Shiga Toxin-producing Escherichia Coli Pathogenicity Research Articles

Transcription levels of hes and their involvement in the biofilm formation of Shiga toxin-producing Escherichia coli O91.

Shiga toxin-producing Escherichia coli (STEC) are recognized as being responsible for many cases of foodborne diseases worldwide. Cattle are the main reservoir of STEC, shedding the microorganisms in their feces. The serogroup STEC O91 has been associated with hemorrhagic colitis and hemolytic uremic syndrome. Locus of Adhesion and Autoaggregation (LAA) and its hes gene are related to the pathogenicity of STEC and the ability to form biofilms. Considering the frequent isolation of STEC O91, the biofilm-forming ability, and the possible role of hes in the pathogenicity of STEC, we propose to evaluate the ability of STEC to form biofilms and to evaluate the expression of hes before and after of biofilm formation. All strains were classified as strong biofilm-forming. The hes expression showed variability between strains before and after biofilm formation, and this may be due to other genes carried by each strain. This study is the first to report the relationship between biofilm formation, and hes expression and proposes that the analysis and diagnosis of LAA, especially hes as STEC O91 virulence factors, could elucidate these unknown mechanisms. Considering that there is no specific treatment for HUS, only supportive care, it is necessary to know the survival and virulence mechanisms of STEC O91.

Pathogenic Factors and Recent Study on the Rapid Detection of Shiga Toxin-Producing Escherichia coli (STEC).

This comprehensive review delves into the pathogenicity and detection of Shiga Toxin-Producing Escherichia coli (STEC), shedding light on its various genetic and clinical manifestations. STEC originating from E. coli acquires pathogenicity through mobility and genetic elements. The pathogenicity of STEC is explored in terms of clinical progression, complications, and key toxins such as Shiga toxin (Stx). Stx1 and Stx2 are two distinct Stx types exhibiting different toxicities, with Stx2 often associated with severe diseases. This review also delves into Subtilase cytotoxin, an additional cytotoxin produced by some STEC strains. Pathogenic mechanisms of STEC, such as attaching and effacing intestinal lesions, are discussed, with a focus on roles of genetic factors. Plasmids in STEC can confer unique pathogenicity. Hybridization with other pathogenic E. coli can create more lethal pathogens. This review covers a range of detection methods, ranging from DNA amplification to antigen detection techniques, emphasizing the need for innovative approaches to improve the sensitivity and speed of STEC diagnosis. In conclusion, understanding diverse aspects of STEC pathogenicity and exploring enhanced diagnostic methods are critical to addressing this foodborne pathogen effectively. Pathology of Shiga toxin toxicity. STEC-derived Shiga toxin consists of one A subunit and five B subunits. Pathological symptoms of the disease can progress to HUS within two weeks after the onset of diarrhea. Shiga toxin intoxication is also associated with many complications, such as neurological and cardiac complications. This figure was reconstructed based on data from Bruyand et al.

Phage Tail Fiber Protein as a Specific Probe for Recognition of Shiga Toxin-Producing Escherichia coli O91, O103, and O111.

The ability to quickly identify specific serotypes of Shiga toxin-producing Escherichia coli (STEC) could facilitate the monitoring and control of STEC pathogens. In this study, we identified the receptors and receptor-binding proteins (RBPs) of three novel phages (pO91, pO103, and pO111) isolated from hospital wastewater. Recombinant versions of these RBPs (pO91-ORF43, pO103-ORF42, and pO111-ORF8) fused to a fluorescent reporter protein were then constructed. Both fluorescence microscopy and transmission electron microscopy showed that all three recombinant RBPs were bound to the bacterial surface. Indirect enzyme-linked immunosorbent assay was used to verify that each recombinant RBP bound specifically to E. coli O91, O103, or O111, but not to any of the 83 strains of E. coli with different O-antigens, nor to 10 other bacterial species that were tested. The recombinant RBPs adsorbed to their respective host bacteria within 10 min of incubation. The minimum concentration of bacteria required for detection by the recombinant RBPs was 33 colony-forming units (CFU)/mL (range: 3.3 × 10 to 3.3 × 108 CFU/mL). Furthermore, each recombinant RBP was also able to detect bacteria in lettuce, chicken breast meat, and infected mice, indicating that their usage will facilitate the detection of STEC and may help to reduce the spread of STEC-related infections and diseases.

Strain- and serotype-dependent affinity of Shiga toxin-producing Escherichia coli for bovine milk fat globules.

Shiga toxin-producing Escherichia coli (STEC) are widely detected in raw milk products intended for human consumption. Although STEC are a worldwide public health problem, the pathogenicity of STEC in cheese remains unclear. In fact, bacterial association with compounds in raw milk cheeses could reduce their pathogenicity. A previous study showed the association of 2 STEC strains with raw milk cream in a natural creaming assay. Different concentrations of each strain were required to saturate the cream. In this study, we hypothesized that all STEC strains could be associated with milk fat globules (MFG) in raw milk and that the bacterial load required for saturation of the cream is serotype dependent. We evaluated the affinity of STEC strains belonging to the O157:H7, O26:H11, and O103:H2 serotypes for bovine raw milk cream and analyzed saturation of the cream layer by natural creaming assay. We used 12 STEC strains and 3 strains belonging to another pathotype to assess the effects of serotypes on this phenomenon. We performed sucrose density gradient centrifugation assays with 2 STEC model strains to confirm the results obtained by natural creaming. The localization of STEC within MFG-enriched creams was observed by confocal and electron microscopy. We recovered approximately 10 times more STEC from the cream layer after natural creaming than from raw bovine milk. The concentration of STEC required to saturate the cream layer (the saturation concentration) was estimated for each strain by nonlinear regression, highlighting a strain and serotype effect. Moreover, the concentration of STEC in the cream was milk fat level dependent. However, even in nonsaturating conditions, a high level of STEC was still present in the aqueous phase, after fat separation. Thus, natural creaming should not be used as the sole preventive measure to remove STEC from naturally contaminated raw milk. The results of our study suggest that cream saturation is a complex mechanism, most likely involving specific interactions between STEC and raw MFG.

Escherichia coli productora de toxina Shiga: el desafío de adherirse para sobrevivir

Shiga Toxin-producing Escherichia coli (STEC) is recognized as being responsible for a large number of foodborne illnesses around the world. The pathogenicity of STEC has been related to Stx toxins. However, the ability of STEC to colonize the host and other surfaces can be essential for developing its pathogenicity. Different virulence profiles detected in STEC could cause the emergence of strains carrying new genes codified in new pathogenicity islands linked to metabolism and adherence. Biofilm formation is a spontaneous mechanism whereby STEC strains resist in a hostile environment being able to survive and consequently infect the host through contaminated food and food contact surfaces. Biofilm formation shows intra-and inter-serotype variability, and its formation does not depend only on the microorganisms involved. Other factors related to the environment (such as pH, temperature) and the surface (stainless steel and polystyrene) influence biofilm expression. The «One Health» concept implies the interrelation between public, animal, and environmental health actors to ensure food safety, prevent cross-contamination and resistance to sanitizers, highlighting the need to identify emerging pathogens through new molecular markers of rapid detection that involve STEC strains carrying the Locus of Enterocyte Effacement or Locus of Adhesion and Autoaggregation.

Shiga Toxin-Producing Escherichia coli in Wheat Grains: Detection and Isolation by Polymerase Chain Reaction and Culture Methods.

Shiga toxin-producing Escherichia coli (STEC) are major foodborne pathogens and seven serogroups, O26, O45, O103, O111, O121, O145, and O157, often called top-7 STEC, account for the majority of the STEC-associated human illnesses in the United States. Two Shiga toxins, Shiga toxins 1 and 2, encoded by stx1 and stx2 genes, are major virulence factors that are involved in STEC infections. Foodborne STEC infections have been linked to a variety of foods of both animal and plant origin, including products derived from cereal grains. In recent years, a few STEC outbreaks have been linked to contaminated wheat flour. The microbiological quality of the wheat grains is a major contributor to the safety of wheat flour. The objective of the study was to utilize polymerase chain reaction (PCR)- and culture-based methods to detect and isolate STEC in wheat grains. Wheat grain samples (n = 625), collected from different regions of the United States, were enriched in modified buffered peptone water with pyruvate (mBPWp) or E. coli (EC) broth, and they were then subjected to PCR- and culture-based methods to detect and isolate STEC. Wheat grains enriched in EC broth yielded more samples positive for stx genes (1.6% vs. 0.32%) and STEC serogroups (5.8% vs. 2.4%) than mBPWp. The four serogroups of top-7 detected and isolated were O26, O45, O103, and O157 and none of the isolates was positive for the Shiga toxin genes. A total of five isolates that carried the stx2 gene were isolated and identified as serogroups O8 (0.6%) and O130 (0.2%). The EC broth was a better medium to enrich wheat grains than mBPWp for the detection and isolation of STEC. The overall prevalence of virulence genes and STEC serogroups in wheat grains was low. The stx2-positive serogroups isolated, O8 and O130, are not major STEC pathogens and have only been implicated in sporadic infections in animals and humans.

Pathogenic potential assessment of the Shiga toxin–producing Escherichia coli by a source attribution–considered machine learning model

Instead of conventional serotyping and virulence gene combination methods, methods have been developed to evaluate the pathogenic potential of newly emerging pathogens. Among them, the machine learning (ML)-based method using whole-genome sequencing (WGS) data are getting attention because of the recent advances in ML algorithms and sequencing technologies. Here, we developed various ML models to predict the pathogenicity of Shiga toxin-producing Escherichia coli (STEC) isolates using their WGS data. The input dataset for the ML models was generated using distinct gene repertoires from positive (pathogenic) and negative (nonpathogenic) control groups in which each STEC isolate was designated based on the source attribution, the relative risk potential of the isolation sources. Among the various ML models examined, a model using the support vector machine (SVM) algorithm, the SVM model, discriminated between the two control groups most accurately. The SVM model successfully predicted the pathogenicity of the isolates from the major sources of STEC outbreaks, the isolates with the history of outbreaks, and the isolates that cannot be assessed by conventional methods. Furthermore, the SVM model effectively differentiated the pathogenic potentials of the isolates at a finer resolution. Permutation importance analyses of the input dataset further revealed the genes important for the estimation, proposing the genes potentially essential for the pathogenicity of STEC. Altogether, these results suggest that the SVM model is a more reliable and broadly applicable method to evaluate the pathogenic potential of STEC isolates compared with conventional methods.

Comparative Transcriptomics of Shiga Toxin-Producing and Commensal Escherichia coli and Cytokine Responses in Colonic Epithelial Cell Culture Infections.

Ingestion of Shiga toxin-producing Escherichia coli (STEC) can result in a range of illness severity from asymptomatic to hemorrhagic colitis and death; thus risk assessment of STEC strains for human pathogenicity is important in the area of food safety. Illness severity depends in part on the combination of virulence genes carried in the genome, which can vary between strains even of identical serotype. To better understand how core genes are regulated differently among strains and to identify possible novel STEC virulence gene candidates that could be added to the risk assessment repertoire, we used comparative transcriptomics to investigate global gene expression differences between two STEC strains associated with severe illness and a commensal E. coli strain during in vitro intestinal epithelial cell (IEC) infections. Additionally, we compared a wide array of concomitant cytokine levels produced by the IECs. The cytokine expression levels were examined for a pattern representing STEC pathogenicity; however, while one STEC strain appeared to elicit a proinflammatory response, infection by the other strain produced a pattern comparable to the commensal E. coli. This result may be explained by the significant differences in gene content and expression observed between the STEC strains. RNA-Seq analysis revealed considerable disparity in expression of genes in the arginine and tryptophan biosynthesis/import pathways between the STEC strains and the commensal E. coli strain, highlighting the important role some amino acids play in STEC colonization and survival. Contrasting differential expression patterns were observed for genes involved in respiration among the three strains suggesting that metabolic diversity is a strategy utilized to compete with resident microflora for successful colonization. Similar temporal expression results for known and putative virulence genes were observed in the STEC strains, revealing strategies used for survival prior to and after initial adherence to IECs. Additionally, three genes encoding hypothetical proteins located in mobile genetic elements were, after interrogation of a large set of E. coli genomes, determined to likely represent novel STEC virulence factors.

Determination of the changes in the gastric fluid endurance of O157 and non-O157 Shiga toxin-producing Escherichia coli during storage of experimentally produced beef frankfurter.

Resistance of Shiga toxin-producing Escherichia coli (STEC) O157:H7 and serogroups O103, O26 and O145 to synthetic gastric fluid (SGF, pH 1.5) were investigated during frankfurter storage. Pathogens were inoculated (5 ± 1 log10cfug-1) on frankfurters and frankfurters were stored at 4°C for 75days in vacuum packages. Population changes of the competitive flora and STEC, changes in the pH of the frankfurters and resistance of STEC to SGF were monitored on days 0, 15, 30, 45, 60 and 75 of frankfurter storage. Direct synthetic gastric fluid (DSGF) challenges were also conducted to assess pathogen resistance without being effected by frankfurters, by inoculating pathogen cultures directly into SGF. Results showed that acid resistance of O145 and O26 was stronger than that of O103 and O157 during frankfurter storage. Resistance of O103 to SGF was better than that of O157 during frankfurter storage but, was similar to that of O157 during DSGF challenges. Results indicate that acid resistance of some strains of STEC pathogens might differentiate during storage of frankfurters. Different resistance capabilities to SGF were observed in the STEC strains when inoculated and stored on frankfurters than directly inoculated in the SGF.

Investigating the Whole-Genome Sequence of a New Locus of Enterocyte Effacement-Positive Shiga Toxin-Producing Escherichia coli O157:H7 Strain Isolated from River Water.

Diverse Shiga toxin-producing Escherichia coli (STEC) strains have been isolated from several environmental samples. Rivers are associated with the distribution of STEC pathogens in the environment. Thus, we report the complete genome sequence of a locus of enterocyte effacement (LEE)-positive STEC O157:H7 strain isolated from the Mississippi River.

Shiga toxin 2 translocation across intestinal epithelium is linked to virulence of Shiga toxin-producing Escherichia coli in humans.

Shiga toxin-producing Escherichia coli (STEC) are characterized by the release of potent Shiga toxins (Stx), which are associated with severe intestinal and renal disease. Although all STEC strains produce Stx, only a few serotypes cause infection in humans. To determine which virulence traits in vitro are linked to human disease in vivo, 13 Stx2a-producing STEC strains of seropathotype (SPT) A or B (associated with severe human intestinal disease and outbreaks) and 6 strains of SPT D or E (rarely or not linked to human disease) were evaluated in a microaerobic human colonic epithelial infection model. All SPT strains demonstrated similar growth, colonization of polarized T84 colon carcinoma cells and Stx release into the medium. In contrast, Stx translocation across the T84 cell monolayer was significantly lower in SPT group DE compared to SPT group AB strains. Further experiments showed that Stx penetration occurred via a transcellular pathway and was independent of bacterial type III secretion and attaching and effacing lesion formation. These results suggest that the extent of Stx transcytosis across the gut epithelium may represent an important indicator of STEC pathogenicity for humans.

Detection, Prevalence, and Pathogenicity of Non-O157 Shiga Toxin-Producing Escherichia coli from Cattle Hides and Carcasses.

Cattle are a major reservoir for Shiga toxin-producing Escherichia coli (STEC) and harbor these bacteria in the intestinal tract. The prevalence, concentration, and STEC serogroup isolated in cattle varies between individuals. Hide removal at slaughter serves as a major point of carcass contamination and ultimately beef products. Certain STEC serogroups, such as O26, O45, O103, O111, O121, O145, and O157, containing the intestinal adherence factor intimin, pose a large economic burden to food producers because of testing and recalls. Human infection with STEC can cause illnesses ranging from diarrhea to hemorrhagic colitis and hemolytic uremic syndrome, and is commonly acquired through ingestion of contaminated foods, often beef products. Previously, most studies focused on O157 STEC, but there is growing recognition of the importance of non-O157 STEC serogroups. This review summarizes detection methods, prevalence, and methods for prediction of pathogenicity of non-O157 STEC from cattle hides and carcasses. A synthesis of procedures is outlined for general non-O157 STEC and targeted detection of specific STEC serogroups. Standardization of sample collection and processing procedures would allow for more robust comparisons among studies. Presence of non-O157 STEC isolated from cattle hides and carcasses and specific factors, such as point of sample collection and season, are summarized. Also, factors that might influence STEC survival on these surfaces, such as the microbial population on hides and microbial adherence genes, are raised as topics for future investigation. Finally, this review gives an overview on studies that have used genetic and cell-based methods to identify specific phenotypes of non-O157 STEC strains isolated from cattle to assess their risk to human health.

Pathogenicity Islands Distribution in Non-O157 Shiga Toxin-Producing Escherichia coli (STEC).

Shiga toxin-producing Escherichia coli (STEC) are foodborne pathogens associated with outbreaks and hemolytic-uremic syndrome. Cattle and meat foods are the main reservoir and infection source, respectively. Pathogenicity islands (PAIs) play an important role in STEC pathogenicity, and non-locus of the enterocyte effacement(LEE) effector (nle) genes present on them encode translocated substrates of the type III secretion system. A molecular risk assessment based on the evaluation of the nle content has been used to predict which STEC strains pose a risk to humans. The goal was to investigate the distribution of the PAIs OI (O-island)-36 (nleB2, nleC, nleH1-1, nleD), OI-57 (nleG2-3, nleG5-2, nleG6-2), OI-71 (nleA, nleF, nleG, nleG2-1, nleG9, nleH1-2) and OI-122 (ent/espL2, nleB, nleE, Z4321, Z4326, Z4332, Z4333) among 204 clinical, food and animal isolates belonging to 52 non-O157:H7 serotypes. Differences in the frequencies of genetic markers and a wide spectrum of PAI virulence profiles were found. In most LEE-negative strains, only module 1 (Z4321) of OI-122 was present. However, some unusual eae-negative strains were detected, which carried other PAI genes. The cluster analysis, excluding isolates that presented no genes, defined two major groups: eae-negative (determined as seropathotypes (SPTs) D, E or without determination, isolated from cattle or food) and eae-positive (mostly identified as SPTs B, C, or not determined).

Inhibition of Shiga toxin-converting bacteriophage development by novel antioxidant compounds

Oxidative stress may be the major cause of induction of Shiga toxin-converting (Stx) prophages from chromosomes of Shiga toxin-producing Escherichia coli (STEC) in human intestine. Thus, we aimed to test a series of novel antioxidant compounds for their activities against prophage induction, thus, preventing pathogenicity of STEC. Forty-six compounds (derivatives of carbazole, indazole, triazole, quinolone, ninhydrine, and indenoindole) were tested. Fifteen of them gave promising results and were further characterized. Eleven compounds had acceptable profiles in cytotoxicity tests with human HEK-293 and HDFa cell lines. Three of them (selected for molecular studies) prevent the prophage induction at the level of expression of specific phage genes. In bacterial cells treated with hydrogen peroxide, expression of genes involved in the oxidative stress response was significantly less efficient in the presence of the tested compounds. Therefore, they apparently reduce the oxidative stress, which prevents induction of Stx prophage in E. coli.

Approaches to treatment of emerging Shiga toxin-producing Escherichia coli infections highlighting the O104:H4 serotype.

Shiga toxin-producing Escherichia coli (STEC) are a group of diarrheagenic bacteria associated with foodborne outbreaks. Infection with these agents may result in grave sequelae that include fatality. A large number of STEC serotypes has been identified to date. E. coli serotype O104:H4 is an emerging pathogen responsible for a 2011 outbreak in Europe that resulted in over 4000 infections and 50 deaths. STEC pathogenicity is highly reliant on the production of one or more Shiga toxins that can inhibit protein synthesis in host cells resulting in a cytotoxicity that may affect various organ systems. Antimicrobials are usually avoided in the treatment of STEC infections since they are believed to induce bacterial cell lysis and the release of stored toxins. Some antimicrobials have also been reported to enhance toxin synthesis and production from these organisms. Various groups have attempted alternative treatment approaches including the administration of toxin-directed antibodies, toxin-adsorbing polymers, probiotic agents and natural remedies. The utility of antibiotics in treating STEC infections has also been reconsidered in recent years with certain modalities showing promise.

Molecular-genetic characterization of shiga-toxin producing Escherichia coli isolated during a food-borne outbreak in St. Petersburg in 2013

Shiga toxin-producing Escherichia coli (STEC) food-borne infections are reported worldwide and represent a serious problem for public healthcare. In the Russian Federation there is little information on epidemiology and etiology of STEC-infections as well as on molecular-genetic peculiarities of STEC pathogens. Our aim was to describe a food-borne outbreak as hemorrhagic colitis (HC) along with hemolytic uremic syndrome (HUS), enterocolitis, and acute gastroenteritis in children in St. Petersburg in 2013. Epidemiological, microbiological, molecular-genetic and bioinformatic methods were applied. Objects to study were clinical specimens, milk and food samples, as well as STEC strains isolated during the outbreak. The outbreak of food-borne infection was found to be caused by STEC-contaminated raw milk as confirmed by epidemiological analysis, detection of STEC DNA and isolation of relevant pathogens in milk and sick children fecal specimens. The whole-genome sequencing revealed two groups ofpathogens, E. coli O157:H7 and E. coli O101:H33 among collected strains. Group I strains were attributed to the previously known sequence type ST24, while group II strains belonged to the previously non-described sequence type ST145. In strain genomes of both groups there were identified nucleotide sequences of VT2-like prophage carrying stx2c gene, plasmid enterohemolysin gene, and gene of the STEC main adhesion factor intimin. Gene of intimin gamma was identified in E. coli O157:H7 strains and intimin iota 2 in E. coli O101:H33 strains. The latter previously was identified only in enteropathogenic E. coli (EPEC) strains. The additional knowledge of epidemiology and biology of STEC pathogens would assist clinicians and epidemiologists in diagnosing, treating and preventing hemorrhagic colitis.

Advances in detection methods for Shiga toxin-producing Escherichia coli (STEC).

Advances in detection methods for Shiga toxin-producing Escherichia coli (STEC).

Pathogenicity Islands in Shiga Toxin–ProducingEscherichia coliO26, O103, and O111 Isolates from Humans and Animals

Non-O157 Shiga toxin-producing Escherichia coli (STEC) are increasingly recognized as foodborne pathogens worldwide. Serogroups O26, O111, and O103 cause most known outbreaks related to non-O157 STEC. Pathogenicity islands (PAIs) play a major role in the evolution of STEC pathogenicity. To determine the distribution of PAIs often associated with highly virulent STECs (OI-122, OI-43/48, OI-57, and high pathogenicity islands) among STEC O26, O103, and O111, a collection of STEC O26 (n=45), O103 (n=29), and O111 (n=52) from humans and animals were included in this study. Pulsed-field gel electrophoresis (PFGE) with XbaI digestion was used to characterize the clonal relationship of the strains. In addition, a polymerase chain reaction-restriction fragment length polymorphism assay was used to determine eae subtypes. Additional virulence genes on PAIs were identified using specific PCR assays, including OI-122: pagC, sen, efa-1, efa-2, and nleB; OI-43/48: terC, ureC, iha, and aidA-1; OI-57: nleG2-3, nleG5-2, and nleG6-2; and HPI: fyuA and irp2. A PFGE dendrogram demonstrated that instead of clustering together with strains from the same O type (O111:H8), the O111:H11 (n=14) strains clustered together with strains of the same H type (O26:H11, n=45). In addition, O26:H11 and O111:H11 strains carried eae subtype β, whereas O111:H8 strains had eae γ2/θ. The O26:H11 and O111:H11 stains contained an incomplete OI-122 lacking pagC and a complete HPI. However, a complete OI-122 but no HPI was found in the O111:H8 strains. Additionally, aidA-1 of OI-43/48 and nleG6-2 of OI-57 were significantly associated with O26:H11 and O111:H11 strains but were almost missing in O111:H8 strains (p<0.001). This study demonstrated that H11 (O111:H11 and O26:H11) strains were closely related and may have come from the same ancestor.

Effect of pH on the effectiveness of whey protein/glycerol edible films containing potassium sorbate to control non-O157 shiga toxin-producing Escherichia coli in ready-to-eat foods

Potassium sorbate (PS) (0.5%, 1.0% and 1.5% w/w) was included into whey protein concentrate (WPC)/glycerol (Gly) edible films at pH 5.2 and 6.0. The films inhibited or retarded the growth of Shiga toxin-producing Escherichia coli (STEC) pathogens in both diffusion and barrier tests. Bacterial growth inhibition was dependent on PS content at both pH values. PS release was not affected by pH. Scanning electron microscopy (SEM) was used to analyze the microstructure of the films and gain a better understanding of their optical parameters. Acidic control films (pH 5.2) prepared without PS were the least transparent. SEM micrographs confirmed the greater structural heterogeneity of these films, coinciding with opacity. The incorporation of PS into WPC/Gly films improved transparency and produced a smoother surface than acidic control ones. The utilization of active packaging based on whey proteins and organic acids to control and prevent the dissemination of STEC pathogens may be an effective, safe, ecological and relatively inexpensive alternative to be used in the food packaging industry.

Differentiation of big-six non-O157 Shiga-toxin producing Escherichia coli (STEC) on spread plates of mixed cultures using hyperspectral imaging

There have been considerable recent advances in the technology for rapidly detecting foodborne pathogens. However, a traditional culture method is still the “gold standard” for presumptive-positive pathogen screening although it is labor-intensive, ineffective in testing large amount of food samples, and cannot completely prevent unwanted background microflora from growing together with target microorganisms on agar media. We have developed multivariate classification models based on visible and near-infrared hyperspectral imaging for rapid presumptive-positive screening of six representative non-O157 Shiga-toxin producing Escherichia coli (STEC) serogroups (O26, O45, O103, O111, O121, and O145) on agar plates of pure and mixed cultures. The classification models were developed with spread plates of pure cultures. In this study, we evaluated the performance of the classification models with independent validation samples of mixed cultures that were not used during training and found the best classification model for differentiating non-O157 STEC colonies on spread plates of mixed cultures. A validation protocol appropriate to hyperspectral imaging of mixed cultures was developed. An additional independent validation set of 12 spread plates with pure cultures was used as positive controls to help the validation process with the mixed cultures and to affirm the model performance. One imaging experiment with colonies obtained from two serial dilutions was performed. A total of six agar plates of mixed cultures were prepared, where O45, O111 and O121 serogroups that were relatively easy to differentiate were inoculated into all six plates and then each of O26, O103 and O145 serogroups was added into the mixture of the three common bacterial cultures. The number of mixed colonies grown after 24-h incubation was 331 and the number of pixels associated with the grown colonies was 16,379. The best model found from this validation study was based on pre-processing with standard normal variate and detrending, first derivative, spectral smoothing, and k-nearest neighbor classification (kNN, k = 3) of scores in the principal component subspace spanned by 12 principal components. The results showed 95 % overall detection accuracy at pixel level and 97 % at colony level. The developed model was proven to be still valid even for the independent validation samples although the size of a validation set was small and only one experiment was performed. This study was an important first step in validating and updating multivariate classification models for rapid screening of ground beef samples contaminated by non-O157 STEC pathogens using hyperspectral imaging.