ABSTRACT
BACKGROUND: Compounds of natural origin are potent source of drugs with unique mechanisms of action. Among phytochemicals, trans-cinnamaldehyde (t-CA) exhibits a wide range of biological activity, thus has been used for centuries to fight bacterial and fungal infections. However, the molecular basis of these properties has not been fully covered. Considering that difficult-to-control infections are becoming a rising global problem, there is a need to elucidate the molecular potential of t-CA. PURPOSE: To evaluate the antibacterial activity of t-CA against Shiga-toxigenic E. coli strains and elucidate its mechanism of action based on the inhibition of the virulence factor expression. METHODS: The antimicrobial potential of t-CA was assessed with two-fold microdilution and time-kill assays. Further evaluation included bioluminescence suppression assays, quantification of reactive oxygen species (ROS) and assessment of NAD+/NADH ratios. Morphological changes post t-CA exposure were examined using transmission electron microscopy. RNA sequencing and radiolabeling of nucleotides elucidated the metabolic alterations induced by t-CA. Toxin expression level was monitored through the application of fusion proteins, monitoring of bacteriophage development, and fluorescence microscopy studies. Lastly, the therapeutic efficacy in vivo was assessed using Galleria mellonella infection model. RESULTS: A comprehensive study of t-CA's bioactivity showed unique properties affecting bacterial metabolism and morphology, resulting in significant bacterial cell deformation and effective virulence inhibition. Elucidation of the underlying mechanisms indicated that t-CA activates the global regulatory system, the stringent response, manifested by its alarmone, (p)ppGpp, overproduction mediated by the RelA enzyme, thereby inhibiting bacterial proliferation. Intriguingly, t-CA effectively downregulates Shiga toxin gene expression via alarmone molecules, indicating its potential for therapeutic effect. In vivo validation demonstrated a significant improvement in larval survival rates post- t-CA treatment with 50 mg/kg (p < 0.05), akin to the efficacy observed with azithromycin, thus indicating its effectiveness against EHEC infections (p < 0.05). CONCLUSIONS: Collectively, these results reveal the robust antibacterial capabilities of t-CA, warranting its further exploration as a viable anti-infective agent.
Subject(s)
Acrolein , Anti-Bacterial Agents , Enterohemorrhagic Escherichia coli , Microbial Sensitivity Tests , Acrolein/analogs & derivatives , Acrolein/pharmacology , Anti-Bacterial Agents/pharmacology , Enterohemorrhagic Escherichia coli/drug effects , Animals , Reactive Oxygen Species/metabolism , Virulence FactorsABSTRACT
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.
Subject(s)
Ciprofloxacin , Proteomics , Transcriptome , Ciprofloxacin/pharmacology , Proteomics/methods , Virulence/drug effects , Transcriptome/drug effects , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/pathogenicity , Enterohemorrhagic Escherichia coli/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Anti-Bacterial Agents/pharmacology , Virulence Factors/genetics , Virulence Factors/metabolism , Proteome/metabolism , Gene Expression Profiling , HumansABSTRACT
The indiscriminate use of antimicrobials has led to the emergence of resistant bacteria, especially pathogenic strains of Escherichia coli, which are associated with diseases in animals and humans. The aim of the present study was to characterize E. coli isolates in calves with regards to the presence of virulence genes and investigate the resistance of the isolates to different antimicrobials. Between 2021 and 2023, 456 fecal samples were collected from calves in the Pantanal and Cerrado biomes of the state of Mato Grosso do Sul, Brazil. All samples were subjected to microbiological analysis and disc diffusion antibiogram testing. The polymerase chain reaction method was used to detect virulence genes. Bacterial growth was found in 451 of the 456 samples and biochemically identified as Escherichia coli. All 451 isolates (100 %) exhibited some phenotypic resistance to antimicrobials and 67.62 % exhibited multidrug resistance. The frequency of multidrug-resistant isolates in the Cerrado biome was significantly higher than that in the Pantanal biome (p = 0.0001). In the Cerrado, the most common pathotype was Shiga toxin-producing Escherichia coli (STEC) (28 %), followed by toxigenic Escherichia coli (ETEC) (11 %), enterohemorrhagic Escherichia coli (EHEC) (8 %) and enteropathogenic Escherichia coli (EPEC) (2 %). In most cases, the concomitant occurrence of pathotypes was more common, the most frequent of which were ETEC + STEC (33 %), ETEC + EHEC (15 %) and ETEC + EPEC (3 %). The STEC pathotype (30 %) was also found more frequently in the Pantanal, followed by EHEC (12 %), ETEC (9 %) and EPEC (6 %). The STEC pathotype had a significantly higher frequency of multidrug resistance (p = 0.0486) compared to the other pathotypes identified. The frequency of resistance was lower in strains from the Pantanal biome compared to those from the Cerrado biome. Although some factors are discussed in this paper, it is necessary to clarify the reasons for this difference and the possible impacts of these findings on both animal and human health in the region.
Subject(s)
Anti-Bacterial Agents , Cattle Diseases , Drug Resistance, Multiple, Bacterial , Escherichia coli Infections , Escherichia coli , Feces , Microbial Sensitivity Tests , Virulence Factors , Animals , Cattle , Brazil , Escherichia coli Infections/microbiology , Escherichia coli Infections/veterinary , Escherichia coli Infections/epidemiology , Feces/microbiology , Anti-Bacterial Agents/pharmacology , Escherichia coli/drug effects , Escherichia coli/genetics , Escherichia coli/isolation & purification , Cattle Diseases/microbiology , Cattle Diseases/epidemiology , Virulence Factors/genetics , Shiga-Toxigenic Escherichia coli/isolation & purification , Shiga-Toxigenic Escherichia coli/genetics , Shiga-Toxigenic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/genetics , Enterohemorrhagic Escherichia coli/isolation & purification , Enterohemorrhagic Escherichia coli/drug effects , Enterotoxigenic Escherichia coli/drug effects , Enterotoxigenic Escherichia coli/genetics , Enterotoxigenic Escherichia coli/isolation & purification , Escherichia coli Proteins/geneticsABSTRACT
Bacteria produce and release small signal molecules, autoinducers, as an indicator of their cell density. The system, called a quorum-sensing (QS) system, is used to control not only virulence factors but also antibiotic production, sporulation, competence, and biofilm formation in bacteria. Different from antibiotics, QS inhibitors are expected to specifically repress the virulence factors in pathogenic bacteria without inhibiting growth or bactericidal effects. Therefore, since QS inhibitors have little risk of antibiotic-resistant bacteria emergence, they have been proposed as promising anti-bacterial agents. In the present study, we aimed to find new QS inhibitors that prohibit the signaling cascade of autoinducer 3 (AI-3) recognized by a QseCB two-component system that regulates some virulence factors of pathogens, such as enterohemorrhagic Escherichia coli (EHEC) and Salmonella enterica subsp. enterica serovar Typhimurium. We have established the method for QS-inhibitor screening using a newly constructed plasmid pLES-AQSA. E. coli DH5α transformed with the pLES-AQSA can produce ß-galactosidase that converts 5-bromo-4-chloro-3-indolyl ß-d-galactopyranoside (X-gal) into blue pigment (5-bromo-4-chloro-indoxyl) under the control of the QseCB system. By screening, Heyndrickxia coagulans (formerly Bacillus coagulans) 29-2E was found to produce an exopolysaccharide (EPS)-like water-soluble polymer that prohibits QseCB-mediated ß-galactosidase production without antibacterial activities. Further, the simultaneous injection of the 29-2E strain significantly improves the survival rate of Salmonella Typhimurium-infected silkworm larvae (from 0% to 83.3%), suggesting that the substance may be a promising inhibitor against the virulence of pathogens without risk of the emergence of antibiotic-resistant bacteria.
Subject(s)
Quorum Sensing , Salmonella typhimurium , Quorum Sensing/drug effects , Salmonella typhimurium/drug effects , Virulence , Bacillus/metabolism , Anti-Bacterial Agents/pharmacology , Lactones/pharmacology , Lactones/metabolism , Virulence Factors/metabolism , Virulence Factors/genetics , Animals , Bacterial Proteins/metabolism , Bacterial Proteins/genetics , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/pathogenicity , Enterohemorrhagic Escherichia coli/metabolism , Biofilms/drug effects , Biofilms/growth & development , Homoserine/analogs & derivativesABSTRACT
Enterohemorrhagic Escherichia coli causes watery to bloody diarrhea, which may progress to hemorrhagic colitis and hemolytic-uremic syndrome. While early studies suggested that antibiotic treatment may worsen the pathology of an enterohemorrhagic Escherichia coli (EHEC) infection, recent work has shown that certain non-Shiga toxin-inducing antibiotics avert disease progression. Unfortunately, both intestinal bacterial infections and antibiotic treatment are associated with dysbiosis. This can alleviate colonization resistance, facilitate secondary infections, and potentially lead to more severe illness. To address the consequences in the context of an EHEC infection, we used the established mouse infection model organism Citrobacter rodentium Ïstx2dact and monitored changes in fecal microbiota composition during infection and antibiotic treatment. C. rodentium Ïstx2dact infection resulted in minor changes compared to antibiotic treatment. The infection caused clear alterations in the microbial community, leading mainly to a reduction of Muribaculaceae and a transient increase in Enterobacteriaceae distinct from Citrobacter. Antibiotic treatments of the infection resulted in marked and distinct variations in microbiota composition, diversity, and dispersion. Enrofloxacin and trimethoprim/sulfamethoxazole, which did not prevent Shiga toxin-mediated organ damage, had the least disruptive effects on the intestinal microbiota, while kanamycin and tetracycline, which rapidly cleared the infection without causing organ damage, caused a severe reduction in diversity. Kanamycin treatment resulted in the depletion of all but Bacteroidetes genera, whereas tetracycline effects on Clostridia were less severe. Together, these data highlight the need to address the impact of individual antibiotics in the clinical care of life-threatening infections and consider microbiota-regenerating therapies.IMPORTANCEUnderstanding the impact of antibiotic treatment on EHEC infections is crucial for appropriate clinical care. While discouraged by early studies, recent findings suggest certain antibiotics can impede disease progression. Here, we investigated the impact of individual antibiotics on the fecal microbiota in the context of an established EHEC mouse model using C. rodentium Ïstx2dact. The infection caused significant variations in the microbiota, leading to a transient increase in Enterobacteriaceae distinct from Citrobacter. However, these effects were minor compared to those observed for antibiotic treatments. Indeed, antibiotics that most efficiently cleared the infection also had the most detrimental effect on the fecal microbiota, causing a substantial reduction in microbial diversity. Conversely, antibiotics showing adverse effects or incomplete bacterial clearance had a reduced impact on microbiota composition and diversity. Taken together, our findings emphasize the delicate balance required to weigh the harmful effects of infection and antibiosis in treatment.
Subject(s)
Anti-Bacterial Agents , Citrobacter rodentium , Enterobacteriaceae Infections , Feces , Gastrointestinal Microbiome , Mice, Inbred C57BL , Animals , Citrobacter rodentium/drug effects , Mice , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/therapeutic use , Anti-Bacterial Agents/adverse effects , Feces/microbiology , Enterobacteriaceae Infections/drug therapy , Enterobacteriaceae Infections/microbiology , Gastrointestinal Microbiome/drug effects , Trimethoprim, Sulfamethoxazole Drug Combination/therapeutic use , Trimethoprim, Sulfamethoxazole Drug Combination/pharmacology , Enterohemorrhagic Escherichia coli/drug effects , Enrofloxacin/pharmacology , Enrofloxacin/therapeutic use , Female , Disease Models, Animal , Dysbiosis/microbiologyABSTRACT
Enterohaemorrhagic Escherichia coli (EHEC) comprise a group of intestinal pathogens responsible for a range of illnesses, including kidney failure and neurological compromise. EHEC produce critical virulence factors, Shiga toxin (Stx) 1 or 2, and the synthesis of Stx2 is associated with worse disease manifestations. Infected patients only receive supportive treatment because some conventional antibiotics enable toxin production. Shiga toxin 2 genes (stx2) are carried in λ-like bacteriophages (stx2-phages) inserted into the EHEC genome as prophages. Factors that cause DNA damage induce the lytic cycle of stx2-phages, leading to Stx2 production. The phage Q protein is critical for transcription antitermination of stx2 and phage lytic genes. This study reports that deficiency of two endoribonucleases (RNases), E and G, significantly delayed cell lysis and impaired production of both Stx2 and stx2-phages, unlike deficiency of either enzyme alone. Moreover, scarcity of both enzymes reduced the concentrations of Q and stx2 transcripts and slowed cell growth.
Subject(s)
Bacteriophages/genetics , Enterohemorrhagic Escherichia coli/physiology , Enterohemorrhagic Escherichia coli/virology , Escherichia coli Infections/microbiology , Ribonucleases/metabolism , Shiga Toxin/biosynthesis , Shiga Toxin/genetics , Enterohemorrhagic Escherichia coli/drug effects , Gene Expression Regulation, Bacterial , Mitomycin/pharmacology , Plasmids/genetics , Ribonucleases/genetics , Shiga Toxin 1/genetics , Shiga Toxin 2/genetics , Viral Plaque Assay , Virulence Factors/geneticsABSTRACT
Adherence of bacteria to the human intestinal mucosa can facilitate their internalization and the development of pathological processes. Escherichia coli O104:H4 is considered a hybrid strain (enteroaggregative hemorrhagic E. coli [EAHEC]), sharing virulence factors found in enterohemorrhagic (EHEC), and enteroaggregative (EAEC) E. coli pathotypes. The objective of this study was to analyze the effects of natural and synthetic antimicrobials (carvacrol, oregano extract, brazilin, palo de Brasil extract, and rifaximin) on the adherence of EHEC O157:H7, EAEC 042, and EAHEC O104:H4 to HEp-2 cells and to assess the expression of various genes involved in this process. Two concentrations of each antimicrobial that did not affect (p≤0.05) bacterial viability or damage the bacterial membrane integrity were used. Assays were conducted to determine whether the antimicrobials alter adhesion by affecting bacteria and/or alter adhesion by affecting the HEp-2 cells, whether the antimicrobials could detach bacteria previously adhered to HEp-2 cells, and whether the antimicrobials could modify the adherence ability exhibited by the bacteria for several cycles of adhesion assays. Giemsa stain and qPCR were used to assess the adhesion pattern and gene expression, respectively. The results showed that the antimicrobials affected the adherence abilities of the bacteria, with carvacrol, oregano extract, and rifaximin reducing up to 65% (p≤0.05) of E. coli adhered to HEp-2 cells. Carvacrol (10 mg/ml) was the most active compound against EHAEC O104:H4, even altering its aggregative adhesion pattern. There were changes in the expression of adhesion-related genes (aggR, pic, aap, aggA, and eae) in the bacteria and oxidative stress-related genes (SOD1, SOD2, CAT, and GPx) in the HEp-2 cells. In general, we demonstrated that carvacrol, oregano extract, and rifaximin at sub-minimal bactericidal concentrations interfere with target sites in E. coli, reducing the adhesion efficiency.
Subject(s)
Anti-Infective Agents/pharmacology , Bacterial Adhesion/drug effects , Enterohemorrhagic Escherichia coli/drug effects , Epithelial Cells/drug effects , Escherichia coli Infections/drug therapy , Escherichia coli O104/drug effects , Shiga-Toxigenic Escherichia coli/drug effects , Biofilms/drug effects , Cell Line , Disease Outbreaks/prevention & control , Epithelial Cells/microbiology , Humans , Microbial Viability/drug effects , Serogroup , Virulence/drug effectsABSTRACT
The mammalian gastrointestinal tract is a complex biochemical organ that generates a diverse milieu of host- and microbe-derived metabolites. In this environment, bacterial pathogens sense and respond to specific stimuli, which are integrated into the regulation of their virulence programs. Previously, we identified the transcription factor FadR, a long-chain fatty acid (LCFA) acyl coenzyme A (acyl-CoA) sensor, as a novel virulence regulator in the human foodborne pathogen enterohemorrhagic Escherichia coli (EHEC). Here, we demonstrate that exogenous LCFAs directly inhibit the locus of enterocyte effacement (LEE) pathogenicity island in EHEC through sensing by FadR. Moreover, in addition to LCFAs that are 18 carbons in length or shorter, we introduce host-derived arachidonic acid (C20:4) as an additional LCFA that is recognized by the FadR system in EHEC. We show that arachidonic acid is processed by the acyl-CoA synthetase FadD, which permits binding to FadR and decreases FadR affinity for its target DNA sequences. This interaction enables the transcriptional regulation of FadR-responsive operons by arachidonic acid in EHEC, including the LEE. Finally, we show that arachidonic acid inhibits hallmarks of EHEC disease in a FadR-dependent manner, including EHEC attachment to epithelial cells and the formation of attaching and effacing lesions. Together, our findings delineate a molecular mechanism demonstrating how LCFAs can directly inhibit the virulence of an enteric bacterial pathogen. More broadly, our findings expand the repertoire of ligands sensed by the canonical LFCA sensing machinery in EHEC to include arachidonic acid, an important bioactive lipid that is ubiquitous within host environments.IMPORTANCE Polyunsaturated fatty acids (PUFAs) play important roles in host immunity. Manipulation of lipid content in host tissues through diet or pharmacological interventions is associated with altered severity of various inflammatory diseases. Our work introduces a defined host-pathogen interaction by which arachidonic acid, a host-derived and dietary PUFA, can impact the outcome of enteric infection with the human pathogen enterohemorrhagic Escherichia coli (EHEC). We show that long-chain fatty acids including arachidonic acid act as signaling molecules that directly suppress a key pathogenicity island in EHEC following recognition by the fatty acyl-CoA-responsive transcription factor FadR. Thus, in addition to its established effects on host immunity and its bactericidal activities against other pathogens, we demonstrate that arachidonic acid also acts as a signaling molecule that inhibits virulence in an enteric pathogen.
Subject(s)
Arachidonic Acid/metabolism , Enterohemorrhagic Escherichia coli/physiology , Escherichia coli Infections/metabolism , Escherichia coli Infections/microbiology , Fatty Acids/metabolism , Host-Pathogen Interactions , Arachidonic Acid/pharmacology , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Models, Biological , Transcription Factors/genetics , Transcription Factors/metabolism , Virulence/genetics , Virulence Factors/geneticsABSTRACT
During passage through the human gastrointestinal tract, enterohemorrhagic Escherichia coli (EHEC) is exposed to membrane-damaging bile in the small intestine. We previously reported that EHEC treatment with a physiological bile salt mixture upregulates basRS, encoding a two-component system, and arnBCADTEF, encoding the aminoarabinose lipid A modification pathway (J. V. Kus, A. Gebremedhin, V. Dang, S. L. Tran, A. Serbanescu, and D. Barnett Foster, J Bacteriol 193: 4509-4515, 2011, https://doi.org/10.1128/JB.00200-11). The present study examined the effect of bile salt mix (BSM) treatment on EHEC resistance to three human gastrointestinal defense peptides-HD-5, HNP-1, and LL-37-as well as the role of basRS and arnT in the respective responses. After BSM treatment, EHEC resistance to HD-5 and HNP-1 was significantly increased in a BSM-, defensin dose-dependent manner. The resistance phenotype was dependent on both basRS and arnT However, the BSM treatment did not alter EHEC resistance to LL-37, even when the ompT gene, encoding an LL-37 cleavage protease, was disrupted. Interestingly, enteropathogenic E. coli, a related pathogen that infects the small intestine, showed a similar BSM-induced resistance phenotype. Using a model of EHEC infection in Galleria mellonella, we found significantly lower survival rates in wax moth larvae infected with BSM-treated wild-type EHEC than in those infected with a BSM-treated basS mutant, suggesting that treatment with a physiological BSM enhances virulence through a basS-mediated pathway. The results of this investigation provide persuasive evidence that bile salts typically encountered during transit through the small intestine can serve as an environmental cue for EHEC, enhancing resistance to several key host defense peptides.
Subject(s)
Antimicrobial Cationic Peptides/drug effects , Bile Acids and Salts/pharmacology , Bile Acids and Salts/therapeutic use , Disease Resistance/drug effects , Enterohemorrhagic Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Virulence/drug effects , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Infections/pathology , HumansABSTRACT
Colonization of the gastrointestinal tract by enterohaemorrhagic Escherichia coli (EHEC) is critically dependent on its ability to sense and respond to various microenvironments within the host. EHEC exposure to physiologically relevant levels of bile salts upregulates the two-component system, pmrAB, and the arnBCADTEF operon, resulting in lipopolysaccharide modification and increased resistance to the cationic antimicrobial peptide, polymyxin B (PMB). A similar pmrAB- and arn-dependent PMB resistance has been observed in Salmonella enterica in the presence of ferric iron. Limiting magnesium levels and mild acid can also induce Salmonella resistance to PMB through another two-component system, PhoPQ and the connector protein, PmrD. This study aims to evaluate the relative contributions of a bile-salt mix (BSM), iron, limiting magnesium as well as the roles of pmrAB, phoPQ and pmrD to EHEC's resistance to PMB. Killing assays show that EHEC treatment with the BSM or iron under excess magnesium and neutral pH conditions induces a pmrAB-dependent, phoP-independent PMB resistance. By contrast, exposure to limiting magnesium triggers a pmrB-, phoP- and pmrD-dependent PMB resistance. The iron-induced PMB resistance is independent of phoP and pmrD under limiting magnesium conditions while the bile-salt-induced PMB resistance is independent of pmrD only under non-PhoP-inducing conditions. GFP-pmrD transcriptional reporter studies reveal that the limiting magnesium enhances pmrD expression, which is repressed upon additional exposure to either BSM or iron. Our results also show that exposure to mild acid enhances PMB resistance in a pmrD-independent manner and GFP reporter results confirm minimal expression of pmrD at this pH regardless of the magnesium level. This study provides novel insights into how EHEC differentially employs PmrAB, PhoPQ and PmrD to monitor and respond to bile salts, iron, acidic pH and magnesium typically encountered within the gastrointestinal tract in order to modulate its survival against cationic antimicrobial peptides.
Subject(s)
Antimicrobial Cationic Peptides/metabolism , Bile Acids and Salts/pharmacology , Drug Resistance, Bacterial/drug effects , Enterohemorrhagic Escherichia coli/physiology , Iron/pharmacology , Anti-Bacterial Agents/metabolism , Bacterial Proteins/genetics , Bacterial Proteins/metabolism , Drug Resistance, Bacterial/genetics , Enterohemorrhagic Escherichia coli/drug effects , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Gene Expression Regulation, Bacterial/drug effects , Hydrogen-Ion Concentration , Magnesium/metabolism , Polymyxin B/metabolism , Promoter Regions, Genetic , Transcription Factors/genetics , Transcription Factors/metabolismABSTRACT
The molecular environment of the host can have profound effects on the behavior of resident bacterial species. We recently established how the sensing and response of enterohemorrhagic Escherichia coli (EHEC) to d-serine (d-Ser) resulted in down-regulation of type 3 secretion system-dependent colonization, thereby avoiding unfavorable environments abundant in this toxic metabolite. However, this model ignores a key determinant of the success of bacterial pathogens, adaptive evolution. In this study, we have explored the adaptation of EHEC to d-Ser and its consequences for pathogenesis. We rapidly isolated multiple, independent, EHEC mutants whose growth was no longer compromised in the presence of d-Ser. Through a combination of whole-genome sequencing and transcriptomics, we showed that tolerance could be attributed to disruption of one of two d-Ser transporters and/or activation of a previously nonfunctional d-Ser deaminase. While the implication of cytoplasmic transport in d-Ser toxicity was unsurprising, disruption of a single transporter, CycA, was sufficient to completely overcome the repression of type 3 secretion system activity normally associated with exposure to d-Ser. Despite the fact that this reveals a mechanism by which evolution could drive a pathogen to colonize new niches, interrogation of sequenced E. coli O157:H7 genomes showed a high level of CycA conservation, highlighting a strong selective pressure for functionality. Collectively, these data show that CycA is a critically important conduit for d-Ser uptake that is central to the niche restriction of EHEC.
Subject(s)
Enterohemorrhagic Escherichia coli , Genome, Bacterial , Serine/pharmacology , Adaptation, Biological/genetics , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/genetics , Enterohemorrhagic Escherichia coli/metabolism , Gene Knockdown Techniques , Genome, Bacterial/drug effects , Genome, Bacterial/genetics , HeLa Cells , Humans , Mutation/genetics , Type III Secretion Systems/geneticsABSTRACT
AIMS: The goal was to explore the effects of subinhibitory concentration (SIC) (0·5 MIC = 20 µg l-1 ) of ciprofloxacin on the transcriptome of enterohaemorrhagic Escherichia coli O26:H11 isolate by 60 minutes of exposure. MATERIALS AND RESULTS: We used a combination of comparative genomic and transcriptomic (RNAseq) analyses. The whole genome of the E. coli O26:H11 #30934 strain of bovine origin was sequenced and assembled. This genome was next used as reference for the differential gene expression analysis. A whole-genome-based analysis of 36 publicly available E. coli O26:H11 genomes was performed to define the core and the accessory transcriptome of E. coli O26:H11. Using RNAseq and RT-qPCR analysis we observed overexpression of the SOS response and of T3SS effectors, together with the inhibition of specific motility-associated genes. Among the large set of transposases present, only three were activated, suggesting moderate transposition of genes with low doses of ciprofloxacin. Our results illustrated that transcriptional repressors, such as the CopG family protein, belonging to the core genome of E. coli O26:H11, are altered in response to fluoroquinolone exposure. The gene ontology enrichment analysis showed SIC of ciprofloxacin induced binding functions and catalytic activities, including mostly transferase and hydrolase proteins. The amino acid pathways involved in metabolic processes were significantly enhanced after the treatment. CONCLUSIONS: Although the core genome of E. coli O26:H11 constituted only 54·5% of the whole genome, we demonstrated that most differentially expressed genes were associated with the core genome of E. coli O26:H11, and that effects on the mobile genetic element, phage, and plasmid-related genes were rare. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time the effect of low dose of ciprofloxacin on the core transcriptome of E. coli O26:H11 was described. The effects on the main biological functions and protein classes including transcriptional regulators were illustrated.
Subject(s)
Anti-Bacterial Agents/pharmacology , Ciprofloxacin/pharmacology , Enterohemorrhagic Escherichia coli/drug effects , Transcriptome/drug effects , Animals , Cattle , Enterohemorrhagic Escherichia coli/genetics , Enterohemorrhagic Escherichia coli/isolation & purification , Escherichia coli Infections/microbiology , Escherichia coli Infections/veterinary , Escherichia coli Proteins/genetics , Gene Expression Regulation, Bacterial/genetics , Genome, Bacterial/geneticsABSTRACT
AST-120 (Kremezin) is used to treat progressive chronic kidney disease (CKD) by adsorbing uremic toxin precursors produced by gut microbiota, such as indole and phenols. In this study, we propose that AST-120 reduces indole level, consequently suppresses indole effects on induction of drug tolerance and virulence in Escherichia coli including enterohaemorrhagic strains. In experiments, AST-120 adsorbed both indole and tryptophan, a precursor of indole production, and led to decreased expression of acrD and mdtEF which encode drug efflux pumps, and elevated glpT, which encodes a transporter for fosfomycin uptake and increases susceptibility to aztreonam, rhodamine 6G, and fosfomycin. AST-120 also decreased the production of EspB, which contributes to pathogenicity of enterohaemorrhagic E. coli (EHEC). Aztreonam, ciprofloxacin, minocycline, trimethoprim, and sulfamethoxazole were also adsorbed by AST-120. However, fosfomycin, in addition to rifampicin, colistin and amikacin were not adsorbed, thus AST-120 can be used together with these drugs for therapy to treat infections. These results suggest another benefit of AST-120, i.e., that it assists antibacterial chemotherapy.
Subject(s)
Anti-Bacterial Agents/pharmacology , Carbon/pharmacology , Escherichia coli Infections/drug therapy , Escherichia coli/drug effects , Indoles/metabolism , Oxides/pharmacology , Signal Transduction/drug effects , Drug Resistance, Bacterial/drug effects , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/metabolism , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli/metabolism , Escherichia coli/pathogenicity , Escherichia coli Infections/microbiology , Humans , Virulence/drug effectsABSTRACT
Most Escherichia coli strains are innocuous to human beings; however, some strains can cause diarrhea and are grouped into pathotypes. Since current trends promote the use of natural-origin compounds to control bacteria, in this study, the effects of the phenolic compounds (PCs) tannic acid (TA), gallic acid (GA), methyl gallate (MG), and epigallocatechin gallate (EG) on the growth, swarming motility, biofilm formation, and expression of selected virulence genes of three E. coli pathotypes (enteropathogenic Escherichia coli [EPEC], enterohemorrhagic Escherichia coli [EHEC], and enterotoxigenic Escherichia coli [ETEC]) were evaluated. Minimum bactericidal concentrations (MBCs) were determined by using microtiter plates, and the effects of sublethal PC concentrations on swarming motility were evaluated on Luria-Bertani agar. Biofilm formation was assessed in microtiter plates via crystal violet staining, and the expression levels of genes involved in biofilm formation (flhC, fliA, fliC, and csgA) and swarming motility (csgD and cyaA) were evaluated via quantitative PCR. All PC were bactericidal with minimal bactericidal concentrations ranging from 0.07 to 2.1 mg/mL. At concentrations lower than the MBC, PCs decreased swarming motility (14.8-100%). GA reduced biofilm formation in all of the tested strains; however, TA, MG, and EG induced biofilm formation in some strains at specific concentrations. TA induced the overexpression of csgA, csgD, and cyaA, whereas the other PCs did not have any effects or reduced their expression levels. The PCs tested in this study showed potential to control E. coli strains belonging to the EHEC, ETEC, and EPEC pathotypes by affecting their growth, swarming motility, and virulence gene expression; however, proper concentrations must be used to avoid the induction of undesirable virulence factor genes.
Subject(s)
Biofilms/drug effects , Enterohemorrhagic Escherichia coli/drug effects , Enteropathogenic Escherichia coli/drug effects , Enterotoxigenic Escherichia coli/drug effects , Polyphenols/pharmacology , Anti-Bacterial Agents/pharmacology , Escherichia coli Proteins/genetics , Genes, Bacterial , Microbial Sensitivity Tests , VirulenceABSTRACT
Infections with enterohemorrhagic Escherichia coli (EHEC) cause disease ranging from mild diarrhea to hemolytic-uremic syndrome (HUS) and are the most common cause of renal failure in children in high-income countries. The severity of the disease derives from the release of Shiga toxins (Stx). The use of antibiotics to treat EHEC infections is generally avoided, as it can result in increased stx expression. Here, we systematically tested different classes of antibiotics and found that their influence on stx expression and release varies significantly. We assessed a selection of these antibiotics in vivo using the Citrobacter rodentium Ïstx2dact mouse model and show that stx2d-inducing antibiotics resulted in weight loss and kidney damage despite clearance of the infection. However, several non-Stx-inducing antibiotics cleared bacterial infection without causing Stx-mediated pathology. Our results suggest that these antibiotics might be useful in the treatment of EHEC-infected human patients and decrease the risk of HUS development.
Subject(s)
Acute Kidney Injury/prevention & control , Anti-Bacterial Agents/therapeutic use , Enterohemorrhagic Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Shiga Toxin 2/metabolism , Acute Kidney Injury/microbiology , Animals , Citrobacter rodentium/genetics , Citrobacter rodentium/metabolism , Disease Models, Animal , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Infections/microbiology , Escherichia coli Infections/pathology , Female , Hemolytic-Uremic Syndrome/drug therapy , Hemolytic-Uremic Syndrome/microbiology , Mice , Mice, Inbred C57BL , Shiga Toxin 2/genetics , Shiga Toxin 2/toxicityABSTRACT
Proadrenomedullin N-terminal 20 peptide (PAMP) is elevated in sepsis, but the function and possible mechanism of PAMP in bacterial infection is elusive. This study is aim to evaluate the role of PAMP in the interaction between the Enterohemorrhagic E. coli (EHEC) and the host barrier. Our results showed that PAMP alleviated the EHEC-induced disruption of goblet cells and mucosal damage in the intestine, increased the expression of occludin in the colon of EHEC-infected mice, and reduced the proinflammatory cytokines level in serum significantly compared with the control group. Meanwhile, lipopolysaccharide (LPS) stimulation could dose-dependently induce the expression of preproADM, the precursor of PAMP, in human intestinal epithelial cell (HIEC) and human umbilical vein endothelial cell (HUVEC). In addition, PAMP inhibited the growth of EHEC O157:H7 and destroyed the inner and outer membrane. At low concentration, PAMP attenuated the EHEC virulence genes including hlyA and eaeA, which was also confirmed from reduced hemolysis to red cells and adhesion to HIEC. These results indicated that EHEC infection would modulate the expression of PAMP in intestinal epithelium or vascular endothelium, and in turn exerted a protective effect in EHEC induced infection by rupturing the bacterial cell membrane and attenuating the bacterial virulence.
Subject(s)
Adrenomedullin/therapeutic use , Cell Membrane/metabolism , Enterohemorrhagic Escherichia coli/physiology , Escherichia coli Infections/drug therapy , Escherichia coli Infections/microbiology , Inflammation/microbiology , Intestines/microbiology , Protective Agents/pharmacology , Adrenomedullin/chemistry , Adrenomedullin/pharmacology , Amino Acid Sequence , Animals , Anti-Infective Agents/pharmacology , Anti-Infective Agents/therapeutic use , Cell Membrane/drug effects , Cytokines/metabolism , Enterohemorrhagic Escherichia coli/drug effects , Gene Expression Regulation/drug effects , Human Umbilical Vein Endothelial Cells , Humans , Inflammation/pathology , Inflammation Mediators/metabolism , Lipopolysaccharides/pharmacology , Male , Mice, Inbred C57BL , Virulence/geneticsABSTRACT
Shiga toxins (Stx) induce the symptoms of the life-threatening hemolytic uremic syndrome (HUS) and are the main virulence factors of enterohemorrhagic Escherichia coli (EHEC). The bacterial SOS response is the essential signal for high level production and release of Stx1/2. To assess the potential effectiveness of different antibiotics in blocking SOS response and Stx1/2 production, we constructed a reporter gene based test system that allows for the time-resolved, simultaneous read-out of the SOS response (recAP-cfp) and Stx1 production (stx1::yfp) in EHEC O157:H7 EDL933. We find that cells exposed to inhibitory or subinhibitory concentrations of ciprofloxacin did induce the SOS response, but not when the cells were exposed to rifaximine, azithromycin, tetracycline, gentamicin or ampicillin. Cell lysis and the peak in Stx1 production were substantially delayed with respect to the peak of the SOS response. We used this feature to show that adding transcriptional or translational inhibitors can block Stx1 production even after the SOS response is fully induced. RT-qPCR based tests with other clinically relevant EHEC isolates showed similar results for both Stx1 and Stx2. These observations suggest that transcriptional and translational inhibitors may be of value in treating EHEC infections.
Subject(s)
Anti-Bacterial Agents/pharmacology , Enterohemorrhagic Escherichia coli/genetics , SOS Response, Genetics/drug effects , Shiga Toxin 1/genetics , Ampicillin/pharmacology , Cell Wall/drug effects , Ciprofloxacin/pharmacology , Enterohemorrhagic Escherichia coli/drug effects , Escherichia coli Infections/drug therapy , Genes, Reporter , Protein Biosynthesis/drug effects , Protein Synthesis Inhibitors/pharmacology , Shiga Toxin 1/biosynthesis , Transcription, Genetic/drug effectsABSTRACT
In the field, foodborne pathogens such as enterohemorrhagic Escherichia coli (EHEC) are capable of surviving on produce over time, yet little is known about how these pathogens adapt to this environment. To assess the impact of pre-harvest environmental conditions on EHEC survival, we quantified survival on romaine lettuce under two relative humidity (75% and 45%) and seasonal conditions (March and June). Greenhouse-grown lettuce was spray-inoculated with EHEC and placed in a growth chamber, mimicking conditions typical for June and March in Salinas Valley, California. Bacteria were enumerated on days 0, 1, 3, and 5 post-inoculation. Overall, we found that the effect of relative humidity on EHEC survival depended on the seasonal conditions. Under June seasonal conditions, higher relative humidity led to lower survival, and lower relative humidity led to greater survival, five days post-inoculation. Under March seasonal conditions, the impact of relative humidity on EHEC survival was minimal over the five days. The bacteria were also tested for their ability to survive a chlorine decontamination wash. Inoculated lettuce was incubated under the June 75% relative humidity conditions and then washed with a 50 ppm sodium hypochlorite solution (40 ppm free chlorine). When incubated under June seasonal conditions for three to five days, EHEC strains showed increased tolerance to chlorine (adj. p < 0.05) compared to chlorine tolerance upon inoculation onto lettuce. This indicated that longer incubation on lettuce led to greater EHEC survival upon exposure to chlorine. Subsequent transcriptome analysis identified the upregulation of osmotic and oxidative stress response genes by EHEC after three and five days of incubation on pre-harvest lettuce. Assessing the physiological changes in EHEC that occur during association with pre-harvest lettuce is important for understanding how changing tolerance to post-harvest control measures may occur.
Subject(s)
Chlorine/pharmacology , Enterohemorrhagic Escherichia coli/drug effects , Food Microbiology , Lactuca/microbiology , Colony Count, Microbial , Enterohemorrhagic Escherichia coli/isolation & purification , Enterohemorrhagic Escherichia coli/physiology , Food HandlingABSTRACT
Shiga toxin (Stx) is the main virulence factor of enterohemorrhagic Escherichia coli (EHEC), that cause gastrointestinal infection leading to hemolytic uremic syndrome. The aim of this study was to investigate if Stx signals via ATP and if blockade of purinergic receptors could be protective. Stx induced ATP release from HeLa cells and in a mouse model. Toxin induced rapid calcium influx into HeLa cells, as well as platelets, and a P2X1 receptor antagonist, NF449, abolished this effect. Likewise, the P2X antagonist suramin blocked calcium influx in Hela cells. NF449 did not affect toxin intracellular retrograde transport, however, cells pre-treated with NF449 exhibited significantly higher viability after exposure to Stx for 24 hours, compared to untreated cells. NF449 protected HeLa cells from protein synthesis inhibition and from Stx-induced apoptosis, assayed by caspase 3/7 activity. The latter effect was confirmed by P2X1 receptor silencing. Stx induced the release of toxin-positive HeLa cell- and platelet-derived microvesicles, detected by flow cytometry, an effect significantly reduced by NF449 or suramin. Suramin decreased microvesicle levels in mice injected with Stx or inoculated with Stx-producing EHEC. Taken together, we describe a novel mechanism of Stx-mediated cellular injury associated with ATP signaling and inhibited by P2X receptor blockade.
Subject(s)
Escherichia coli Infections/drug therapy , Hemolytic-Uremic Syndrome/drug therapy , Receptors, Purinergic P2X1/genetics , Shiga Toxin/genetics , Adenosine Triphosphate/metabolism , Animals , Benzenesulfonates/pharmacology , Blood Platelets/microbiology , Enterohemorrhagic Escherichia coli/drug effects , Enterohemorrhagic Escherichia coli/pathogenicity , Escherichia coli Infections/genetics , Escherichia coli Infections/microbiology , Escherichia coli Infections/pathology , HeLa Cells , Hemolytic-Uremic Syndrome/genetics , Hemolytic-Uremic Syndrome/microbiology , Hemolytic-Uremic Syndrome/pathology , Humans , Mice , Purinergic P2X Receptor Antagonists/pharmacology , Shiga Toxin/antagonists & inhibitorsABSTRACT
BACKGROUND: Several serious vegetable-associated outbreaks of enterohemorrhagic Escherichia coli (EHEC) infections have occurred during the last decades. In this context, vegetables have been suggested to function as secondary reservoirs for EHEC strains. Increased knowledge about the interaction of EHEC with plants including gene expression patterns in response to plant-derived compounds is required. In the current study, EHEC O157:H7 strain Sakai, EHEC O157:H- strain 3072/96, and the EHEC/enteroaggregative E. coli (EAEC) hybrid O104:H4 strain C227-11φcu were grown in lamb's lettuce medium and in M9 minimal medium to study the differential transcriptional response of these strains to plant-derived compounds with RNA-Seq technology. RESULTS: Many genes involved in carbohydrate degradation and peptide utilization were similarly upregulated in all three strains, suggesting that the lamb's lettuce medium provides sufficient nutrients for proliferation. In particular, the genes galET and rbsAC involved in galactose metabolism and D-ribose catabolism, respectively, were uniformly upregulated in the investigated strains. The most prominent differences in shared genome transcript levels were observed for genes involved in the expression of flagella. Transcripts of all three classes of the flagellar hierarchy were highly abundant in strain C227-11φcu. Strain Sakai expressed only genes encoding the basal flagellar structure. In addition, both strains showed increased motility in presence of lamb's lettuce extract. Moreover, strain 3072/96 showed increased transcription activity for genes encoding the type III secretion system (T3SS) including effectors, and was identified as a powerful biofilm-producer in M9 minimal medium. CONCLUSION: The current study provides clear evidence that EHEC and EHEC/EAEC strains are able to adjust their gene expression patterns towards metabolization of plant-derived compounds, demonstrating that they may proliferate well in a plant-associated environment. Moreover, we propose that flagella and other surface structures play a fundamental role in the interaction of EHEC and EHEC/EAEC with plants.