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Enterohemorrhagic Escherichia coli O157:H7 (EHEC) has caused foodborne outbreaks worldwide and the bacterium forms antimicrobial-tolerant biofilms. We investigated the abilities of various plant essential oils and their components to inhibit biofilm formation by EHEC. Bay, clove, pimento berry oils and their major common constituent eugenol at 0.005% (v/v) were found to markedly inhibit EHEC biofilm formation without affecting planktonic cell growth. In addition, three other eugenol derivatives isoeugenol, 2-methoxy-4-propylphenol, and 4-ethylguaiacol had antibiofilm activity, indicating that the C-1 hydroxyl unit, the C-2 methoxy unit, and C-4 alkyl or alkane chain on the benzene ring of eugenol play important roles in antibiofilm activity. Interestingly, these essential oils and eugenol did not inhibit biofilm formation by three laboratory E. coli K-12 strains that reduced curli fimbriae production. Transcriptional analysis showed that eugenol down-regulated 17 of 28 genes analysed, including curli genes ( csgABDFG ), type I fimbriae genes ( fimCDH ) and ler -controlled toxin genes ( espD , escJ , escR , and tir ), which are required for biofilm formation and the attachment and effacement phenotype. In addition, biocompatible poly(lactic-co-glycolic acid) coatings containing clove oil or eugenol exhibited efficient biofilm inhibition on solid surfaces. In a Caenorhabditis elegans nematode model, clove oil and eugenol attenuated the virulence of EHEC.

In the United States, Shiga toxin-producing Escherichia coli (STEC) outbreaks cause >265,000 infections and cost $280 million annually. We investigated REPEXH01, a persistent strain of STEC O157:H7 associated with multiple sources, including romaine lettuce and recreational water, that has caused multiple outbreaks since emerging in late 2015. By comparing the genomes of 729 REPEXH01 isolates with those of 2,027 other STEC O157:H7 isolates, we identified a highly conserved, single base pair deletion in espW that was strongly linked to REPEXH01 membership. The biological consequence of that deletion remains unclear; further studies are needed to elucidate its role in REPEXH01. Additional analyses revealed that REPEXH01 isolates belonged to Manning clade 8; possessed the toxins stx2a, stx2c, or both; were predicted to be resistant to several antimicrobial compounds; and possessed a diverse set of plasmids. Those factors underscore the need to continue monitoring REPEXH01 and clarify aspects contributing to its emergence and persistence.

Escherichia coli O157:H7, a toxin-producing food and waterborne bacterial pathogen, has been linked to large outbreaks of gastrointestinal illness for more than two decades. E. coli O157 causes a wide range of clinical illness that varies by outbreak, although factors that contribute to variation in disease severity are poorly understood. Several recent outbreaks involving O157 contamination of fresh produce (e.g., spinach) were associated with more severe disease, as defined by higher hemolytic uremic syndrome and hospitalization frequencies, suggesting that increased virulence has evolved. To test this hypothesis, we developed a system that detects SNPs in 96 loci and applied it to >500 E. coli O157 clinical strains. Phylogenetic analyses identified 39 SNP genotypes that differ at 20% of SNP loci and are separated into nine distinct clades. Differences were observed between clades in the frequency and distribution of Shiga toxin genes and in the type of clinical disease reported. Patients with hemolytic uremic syndrome were significantly more likely to be infected with clade 8 strains, which have increased in frequency over the past 5 years. Genome sequencing of a spinach outbreak strain, a member of clade 8, also revealed substantial genomic differences. These findings suggest that an emergent subpopulation of the clade 8 lineage has acquired critical factors that contribute to more severe disease. The ability to detect and rapidly genotype O157 strains belonging to such lineages is important and will have a significant impact on both disease diagnosis and treatment guidelines.

Significance Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen that can cause bloody diarrhea and hemolytic uremic syndrome, which can lead to severe clinical complications such as kidney failure. The main factors triggering disease are well known and include type 3 secreted effectors, adhesins, and Shiga toxins. Much less is known about how these factors are induced in response to the environmental transition that bacteria experience during transfer into and passage through the host. We show here that although positive regulators of virulence are induced during passage through the host, they are only activated to increase virulence as a result of force generated by host cell contact. Thus, mechanosensation is a way of integrating multifactorial environmental cues to fine-tune virulence regulation. Enterohemorrhagic Escherichia coli (EHEC) is a foodborne pathogen causing hemorrhagic colitis and hemolytic uremic syndrome. EHEC colonizes the intestinal tract through a range of virulence factors encoded by the locus of enterocyte effacement (LEE), as well as Shiga toxin. Although the factors involved in colonization and disease are well characterized, how EHEC regulates its expression in response to a host encounter is not well understood. Here, we report that EHEC perceives attachment to host cells as a mechanical cue that leads to expression of LEE-encoded virulence genes. This signal is transduced via the LEE-encoded global regulator of LEE-encoded regulator (Ler) and global regulator of Ler and is further enhanced by levels of shear force similar to peristaltic forces in the intestinal tract. Our data suggest that, in addition to a range of chemical environmental signals, EHEC is capable of sensing and responding to mechanical cues to adapt to its host’s physiology.

Background Magnesium oxide nanoparticles (MgO nanoparticles, with average size of 20 nm) have considerable potential as antimicrobial agents in food safety applications due to their structure, surface properties, and stability. The aim of this work was to investigate the antibacterial effects and mechanism of action of MgO nanoparticles against several important foodborne pathogens. Results Resazurin (a redox sensitive dye) microplate assay was used for measuring growth inhibition of bacteria treated with MgO nanoparticles. The minimal inhibitory concentrations of MgO nanoparticles to 10 4 colony-forming unit/ml (CFU/ml) of Campylobacter jejuni , Escherichia coli O157:H7, and Salmonella Enteritidis were determined to be 0.5, 1 and 1 mg/ml, respectively. To completely inactivate 10 8−9 CFU/ml bacterial cells in 4 h, a minimal concentration of 2 mg/ml MgO nanoparticles was required for C. jejuni whereas E. coli O157:H7 and Salmonella Enteritidis required at least 8 mg/ml nanoparticles. Scanning electron microscopy examination revealed clear morphological changes and membrane structural damage in the cells treated with MgO nanoparticles. A quantitative real-time PCR combined with ethidium monoazide pretreatment confirmed cell membrane permeability was increased after exposure to the nanoparticles. In a cell free assay, a low level (1.1 μM) of H 2 O 2 was detected in the nanoparticle suspensions. Consistently, MgO nanoparticles greatly induced the gene expression of KatA, a sole catalase in C. jejuni for breaking down H 2 O 2 to H 2 O and O 2 . Conclusions MgO nanoparticles have strong antibacterial activity against three important foodborne pathogens. The interaction of nanoparticles with bacterial cells causes cell membrane leakage, induces oxidative stress, and ultimately leads to cell death.

Background. Escherichia coli O157:H7 is a Shiga toxin-producing E. coli (STEC) associated with numerous foodborne outbreaks in the United States and is an important cause of bacterial gastrointestinal illness. In May 2009, we investigated a multistate outbreak of E. coli O157:H7 infections. Methods. Outbreak-associated cases were identified using serotyping and molecular subtyping procedures. Traceback investigation and product testing were performed. A matched case-control study was conducted to identify exposures associated with illness using age-, sex-, and state-matched controls. Results. Seventy-seven patients with illnesses during the period 16 March-8 July 2009 were identified from 30 states; 35 were hospitalized, 10 developed hemolytic-uremic syndrome, and none died. Sixty-six percent of patients were < 19 years; 71% were female. In the case-control study, 33 of 35 case patients (94%) consumed ready-to-bake commercial prepackaged cookie dough, compared with 4 of 36 controls (11%) (matched odds ratio = 41.3; P < .001); no other reported exposures were significantly associated with illness. Among case patients consuming cookie dough, 94% reported brand A. Three nonoutbreak STEC strains were isolated from brand A cookie dough. The investigation led to a recall of 3.6 million packages of brand A cookie dough and a product reformulation. Conclusions. This is the first reported STEC outbreak associated with consuming ready-to-bake commercial prepackaged cookie dough. Despite instructions to bake brand A cookie dough before eating, case patients consumed the product uncooked. Manufacturers should consider formulating ready-to-bake commercial prepackaged cookie dough to be as safe as a ready-to-eat product. More effective consumer education about the risks of eating unbaked cookie dough is needed.

Contaminated lake water and fish can be sources of bacterial pathogens of public health concern, including pathogenic E. coli . Within Ethiopia, specifically, Central Oromia, raw fish consumption is a common practice. Although there are few reports on occurrence of E. coli O157 in fish destined for human consumption and children under five years, information on the transmission pathways of E. coli O157 and other sorbitol non-fermenting (SN-F) E. coli from water-to-fish-to-human, and their virulence factors and antimicrobial resistant determinants along the fish supply chain is lacking. The study aimed to investigate the occurrence, molecular characteristics, and antimicrobial susceptibility of E. coli O157 and other SN-F E. coli strains in fish, lake water and humans in central Oromia, Ethiopia. A total of 750 samples (450 fish samples, 150 water samples, 150 human stool samples) were collected from five lakes and three health facilities. The samples were processed following the standard protocol recommended by European Food Safety Authority and Kirby–Bauer disc diffusion method for detection of the bacteria, and antimicrobial susceptibility tests, respectively. Molecular characterization of presumptive isolates was performed using Whole-Genome Sequencing ( WGS) for serotyping, determination of virulence factors, antimicrobial resistance traits, and genetic linkage of the isolates. Overall, 3.9% (29/750) of the samples had SN-F E. coli; of which 6.7% (n = 10), 1.8% (n = 8) and 7.3% (n = 11) were retrieved from water, fish, and diarrheic human patients, respectively. The WGS confirmed that all the isolates were SN-F non-O157: H7 E. coli strains. We reported two new E. coli strains with unknown O-antigen from fish and human samples. All the strains have multiple virulence factors and one or more genes encoding for them. Genetic relatedness was observed among strains from the same sources (water, fish, and humans). Most isolates were resistant to ampicillin (100%), tetracycline (100%), cefotaxime (100%), ceftazidime (100%), meropenem (100%), nalidixic acid (93.1%) and sulfamethoxazole/trimethoprim (79.3%). Majority of the strains were resistant to chloramphenicol (58.6%) and ciprofloxacin (48.3%), while small fraction showed resistance to azithromycin (3.45%). Isolates had an overall MDR profile of 87.5%. Majority, (62.1%; n = 18) of the strains had acquired MDR traits. Genes encoding for mutational resistance and Extended-spectrum beta-lactamases (ESBL) were also detected. In conclusion, our study revealed the occurrence of virulent and MDR SN-F E. coli strains in water, fish, and humans. Although no genetic relatedness was observed among strains from various sources, the genomic clustering among strains from the same sources strongly suggests the potential risk of transmission along the supply chain at the human–fish-environment interface if strict hygienic fish production is not in place. Further robust genetic study of the new strains with unknown O-antigens, and the epidemiology of SN-F E. coli is required to elucidate the molecular profile and public health implications of the pathogens.

The formation of viable but nonculturable (VBNC) Escherichia coli O157:H7 induced by high-pressure CO 2 (HPCD) was investigated using RNA sequencing (RNA-Seq) transcriptomics and isobaric tag for relative and absolute quantitation (iTRAQ) proteomic methods. The analyses revealed that 97 genes and 56 proteins were significantly changed upon VBNC state entry. Genes and proteins related to membrane transport, central metabolisms, DNA replication, and cell division were mainly downregulated in the VBNC cells. This caused low metabolic activity concurrently with a division arrest in cells, which may be related to VBNC state formation. Cell division repression and outer membrane overexpression were confirmed to be involved in VBNC state formation by homologous expression of z2046 coding for transcriptional repressor and ompF encoding outer membrane protein F. Upon VBNC state entry, pyruvate catabolism in the cells shifted from the tricarboxylic acid (TCA) cycle toward the fermentative route; this led to a low level of ATP. Combating the low energy supply, ATP production in the VBNC cells was compensated by the degradation of l -serine and l -threonine, the increased AMP generation, and the enhanced electron transfer. Furthermore, tolerance of the cells with respect to HPCD-induced acid, oxidation, and high CO 2 stresses was enhanced by promoting the production of ammonia and NADPH and by reducing CO 2 production during VBNC state formation. Most genes and proteins related to pathogenicity were downregulated in the VBNC cells. This would decrease the cell pathogenicity, which was confirmed by adhesion assays. In conclusion, the decreased metabolic activity, repressed cell division, and enhanced survival ability in E. coli O157:H7 might cause HPCD-induced VBNC state formation. IMPORTANCE Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO 2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment. Escherichia coli O157:H7 has been implicated in large foodborne outbreaks worldwide. It has been reported that the presence of as few as 10 cells in food could cause illness. However, the presence of only 0.73 to 1.5 culturable E. coli O157:H7 cells in salted salmon roe caused infection in Japan. Investigators found that E. coli O157:H7 in the viable but nonculturable (VBNC) state was the source of the outbreak. So far, formation mechanisms of VBNC state are not well known. In a previous study, we demonstrated that high-pressure CO 2 (HPCD) could induce the transition of E. coli O157:H7 into the VBNC state. In this study, we used RNA-Seq transcriptomic analysis combined with the iTRAQ proteomic method to investigate the formation of VBNC E. coli O157:H7 induced by HPCD treatment. Finally, we proposed a putative formation mechanism of the VBNC cells induced by HPCD, which may provide a theoretical foundation for controlling the VBNC state entry induced by HPCD treatment.

Escherichia coli is a single species consisting of many biotypes, some of which are commensal colonizers of mammals and others that cause disease. Humans are colonized on average with five commensal biotypes, and it is widely thought that the commensals serve as a barrier to infection by pathogens. Previous studies showed that a combination of three pre-colonized commensal E. coli strains prevents colonization of E. coli O157:H7 in a mouse model (Leatham, et al., 2010, Infect Immun 77: 2876-7886). The commensal biotypes included E. coli HS, which is known to successfully colonize humans at high doses with no adverse effects, and E. coli Nissle 1917, a human commensal strain that is used in Europe as a preventative of traveler's diarrhea. We hypothesized that commensal biotypes could exert colonization resistance by consuming nutrients needed by E. coli O157:H7 to colonize, thus preventing this first step in infection. Here we report that to colonize streptomycin-treated mice E. coli HS consumes six of the twelve sugars tested and E. coli Nissle 1917 uses a complementary yet divergent set of seven sugars to colonize, thus establishing a nutritional basis for the ability of E. coli HS and Nissle 1917 to occupy distinct niches in the mouse intestine. Together these two commensals use the five sugars previously determined to be most important for colonization of E. coli EDL933, an O157:H7 strain. As predicted, the two commensals prevented E. coli EDL933 colonization. The results support a model in which invading pathogenic E. coli must compete with the gut microbiota to obtain the nutrients needed to colonize and establish infection; accordingly, the outcome of the challenge is determined by the aggregate capacity of the native microbiota to consume the nutrients required by the pathogen.