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Avian pathogenic Escherichia coli (APEC) is a notable pathogen that frequently leads to avian colibacillosis, posing a substantial risk to both the poultry industry and public health. The commercial vaccines against avian colibacillosis are primarily inactivated vaccines, but their effectiveness is limited to specific serotypes. Recent advances have highlighted bacterial membrane vesicles (MV) as a promising candidate in vaccine research. How to produce bacterial MVs vaccines on a large scale is a significant challenge for the industrialization of MVs. The msbB gene encodes an acyltransferase and has been implicated in altering the acylation pattern of lipid A, leading to a decrease in lipid A content in lipopolysaccharides (LPS). Here, we evaluated the immunoprotective efficacy of MVs derived from the LPS low-expressed APEC strain FY26ΔmsbB, which was an APEC mutant strain with a deletion of the msbB gene. The nitrogen cavitation technique was employed to extract APEC MVs, with results indicating a significant increase in MVs yield compared to that obtained under natural culture. The immunization effectiveness was assessed, revealing that FY26ΔmsbB MVs elicited an antibody response of laying hens and facilitated bacterial clearance. Protective efficacy studies demonstrated that immunization with FY26ΔmsbB MVs conferred the immune protection in chickens challenged with the wild-type APEC strain FY26. Notably, LPS low-carried MVs recovered from the mutant FY26ΔmsbB also displayed cross-protective capabilities, and effectively safeguarding against infections caused by O1, O7, O45, O78, and O101 serotypes virulent APEC strains. These findings suggest that MVs generated from the LPS low-expressed APEC strain FY26ΔmsbB represent a novel and empirically validated subunit vaccine for the prevention and control of infections by various APEC serotypes.
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Avian pathogenic Escherichia coli (APEC) is a notable subpathotype of the nonhuman extraintestinal pathogenic E. coli (ExPEC). Recognized as an extraintestinal foodborne pathogen, the zoonotic potential of APEC/ExPEC allows for cross-host transmission via APEC-contaminated poultry meat and eggs. ProQ, an RNA binding protein, is evolutionarily conserved in E. coli. However, its regulatory roles in the biofilm formation and virulence of APEC/ExPEC have not been explored. In this study, proQ deletion in the APEC strain FY26 significantly compromised its biofilm-forming ability. Furthermore, animal tests and cellular infection experiments showed that ProQ depletion significantly attenuated APEC virulence, thereby diminishing its capacity for bloodstream infection and effective adherence to and persistence within host cells. Transcriptome analysis revealed a decrease in the transcription level of the small RNA (sRNA) RyfA in the mutant FY26ΔproQ, suggesting a direct interaction between the sRNA RyfA and ProQ. This interaction might indicate that sRNA RyfA is a novel ProQ-associated sRNA. Moreover, the direct binding of ProQ to the sRNA RyfA was crucial for APEC biofilm formation, pathogenicity, adhesion, and intracellular survival. In conclusion, our findings provide detailed insight into the interaction between ProQ and sRNA RyfA and deepen our understanding of the regulatory elements that dictate APEC virulence and biofilm development. Such insights are instrumental in developing strategies to counteract APEC colonization within hosts and impede APEC biofilm establishment on food surfaces.
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Infecciones por Escherichia coli , Proteínas de Escherichia coli , Enfermedades de las Aves de Corral , ARN Pequeño no Traducido , Animales , Escherichia coli , Virulencia , Infecciones por Escherichia coli/veterinaria , Pollos/genética , Enfermedades de las Aves de Corral/patología , Factores de Virulencia/genética , Biopelículas , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Unión al ARNRESUMEN
BACKGROUND: Avian pathogenic Escherichia coli (APEC) is the major pathogen causing important avian diseases in poultry. As an important subtype of extraintestinal pathogenic E. coli, APEC has zoonotic potential and is considered a foodborne pathogen. APEC extracellular vesicles (EVs) may play vital roles in the interaction of the pathogen with its host cells. However, the precise roles played by APEC EVs are still not completely clear, especially in immune cells. RESULTS: In this study, we investigated the relationships between APEC EVs and immune cells. The production and characteristics of the EVs of APEC isolate CT265 were identified. Toll like receptor 4 (TLR4) triggered the cellular immune responses when it interacted with APEC EVs. APEC EVs induced a significant release of proinflammatory cytokines in THP-1 macrophages. APEC EVs induced the macrophage inflammatory response via the TLR4/MYD88/NF-κB signaling pathway, which participated in the activation of the APEC-EV-induced NLRP3 inflammasome. However, the loss of lipopolysaccharide (LPS) from APEC EVs reduced the activation of the NLRP3 inflammasome mediated by TLR4/MYD88/NF-κB signaling. Because APEC EVs activated the macrophage inflammatory response and cytokines release, we speculated that the interaction between APEC EVs and macrophages activated and promoted neutrophil migration during APEC extraintestinal infection. This study is the first to report that APEC EVs induce the formation of neutrophil extracellular traps (NETs) and chicken heterophil extracellular traps. Treatment with APEC EVs induced SAPK/JNK activation in neutrophils. The inhibition of TLR4 signaling suppressed APEC-EV-induced NET formation. However, although APEC EVs activated the immune response of macrophages and initiated NET formation, they also damaged macrophages, causing their apoptosis. The loss of LPS from APEC EVs did not prevent this process. CONCLUSION: APEC-derived EVs induced inflammatory responses in macrophages and NETs in neutrophils, and that TLR4 was involved in the APEC-EV-activated inflammatory response. These findings provided a basis for the further study of APEC pathogenesis.
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Infecciones por Escherichia coli , Trampas Extracelulares , Vesículas Extracelulares , Humanos , Escherichia coli , Receptor Toll-Like 4 , FN-kappa B , Inflamasomas , Lipopolisacáridos , Factor 88 de Diferenciación Mieloide , Proteína con Dominio Pirina 3 de la Familia NLR , Transducción de Señal , Proteínas Adaptadoras Transductoras de Señales , Infecciones por Escherichia coli/veterinariaRESUMEN
Carbapenem-resistant Klebsiella pneumoniae (CRKP), particularly those with high virulence, cause invasive disease in clinical settings. An epidemiological investigation was conducted on the evolution, virulence, and antimicrobial resistance of CRKP isolates in two tertiary teaching hospitals in Jiangsu, China from November 2020 to December 2021. There were 31 different CRKP strains discovered. We performed whole genome sequencing (WGS) on 13 SHV, cmlv, and FosA6-producing CRKP to reveal molecular characteristics. Five ST15/ST11 isolates had CRISPR-Cas systems. By conjugation tests, KPC-2 can be transmitted horizontally to E. coil. A conjugative pHN7A8-related multi-resistance plasmid (KPC-2, blaCTX-M-65, blaTEM-1, fosA3, catII, and rmtB) was first discovered in CRKP clinical isolates. Using bacteriological testing, a serum killing assay, and an infection model with Galleria mellonella, three ST11-K64 KPC-2 generating carbapenem-resistant hypervirulent Klebsiella pneumoniae (CR-hvKP) were identified. These strains harbored a virulent plasmid and an IncFII-family pKPC/pHN7A8 conjugative plasmid, which led to hypervirulence and resistance. One of these CR-hvKPs, which co-harbored KPC-2, NDM-6, SHV-182, SHV-64, and blaCTX-M-122 genes, was first discovered. Importantly, this CR-hvKP strain also produced biofilm and had non-inferior fitness. The widespread use of ceftazidime/avibactam might provide this CR-hvKP with a selective advantage; hence, immediate action is required to stop its dissemination. Another important finding is the novel ST6136 in K. pneumoniae. Finally, the sterilization efficiency rates of Fe2C nanoparticles in CRKP were more than 98%. Furthermore, our novel antibacterial Fe2C nanoparticles may also provide a therapeutic strategy for infections.
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The emergence of TMexCD1-TOprJ1, a novel transferable resistance-nodulation-division (RND)-type efflux pump conferring resistance to tigecycline, is now a serious public health issue in the world. Here, we found that melatonin synergistically enhanced the antibacterial efficacy of tigecycline against tmexCD1-toprJ1-positive Klebsiella pneumoniae by disrupting the proton driving force and efflux function to promote the accumulation of tigecycline into cells, damaging cell membrane integrity and causing the leakage of cell contents. The synergistic effect was further validated by a murine thigh infection model. The results revealed that the melatonin/tigecycline combination is a potential therapy to combat resistant bacteria carrying the tmexCD1-toprJ1 gene.
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Infecciones por Klebsiella , Melatonina , Animales , Ratones , Tigeciclina/farmacología , Melatonina/farmacología , Melatonina/metabolismo , Minociclina/farmacología , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Farmacorresistencia Bacteriana/genética , Proteínas de Transporte de Membrana/genética , Antibacterianos/uso terapéutico , Adyuvantes Inmunológicos , Adyuvantes Farmacéuticos , Pruebas de Sensibilidad Microbiana , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismoRESUMEN
Although electrochemical detection of microliters-level solutions is attractive for analysis of low-amount biological samples, its performance could be weakened by limited mass transfer due to low Reynolds number and laminar flow. Herein we designed a 3D-printed electroanalytical device to apply vibration for improvement of mass transfer during electrochemical detection. In our approach, the droplet-size sample solution containing Indole-3-acetic acid (IAA, as a model) was directly applied on the effective surface of a disposable working electrode. We demonstrated that vibration could enhance electrochemical responses of IAA more on the rough surface than on the smooth surface of the working electrodes. After optimization, the sensitivity for electrochemical detection of a 20-µL droplet under vibration with the voltage of 7 V increased more than 100% compared with the static condition. The enhanced electrochemical responses brought by vibration could be achieved reproducibly, which could be ascribed to improved mass transfer. Our strategy could be practically applied for differentiation of IAA in different tissues of Marchantia polymorpha with enhanced responses. This study suggested that vibration might become a simple and effective method to improve mass transfer in analysis of microliter-volume solutions, which might be extended for more biochemical assays.
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Técnicas Electroquímicas , Vibración , Técnicas Electroquímicas/métodos , ElectrodosRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) is a pathogen that causes host extraintestinal diseases. The ST95 E. coli lineage is one of the dominant ExPEC lineages in humans and poultry. In this study, we took advantage of extensive E. coli genomes available through public open-access databases to construct a detailed understanding of the phylogeny and evolution of ST95. We used a high variability of accessory genomes to highlight the diversity and dynamic traits of ST95. Isolates from diverse hosts and geographic sources were randomly located on the phylogenetic tree, which suggested that there is no host specificity for ST95. The time-scaled phylogeny showed that ST95 is an ancient and long-lasting lineage. The virulence genes, resistance genes, and pathogenicity islands (PAIs) were characterized in ST95 pan-genomes to provide novel insights into the pathogenicity and multidrug resistance (MDR) genotypes. We found that a pool of large plasmids drives virulence and MDR. Based on the unique genes in the ST95 pan-genome, we designed a novel multiplex PCR reaction to rapidly detect ST95. Overall, our study addressed a gap in the current understanding of ST95 ExPEC genomes, with significant implications for recognizing the success and spread of ST95.
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Extra-intestinal Pathogenic Escherichia coli (ExPEC) is defined as an extra-intestinal foodborne pathogen, and several dominant sequence types (STs) ExPEC isolates are highly virulent, with zoonotic potential. Bacteria extracellular vesicles (EVs) carry specific subsets of molecular cargo, which affect various biological processes in bacteria and host. The mechanisms of EVs formation in ExPEC remains to be elucidated. Here, the purified EVs of ExPEC strains of different STs were isolated with ultracentrifugation processes. A comparative analysis of the strain proteomes showed that cytoplasmic proteins accounted for a relatively high proportion of the proteins among ExPEC EVs. The proportion of cytoplasm-carrying vesicles in ExPEC EVs was calculated with a simple green fluorescent protein (GFP) expression method. The RecA/LexA-dependent SOS response is a critical mediator of generation of cytoplasm-carrying EVs. The SOS response activates the expression of prophage-associated endolysins, Epel1, Epel2.1, and Epel2.2, which triggered cell lysis, increasing the production of ExPEC cytoplasm-carrying EVs. The repressor LexA controlled directly the expression of these endolysins by binding to the SOS boxes in the endolysin promoter regions. Reducing bacterial viability stimulated the production of ExPEC EVs, especially cytoplasm-carrying EVs. The imbalance in cell division caused by exposure to H2O2, the deletion of ftsK genes, or t6A synthesis defects activated the RecA/LexA-dependent SOS response, inducing the expression of endolysins, and thus increasing the proportion of cytoplasm-carrying EVs in the total ExPEC EVs. Antibiotics, which decreased bacterial viability, also increase the production of ExPEC cytoplasm-carrying EVs through the SOS response. Changes in the proportion of cytoplasm-carrying EVs affected the total DNA content of ExPEC EVs. When macrophages are exposed to a higher proportion of cytoplasm-carrying vesicles, ExPEC EVs were more cytotoxic to macrophages, accompanied with more-severe mitochondrial disruption and a higher level of induced intrinsic apoptosis. In summary, we offered comprehensive insight into the proteome analysis of ExPEC EVs. This study demonstrated the novel formation mechanisms of E. coli cytoplasm-carrying EVs.
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Proteínas de Escherichia coli , Vesículas Extracelulares , Escherichia coli Patógena Extraintestinal , Viabilidad Microbiana , Citoplasma/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Vesículas Extracelulares/metabolismo , Escherichia coli Patógena Extraintestinal/genética , Peróxido de Hidrógeno/metabolismo , Proteínas de la Membrana/metabolismoRESUMEN
Avian pathogenic Escherichia coli (APEC) is recognized as a primary source of foodborne extraintestinal pathogenic E. coli (ExPEC), which poses a significant risk of extraintestinal infections in humans. The potential of human infection with ST117 lineage APEC/ExPEC from poultry is particularly concerning. However, relatively few whole-genome studies have focused on ST117 as an emerging ExPEC lineage. In this study, the complete genomes of 11 avian ST117 isolates and the draft genomes of 20 ST117 isolates in China were sequenced to reveal the genomic islands and large plasmid composition of ST117 APEC. With reference to the extensive E. coli genomes available in public databases, large-scale comprehensive genomic analysis of the ST117 lineage APEC/ExPEC was performed to reveal the features of the ST117 pan-genome and population. The high variability of the accessory genome emphasized the diversity and dynamic traits of the ST117 pan-genome. ST117 isolates recovered from different hosts and geographic sources were randomly located on a phylogeny tree, suggesting that ST117 E. coli lacked host specificity. A time-scaled phylogeny tree showed that ST117 was a recent E. coli lineage with a relatively short evolutionary period. Further characterization of a wide diversity of ExPEC-related virulence genes, pathogenicity islands (PAIs), and resistance genes of the ST117 pan-genome provided insights into the virulence and resistance of ST117 APEC/ExPEC. The results suggested zoonotic potential of ST117 APEC/ExPEC between birds and humans. Moreover, genomic analysis showed that a pool of diverse plasmids drove the virulence and multidrug resistance of ST117 APEC/ExPEC. Several types of large plasmids were scattered across the ST117 isolates, but there was no strong plasmid-clade adaptation. Combined with the pan-genome analysis, a double polymerase chain reaction (PCR) method was designed for rapid and cost-effective detection of ST117 isolates from various avian and human APEC/ExPEC isolates. Overall, this study addressed a gap in current knowledge about the ST117 APEC/ExPEC genome, with significant implications to understand the success and spread of ST117 APEC/ExPEC.
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Infecciones por Escherichia coli , Escherichia coli Patógena Extraintestinal , Enfermedades de las Aves de Corral , Animales , Humanos , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/veterinaria , Aves , Genómica , Enfermedades de las Aves de Corral/epidemiología , Filogenia , Pollos , Factores de Virulencia/genéticaRESUMEN
Klebsiella pneumoniae is well-known opportunistic enterobacteria involved in complex clinical infections in humans and animals. The domestic animals might be a source of the multidrug-resistant virulent K. pneumoniae to humans. K. pneumoniae infections in domestic animals are considered as an emergent global concern. The horizontal gene transfer plays essential roles in bacterial genome evolution by spread of virulence and resistance determinants. However, the virulence genes can be transferred horizontally via K. pneumoniae-derived outer membrane vesicles (OMVs) remains to be unreported. In this study, we performed complete genome sequencing of two K. pneumoniae HvK2115 and CRK3022 with hypervirulent or carbapenem-resistant traits. OMVs from K. pneumoniae HvK2115 and CRK3022 were purified and observed. The carriage of virulence or resistance genes in K. pneumoniae OMVs was identified. The influence of OMVs on the horizontal transfer of virulence-related or drug-resistant plasmids among K. pneumoniae strains was evaluated thoroughly. The plasmid transfer to recipient bacteria through OMVs was identified by polymerase chain reaction, pulsed field gel electrophoresis and Southern blot. This study revealed that OMVs could mediate the intraspecific and interspecific horizontal transfer of the virulence plasmid phvK2115. OMVs could simultaneously transfer two resistance plasmids into K. pneumoniae and Escherichia coli recipient strains. OMVs-mediated horizontal transfer of virulence plasmid phvK2115 could significantly enhance the pathogenicity of human carbapenem-resistant K. pneumoniae CRK3022. The CRK3022 acquired the virulence plasmid phvK2115 could become a CR-hvKp strain. It was critically important that OMVs-mediated horizontal transfer of phvK2115 lead to the coexistence of virulence and carbapenem-resistance genes in K. pneumoniae, resulting in the emerging of carbapenem-resistant hypervirulent K. pneumoniae.
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Enterobacteriaceae Resistentes a los Carbapenémicos , Infecciones por Klebsiella , Animales , Antibacterianos/farmacología , Antibacterianos/uso terapéutico , Carbapenémicos/farmacología , Escherichia coli/genética , Humanos , Infecciones por Klebsiella/microbiología , Infecciones por Klebsiella/veterinaria , Klebsiella pneumoniae/genética , Plásmidos/genética , Virulencia/genética , beta-LactamasasRESUMEN
As the leading cause of bovine respiratory disease (BRD), bacterial pneumonia can result in tremendous losses in the herd farming industry worldwide. N-acetylcysteine (NAC), an acetylated precursor of the amino acid L-cysteine, has been reported to have anti-inflammatory and antioxidant properties. To explore the protective effect and underlying mechanisms of NAC in ALI, we investigated its role in lipopolysaccharide (LPS)-induced bovine embryo tracheal cells (EBTr) and mouse lung injury models. We found that NAC pretreatment attenuated LPS-induced inflammation in EBTr and mouse models. Moreover, LPS suppressed the expression of oxidative-related factors in EBTr and promoted gene expression and the secretion of inflammatory cytokines. Conversely, the pretreatment of NAC alleviated the secretion of inflammatory cytokines and decreased their mRNA levels, maintaining stable levels of antioxidative gene expression. In vivo, NAC helped LPS-induced inflammatory responses and lung injury in ALI mice. The relative protein concentration, total cells, and percentage of neutrophils in BALF; the level of secretion of IL-6, IL-8, TNF-α, and IL-1ß; MPO activity; lung injury score; and the expression level of inflammatory-related genes were decreased significantly in the NAC group compared with the LPS group. NAC also ameliorated LPS-induced mRNA level changes in antioxidative genes. In conclusion, our findings suggest that NAC affects the inflammatory and oxidative response, alleviating LPS-induced EBTr inflammation and mouse lung injury, which offers a natural therapeutic strategy for BRD.
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The extended-spectrum cephalosporin resistant E. coli from food animals transferring to community settings of humans causes a serious threat to public health. Unlike phylogroup B2 E. coli strains, the clinical significance of isolates in phylogroup F is not well revealed. Here, we report on a collection (n = 563) of phylogroup F E. coli isolates recovered from chicken colibacillosis tissues and retail raw chicken meat samples in Eastern China. There was an overlapped distribution of MLST types between chicken colibacillosis-origin and meat-source phylogroup F E. coli, including dominant STs (ST648, ST405, ST457, ST393, ST1158, etc). This study further investigated the presence of extended-spectrum ß-lactamase (ESBL/pAmpC) producers in these chicken-source phylogroup F E. coli strains. The prevalence of extended-spectrum cephalosporin resistant strains in phylogroup F E. coli from chicken colibacillosis and raw meat separately accounted for 66.1 and 71.2%. The resistance genotypes and plasmid replicon types of chicken-source phylogroup F E. coli isolates were characterized by multiplex PCR. Our results revealed ß-lactamase CTX-M, OXA, CMY and TEM genes were widespread in chicken-source phylogroup F E. coli, and blaCTX-M was the most predominant ESBL gene. Moreover, there was a high prevalence of non-lactamase resistance genes in these ß-lactam-resistant isolates. The replicons IncB/O/K/Z, IncI1, IncN, IncFIC, IncQ1, IncX4, IncY, and p0111, associated with antibiotic-resistant large plasmids, were widespread in chicken-source phylogroup F E. coli. There was no obvious difference for the populations, resistance spectrums, and resistance genotypes between phylogroup F E. coli from chicken colibacillosis tissues and retail meats. This detail assessment of the population and resistance genotype showed chicken-source phylogroup F E. coli might hold zoonotic risk and contribute the spread of multidrug-resistant E. coli to humans.
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Infecciones por Escherichia coli , Escherichia coli , Animales , Antibacterianos/farmacología , Pollos , Farmacorresistencia Bacteriana/genética , Escherichia coli/genética , Infecciones por Escherichia coli/epidemiología , Infecciones por Escherichia coli/veterinaria , Carne , Tipificación de Secuencias Multilocus/veterinariaRESUMEN
ExPEC is an important pathogen that causes diverse infection in the human extraintestinal sites. Although avian-source phylogroup F Escherichia coli isolates hold a high level of virulence traits, few studies have systematically assessed the pathogenicity and zoonotic potential of E. coli isolates within phylogroup F. A total of 1,332 E. coli strains were recovered from chicken colibacillosis in China from 2012 to 2017. About 21.7% of chicken-source E. coli isolates were presented in phylogroup F. We characterized phylogroup F E. coli isolates both genotypically and phenotypically. There was a widespread prevalence of ExPEC virulence-related genes among chicken-source E. coli isolates within phylogroup F. ColV/BM plasmid-related genes (i.e. hlyF, mig-14p, ompTp, iutA and tsh) occurred in the nearly 65% of phylogroup F E. coli isolates. Population structure of chicken-source E. coli isolates within phylogroup F was revealed and contained several dominant STs (such as ST59, ST354, ST362, ST405, ST457 and ST648). Most chicken-source phylogroup F E. coli held the property to produce biofilm and exhibited strongly swimming and swarming motilities. Our result showed that the complement resistance of phylogroup F E. coli isolates was closely associated with its virulence genotype. Our research further demonstrated the zoonotic potential of chicken-source phylogroup F E. coli isolates. The phylogroup F E. coli isolates were able to cause multiple diseases in animal models of avian colibacillosis and human infections (sepsis, meningitis and UTI). The chicken-source phylogroup F isolates, especially dominant ST types, might be recognized as a high-risk food-borne pathogen. This was the first study to identify that chicken-source E. coli isolates within phylogroup F were associated with human ExPEC pathotypes and exhibited zoonotic potential.
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Pollos/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/genética , Escherichia coli Patógena Extraintestinal/genética , Animales , China , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli , Genotipo , Humanos , Virulencia , Factores de Virulencia/genéticaRESUMEN
Avian pathogenic Escherichia coli (APEC), a pathotype of extraintestinal pathogenic E. coli, causes one of the most serious infectious diseases of poultry and shares some common virulence genes with neonatal meningitis-associated E. coli. TonB-dependent receptors (TBDRs) are ubiquitous outer membrane ß-barrel proteins; they play an important role in the recognition of siderophores during iron uptake. Here, in the APEC strain DE205B, we investigated the role of four putative TBDRs-ireA, 0007, 0008, and 2235-in iron uptake. Glutathione-S-transferase pulldown assays indicated that the proteins encoded by these genes directly interact with TonB. Moreover, the expression levels of all four genes were significantly upregulated under iron-depleted conditions compared with iron-rich conditions. The expression levels of several iron uptake-related genes were significantly increased in the ireA, 0007, 0008, and 2235 deletion strains, with the upregulation being the most prominent in the ireA deletion mutant. Furthermore, iron uptake by the ireA deletion strain was significantly increased compared to that by the wild-type strain. Moreover, a tonB mutant strain was constructed to study the effect of tonB deletion on the TBDRs. We found that regardless of the presence of tonB, the expression levels of the genes encoding the four TBDRs were regulated by fur. In conclusion, our findings indicated that ireA, 0007, 0008, and 2235 indeed encode TBDRs, with ireA having the most important role in iron uptake. These results should help future studies explore the mechanisms underlying the TonB-dependent iron uptake pathway.
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Proteínas de la Membrana Bacteriana Externa/genética , Infecciones por Escherichia coli/veterinaria , Proteínas de Escherichia coli/genética , Escherichia coli/genética , Enfermedades de las Aves de Corral/metabolismo , Animales , Proteínas de la Membrana Bacteriana Externa/metabolismo , Pollos , Escherichia coli/metabolismo , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Enfermedades de las Aves de Corral/microbiologíaRESUMEN
Extraintestinal pathogenic Escherichia coli (ExPEC) causes bloodstream infections in humans and animals. Complement escape is a prerequisite for bacteria to survive in the bloodstream. Factor H (FH) is an important regulatory protein of the complement system. In this study, ExPEC was found to bind FH from serum. However, the mechanisms of ExPEC binding to FH and then resistance to complement-mediated attacks remain unclear. Here, a method that combined desthiobiotin pull-down and liquid chromatography-tandem mass spectrometry was used to identify the FH-binding membrane proteins of ExPEC. Seven identified proteins, which all were carbohydrate metabolic enzymes (CMEs), including acetate kinase, fructose-bisphosphate aldolase, fumarate reductase flavoprotein subunit, L-lactate dehydrogenase, dihydrolipoamide dehydrogenase, phosphoenolpyruvate synthase, and pyruvate dehydrogenase, were verified to recruit FH from serum using GST pull-down and ELISA plate binding assay. The ELISA plate binding assay determined that these seven proteins bind to FH in a dose-dependent manner. Magnetic beads coupled with any one of seven proteins significantly reduced the FH recruitment of ExPEC (p < 0.05) Subsequently, immunofluorescence, colony blotting, and Western blotting targeting outer membrane proteins determined that these seven CMEs were located on the outer membrane of ExPEC. Furthermore, the FH recruitment levels and C3b deposition levels on bacteria were significantly increased and decreased in an FH-concentration-dependent manner, respectively (p < 0.05). The FH recruitment significantly enhanced the ability of ExPEC to resist the opsonophagocytosis of human macrophage THP-1 in an FH-concentration-dependent manner (p < 0.05), which revealed a new mechanism for ExPEC to escape complement-mediated killing. The identification of novel outer membrane-displayed CMEs which played a role in the FH recruitment contributes to the elucidation of the molecular mechanism of ExPEC pathogenicity.
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Infecciones por Escherichia coli , Proteínas de Escherichia coli , Escherichia coli Patógena Extraintestinal , Animales , Metabolismo de los Hidratos de Carbono , Factor H de Complemento , Proteínas de Escherichia coli/metabolismo , Humanos , VirulenciaRESUMEN
Colisepticemia caused by bloodstream infection of the extraintestinal pathogenic Escherichia coli (ExPEC) has become a serious public health problem. The recent emergence of the colistin-resistant Enterobacteriaceae, especially mcr-1-positive E. coli (MCRPEC) exerts great concern around the world. The molecular epidemiology and zoonosis risk of avian-origin MCRPEC are reported to be substantially lower. Here, we presented a system-wide analysis of emerging trends and zoonotic risk of MCRPEC recovered from avian colibacillosis in China. Our results showed the majority of avian-source MCRPEC isolates were classified as ExPECs. We also found that not only MCRPEC in phylogroups B2 and D, but also several E. coli populations in groups B1 and F possessed high virulence in the two models of avian colibacillosis and three rodent models for ExPEC-associated human infections. The high-virulent MCRPEC clones belong to ST131, as well as ST-types (such as ST48, ST117, ST162, ST501, ST648, and ST2085). Our data suggested the zoonotic risk of MCRPEC appeared to be a close association with ColV/ColBM type virulence plasmids. A comprehensive genomic analysis showed the overlapped of ColV/ColBM plasmids contents between MCRPEC isolates from humans and poultry. Identification of ColV/ColBM plasmids among human MCRPEC isolates revealed the potential transmission of avian-source mcr-1-positive ExPECs to humans. Moreover, the presence of ColV/ColBM plasmid-encoded virulence determinants, could be used as a predictive label for pathogenic MCRPEC. These findings highlighted avian-origin MCRPEC isolates could be recognized as a foodborne pathogen.
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Infecciones por Escherichia coli/microbiología , Proteínas de Escherichia coli/metabolismo , Escherichia coli/clasificación , Escherichia coli/patogenicidad , Filogenia , Animales , Enfermedades de las Aves/microbiología , Aves , Escherichia coli/genética , Humanos , Factores de Virulencia/genéticaRESUMEN
Avian pathogenic Escherichia coli (APEC) is a facultative intracellular pathogen, and intracellular persistence in macrophages is essential for APEC extraintestinal dissemination. Until now, there is still no systematic interpretation of APEC intracellular proliferation. Intracellular survival factors, especially involved in pathometabolism, need to be further revealed. Acetate plays critical roles in supporting energy homeostasis and acts as a metabolic signal in the inflammatory response of eukaryotes. In this study, we identified that APEC acs-yjcH-actP operon, encoding acetate assimilation system, presented the host-induced transcription during its proliferation in macrophages. Our result showed that this acetate assimilation system acted as a novel intracellular survival factor to promote APEC replication within macrophages. Furthermore, deletion of acs-yjcH-actP operon in APEC decreased its cytotoxic level to macrophages. qRT-PCR results showed that the production of pro-inflammatory cytokines (IL-1ß, IL-6, IL-8, IL-12ß, and TNF-α) and iNOS in FY26∆acs-yjcH-actP infected macrophages were obviously down-regulated compared to that in wild-type FY26 infected cells. Deletion of actP/yjcH/acs genes attenuated APEC virulence and colonization capability in avian lungs in vivo for colibacillosis infection models. And acetate assimilation system acted as a virulence factor and conferred a fitness advantage during APEC early colonization. Taken together, our research unravelled the metabolic requirement of APEC intracellular survival/replication within macrophages, and acetate metabolic requirement acted as an important strategy of APEC pathometabolism. The intracellular acetate consumption during facultative intracellular bacteria replication within macrophages promoted immunomodulatory disorders, resulting in excessively pro-inflammatory responses of host macrophages.
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Acetatos/metabolismo , Enfermedades de las Aves/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/metabolismo , Macrófagos/microbiología , Animales , Línea Celular , Pollos , Citocinas/metabolismo , Patos/microbiología , Ensayo de Inmunoadsorción Enzimática , Escherichia coli/crecimiento & desarrollo , Escherichia coli/fisiología , Infecciones por Escherichia coli/metabolismo , Infecciones por Escherichia coli/microbiología , Enfermedades de las Aves de Corral/microbiología , Reacción en Cadena en Tiempo Real de la PolimerasaRESUMEN
Recent emergence of mcr-1-positive Escherichia coli (MCRPEC) is causing serious concern around the world. Due to poultry-origin E. coli holding zoonotic potential, the improved understandings of MCRPEC population structure and antimicrobial resistance are critical to public health purposes. This study provided novel insights into the molecular epidemiology of avian-origin MCRPEC. For the mcr genes prevalence study, we analysed 1,360 E. coli recovered from avian colibacillosis in eastern China from 2015 to 2017. The mcr-1 was present in 172 (12.6%) E. coli isolates. For all of MCRPEC isolates, MICs of colistin were ≥4 mg/L. Avian-origin MCRPEC was widely distributed throughout phylogroups A, B1, B2, D, and F. Moreover, those isolates were assigned to 52 unique STs, such as ST48, ST117, ST131, and ST648, suggesting substantial horizontal dissemination of mcr-1 gene through avian-origin E. coli populations. The susceptibility of MCRPEC isolates was tested with 26 antimicrobial agents from 16 antimicrobial categories. There were high resistance rates of MCRPEC isolates against the clinically used antibiotics. All MCRPEC isolates in this study presented the multidrug-resistant (MDR) trait were even considered as extensively drug-resistant (XDR) strains. Resistance genotypes and plasmid replicon profiling showed that a majority of MCRPEC isolates contained plasmid-mediated resistance genes and exhibited the co-existence of mcr-1 with ESBLs and pAmpCs genes. Furthermore, the overlapped distribution of ST types and resistance gene contents was detected among MCRPEC isolates from humans and poultry. Besides mcr-1, our findings highlighted a significant prevalence of plasmid-mediated resistance genes among avian-origin MCRPEC isolates.
Asunto(s)
Antibacterianos/farmacología , Pollos , Farmacorresistencia Bacteriana/genética , Patos , Infecciones por Escherichia coli/microbiología , Escherichia coli/fisiología , Enfermedades de las Aves de Corral/microbiología , Animales , China , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Proteínas de Escherichia coli/análisis , Pruebas de Sensibilidad Microbiana/veterinariaRESUMEN
BACKGROUND: Escherichia coli is an important pathogen that causes diarrhea in both humans and animals. To determine the relationships between putative virulence factors and pathotypes or host taxa, many molecular studies on diarrhea-associated E. coli have been reported. However, little is known regarding genome-wide variation of E. coli from animal hosts. In this study, we performed whole genome sequencing of 127 E. coli isolates from sheep and swine with diarrhea in China. We compared isolates to explore the phylogenomic relatedness based on host origin. We explored the relationships of putative virulence factors across host taxa and pathotypes. Antimicrobial resistance was also tested. RESULTS: The E. coli genomes in this study were diverse with clear differences in the SNP, MLST, and O serotypes. Seven putative virulence factors (VFs) were prevalent (> 95%) across the isolates, including Hcp, csgC, dsdA, feoB, fepA, guaA, and malX. Sixteen putative VFs showed significantly different distributions (P < 0.05) in strains from sheep and swine and were primarily adhesion- and toxin-related genes. Some putative VFs were co-occurrent in some specific pathotypes and O serotypes. The distribution of 4525 accessory genes of the 127 strains significantly differed (P < 0.05) between isolates obtained from the two animal species. The 127 animal isolates sequenced in this study were each classified into one of five pathotypes: EAEC, ETEC, STEC, DAEC, and EPEC, with 66.9% of isolates belonging to EAEC. Analysis of stx subtypes and a minimum spanning tree based on MLST revealed that STEC isolates from sheep and EAEC isolates from sheep and swine have low potential to infect humans. Antibiotic resistance analysis showed that the E. coli isolates were highly resistant to ampicillin and doxycycline. Isolates from southeast China were more resistant to antibiotics than isolates from northwest China. Additionally, the plasmid-mediated colist in resistance gene mcr-1 was detected in 15 isolates, including 4 from sheep in Qinghai and 11 from swine in Jiangsu. CONCLUSIONS: Our study provides insight into the genomes of E. coli isolated from animal sources. Distinguishable differences between swine and sheep isolates at the genomic level provides a baseline for future investigations of animal E. coli pathogens.
Asunto(s)
Animales Domésticos/microbiología , Diarrea/microbiología , Infecciones por Escherichia coli/veterinaria , Escherichia coli/clasificación , Genómica/métodos , Animales , Técnicas de Tipificación Bacteriana , China , Diarrea/veterinaria , Farmacorresistencia Bacteriana , Escherichia coli/genética , Escherichia coli/aislamiento & purificación , Escherichia coli/patogenicidad , Proteínas de Escherichia coli/genética , Tipificación de Secuencias Multilocus , Filogenia , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Serogrupo , Ovinos/microbiología , Porcinos/microbiología , Factores de Virulencia/genéticaRESUMEN
Salmonella are causes of livestock, poultry, and other animal diseases but they also have the potential to infect people. Currently, antibiotics are the first choice for treatment of Salmonella infections. Thus, the utility of phage has become the research focus for scientists for several reasons. There are efficient, non-toxic, ubiquitous, easy to prepare and can result in the lysis of host bacteria. In this study, a broad spectrum bacteriophage of Salmonella was isolated from the fecal samples of a poultryfarm and we studied the morphological aspects, thermal stability, pH stability, optimal multiplicity of infection (MOI), and one-step growth curve of this phage. This phage was named Salmonella phage SaFB14 and lysed 54.12%(105/194)Salmonella spp. SaFB14 belongs to the Siphoviridae and has a polyhedron head with a diameter approximating 60â¯×â¯60â¯nm and a tail approximating 140â¯nm. The optimum growth temperature was 37⯰C and maintained high activity over a widepH range(pH3-10) with an optimum of pH 7.0. The optimal MOI was 0.1. A one-step growth curve showed that its latency time was 10â¯min, burst time was 70â¯min, and burst was 23 particles. In order to study the therapeutic effect of phage SaFB14 in infected mice, mice were challenged with 2â¯×â¯109â¯CFU/mouse Salmonella (cs20130523-001-1). Each mouse was injected to 2â¯×â¯1010â¯PFU SaFB14 1â¯h later. SaFB14 protected 40% of mice from infection. Then, the same dose of phage was given to mice for 3 days continuously. After 3 days treatment, the survival rate increased to 60%.In conclusion, phage SaFB14 showed wide host range and good activity in vivo, it is promising against diseases caused by Salmonella.