência , Animais , Brasil , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Animais de Zoológico/microbiologia , Doenças das Aves/microbiologia , Escherichia coli/genética , Escherichia coli/patogenicidade , Escherichia coli/isolamento & purificação , Escherichia coli/classificação , Proteínas de Escherichia coli/genética , Fatores de Virulência/genética , Virulência/genética , Escherichia coli Enteropatogênica/genética , Escherichia coli Enteropatogênica/patogenicidade , Escherichia coli Enteropatogênica/isolamento & purificação , Escherichia coli Enteropatogênica/classificação , Reação em Cadeia da Polimerase Multiplex , Psittaciformes/microbiologia , Cloaca/microbiologia , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/patogenicidade , Escherichia coli Enterotoxigênica/isolamento & purificação , Escherichia coli Enterotoxigênica/classificaçãoRESUMO
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
BackgroundHaemolytic uremic syndrome (HUS) is a severe complication of infection with Shiga toxin-producing Escherichia coli (STEC). Although the reservoirs of STEC are known, the source of the infection of sporadic cases is often unknown. In 2023, we observed several cases of bloody diarrhoea with STEC infection in children and adolescents returning from vacations.AimWe aimed to explore the association between travel and bloody diarrhoea with STEC infection in children and adolescents.MethodsWe included all children and adolescents with bloody diarrhoea with STEC infection identified in 2023 by the ItalKid-HUS Network surveillance system in northern Italy. We interviewed children's families and sent a questionnaire on recent travels abroad. The exposure time was between 3â¯days after arrival abroad and 5â¯days after return home. A self-controlled case series (SCCS) design was used in the analysis.ResultsOf the 43 cases, 11 developed HUS. Twenty-three cases did not travel abroad, while 20 had travelled to several destinations. The incidence rate ratio (IRR) associated with travel to Egypt was 88.6 (95% confidence interval (CI): 17.0-462). Serotype analysis excluded the possibility of a single strain causing the infections. We did not find the source of the infections.ConclusionThere is an elevated risk of acquiring STEC infection with bloody diarrhoea and HUS associated with travel to Egypt. Specific investigations to identify the source are needed to implement effective preventive measures.
Assuntos
Diarreia , Infecções por Escherichia coli , Síndrome Hemolítico-Urêmica , Escherichia coli Shiga Toxigênica , Viagem , Humanos , Egito/epidemiologia , Escherichia coli Shiga Toxigênica/isolamento & purificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/diagnóstico , Adolescente , Criança , Feminino , Masculino , Síndrome Hemolítico-Urêmica/epidemiologia , Síndrome Hemolítico-Urêmica/microbiologia , Itália/epidemiologia , Diarreia/microbiologia , Diarreia/epidemiologia , Pré-Escolar , Lactente , Incidência , Vigilância da PopulaçãoRESUMO
Lymph nodes (LN) harboring bacteria, when being incorporated into ground beef, may impact the microbial safety and quality of such products. We tested two main foodborne pathogens Salmonella and Shiga toxin-producing Escherichia coli (STEC) and profiled the microbiota in LNs (n = 160) of cattle harvested at a Canadian abattoir, by conventional plating methods, PCR, and high throughput sequencing. LNs at two anatomical locations, subiliac and popliteal from 80 cattle were included. All cattle had bacteria detected in popliteal and/or subiliac LNs with the maximum bacterial load of 5.4 and 2.8 log10CFU/g in popliteal and subiliac LNs, respectively. Neither Salmonella nor STEC was found in LNs although STEC was detected in a significant percentage of samples from beef hides (50.6 %) by plating and/or PCR. Both 16S rRNA gene amplicon and metagenome sequencing found the predominance of Escherichia (13-34.6 % among bacterial community), Clostridium (12.6-20.6 %) and Streptococcus (9.7-10 %) in popliteal LNs. Metagenomic sequencing was able to identify the predominant taxa at species level with E. coli (13 %), Clostridium perfringens (11.1 %) and Streptococcus uberis (6 %) predominant in LNs. Low prevalence/abundance of Salmonella was found by metagenomic sequencing. In conclusion, the relatively high bacterial load and diversity in LNs may affect the shelf life of ground beef and high relative abundance of E. coli would warrant further monitoring.
Assuntos
Matadouros , Linfonodos , Microbiota , RNA Ribossômico 16S , Salmonella , Escherichia coli Shiga Toxigênica , Animais , Bovinos , Canadá , Linfonodos/microbiologia , RNA Ribossômico 16S/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/genética , Salmonella/isolamento & purificação , Salmonella/genética , Salmonella/classificação , Carne Vermelha/microbiologia , Microbiologia de AlimentosRESUMO
Shiga toxin-producing Escherichia coli (STEC) causes a wide spectrum of diseases including hemorrhagic colitis and hemolytic uremic syndrome (HUS). Previously, we developed a rapid, sensitive, and potentially portable assay that identified STEC by detecting Shiga toxin (Stx) using a B-cell based biosensor platform. We applied this assay to detect Stx2 present in food samples that have been implicated in previous STEC foodborne outbreaks (milk, lettuce, and beef). The STEC enrichment medium, modified Tryptone Soy Broth (mTSB), inhibited the biosensor assay, but dilution with the assay buffer relieved this effect. Results with Stx2a toxoid-spiked food samples indicated an estimated limit of detection (LOD) of ≈4 ng/mL. When this assay was applied to food samples inoculated with STEC, it was able to detect 0.4 CFU/g or 0.4 CFU/mL of STEC at 16 h post incubation (hpi) in an enrichment medium containing mitomycin C. Importantly, this assay was even able to detect STEC strains that were high expressors of Stx2 at 8 hpi. These results indicate that the STEC CANARY biosensor assay is a rapid and sensitive assay applicable for detection of STEC contamination in food with minimal sample processing that can complement the current Food Safety Inspection Service (US) methodologies for STEC.
Assuntos
Técnicas Biossensoriais , Microbiologia de Alimentos , Lactuca , Escherichia coli Shiga Toxigênica , Escherichia coli Shiga Toxigênica/isolamento & purificação , Técnicas Biossensoriais/métodos , Lactuca/microbiologia , Contaminação de Alimentos/análise , Leite/microbiologia , Animais , Toxina Shiga II/análise , Toxina Shiga II/genética , Limite de Detecção , Carne Vermelha/microbiologia , BovinosRESUMO
AIM: To investigate the possible contamination of raw flour and raw flour-based products, such as pancake/batter mixes, with Salmonella, generic Escherichia coli, and Shiga-toxin-producing E. coli (STEC). Samples included flours available for sale in the UK over a period of four months (January to April 2020). The Bread and Flour regulations, 1998 state the permitted ingredients in flour and bread but it does not specify the regular monitoring of the microbiological quality of flour and flour-based products. METHODS AND RESULTS: Samples of raw flour were collected by local authority sampling officers in accordance with current guidance on microbiological food sampling then transported to the laboratory for examination. Microbiological testing was performed to detect Salmonella spp., generic E. coli, and STEC characterized for the presence of STEC virulence genes: stx1, stx2, and subtypes, eae, ipah, aggR, lt, sth, and stp, using molecular methods Polymerase Chain Reaction (PCR). Of the 882 flours sampled, the incidence of Salmonella was 0.1% (a single positive sample that contained multiple ingredients such as flour, dried egg, and dried milk, milled in the UK), and 68 samples (7.7%) contained generic E. coli at a level of >20 CFU/g. Molecular characterization of flour samples revealed the presence of the Shiga-toxin (stx) gene in 10 samples (5 imported and 5 from the UK) (1.1%), from which STEC was isolated from 7 samples (0.8%). Salmonella and STEC isolates were sequenced to provide further characterization of genotypes and to compare to sequences of human clinical isolates held in the UKHSA archive. Using our interpretive criteria based on genetic similarity, none of the STEC flour isolates correlated with previously observed human cases, while the singular Salmonella serotype Newport isolate from the mixed ingredient product was similar to a human case in 2019, from the UK, of S. Newport. Although there have been no reported human cases of STEC matching the isolates from these flour samples, some of the same serotypes and stx subtypes detected are known to have caused illness in other contexts. CONCLUSION: Results indicate that while the incidence was low, there is a potential for the presence of Salmonella and STEC in flour, and a genetic link was demonstrated between a Salmonella isolate from a flour-based product and a human case of salmonellosis.
Assuntos
Farinha , Microbiologia de Alimentos , Salmonella , Escherichia coli Shiga Toxigênica , Farinha/microbiologia , Farinha/análise , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/genética , Salmonella/genética , Salmonella/isolamento & purificação , Reino Unido , Contaminação de Alimentos/análise , HumanosRESUMO
Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens frequently carried by cattle, responsible in humans of mild to bloody diarrhoea, haemolytic uraemic syndrome (HUS) and even death. In 2023-2024, a study on STEC contamination of hide and carcasses of dairy cattle at slaughter was planned in Emilia-Romagna region (northern Italy). When the study was still in progress and 60 animals were sampled, the detection of STEC O177 isolates reached high rates and gained our attention. A total of five O177 STEC strains were detected, namely four from three carcasses (5.0 %) and one from a hide sample (1.7 %). The isolates were typed by WGS as following: 1) STEC O177:H11 sequence type (ST) 765 (stx2a+, eae+), detected from one carcass; 2) STEC O177:H25 ST659 (stx2c+, eae+) detected from three carcasses and one hide sample. One carcass was contaminated by both STEC serotypes. The isolates carried other virulence determinants often found in STEC strains associated with HUS, namely the exha, astA and espP genes, together with genes for adhesion to the epithelial cells of the gut (lpfA, fdeC, fimH) and non-Locus for Enterocyte Effacement (LEE) effector protein genes (nleA, nleB). The STEC O177:H11 isolate harboured antimicrobial resistance (AMR) genes to ß-lactams (blaTEM-1A), aminoglycosides (aadA1, aph(3â³)-Ib, aph(6)-Id), trimethoprim (dfrA1), sulphonamides (sul1, sul2), tetracyclines (tetA), (tetB), streptothricin (sat2), and quaternary ammonium compounds (qacEdelta1). On the contrary, the STEC O177:H25 isolates carried no AMR genes. Persistent carriage of STEC O177:H25 ST659 (stx2c+, eae+) at farm level was assessed by testing animals of the same herd sent to slaughter. Interestingly, the colonies of STEC O177:H11 and STEC O177:H25 had different morphology on CHROMagar™ STEC plates, being mauve and colourless, respectively. Since mauve is the colour STEC colonies commonly have on the CHROMagar™ STEC medium, our findings can help microbiologists in the selection of uncommon serotypes. To the best of our knowledge, this is the first detection of STEC O177 from carcasses and hides of dairy cattle at slaughter. Noteworthy, the STEC-positive hide was classified as "very dirty" thus stressing the need of clean animals entering the slaughter chain, as required by Regulation (EC) No 853/2004. Since STEC O177 has been responsible of HUS in Europe, our data could add information on the source of uncommon serogroups in human infections.
Assuntos
Matadouros , Escherichia coli Shiga Toxigênica , Bovinos , Animais , Itália , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/classificação , Fatores de Virulência/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Antibacterianos/farmacologia , Virulência , Doenças dos Bovinos/microbiologia , SorogrupoRESUMO
Shiga toxin-producing Escherichia coli (STEC) are zoonotic pathogens causing hemorrhagic colitis and hemolytic uremic syndrome (HUS) in children and the elderly. Stool samples were collected from 180 children hospitalized in five pediatric centers in Poland in 2018-2022. Direct stx1/stx2 gene detection by PCR in feces and E. coli isolates was performed. Antibiotic susceptibility was tested according to EUCAST v.12. Randomly selected isolates were serotyped with O157 antiserum and genotyped by pulsed-field gel electrophoresis (PFGE). A total of 44 E. coli isolates were confirmed as STEC by PCR. Among them, 84.4% were positive for stx2, and equally 6,8% for only stx1 and both stx1 and stx2 genes. The stx1 gene was also found in one Citrobacter freundii isolate. E. coli serotype O157 was present in 97.6% of the isolates. STEC infections most often occurred between June-October with a peak in July and August (51%). The highest, 77.8% of STEC isolates were found in the 1-5 years old group. No extended-spectrum ß-lactamases (ESBL) were found. Resistance only to amoxicillin/clavulanic acid (24.4%), piperacillin/tazobactam (3%), cefotaxime (6%), gentamicin (6%), ciprofloxacin (3%), azithromycin (3%), trimethoprim/sulfamethoxazole (24,2%) was detected. PFGE analysis showed 18 PFGE types with no clonal distribution. Eight isolates with A, B, and C PFGE types showed genetic relatedness in the type with no detection of transmission way of distribution. STEC strains pose a serious threat to human health, therefore demographic and epidemiological characteristics are crucial for their surveillance.
Assuntos
Antibacterianos , Infecções por Escherichia coli , Fezes , Escherichia coli Shiga Toxigênica , Humanos , Polônia/epidemiologia , Pré-Escolar , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/classificação , Infecções por Escherichia coli/epidemiologia , Infecções por Escherichia coli/microbiologia , Criança , Lactente , Antibacterianos/farmacologia , Fezes/microbiologia , Feminino , Masculino , Testes de Sensibilidade Microbiana , Adolescente , Eletroforese em Gel de Campo Pulsado , Genótipo , Recém-NascidoRESUMO
The emerging heteropathotype shigatoxigenic (STEC) and extra-intestinal pathogenic Escherichia coli (ExPEC) O80:H2 has been the second leading cause of pediatric HUS in France since the mid-2010s. In contrast with other highly pathogenic STEC serotypes, for which ruminants have clearly been identified as the main human infection source, this heteropathotype's reservoir remains unknown. In this context, we describe for the first time the isolation of seven STEC O80:H2 strains from healthy cattle on a single cattle farm in France. This study aimed at (i) characterizing the genome and (ii) investigating the phylogenetic positions of these O80:H2 STEC strains. The virulomes, resistomes, and phylogenetic positions of the seven bovine isolates were investigated using in silico typing tools, antimicrobial susceptibility testing and cgMLST analysis after short-read whole genome sequencing (WGS). One representative isolate (A13P112V1) was also subjected to long-read sequencing. The seven isolates possessed ExPEC-related virulence genes on a pR444_A-like mosaic plasmid, previously described in strain RDEx444 and known to confer multi-drug resistance. All isolates were clonally related and clustered with human clinical strains from France and Switzerland with a range of locus differences of only one to five. In conclusion, our findings suggest that healthy cattle in France could potentially act as a reservoir of the STEC-ExPEC O80:H2 pathotype.
Assuntos
Infecções por Escherichia coli , Genoma Bacteriano , Filogenia , Escherichia coli Shiga Toxigênica , Sequenciamento Completo do Genoma , Animais , Bovinos , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/patogenicidade , Escherichia coli Shiga Toxigênica/classificação , França , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Sequenciamento Completo do Genoma/métodos , Escherichia coli Extraintestinal Patogênica/genética , Escherichia coli Extraintestinal Patogênica/isolamento & purificação , Escherichia coli Extraintestinal Patogênica/patogenicidade , Doenças dos Bovinos/microbiologia , Fatores de Virulência/genética , Virulência/genética , Sorogrupo , Genômica/métodos , Plasmídeos/genéticaRESUMO
BACKGROUND: A number of studies have detected relationships between weather and diarrhea. Few have investigated associations with specific enteric pathogens. Understanding pathogen-specific relationships with weather is crucial to inform public health in low-resource settings that are especially vulnerable to climate change. OBJECTIVES: Our objectives were to identify weather and environmental risk factors associated with diarrhea and enteropathogen prevalence in young children in rural Bangladesh, a population with high diarrheal disease burden and vulnerability to weather shifts under climate change. METHODS: We matched temperature, precipitation, surface water, and humidity data to observational longitudinal data from a cluster-randomized trial that measured diarrhea and enteropathogen prevalence in children 6 months-5.5 years from 2012-2016. We fit generalized additive mixed models with cubic regression splines and restricted maximum likelihood estimation for smoothing parameters. RESULTS: Comparing weeks with 30°C versus 15°C average temperature, prevalence was 3.5% higher for diarrhea, 7.3% higher for Shiga toxin-producing Escherichia coli (STEC), 17.3% higher for enterotoxigenic E. coli (ETEC), and 8.0% higher for Cryptosporidium. Above-median weekly precipitation (median: 13mm; range: 0-396mm) was associated with 29% higher diarrhea (adjusted prevalence ratio 1.29, 95% CI 1.07, 1.55); higher Cryptosporidium, ETEC, STEC, Shigella, Campylobacter, Aeromonas, and adenovirus 40/41; and lower Giardia, sapovirus, and norovirus prevalence. Other associations were weak or null. DISCUSSION: Higher temperatures and precipitation were associated with higher prevalence of diarrhea and multiple enteropathogens; higher precipitation was associated with lower prevalence of some enteric viruses. Our findings emphasize the heterogeneity of the relationships between hydrometeorological variables and specific enteropathogens, which can be masked when looking at composite measures like all-cause diarrhea. Our results suggest that preventive interventions targeted to reduce enteropathogens just before and during the rainy season may more effectively reduce child diarrhea and enteric pathogen carriage in rural Bangladesh and in settings with similar meteorological characteristics, infrastructure, and enteropathogen transmission.
Assuntos
Diarreia , População Rural , Humanos , Bangladesh/epidemiologia , Diarreia/epidemiologia , Diarreia/microbiologia , Lactente , Pré-Escolar , Fatores de Risco , População Rural/estatística & dados numéricos , Prevalência , Masculino , Feminino , Tempo (Meteorologia) , Escherichia coli Enterotoxigênica/isolamento & purificação , Cryptosporidium/isolamento & purificação , Temperatura , Escherichia coli Shiga Toxigênica/isolamento & purificação , Mudança Climática , Criptosporidiose/epidemiologiaRESUMO
Shiga toxin-producing Escherichia coli (STEC) are foodborne enteric pathogens. STEC are differentiated from other E. coli by detection of Shiga toxin (Stx) or its gene (stx). The established nomenclature of Stx identifies ten subtypes (Stx1a, Stx1c, Stxd, Stx2a to Stx2g). An additional nine subtypes have been reported and described (Stx1e, Stx2h to Stx2o). Many PCR protocols only detect a subset of Stx subtypes which limits their inclusivity. Here we describe a real-time PCR assay inclusive of the DNA sequences of representatives of all currently described Stx subtypes. A multiplex real-time PCR assay for detection of stx was developed using nine primers and four probes. Since the identification of STEC does not require differentiation of stx subtypes, the probes use the same fluorescent reporter to enable detection of multiple possible targets in a single reaction. The PCR mixture includes an internal positive control to detect inhibition of the reaction. Thus, the protocol can be performed on a two-channel real-time PCR platform. To reduce the biosafety risk inherent in the use of STEC cultures as process controls, the protocol also includes the option of a non-pathogenic E. coli transformant carrying a plasmid encoding the targeted fragment of the stx2a sequence. The inclusivity of the PCR was assessed against colonies of 137 STEC strains and one strain of Shigella dysenteriae, including strains carrying single copies of stx representing fourteen subtypes (stx1 a, c, d; stx2 a-j and o). Five additional subtypes (stx1e, 2k, 2l, 2m and 2n) were represented by E. coli transformed with plasmids encoding toxoid (enzymatically inactive A subunit) sequences. The exclusivity panel consisted of 70 bacteria, including 21 stx-negative E. coli. Suitability for food analysis was assessed with artificially inoculated ground beef, spinach, cheese, and apple cider. The real-time PCR generated positive results for all 19 stx subtypes, represented by colonies of STEC, S. dysenteriae and E. coli transformants carrying stx toxoid plasmids. Tests of exclusivity panel colonies were all negative. The real-time PCR detected the presence of stx in all inoculated food enrichments tested, and the presence of STEC was confirmed by isolation.
Assuntos
Primers do DNA , Reação em Cadeia da Polimerase em Tempo Real , Escherichia coli Shiga Toxigênica , Reação em Cadeia da Polimerase em Tempo Real/métodos , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/classificação , Escherichia coli Shiga Toxigênica/isolamento & purificação , Primers do DNA/genética , Microbiologia de Alimentos , Contaminação de Alimentos/análise , Toxina Shiga/genética , Reação em Cadeia da Polimerase Multiplex/métodosRESUMO
The indiscriminate use of antimicrobials has led to the emergence of resistant bacteria, especially pathogenic strains of Escherichia coli, which are associated with diseases in animals and humans. The aim of the present study was to characterize E. coli isolates in calves with regards to the presence of virulence genes and investigate the resistance of the isolates to different antimicrobials. Between 2021 and 2023, 456 fecal samples were collected from calves in the Pantanal and Cerrado biomes of the state of Mato Grosso do Sul, Brazil. All samples were subjected to microbiological analysis and disc diffusion antibiogram testing. The polymerase chain reaction method was used to detect virulence genes. Bacterial growth was found in 451 of the 456 samples and biochemically identified as Escherichia coli. All 451 isolates (100 %) exhibited some phenotypic resistance to antimicrobials and 67.62 % exhibited multidrug resistance. The frequency of multidrug-resistant isolates in the Cerrado biome was significantly higher than that in the Pantanal biome (p = 0.0001). In the Cerrado, the most common pathotype was Shiga toxin-producing Escherichia coli (STEC) (28 %), followed by toxigenic Escherichia coli (ETEC) (11 %), enterohemorrhagic Escherichia coli (EHEC) (8 %) and enteropathogenic Escherichia coli (EPEC) (2 %). In most cases, the concomitant occurrence of pathotypes was more common, the most frequent of which were ETEC + STEC (33 %), ETEC + EHEC (15 %) and ETEC + EPEC (3 %). The STEC pathotype (30 %) was also found more frequently in the Pantanal, followed by EHEC (12 %), ETEC (9 %) and EPEC (6 %). The STEC pathotype had a significantly higher frequency of multidrug resistance (p = 0.0486) compared to the other pathotypes identified. The frequency of resistance was lower in strains from the Pantanal biome compared to those from the Cerrado biome. Although some factors are discussed in this paper, it is necessary to clarify the reasons for this difference and the possible impacts of these findings on both animal and human health in the region.
Assuntos
Antibacterianos , Doenças dos Bovinos , Farmacorresistência Bacteriana Múltipla , Infecções por Escherichia coli , Escherichia coli , Fezes , Testes de Sensibilidade Microbiana , Fatores de Virulência , Animais , Bovinos , Brasil , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/veterinária , Infecções por Escherichia coli/epidemiologia , Fezes/microbiologia , Antibacterianos/farmacologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/isolamento & purificação , Doenças dos Bovinos/microbiologia , Doenças dos Bovinos/epidemiologia , Fatores de Virulência/genética , Escherichia coli Shiga Toxigênica/isolamento & purificação , Escherichia coli Shiga Toxigênica/genética , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Êntero-Hemorrágica/genética , Escherichia coli Êntero-Hemorrágica/isolamento & purificação , Escherichia coli Êntero-Hemorrágica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Escherichia coli Enterotoxigênica/genética , Escherichia coli Enterotoxigênica/isolamento & purificação , Proteínas de Escherichia coli/genéticaRESUMO
During July-September 2023, an outbreak of Shiga toxin-producing Escherichia coli O157:H7 illness among children in city A, Utah, caused 13 confirmed illnesses; seven patients were hospitalized, including two with hemolytic uremic syndrome. Local, state, and federal public health partners investigating the outbreak linked the illnesses to untreated, pressurized, municipal irrigation water (UPMIW) exposure in city A; 12 of 13 ill children reported playing in or drinking UPMIW. Clinical isolates were genetically highly related to one another and to environmental isolates from multiple locations within city A's UPMIW system. Microbial source tracking, a method to indicate possible contamination sources, identified birds and ruminants as potential sources of fecal contamination of UPMIW. Public health and city A officials issued multiple press releases regarding the outbreak reminding residents that UPMIW is not intended for drinking or recreation. Public education and UPMIW management and operations interventions, including assessing and mitigating potential contamination sources, covering UPMIW sources and reservoirs, indicating UPMIW lines and spigots with a designated color, and providing conspicuous signage to communicate risk and intended use might help prevent future UPMIW-associated illnesses.