Detection and Isolation of Escherichia coli O157:H7 in Beef from Food Markets and Fecal Samples of Dairy Calves in the Peruvian Central Highlands.

Shiga toxin-producing Escherichia coli (STEC) O157:H7 is a food and waterborne pathogen with severe public health implications. We report the first-time isolation of this pathogen in the Central Highlands of Peru through standardized culture procedures and polymerase chain reaction (PCR). Escherichia coli strains were cultured from rectal-anal swabs from dairy calves and beef from food markets. The latex agglutination test was used to detect O157 and H7 antigens, and multiplex real-time PCR was carried out to detect virulence-related genes. The STEC O157:H7 strains were isolated from 3.5% (1/28) of beef samples and from 6.0% (3/50) of dairy calves that also carried both eaeA and stx1 genes. Therefore, this pathogen is a potential cause of food/waterborne disease in the region, and its surveillance in both livestock and their products should be improved to characterize the impact of its zoonotic transmission. From 2010 to 2020, E. coli was suspected in 10 outbreaks reported to the Peruvian Ministry of Health. Isolates from future outbreaks should be characterized to assess the burden posed by STEC O157:H7 in Peru.

A point-of-care detection platform for Escherichia coli O157:H7 by integration of smartphone and the structural colour of photonic microsphere.

A smartphone-based sensitive, rapid, label-free and high-throughput detection platform for Escherichia coli O157:H7 was established. The specific recognition capability of this platform was dependent of the aptamer modified on the silica photonic microsphere (SPM), whose structural colour was utilized for the quantification of the target bacterium. Gold nanoparticles and silver staining technique were employed to improve the sensitivity of the detection platform. Such smartphone-based detection platform gave a wide linear detection range of 102 âˆ¼ 108 CFU/mL with a low limit of detection (LOD) of 68 CFU/mL and high specificity for Escherichia coli O157:H7. Moreover, the recovery rates of the detection method were measured in the range of 99 âˆ¼ 108% in the milk, pork and purified water samples. Furthermore, the developed detection platform did not require complex sample pretreatment and could be easily manipulated, displaying great application potential in the fields of food safety, environmental monitoring and disease diagnosis.

Síndrome urémico hemolítico del adulto / Adult hemolytic uremic syndrome

El síndrome urémico hemolítico (SUH), descripto en 1955, se caracteriza por la tríada de anemia hemolítica no inmunomediada, trombocitopenia y lesión renal aguda. En su patogenia interviene la toxina Shiga, producida con mayor frecuencia por E. coli O157:H. Puede manifestarse a cualquier edad, aunque es infrecuente en adultos, y se desarrolla en forma esporádica o en brote. Se presenta con un cuadro de dolor abdominal, diarrea, fiebre y vómitos. Puede afectar el sistema nervioso central, pulmones, páncreas y corazón. En adultos, el síndrome evoluciona tras un período de incubación de 1 semana posterior a la diarrea y tiene alta morbimortalidad, a diferencia de los casos pediátricos. Presentamos el caso de una paciente adulta, que cursó internación por síndrome urémico hemolítico. (AU)

Hemolytic uremic syndrome (HUS), described in 1955, is characterized by the triad of non-immune mediated hemolytic anemia, thrombocytopenia, and acute kidney injury. Shiga toxin, produced most frequently by E coli O157:H, is involved in its pathogenesis. Hus can manifest at any age, although it is rare in adults and develops sporadically or in outbreaks. HUS presents with a picture of abdominal pain, diarrhea, fever and vomiting. It can affect the central nervous system, lungs, pancreas, and heart.In adults, the syndrome evolves after an incubation period of 1 week after diarrhea, with high morbidity and mortality, unlike pediatric cases.We present the case of an adult patient who was hospitalized for hemolytic uremic syndrome. (AU)

A Novel Activated Biochar-Based Immunosensor for Rapid Detection of E. coli O157:H7.

E. coli O157:H7, one of the major foodborne pathogens, can cause a significant threat to the safety of foods. The aim of this research is to develop an activated biochar-based immunosensor that can rapidly detect E. coli O157:H7 cells without incubation in pure culture. Biochar was developed from corn stalks using proprietary reactors and then activated using steam-activation treatment. The developed activated biochar presented an enhanced surface area of 830.78 m2/g. To develop the biosensor, the gold electrode of the sensor was first coated with activated biochar and then functionalized with streptavidin as a linker and further immobilized with biotin-labeled anti-E. coli polyclonal antibodies (pAbs). The optimum concentration of activated biochar for sensor development was determined to be 20 mg/mL. Binding of anti-E. coli pAbs with E. coli O157:H7 resulted in a significant increase in impedance amplitude from 3.5 to 8.5 kΩ when compared to an only activated biochar-coated electrode. The developed immunosensor was able to detect E. coli O157:H7 cells with a limit of detection of 4 log CFU/mL without incubation. Successful binding of E. coli O157:H7 onto an activated biochar-based immunosensor was observed on the microelectrode surface in scanning electron microscopy (SEM) images.

Polydopamine nanospheres-assisted direct PCR for rapid detection of Escherichia coli O157:H7.

Polymerase chain reaction (PCR) is one of the most common methods for rapid monitoring of foodborne pathogens; however, it requires purified nucleic acid as a template. Conventional nucleic acid purification is a time-consuming and laborious process. To overcome this, we developed polydopamine nanospheres (PDA NPs)-assisted direct PCR for detecting Escherichia coli O157:H7 (E. coli O157:H7). PDA NPs significantly enhanced PCR efficiency because of their strong interaction with PCR reagents, including polymerase and primers, thereby enabling regulation of the PCR performance. The optimal concentration and diameter for PDA NPs were 0.10 µg/µL and 504 nm, respectively. The PDA NPs-assisted direct PCR exhibited high sensitivity in E.coli O157:H7 detection. The detection limit of PDA NPs-assisted direct PCR was 6.7 × 104 CFU/mL, which was 10-fold lower than that of direct PCR (6.7 × 105 CFU/mL). Moreover, the sensor demonstrated excellent selectivity against E. coli O157:H7, with a negative reaction to eight other common pathogens. Most importantly, the PDA NPs-assisted direct PCR detected the order of 104-5 CFU/mL E.coli O157:H7 in milk, beef, and watermelon samples. No cultural enrichment was required, with the whole process taking <3 h. Therefore, PDA NPs-assisted direct PCR has tremendous potential in the rapid and sensitive detection of pathogens.

Bacteriological Survey of Fresh Minced Beef on Sale at Retail Outlets in Scotland in 2019: Three Foodborne Pathogens, Hygiene Process Indicators, and Phenotypic Antimicrobial Resistance.

ABSTRACT: The health and economic burden of foodborne illness is high, with approximately 2.4 million cases occurring annually in the United Kingdom. A survey to understand the baseline microbial quality and prevalence of food-related hazards of fresh beef mince on retail sale could inform risk assessment, management, and communication to ensure the safety of this commodity. In such a survey, a two-stage sampling design was used to reflect variations in population density and the market share of five categories of retail outlets in Scotland. From January to December 2019, 1,009 fresh minced beef samples were collected from 15 geographic areas. The microbial quality of each sample was assessed using aerobic colony count and Escherichia coli count. Samples were cultured for Campylobacter and Salmonella, and PCR was used to detect target genes (stx1 all variants, stx2 a to g, and rfbO157) for Shiga toxin-producing E. coli (STEC). The presence of viable E. coli O157 and STEC in samples with a positive PCR signal was confirmed via culture and isolation. Phenotypic antimicrobial sensitivity patterns of cultured pathogens and 100 E. coli isolates were determined, mostly via disk diffusion. The median aerobic colony count and E. coli counts were 6.4 × 105 (interquartile range, 6.9 × 104 to 9.6 × 106) and <10 CFU/g (interquartile range, <10 to 10) of minced beef, respectively. The prevalence was 0.1% (95% confidence interval [CI], 0 to 0.7%) for Campylobacter, 0.3% (95% CI, 0 to 1%) for Salmonella, 22% (95% CI, 20 to 25%) for PCR-positive STEC, and 4% (95% CI, 2 to 5%) for culture-positive STEC. The evidence for phenotypic antimicrobial resistance detected did not give cause for concern, mainly occurring in a few E. coli isolates as single nonsusceptibilities to first-line active substances. The low prevalence of pathogens and phenotypic antimicrobial resistance is encouraging, but ongoing consumer food safety education is necessary to mitigate the residual public health risk.

Escherichia coli serogroups in slaughterhouses: Antibiotic susceptibility and molecular typing of isolates.

This study aimed to investigate the contamination of carcasses and slaughterhouse environment with Escherichia coli O157:H7 and non-O157 serogroups (O45:H2, O103:H2, O121:H19, O145:H28, O26:H11, O111:H8). For this purpose, a total of 150 samples (30 carcasses, 30 shredding units, 30 knives, 30 slaughterhouse waste water and 30 wall surfaces) were collected from 5 different slaughterhouses in Kayseri, Turkey. The conventional and molecular methods were performed in order to detect Escherichia coli and its serogroups. Of the 150 samples, 55 (36%) were found to be contaminated with E. coli. Among isolates, E. coli serogroup (O157:H7) were detected in 2 (11%) carcass and 2 (11%) wastewater samples. None of the E. coli isolates harbored tested genes (stx1, stx2, eaeA, and hylA). Effective infection control measures and antibiotic stewardship programs should be adopted to limit the spread of multidrug-resistant bacteria. It was also deduced that these isolates resistance to different antibiotics could be hazardous for public health.

A microfluidic biosensor for rapid simultaneous detection of waterborne pathogens.

A microfluidic based biosensor was investigated for rapid and simultaneous detection of Salmonella, Legionella, and Escherichia coli O157:H7 in tap water and wastewater. The biosensor consisted of two sets of focusing electrodes connected in parallel and three sets of interdigitated electrodes (IDE) arrays. The electrodes enabled the biosensor to concentrate and detect bacteria at both low and high concentrations. The focusing region was designed with vertical metal sidewall pairs and multiple tilted thin-film finger pairs to generate positive dielectrophoresis (p-DEP) to force the bacteria moving toward the microchannel centerline. As a result, the bacterial pathogens were highly concentrated when they reached the detection electrode arrays. The detection IDE arrays were coated with three different antibodies against the target bacterial pathogens and a cross-linker to enhance the binding of antibodies to the detection electrode. As the binding of bacterial pathogen to its specific antibodies took place, the impedance value changed. The results demonstrated that the biosensors were capable of detecting Salmonella, Legionella, and E. coli 0157:H7 simultaneously with a detection limit of 3 bacterial cells/ml in 30 - 40 min.

Genomic analysis of shiga toxin-containing Escherichia coli O157:H7 isolated from Argentinean cattle.

Cattle are the main reservoir of Enterohemorrhagic Escherichia coli (EHEC), with O157:H7 the distinctive serotype. EHEC is the main causative agent of a severe systemic disease, Hemolytic Uremic Syndrome (HUS). Argentina has the highest pediatric HUS incidence worldwide with 12-14 cases per 100,000 children. Herein, we assessed the genomes of EHEC O157:H7 isolates recovered from cattle in the humid Pampas of Argentina. According to phylogenetic studies, EHEC O157 can be divided into clades. Clade 8 strains that were classified as hypervirulent. Most of the strains of this clade have a Shiga toxin stx2a-stx2c genotype. To better understand the molecular bases related to virulence, pathogenicity and evolution of EHEC O157:H7, we performed a comparative genomic analysis of these isolates through whole genome sequencing. The isolates classified as clade 8 (four strains) and clade 6 (four strains) contained 13 to 16 lambdoid prophages per genome, and the observed variability of prophages was analysed. An inter strain comparison show that while some prophages are highly related and can be grouped into families, other are unique. Prophages encoding for stx2a were highly diverse, while those encoding for stx2c were conserved. A cluster of genes exclusively found in clade 8 contained 13 genes that mostly encoded for DNA binding proteins. In the studied strains, polymorphisms in Q antiterminator, the Q-stx2A intergenic region and the O and P γ alleles of prophage replication proteins are associated with different levels of Stx2a production. As expected, all strains had the pO157 plasmid that was highly conserved, although one strain displayed a transposon interruption in the protease EspP gene. This genomic analysis may contribute to the understanding of the genetic basis of the hypervirulence of EHEC O157:H7 strains circulating in Argentine cattle. This work aligns with other studies of O157 strain variation in other populations that shows key differences in Stx2a-encoding prophages.

Molecular detection of Shiga toxin-producing Escherichia coli (STEC) O157 in sheep, goats, cows and buffaloes.

BACKGROUND: Shiga toxin-producing E. coli (STEC) are important foodborne pathogens that causing serious public health consequences worldwide. The present study aimed to estimate the prevalence ratio and to identify the zoonotic potential of E. coli O157 isolates in slaughtered adult sheep, goats, cows and buffaloes. MATERIALS AND METHODS: A total of 400 Recto-anal samples were collected from two targeted sites Rawalpindi and Islamabad. Among them, 200 samples were collected from the slaughterhouse of Rawalpindi included sheep (n = 75) and goats (n = 125). While, 200 samples were collected from the slaughterhouse of Islamabad included cows (n = 120) and buffalos (n = 80). All samples were initially processed in buffered peptone water and then amplified by conventional PCR. Samples positive for E. coli O157 were then streaked onto SMAC media plates. From each positive sample, six different Sorbitol fermented pink-colored colonies were isolated and analyzed again via conventional PCR to confirm the presence of rfbE O157 gene. Isolates positive for rfbE O157 gene were then further analyzed by multiplex PCR for the presence of STEC other virulent genes (sxt1, stx2, eae and ehlyA) simultaneously. RESULTS: Of 400 RAJ samples only 2 (0.5%) showed positive results for E. coli O157 gene, included sheep 1/75 (1.33%) and buffalo 1/80 (1.25%). However, goats (n = 125) and cows (n = 120) found negative for E. coli O157. Only 2 isolates from each positive sample of sheep (1/6) and buffalo (1/6) harbored rfbE O157 genes, while five isolates could not. The rfbE O157 isolate (01) of sheep sample did not carry any of STEC genes, while the rfbE O157 isolate (01) of buffalo sample carried sxt1, stx2, eae and ehlyA genes simultaneously. CONCLUSION: It was concluded that healthy adult sheep and buffalo are possibly essential carriers of STEC O157. However, rfbE O157 isolate of buffalo RAJ sample carried 4 STEC virulent genes, hence considered an important source of STEC infection to humans and environment which should need to devise proper control systems.

Transition-Metal Dichalcogenide Artificial Antibodies with Multivalent Polymeric Recognition Phases for Rapid Detection and Inactivation of Pathogens.

Antibodies are recognition molecules that can bind to diverse targets ranging from pathogens to small analytes with high binding affinity and specificity, making them widely employed for sensing and therapy. However, antibodies have limitations of low stability, long production time, short shelf life, and high cost. Here, we report a facile approach for the design of luminescent artificial antibodies with nonbiological polymeric recognition phases for the sensitive detection, rapid identification, and effective inactivation of pathogenic bacteria. Transition-metal dichalcogenide (TMD) nanosheets with a neutral dextran phase at the interfaces selectively recognized S. aureus, whereas the nanosheets bearing a carboxymethylated dextran phase selectively recognized E. coli O157:H7 with high binding affinity. The bacterial binding sites recognized by the artificial antibodies were thoroughly identified by experiments and molecular dynamics simulations, revealing the significance of their multivalent interactions with the bacterial membrane components for selective recognition. The luminescent WS2 artificial antibodies could rapidly detect the bacteria at a single copy from human serum without any purification and amplification. Moreover, the MoSe2 artificial antibodies selectively killed the pathogenic bacteria in the wounds of infected mice under light irradiation, leading to effective wound healing. This work demonstrates the potential of TMD artificial antibodies as an alternative to antibodies for sensing and therapy.

Evidence of on-going transmission of Shiga toxin-producing Escherichia coli O157:H7 following a foodborne outbreak.

In August 2019, public health surveillance systems in Scotland and England identified seven, geographically dispersed cases infected with the same strain (defined as isolates that fell within the same five single nucleotide polymorphism single linage cluster) of Shiga toxin-producing Escherichia coli O157:H7. Epidemiological analysis of enhanced surveillance questionnaire data identified handling raw beef and shopping from the same national retailer (retailer A) as the common exposure. Concurrently, a microbiological survey of minced beef at retail identified the same strain in a sample of minced beef sold by retailer A, providing microbiological evidence of the link. Between September and November 2019, a further four primary and two secondary cases infected with the same strain were identified; two cases developed haemolytic uraemic syndrome. None of the four primary cases reported consumption of beef from retailer A and the transmission route of these subsequent cases was not identified, although all four primary cases visited the same petting farm. Generally, outbreaks of STEC O157:H7 in the UK appear to be distinct, short-lived events; however, on-going transmission linked to contaminated food, animals or environmental exposures and person-to-person contact do occur. Although outbreaks of STEC caused by contaminated fresh produce are increasingly common, undercooked meat products remain a risk of infection.

Review: Trends in point-of-care diagnosis for Escherichia coli O157:H7 in food and water.

Escherichia coli O157:H7, a Shiga-producing E. coli is a major pathogenic E. coli strain which since the early 1980s has become a crucial food and water-borne pathogen. Several management strategies can be applied to control the spread of infection; however early diagnosis represents the optimum preventive strategy to minimize the infection. Therefore, it is crucial to detect this pathogen in a fast and efficient manner in order to reduce the morbidity and mortality. Currently used gold standard tests rely on culture and pre-enrichment of E. coli O157:H7 from the contaminated source; they are time consuming and laborious. Molecular methods such as polymerase chain reaction are sensitive; however, they require expensive instrumentation. Therefore, there is a requirement for Accurate, Sensitive, Specific, User friendly, Rapid, Equipment free and Deliverable (ASSURED) detection methods for use in the laboratory and in the field. Emerging technologies such as isothermal amplification methods, biosensors, surface enhanced Raman Spectroscopy, paper-based diagnostics and smartphone-based digital methods are recognized as new approaches in the field of E. coli O157:H7 diagnostics and are discussed in this review. Mobile PCR and CRISPR-Cas diagnostic platforms have been identified as new tools in E. coli O157:H7 POC diagnostics with the potential for implementation by industry. This review describes advances and progress in the field of E. coli O157:H7 diagnosis in the context of food and water industry. The focus is on emerging high throughput point-of-care (POC) E. coli O157:H7 diagnostics and the requirement for the transformation to service routine diagnostics in the food and water industry.

A cluster of Shiga Toxin-producing Escherichia coli O157:H7 highlights raw pet food as an emerging potential source of infection in humans.

In August 2017, a cluster of four persons infected with genetically related strains of Shiga toxin-producing Escherichia coli (STEC) O157:H7 was identified. These strains possessed the Shiga toxin (stx) subtype stx2a, a toxin type known to be associated with severe clinical outcome. One person died after developing haemolytic uraemic syndrome. Interviews with cases revealed that three of the cases had been exposed to dogs fed on a raw meat-based diet (RMBD), specifically tripe. In two cases, the tripe had been purchased from the same supplier. Sampling and microbiological screening of raw pet food was undertaken and indicated the presence of STEC in the products. STEC was isolated from one sample of raw tripe but was different from the strain causing illness in humans. Nevertheless, the detection of STEC in the tripe provided evidence that raw pet food was a potential source of human STEC infection during this outbreak. This adds to the evidence of raw pet food as a risk factor for zoonotic transmission of gastrointestinal pathogens, which is widely accepted for Salmonella, Listeria and Campylobacter spp. Feeding RMBD to companion animals has recently increased in popularity due to the belief that they provide health benefits to animals. Although still rare, an increase in STEC cases reporting exposure to RMBDs was detected in 2017. There has also been an increased frequency of raw pet food incidents in 2017, suggesting an increasing trend in potential risk to humans from raw pet food. Recommendations to reduce the risk of infection included improved awareness of risk and promotion of good hygiene practices among the public when handling raw pet food.

Visual and quantitative detection of E. coli O157:H7 by coupling immunomagnetic separation and quantum dot-based paper strip.

Simple and visual quantitative detection of foodborne pathogens can effectively reduce the outbreaks of foodborne diseases. Herein, we developed a simple and sensitive quantum dot (QD)-based paper device for visual and quantitative detection of Escherichia coli (E. coli) O157:H7 based on immunomagnetic separation and nanoparticle dissolution-triggered signal amplification. In this study, E. coli O157:H7 was magnetically separated and labeled with silver nanoparticles (AgNPs), and the AgNP labels can be converted into millions of Ag ions, which subsequently quench the fluorescence of QDs in the paper strip, which along with the readout can be visualized and quantified by the change in length of fluorescent quenched band. Owing to the high capture efficiency and effective signal amplification, as low as 500 cfu mL-1 of E. coli O157:H7 could be easily detected by naked eyes. Furthermore, this novel platform was successfully applied to detect E. coli O157:H7 in spiked milk samples with good accuracy, indicating its potential in the detection of foodborne pathogens in real samples.

Escherichia coli O15 : H7 from Food of Animal Origin in Arsi: Occurrence at Catering Establishments and Antimicrobial Susceptibility Profile.

Escherichia coli O157 : H7 (E. coli O157 : H7) has been found to be the major cause of food-borne diseases and a serious public health problem in the world, with an increasing concern for the emergence and spread of antimicrobial-resistant strains. Hitherto, little is known about the carriage of E. coli O157 : H7 and its antimicrobial susceptibility profile in the food of animal origin in Ethiopia. This study aimed to determine the occurrence and multidrug resistance profile of E. coli O157 : H7 from food of animal origin at different catering establishments in the selected study settings of Arsi Zone. One hundred ninety-two animal origin food items, namely, raw/minced meat (locally known as "Kitfo," "Kurt," and "Dulet"), raw milk, egg sandwich, and cream cake samples were collected and processed for microbiological detection of E. coli O157 : H7. Out of 192 samples, 2.1% (4/192) were positive for E. coli O157 : H7. Two E. coli O157 : H7 isolates were obtained from "Dulet" (6.3%) followed by "Kurt" (3.1%, 1/32) and raw milk (3.1%, 1/32), whereas no isolate was obtained from "Kitfo," egg sandwich, and cream cake samples. Of the 4 E. coli O157 : H7 isolates subjected to 10 panels of antimicrobial discs, 3 (75%) were highly resistant to kanamycin, streptomycin, and nitrofurantoin. Besides, all the isolates displayed multidrug resistance phenotypes, 3 to 5 antimicrobial resistance, amid kanamycin, streptomycin, nitrofurantoin, tetracycline, and chloramphenicol. The occurrence of multidrug-resistant E. coli O157 : H7 isolates from foods of animal origin sampled from different catering establishments reveals that the general sanitary condition of the catering establishments, utensils used, and personnel hygienic practices did not comply with the recommended standards. Thus, this finding calls for urgent attention toward appropriate controls and good hygienic practices in different catering establishments dealing with consuming raw/undercooked foods of animal origin.

Genetic diversity and pathogenic potential of Shiga toxin-producing Escherichia coli (STEC) derived from German flour.

Shiga toxin-producing Escherichia coli (STEC) can cause severe human illness, which are frequently linked to the consumption of contaminated beef or dairy products. However, recent outbreaks associated with contaminated flour and undercooked dough in the United States and Canada, highlight the potential of plant based food as transmission routes for STEC. In Germany STEC has been isolated from flour, but no cases of illness have been linked to flour. In this study, we characterized 123 STEC strains isolated from flour and flour products collected between 2015 and 2019 across Germany. In addition to determination of serotype and Shiga toxin subtype, whole genome sequencing (WGS) was used for isolates collected in 2018 to determine phylogenetic relationships, sequence type (ST), and virulence-associated genes (VAGs). We found a high diversity of serotypes including those frequently associated with human illness and outbreaks, such as O157:H7 (stx2c/d, eae), O145:H28 (stx2a, eae), O146:H28 (stx2b), and O103:H2 (stx1a, eae). Serotypes O187:H28 (ST200, stx2g) and O154:H31 (ST1892, stx1d) were most prevalent, but are rarely linked to human cases. However, WGS analysis revealed that these strains, as well as, O156:H25 (ST300, stx1a) harbour high numbers of VAGs, including eae, nleB and est1a/sta1. Although STEC-contaminated flour products have yet not been epidemiologically linked to human clinical cases in Germany, this study revealed that flour can serve as a vector for STEC strains with a high pathogenic potential. Further investigation is needed to determine the sources of STEC contamination in flour and flour products particularly in regards to these rare serotypes.

Survival rate of Escherichia coli O157 in artificially contaminated raw and thermized ewe milk in different Pecorino cheese production processes.

Pecorino is a typical Italian cheese, mostly produced in central and southern Italy regions using ewe raw milk and following traditional procedures. The use of raw milk constitutes a risk linked to the potential survival or multiplication of pathogenic microorganisms, as Shiga toxin-producing Escherichia coli (STEC). The aim of this study was to compare different Italian traditional Pecorino production methods to determine if there were any phases that could influence the Escherichia coli O157 survival rate, but also if they could negatively influence lactic acid bacteria survival rate, during the phases of production and ripening. Therefore batches of Pecorino cheese were prepared using different production methods, representing the real and typical cheese production in southern and central Italy regions: 1) heating the milk at 37 °C for about 40 min before curding, 2) heating the milk at 60 °C (thermization) for 13 min, so that the alkaline phosphatase reaction is still positive before curding, 3) cooking curd at 41 °C and 4) at 45 °C, both for 5 min. Our results demonstrated that traditional milk treatments different from pasteurization can help but do not eliminate serious microbiological treats, as E. coli O157, especially if the raw milk is heavily contaminated. The heat treatment at 60 °C applied to raw milk was able to decrease the concentration of E. coli O157 of 1.7 log10CFU/ml and, according to the inactivation slope, it would be further reduced prolonging the heating treatment. The results obtained also showed that, during the Pecorino cheese ripening, E. coli O157 was always enumerable for 60 days, remaining detectable after 90 days of ripening.

Prevalence, characterization and antibiotic resistance of Shiga toxigenic Escherichia coli serogroups isolated from fresh beef and locally processed ready-to-eat meat products in Lagos, Nigeria.

Fresh beef and meat products have been implicated in outbreaks of Shiga toxin-producing Escherichia coli (STEC) worldwide. This study investigated the prevalence of E. coli O157: H7 and non-O157 STEC serogroups in fresh beef in the open market and street vended meat products (n = 180) in Lagos metropolis, Nigeria. A combination of culture media and immunomagnetic separation followed by typing for associated virulence factors and serotypes was performed. Antimicrobial susceptibility testing was performed on the isolated STEC serotypes using the disk diffusion method. A total of 72 STEC serogroup isolates were detected from 61 out of 180 samples. The O157 STEC serotypes were detected in fresh beef, suya, minced meat and tsire with prevalence of 20.8% while non-O157 STEC serogroups were detected in all the samples. Molecular typing revealed 25% (n = 18) of the STEC serogroups showed presence of all the stx1, stx2, eaeA, fliCH7 and rfbEO157 virulence factors while 54.2% (n = 39) possessed a combination of two virulence genes. Multidrug resistance was discovered in 23.6% (n = 17) of the total STEC serogroups. Locally processed ready-to-eat meat products in Lagos metropolis, Nigeria harbour potentially pathogenic multi-drug resistant STEC serogroups that can constitute public health hazard.

Analysis of a small outbreak of Shiga toxin-producing Escherichia coli O157:H7 using long-read sequencing.

Compared to short-read sequencing data, long-read sequencing facilitates single contiguous de novo assemblies and characterization of the prophage region of the genome. Here, we describe our methodological approach to using Oxford Nanopore Technology (ONT) sequencing data to quantify genetic relatedness and to look for microevolutionary events in the core and accessory genomes to assess the within-outbreak variation of four genetically and epidemiologically linked isolates. Analysis of both Illumina and ONT sequencing data detected one SNP between the four sequences of the outbreak isolates. The variant calling procedure highlighted the importance of masking homologous sequences in the reference genome regardless of the sequencing technology used. Variant calling also highlighted the systemic errors in ONT base-calling and ambiguous mapping of Illumina reads that results in variations in the genetic distance when comparing one technology to the other. The prophage component of the outbreak strain was analysed, and nine of the 16 prophages showed some similarity to the prophage in the Sakai reference genome, including the stx2a-encoding phage. Prophage comparison between the outbreak isolates identified minor genome rearrangements in one of the isolates, including an inversion and a deletion event. The ability to characterize the accessory genome in this way is the first step to understanding the significance of these microevolutionary events and their impact on the evolutionary history, virulence and potentially the likely source and transmission of this zoonotic, foodborne pathogen.