Transcriptomic and proteomic analysis of the virulence inducing effect of ciprofloxacin on enterohemorrhagic Escherichia coli.

Enterohemorrhagic E. coli (EHEC) is considered to be the most dangerous pathotype of E. coli, as it causes severe conditions such as hemorrhagic colitis (HC) and hemolytic uremic syndrome (HUS). Antibiotic treatment of EHEC infections is generally not recommended since it may promote the production of the Shiga toxin (Stx) and lead to worsened symptoms. This study explores how exposure to the fluoroquinolone ciprofloxacin reorganizes the transcriptome and proteome of EHEC O157:H7 strain EDL933, with special emphasis on virulence-associated factors. As expected, exposure to ciprofloxacin caused an extensive upregulation of SOS-response- and Stx-phage proteins, including Stx. A range of other virulence-associated factors were also upregulated, including many genes encoded by the LEE-pathogenicity island, the enterohemolysin gene (ehxA), as well as several genes and proteins involved in LPS production. However, a large proportion of the genes and proteins (17 and 8%, respectively) whose expression was upregulated upon ciprofloxacin exposure (17 and 8%, respectively) are not functionally assigned. This indicates a knowledge gap in our understanding of mechanisms involved in EHECs response to antibiotic-induced stress. Altogether, the results contribute to better understanding of how exposure to ciprofloxacin influences the virulome of EHEC and generates a knowledge base for further studies on how EHEC responds to antibiotic-induced stress. A deeper understanding on how EHEC responds to antibiotics will facilitate development of novel and safer treatments for EHEC infections.

Freshwater plastispheres as a vector for foodborne bacteria and viruses.

There is growing evidence that plastic particles can accumulate microorganisms that are pathogenic to humans or animals. In the current study, the composition of the plastispheres that accumulated on polypropylene (PP), polyvinyl chloride (PVC), and high-density polyethylene (HDPE) pieces submerged in a river in the southeast Norway was characterized by 16S rRNA amplicon sequencing. Seasonal and geographical effects on the bacterial composition of the plastisphere were identified, in addition to the detection of potential foodborne pathogenic bacteria and viruses as part of the plastisphere. The diversity and taxonomic composition of the plastispheres were influenced by the number of weeks in the river, the season, and the location. The bacterial diversity differed significantly in the plastisphere from June and September, with a generally higher diversity in June. Also, the community composition of the plastisphere was significantly influenced by the geographical location, while the type of plastic had less impact. Plastics submerged in river water assembled a variety of microorganisms including potentially pathogenic bacteria and viruses (noro- and adenovirus) detected by qPCR. Cultivation methods detected viable bacteria such as Escherichia coli and Listeria monocytogenes. The results highlight the need for additional research on the risk of contaminating food with plastic particles colonized with human pathogens through irrigation water.

In vitro digestion of ESC-resistant Escherichia coli from poultry meat and evaluation of human health risk.

Introduction: The spread of antimicrobial resistance (AMR) has become a threat against human and animal health. Third and fourth generation cephalosporins have been defined as critically important antimicrobials by The World Health Organization. Exposure to Extended spectrum cephalosporin-resistant E. coli may result in consumers becoming carriers if these bacteria colonize the human gut or their resistance genes spread to other bacteria in the gut microbiota. In the case that these resistant bacteria at later occasions cause disease, their resistance characteristics may lead to failure of treatment and increased mortality. We hypothesized that ESC-resistant E. coli from poultry can survive digestion and thereby cause infections and/or spread their respective resistance traits within the gastro-intestinal tract. Methods: In this study, a selection of 31 ESC-resistant E. coli isolates from retail chicken meat was exposed to a static in vitro digestion model (INFOGEST). Their survival, alteration of colonizing characteristics in addition to conjugational abilities were investigated before and after digestion. Whole genome data from all isolates were screened through a custom-made virulence database of over 1100 genes for virulence- and colonizing factors. Results and discussion: All isolates were able to survive digestion. Most of the isolates (24/31) were able to transfer their bla CMY2-containing plasmid to E. coli DH5-á, with a general decline in conjugation frequency of digested isolates compared to non-digested. Overall, the isolates showed a higher degree of cell adhesion than cell invasion, with a slight increase after digestion compared non-digested, except for three isolates that displayed a major increase of invasion. These isolates also harbored genes facilitating invasion. In the virulence-associated gene analysis two isolates were categorized as UPEC, and one isolate was considered a hybrid pathogen. Altogether the pathogenic potential of these isolates is highly dependent on the individual isolate and its characteristics. Poultry meat may represent a reservoir and be a vehicle for dissemination of potential human pathogens and resistance determinants, and the ESC-resistance may complicate treatment in the case of an infection.

Population structure and uropathogenic potential of extended-spectrum cephalosporin-resistant Escherichia coli from retail chicken meat.

BACKGROUND: Food-producing animals and their products are considered a source for human acquisition of antimicrobial resistant (AMR) bacteria, and poultry are suggested to be a reservoir for Escherichia coli resistant to extended-spectrum cephalosporins (ESC), a group of antimicrobials used to treat community-onset urinary tract infections in humans. However, the zoonotic potential of ESC-resistant E. coli from poultry and their role as extraintestinal pathogens, including uropathogens, have been debated. The aim of this study was to characterize ESC-resistant E. coli isolated from domestically produced retail chicken meat regarding their population genetic structure, the presence of virulence-associated geno- and phenotypes as well as their carriage of antimicrobial resistance genes, in order to evaluate their uropathogenic potential. RESULTS: A collection of 141 ESC-resistant E. coli isolates from retail chicken in the Norwegian monitoring program for antimicrobial resistance in bacteria from food, feed and animals (NORM-VET) in 2012, 2014 and 2016 (n = 141) were whole genome sequenced and analyzed. The 141 isolates, all containing the beta-lactamase encoding gene blaCMY-2, were genetically diverse, grouping into 19 different sequence types (STs), and temporal variations in the distribution of STs were observed. Generally, a limited number of virulence-associated genes were identified in the isolates. Eighteen isolates were selected for further analysis of uropathogen-associated virulence traits including expression of type 1 fimbriae, motility, ability to form biofilm, serum resistance, adhesion- and invasion of eukaryotic cells and colicin production. These isolates demonstrated a high diversity in virulence-associated phenotypes suggesting that the uropathogenicity of ESC-resistant E. coli from chicken meat is correspondingly highly variable. For some isolates, there was a discrepancy between the presence of virulence-associated genes and corresponding expected phenotype, suggesting that mutations or regulatory mechanisms could influence their pathogenic potential. CONCLUSION: Our results indicate that the ESC-resistant E. coli from chicken meat have a low uropathogenic potential to humans, which is important knowledge for improvement of future risk assessments of AMR in the food chains.

Vitamin K Analogs Influence the Growth and Virulence Potential of Enterohemorrhagic Escherichia coli.

Enterohemorrhagic Escherichia coli (EHEC) causes serious foodborne disease worldwide. It produces the very potent Shiga toxin 2 (Stx2). The Stx2-encoding genes are located on a prophage, and production of the toxin is linked to the synthesis of Stx phages. There is, currently, no good treatment for EHEC infections, as antibiotics may trigger lytic cycle activation of the phages and increased Stx production. This study addresses how four analogs of vitamin K, phylloquinone (K1), menaquinone (K2), menadione (K3), and menadione sodium bisulfite (MSB), influence growth, Stx2-converting phage synthesis, and Stx2 production by the EHEC O157:H7 strain EDL933. Menadione and MSB conferred a concentration-dependent negative effect on bacterial growth, while phylloquinone or menaquinone had little and no effect on bacterial growth, respectively. All four vitamin K analogs affected Stx2 phage production negatively in uninduced cultures and in cultures induced with either hydrogen peroxide (H2O2), ciprofloxacin, or mitomycin C. Menadione and MSB reduced Stx2 production in cultures induced with either H2O2 or ciprofloxacin. MSB also had a negative effect on Stx2 production in two other EHEC isolates tested. Phylloquinone and menaquinone had, on the other hand, variable and concentration-dependent effects on Stx2 production. MSB, which conferred the strongest inhibitory effect on both Stx2 phage and Stx2 production, improved the growth of EHEC in the presence of H2O2 and ciprofloxacin, which could be explained by the reduced uptake of ciprofloxacin into the bacterial cell. Together, the data suggest that vitamin K analogs have a growth- and potential virulence-reducing effect on EHEC, which could be of therapeutic interest.IMPORTANCE Enterohemorrhagic E. coli (EHEC) can cause serious illness and deaths in humans by producing toxins that can severely damage our intestines and kidneys. There is currently no optimal treatment for EHEC infections, as antibiotics can worsen disease development. Consequently, the need for new treatment options is urgent. Environmental factors in our intestines can affect the virulence of EHEC and help our bodies fight EHEC infections. The ruminant intestine, the main reservoir for EHEC, contains high levels of vitamin K, but the levels are variable in humans. This study shows that vitamin K analogs can inhibit the growth of EHEC and/or production of its main virulence factor, the Shiga toxin. They may also inhibit the spreading of the Shiga toxin encoding bacteriophage. Our findings indicate that vitamin K analogs have the potential to suppress the development of serious disease caused by EHEC.

Zinc and Copper Reduce Conjugative Transfer of Resistance Plasmids from Extended-Spectrum Beta-Lactamase-Producing Escherichia coli.

The present work addresses the effect of excess levels of ZnCl2 and CuSO4 in the growth medium on the conjugative transfer of plasmids carrying the antibiotic resistance gene blaCMY-2 from extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli. Norwegian poultry are not treated prophylactically with antibiotics, but still, ESBL-producing E. coli are found in the chicken populations. Chickens receive higher amounts of Zn and Cu than their biological need, and several metals have been shown to act as drivers of antimicrobial resistance. In the present study, ESBL-producing E. coli strains collected from retail chicken meat were mated in broth containing various concentrations of ZnCl2 and CuSO4. Manual counting of transconjugants showed that ZnCl2 and CuSO4 reduced the conjugation frequency between E. coli strains in a concentration-dependent manner. Quantitative real-time PCR analyses showed that the presence of ZnCl2 and CuSO4 in the growth media reduced expression of the conjugation genes traB and nikB. By propagating monocultures over several generations, it was found that the blaCMY-2 plasmids remained stable in the recipient strains. Together the results show that exposure of ESBL-producing E. coli to Zn and Cu reduce horizontal transfer of the blaCMY-2 resistance plasmid by reducing expression of genes involved in conjugation in the plasmid donor strain.

Thiophenone Attenuates Enteropathogenic Escherichia coli O103:H2 Virulence by Interfering with AI-2 Signaling.

Interference with bacterial quorum sensing communication provides an anti-virulence strategy to control pathogenic bacteria. Here, using the Enteropathogenic E. coli (EPEC) O103:H2, we showed for the first time that thiophenone TF101 reduced expression of lsrB; the gene encoding the AI-2 receptor. Combined results of transcriptional and phenotypic analyses suggested that TF101 interfere with AI-2 signalling, possibly by competing with AI-2 for binding to LsrB. This is supported by in silico docking prediction of thiophenone TF101 in the LsrB pocket. Transcriptional analyses furthermore showed that thiophenone TF101 interfered with expression of the virulence genes eae and fimH. In addition, TF101 reduced AI-2 induced E. coli adhesion to colorectal adenocarcinoma cells. TF101, on the other hand, did not affect epinephrine or norepinephrine enhanced E. coli adhesion. Overall, our results showed that thiophenone TF101 interfered with virulence expression in E. coli O103:H2, suggestedly by interfering with AI-2 mediated quorum sensing. We thus conclude that thiophenone TF101 might represent a promising future anti-virulence agent in the fight against pathogenic E. coli.

Biofilm formation as a function of adhesin, growth medium, substratum and strain type.

Biofilm formation is involved in the majority of bacterial infections. Comparing six Escherichia coli and Klebsiella pneumoniae isolates revealed significant differences in biofilm formation depending on the growth medium. Fimbriae are known to be involved in biofilm formation, and type 1, F1C and P fimbriae were seen to influence biofilm formation significantly different depending on strain background, growth media and aeration as well as surface material. Altogether, this report clearly demonstrates that biofilm formation of a given strain is highly dependent on experimental design and that specific mechanisms involved in biofilm formation such as fimbrial expression only play a role under certain environmental conditions. This study underscores the importance of careful selection of experimental conditions when investigating bacterial biofilm formation and to take great precaution/care when comparing results from different biofilm studies.