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The importance of bacteria detection lies in its role in enabling early intervention, disease prevention, environmental protection, and effective treatment strategies. Advancements in technology continually enhance the speed, accuracy, and sensitivity of detection methods, aiding in addressing these critical issues. This study first reports the fabrication of an inverter constructed using crosslinked-poly(4-vinylphenol) (C-PVP) as the dielectric layer and an organic complementary metal-oxide semiconductor (O-CMOS) based on pentacene and N,N'-ditridecylperylene-3,4,9,10-tetracarboxylic diimide (PTCDI-C13) as a diagnostic biosensor to rapidly detect bacterial concentration. Bacteria including Escherichia coli O157, Staphylococcus aureus ATCC25922, and Enterococcus faecalis SH-1051210 were analysed on the inverters at an ultra-low operating voltage of 2 V. The high density of negative charge on bacteria surfaces strongly modulates the accumulated negative carriers within the inverter channel, resulting in a shift of the switching voltage. The inverter-based bacteria sensor exhibits a linear-like response to bacteria concentrations ranging from 102 to 108 CFU/mL, with a sensitivity above 60%. Compared to other bacterial detectors, the advantage of using an inverter lies in its ability to directly read the switching voltage without requiring an external computing device. This facilitates rapid and accurate bacterial concentration measurement, offering significant ease of use and potential for mass production.
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BACKGROUND: Urinary tract infections (UTIs) are common bacterial infections, primarily caused by uropathogenic Escherichia coli (UPEC), leading to significant health issues and economic burden. Although antibiotics have been effective in treating UPEC infections, the rise of antibiotic-resistant strains hinders their efficacy. Hence, identifying novel bacterial targets for new antimicrobial approaches is crucial. Bacterial factors required for maintaining the full virulence of UPEC are the potential target. MepM, an endopeptidase in E. coli, is involved in the biogenesis of peptidoglycan, a major structure of bacterial envelope. Given that the bacterial envelope confronts the hostile host environment during infections, MepM's function could be crucial for UPEC's virulence. This study aims to explore the role of MepM in UPEC pathogenesis. RESULTS: MepM deficiency significantly impacted UPEC's survival in urine and within macrophages. Moreover, the deficiency hindered the bacillary-to-filamentous shape switch which is known for aiding UPEC in evading phagocytosis during infections. Additionally, UPEC motility was downregulated due to MepM deficiency. As a result, the mepM mutant displayed notably reduced fitness in causing UTIs in the mouse model compared to wild-type UPEC. CONCLUSIONS: This study provides the first evidence of the vital role of peptidoglycan endopeptidase MepM in UPEC's full virulence for causing UTIs. MepM's contribution to UPEC pathogenesis may stem from its critical role in maintaining the ability to resist urine- and immune cell-mediated killing, facilitating the morphological switch, and sustaining motility. Thus, MepM is a promising candidate target for novel antimicrobial strategies.
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Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Infecções Urinárias/microbiologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Escherichia coli Uropatogênica/enzimologia , Escherichia coli Uropatogênica/efeitos dos fármacos , Animais , Camundongos , Infecções por Escherichia coli/microbiologia , Virulência , Endopeptidases/genética , Endopeptidases/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Feminino , Peptidoglicano/metabolismo , Macrófagos/microbiologia , Macrófagos/imunologia , Humanos , Modelos Animais de DoençasRESUMO
INTRODUCTION: Carbon dioxide-dependent Proteus mirabilis has been isolated from clinical specimens. It is not clear whether mutations in carbonic anhydrase are responsible for the carbon dioxide dependence of P. mirabilis. The pathogenicity of carbon dioxide-dependent P. mirabilis also remains unclear. The purpose of this study was to determine the cause carbon dioxide dependence of P. mirabilis and its pathogenicity. METHODS: The DNA sequence of can encoding carbonic anhydrase of a carbon dioxide-dependent P. mirabilis small colony variant (SCV) isolate was analyzed. To confirm that impaired carbonic anhydrase activity is responsible for the formation of the carbon dioxide-dependent SCV phenotype of P. mirabilis, we performed complementation experiments using plasmids with intact can. Additionally, mouse infection experiments were performed to confirm the change in virulence due to the mutation of carbonic anhydrase. RESULTS: We found that the can gene of the carbon dioxide-dependent P. mirabilis SCV isolate showed had a frameshift mutation with a deletion of 1 bp (c. 173delC). The can of P. mirabilis encodes carbonic anhydrase was also found to function in Escherichia coli. The cause of the carbon dioxide-dependent SCV phenotype of P. mirabilis was an abnormality in carbonic anhydrase. Nevertheless, no changes were observed in virulence due to the mutation of carbonic anhydrase in mouse infection experiments. CONCLUSIONS: The can gene is essential for the growth of P. mirabilis in ambient air. The mechanisms underlying this fitness advantage in terms of infection warrant further investigation.
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Dióxido de Carbono , Anidrases Carbônicas , Infecções por Proteus , Proteus mirabilis , Proteus mirabilis/genética , Proteus mirabilis/patogenicidade , Proteus mirabilis/isolamento & purificação , Dióxido de Carbono/metabolismo , Animais , Camundongos , Infecções por Proteus/microbiologia , Anidrases Carbônicas/genética , Anidrases Carbônicas/metabolismo , Humanos , Virulência/genética , Feminino , Mutação da Fase de LeituraRESUMO
BACKGROUND: Our objective was to investigate the prevalence of plasmid-mediated quinolone resistance (PMQR) genes in fluoroquinolone-nonsusceptible Klebsiella pneumoniae (FQNSKP) in Taiwan, 1999-2022. METHODS: A total of 938 FQNSKP isolates were identified from 1966 isolates. The presence of PMQR and virulence genes, antimicrobial susceptibility, capsular types, and PMQR-plasmid transferability were determined. RESULTS: An increasing number of PMQR-containing FQNSKP isolates were observed over the study period. Our results showed that 69.0% (647 isolates) of FQNSKP isolates contained at least one PMQR gene, and 40.6%, 37.0%, and 33.9% of FQNSKP carried aac(6')-Ib-cr, qnrB, and qnrS, respectively. None of FQNSKP carried qepA and qnrC. The most common combination of PMQR genes was aac(6')-Ib-cr and qnrB (12.3%). The presence of PMQR genes is strongly related to resistance to aminoglycoside, cephalosporin, tetracycline, and sulfamethoxazole/trimethoprim in FQNSKP. The capsular serotype K64 is the most common serotype we tested in both the non-PMQR and PMQR FQNSKP isolates, while K20 showed a higher prevalence in PMQR isolates. The magA and peg-344 genes showed a significantly higher prevalence rate in non-PMQR isolates than in PMQR isolates. Eleven isolates that carried the PMQR and carbapenemase genes were identified; however, three successful transconjugants showed that the PMQR and carbapenemase genes were not located on the same plasmid. CONCLUSIONS: Our results indicated an increasing prevalence of PMQR genes, especially qnrB and qnrS, in FQNSKP in Taiwan. Moreover, the distribution of PMQR genes was associated with capsular serotypes and antimicrobial resistance gene and virulence gene distribution in FQNSKP.
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Klebsiella pneumoniae , Quinolonas , Humanos , Fluoroquinolonas/farmacologia , Prevalência , Taiwan/epidemiologia , Plasmídeos/genética , Quinolonas/farmacologia , Antibacterianos/farmacologia , Testes de Sensibilidade Microbiana , Farmacorresistência Bacteriana/genéticaRESUMO
BACKGROUND: Escherichia coli is the leading pathogen responsible for urinary tract infection (UTI) and recurrent UTI (RUTI). Few studies have dealt with the characterization of host and bacteria in RUTI caused by E. coli with genetically identical or different strains. This study aimed to investigate the host and bacterial characteristics of E. coli RUTI based on molecular typing. RESULTS: Patients aged 20 years or above who presented with symptoms of UTI in emergency department or outpatient clinics between August 2009 and December 2010 were enrolled. RUTI was defined as patients had 2 or more infections in 6 months or 3 or more in 12 months during the study period. Host factors (including age, gender, anatomical/functional defect, and immune dysfunction) and bacterial factors (including phylogenicity, virulence genes, and antimicrobial resistance) were included for analysis. There were 41 patients (41%) with 91 episodes of E. coli RUTI with highly related PFGE (HRPFGE) pattern (pattern similarity > 85%) and 58 (59%) patients with 137 episodes of E. coli RUTI with different molecular typing (DMT) pattern, respectively. There was a higher prevalence of phylogenetic group B2 and neuA and usp genes in HRPFGE group if the first episode of RUTI caused by HRPFGE E. coli strains and all episodes of RUTI caused by DMT E. coli strains were included for comparison. The uropathogenic E. coli (UPEC) strains in RUTI were more virulent in female gender, age < 20 years, neither anatomical/ functional defect nor immune dysfunction, and phylogenetic group B2. There were correlations among prior antibiotic therapy within 3 months and subsequent antimicrobial resistance in HRPFGE E. coli RUTI. The use of fluoroquinolones was more likely associated with subsequent antimicrobial resistance in most types of antibiotics. CONCLUSIONS: This study demonstrated that the uropathogens in RUTI were more virulent in genetically highly-related E. coli strains. Higher bacterial virulence in young age group (< 20 years) and patients with neither anatomical/functional defect nor immune dysfunction suggests that virulent UPEC strains are needed for the development of RUTI in healthy populations. Prior antibiotic therapy, especially the fluoroquinolones, within 3 months could induce subsequent antimicrobial resistance in genetically highly-related E. coli RUTI.
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Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Humanos , Feminino , Infecções por Escherichia coli/microbiologia , Filogenia , Infecções Urinárias/microbiologia , Antibacterianos/farmacologia , Tipagem Molecular , Bactérias/genética , Fluoroquinolonas , Fatores de Virulência/genéticaRESUMO
Recurrent urinary tract infection (RUTI) can damage renal function and has impact on healthcare costs and patients' quality of life. There were 2 stages for development of prediction models for RUTI. The first stage was a scenario in the clinical visit. The second stage was a scenario after hospitalization for urinary tract infection caused by Escherichia coli. Three machine learning models, logistic regression (LR), decision tree (DT), and random forest (RF) were built for the RUTI prediction. The RF model had higher prediction accuracy than LR and DT (0.700, 0.604, and 0.654 in stage 1, respectively; 0.709, 0.604, and 0.635 in stage 2, respectively). The decision rules constructed by the DT model could provide high classification accuracy (up to 0.92 in stage 1 and 0.94 in stage 2) in certain subgroup patients in different scenarios. In conclusion, this study provided validated machine learning models and RF could provide a better accuracy in predicting the development of single uropathogen (E. coli) RUTI. Both host and bacterial characteristics made important contribution to the development of RUTI in the prediction models in the 2 clinical scenarios, respectively. Based on the results, physicians could take action to prevent the development of RUTI.
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Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli , Infecções por Escherichia coli/microbiologia , Humanos , Aprendizado de Máquina , Qualidade de Vida , Infecções Urinárias/microbiologiaRESUMO
Rationale and Objective: Gut microbiota have been targeted by alternative therapies for non-communicable diseases. We examined the gut microbiota of a healthy Taiwanese population, identified various bacterial drivers in different demographics, and compared them with dialysis patients to associate kidney disease progression with changes in gut microbiota. Study Design: This was a cross-sectional cohort study. Settings and Participants: Fecal samples were obtained from 119 healthy Taiwanese volunteers, and 16S rRNA sequencing was done on the V3-V4 regions to identify the bacterial enterotypes. Twenty-six samples from the above cohort were compared with fecal samples from 22 peritoneal dialysis and 16 hemodialysis patients to identify species-level bacterial biomarkers in the dysbiotic gut of chronic kidney disease (CKD) patients. Results: Specific bacterial species were identified pertaining to different demographics such as gender, age, BMI, physical activity, and sleeping habits. Dialysis patients had a significant difference in gut microbiome composition compared to healthy controls. The most abundant genus identified in CKD patients was Bacteroides, and at the species level hemodialysis patients showed significant abundance in B. ovatus, B. caccae, B. uniformis, and peritoneal dialysis patients showed higher abundance in Blautia producta (p ≤ 0.05) than the control group. Pathways pertaining to the production of uremic toxins were enriched in CKD patients. The abundance of the bacterial species depended on the type of dialysis treatment. Conclusion: This study characterizes the healthy gut microbiome of a Taiwanese population in terms of various demographics. In a case-control examination, the results showed the alteration in gut microbiota in CKD patients corresponding to different dialysis treatments. Also, this study identified the bacterial species abundant in CKD patients and their possible role in complicating the patients' condition.
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Microbioma Gastrointestinal , Microbiota , Insuficiência Renal Crônica , Toxinas Biológicas , Bactérias/genética , Bactérias/metabolismo , Bacteroides/genética , Estudos Transversais , Disbiose/microbiologia , Feminino , Humanos , Masculino , RNA Ribossômico 16S/genética , Insuficiência Renal Crônica/microbiologia , Insuficiência Renal Crônica/terapia , Taiwan , Toxinas UrêmicasRESUMO
BACKGROUND: Escherichia coli is the most common cause of urinary tract infections (UTIs). It is widely accepted that uropathogenic E. coli (UPEC) mainly emerge from the distal gut microbiota. Identification of bacterial characteristics that are able to differentiate UPEC from fecal commensal strains will facilitate the development of novel strategies to detect and monitor the spread of UPEC. METHODS: Fifty fecal commensal, 83 UTI-associated and 40 biliary tract infection (BTI)-associated E. coli isolates were analyzed. The NotI restriction patterns of chromosomal DNA in the isolates were determined by pulse-field gel electrophoresis. The phylogenetic types and the presence of 9 known virulence genes of each isolate were determined by PCR analyses. Additionally, the susceptibilities of the isolates to antibiotics were revealed. Then the associations of NotI resistance with UTI-associated isolates, phylotypes, and antibiotic resistance were assessed. RESULTS: NotI resistance was correlated with UTI-associated isolates, compared to the fecal isolates. Consistently, NotI-resistant isolates harbored a greater number of virulence factors and mainly belonged to phylotype B2. Additionally NotI resistance was correlated with chloramphenicol resistance among the bacteria. Among the fecal, UTI-associated and BTI-associated groups, the distribution of NotI-resistant group B2 isolates was correlated with UTI-associated bacteria. CONCLUSION: NotI resistance alone is a potential marker for distinguishing fecal strains and UPEC, while the combination of NotI resistance and B2 phylogeny is a candidate marker to differentiate UPEC from fecal and other extraintestinal pathogenic E. coli. Additionally, NotI resistance may be valuable for assessing the potential of chloramphenicol resistance of E. coli.
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Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Antibacterianos , Humanos , Filogenia , Fatores de VirulênciaRESUMO
BACKGROUND: Urinary tract infections (UTIs) caused by uropathogenic Escherichia coli (UPEC) are one of the most common outpatient bacterial infections. Although bacterial and host factors are reported to be associated with UTI pathogenesis, little is known about the host age-related differences in bacterial virulence factors and antimicrobial susceptibility. METHODS: PCRs were carried out to detect K1 capsule antigen, 15 virulence factors, and phylogenetic groups in E. coli isolates. Antimicrobial susceptibility of selected agents was determined by the disk diffusion method. Isolates were divided into 6 groups based on their host age. RESULTS: The results showed that virulence factors PapGII, PapGIII, Cnf1, Aer, Usp, Iha, OmpT, HlyA, and Sat, had highest frequencies in the host age group 0-3. Phylogenetic group B2 dominated in our isolates (59.6%) followed by group D (20.7%). In addition, 77.4% of strains isolated from 0 to 3 age group belonged to phylogenetic group B2. Antimicrobial susceptibility tests showed that E. coli strains isolated were significantly more resistant to antimicrobial agents as host age increased. Phylogenetic group B2 isolates were more susceptible to antimicrobial agents, compared to A, B1, and D isolates. CONCLUSION: We found E. coli isolated from elders were more resistant to antimicrobial agents and had less virulence factors.
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Infecções por Escherichia coli , Infecções Urinárias , Escherichia coli Uropatogênica , Idoso , Antibacterianos/farmacologia , Antibacterianos/uso terapêutico , Infecções por Escherichia coli/microbiologia , Feminino , Humanos , Masculino , Filogenia , Infecções Urinárias/microbiologia , Fatores de Virulência/genéticaRESUMO
BACKGROUND: FimH adhesin is proposed to enhance Escherichia coli kidney infection by acting with PapGII adhesin, but genetic epidemiology study and animal study have not been widely conducted to confirm this hypothesis. METHODS: We compared the prevalence of adhesin gene and their coexistent pattern between upper and lower urinary tract infection (UTI) strains. fimH mutant (EC114FM), papGII mutant (EC114PM) and fimH/papGII double mutant (EC114DM) were constructed from a pylonephritogenic strain (EC114). We compared among these strains for the infection ability in bladders and kidneys of female BALB/c mice challenged transurethrally with these bacteria and assessed 1, 3, and 7 days after inoculation. RESULTS: Strains carrying fimH-only genotype were significantly more prevalent in lower UTI (P < 0.001). Strains carrying the fimH/papGII, but not papGII-only, were significantly associated with upper UTI (P = 0.001). Incidence of kidney infection increased after inoculation with EC114 on days 1 and 3, at both low and high dose, as compared with EC114DM; and the effect was greater than the sum of individual effect of EC114PM and EC114FM. Geometric means of quantitative bacterial counts in the kidneys significantly decreased when challenged with EC114FM on days 3 and 7, EC114PM on day 3 and EC114DM on day 1 after inoculation at high dose, as compared with EC114 (all P < 0.05). CONCLUSIONS: We confirmed the advantage and synergistic action of FimH and PapGII for E. coli kidney infection and concluded that antagonists against FimH and PapGII adhesin may prevent kidney infection and enable its management.
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Adesinas de Escherichia coli , Infecções por Escherichia coli , Proteínas de Fímbrias , Pielonefrite , Infecções Urinárias , Adesinas de Escherichia coli/genética , Animais , Escherichia coli/genética , Infecções por Escherichia coli/microbiologia , Feminino , Proteínas de Fímbrias/genética , Rim , Camundongos , Camundongos Endogâmicos BALB C , Infecções Urinárias/microbiologiaRESUMO
Escherichia coli is one major cause of bacterial infections and can horizontally acquire antimicrobial resistance and virulence genes through conjugation. Because conjugative plasmids can rapidly spread among bacteria of different species, the plasmids carrying both antimicrobial resistance and virulence genes may pose a significant threat to public health. Therefore, the identification and characterization of these plasmids may facilitate a better understanding of E. coli pathogenesis and the development of new strategies against E. coli infections. Because iron uptake ability is a potential virulence trait of bacteria, we screened for E. coli conjugative plasmids able to confer both iron uptake ability and ampicillin resistance. The plasmid pEC41, which was derived from the bacteremia clinical isolate EC41, was identified. EC41, which carried the fimH27 allele, belonged to sequence type (ST) 405 and phylogroup D. According to the sequencing analyses, pEC41 was 86 kb in size, and its backbone structure was almost identical to that of another highly conjugative plasmid, pCTX-M3, in which the extended-spectrum ß-lactamase gene bla CTX-M-3 was originally identified. pEC41 carried bla CTX-M-3 and bla TEM-1. The ferric citrate uptake (fec) system was identified in pEC41 and was responsible for conferring iron uptake ability. The fec system contributes to the pathogenesis of EC41 in systemic infections but not in urinary tract infections (UTIs). However, this system promoted competitive fitness of a cystitis-associated clinical isolate to colonize urinary tracts. Additionally, the distribution of the fec system was related to E. coli isolates associated with human bacteremia and UTIs. In summary, the present study identified a novel conjugative plasmid, pEC41, which conferred both antimicrobial resistance and an extra iron uptake ability to E. coli. The iron uptake ability was encoded in the fec system and contributed to E. coli pathogenesis. This study is the first to show that the fec system is a virulence factor in E. coli.
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BACKGROUND: Colistin is one of the last-line antimicrobial agents against life-threatening infections. The distribution of the colistin resistance gene mcr-1 has been reported worldwide. However, most studies have focused on the distribution of mcr-1-positive bacteria in humans, animals, food, and sewage; few have focused on their distribution in natural environments. METHOD: We conducted a large spatial survey of mcr-1-positive Escherichia coli at 119 sites in 48 rivers, covering the entire island of Taiwan. We investigated the relationship between the livestock or poultry density in the surveyed riverine area and the number of mcr-1-positive E. coli in the river water. We then sequenced and characterized the isolated mcr-1-positive plasmids. RESULTS: Seven mcr-1 positive E. coli were isolated from 5.9% of the sampling sites. The mcr-1-positive sites correlated with high chicken and pig stocking densities but not human population density or other river parameters. Four of the mcr-1-positive E. coli strains harbored epidemic IncX4 plasmids, and three of them exhibited identical sequences with a size of 33,309 bp. One of the plasmids contained identical 33,309 bp sequences but carried an additional 5711-bp transposon (Tn3 family). To our knowledge, this is the first demonstration that mcr-1-carrying IncX4 plasmids can contain an insertion of such transposons. All mcr-1-positive isolates belonged to phylogenetic group A and harbored few known virulence genes. CONCLUSION: This study showed a positive relationship between the number of mcr-1-positive sites and high livestock and poultry density. The sequencing analyses indicated that the epidemic plasmid in the mcr-1 isolates circulates not only in humans, animals, and food but also in the associated environments or natural habitats in Taiwan, suggesting that the surveillance of antibiotics-resistance genes for livestock or poultry farm quality control should include their associated environments.
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BACKGROUND: Flagella-mediated motility is both a crucial virulence determinant of Helicobacter pylori and a factor associated with gastrointestinal diseases. Flagellar formation requires flagellins to be glycosylated with pseudaminic acid (Pse), a process that has been extensively studied. However, the transfer of Pse to flagellins remains poorly understood. Therefore, the aim of this study is to characterize a putative glycosyltransferase jhp0106 in flagellar formation. MATERIALS AND METHODS: Western blotting and chemical deglycosylation were performed to examine FlaA glycosylation. Protein structural analyses were executed to identify the active site residues of Jhp0106, while the Jhp0106-FlaA interaction was examined using a bacterial two-hybrid assay. Lastly, site-directed mutants with mutated active site residues in the jhp0106 gene were generated and investigated using a motility assay, Western blotting, cDNA-qPCR analysis, and electron microscopic examination. RESULTS: Loss of flagellar formation in the Δjhp0106 mutant was confirmed to be associated with non-glycosylated FlaA. Furthermore, three active site residues of Jhp0106 (S350, F376, and E415) were identified within a potential substrate-binding region. The interaction between FlaA and Jhp0106, Jhp0106::S350A, Jhp0106::F376A, or Jhp0106::E415A was determined to be significant. As well, the substitution of S350A, F376A, or E415A in the site-directed Δjhp0106 mutants resulted in impaired motility, deficient FlaA glycosylation, and lacking flagella. However, these phenotypic changes were regardless of flaA expression, implying an indefinite proteolytic degradation of FlaA occurred. CONCLUSIONS: This study demonstrated that Jhp0106 (PseE) binds to FlaA mediating FlaA glycosylation and flagellar formation. Our discovery of PseE has revealed a new glycosyltransferase family responsible for flagellin glycosylation in pathogens.
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Glicosiltransferases , Infecções por Helicobacter , Helicobacter pylori , Sequência de Aminoácidos , Flagelos , Flagelina , HumanosRESUMO
Uropathogenic Escherichia coli (UPEC) is a major bacterial pathogen that causes urinary tract infections (UTIs). The mouse is an available UTI model for studying the pathogenicity; however, Caenorhabditis elegans represents as an alternative surrogate host with the capacity for high-throughput analysis. Then, we established a simple assay for a UPEC infection model with C. elegans for large-scale screening. A total of 133 clinically isolated E. coli strains, which included UTI-associated and fecal isolates, were applied to demonstrate the simple pathogenicity assay. From the screening, several virulence factors (VFs) involved with iron acquisition (chuA, fyuA, and irp2) were significantly associated with high pathogenicity. We then evaluated whether the VFs in UPEC were involved in the pathogenicity. Mutants of E. coli UTI89 with defective iron acquisition systems were applied to a solid killing assay with C. elegans. As a result, the survival rate of C. elegans fed with the mutants significantly increased compared to when fed with the parent strain. The results demonstrated, the simple assay with C. elegans was useful as a UPEC infectious model. To our knowledge, this is the first report of the involvement of iron acquisition in the pathogenicity of UPEC in a C. elegans model.
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Microbial proteases play pivotal roles in many aspects of bacterial physiological processes. Because a protease exerts its biological function by proteolytically regulating its substrates, the identification and characterization of the physiological substrates of a protease advance our understanding of the biological roles of the protease. Prc (also named Tsp) is an Escherichia coli periplasmic protease thought to be indispensable for E. coli to survive under low osmolality at 42°C. The accumulation of the Prc substrate MepS due to Prc deficiency contributes to the conditional growth defect. Because preventing MepS accumulation only partially restored the growth of Prc-deficient E. coli, we hypothesized that other unidentified Prc substrates intracellularly accumulate due to Prc deficiency and contribute to the conditional growth defect. To identify previously undiscovered substrates, 85 E. coli proteins able to physically interact with Prc were identified using E. coli proteome arrays. Ten proteins were shown to be cleavable by Prc in vitro. Among these candidates, MltG was able to interact with Prc in E. coli. Prc regulated the intracellular level of MltG, indicating that MltG is a physiological substrate of Prc. Prc deficiency induced the accumulation of MltG in the bacteria. Blocking MltG accumulation by deleting mltG partially restored the growth of Prc-deficient E. coli. In addition, Prc-deficient E. coli with blocked MltG and MepS expression exhibited higher growth levels than those with only the MltG or MepS expression blocked under low osmolality at 42°C, suggesting that these accumulated substrates additively contributed to the conditional growth defect. MltG is a lytic transglycosylase involved in the biogenesis of peptidoglycan (PG). In addition to MltG, the previously identified physiological Prc substrates MepS and PBP3 are involved in PG biogenesis, suggesting a potential role of Prc in regulating PG biogenesis.
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BACKGROUND: Extraintestinal pathogenic E. coli (ExPEC) is a common gram-negative organism causing various infections, including urinary tract infections (UTIs), bacteremia, and neonatal meningitis. The cjrABC-senB gene cluster of E. coli contributes to ExPEC virulence in the mouse model of UTIs. Consistently, the distribution of cjrABC-senB is epidemiologically associated with human UTIs caused by E. coli. cjrABC-senB, which has previously been proposed to encode an iron uptake system, may facilitate ExPEC survival in the iron availability-restricted urinary tract. Given that the bloodstream is also an iron limited environment to invading bacteria, the pathogenic role of cjrABC-senB in ExPEC bacteremia, however, remains to be investigated. METHODS: The ability of ExPEC RS218 strains with and without cjrABC-senB to survive in the mouse bloodstream and human serum was evaluated. Subsequently, the role of this gene cluster in the ExPEC interaction with the complement system was evaluated. Finally, the distribution of cjrABC-senB in human clinical E. coli isolates was determined by PCR. The frequency of cjrABC-senB in bacteremia isolates that were not associated with UTIs (non-UTI bacteremia isolates) was compared with that in UTI-associated isolates and fecal isolates. RESULTS: Expression of cjrABC-senB attenuated the survival of RS218 in the mouse bloodstream and human serum. The cjrABC-senB-harboring strains triggered enhanced classical- and alternative-complement pathway activation and became more vulnerable to complement-mediated killing in serum. cjrA was identified as the major gene responsible for the attenuated serum survival. Expressing cjrABC-senB and cjrA increased bacterial susceptibility to detergent and induced periplasmic protein leakage, suggesting that the expression of these genes compromises the integrity of the outer membrane of ExPEC. In addition, the frequency of cjrABC-senB in non-UTI bacteremia isolates was significantly lower than that in UTI-associated isolates, while the frequencies in non-UTI bacteremia isolates and fecal isolates showed no significant difference. Consistently, this epidemiological investigation suggests that cjrABC-senB does not contribute to E. coli bacteremia in humans. CONCLUSION: The contribution of cjrABC-senB to the pathogenesis of ExPEC is niche dependent and contradictory because the genes facilitate ExPEC UTIs but hinder bacteremia. The contradictory niche-dependent characteristic may benefit the development of novel strategies against E. coli-caused infections.
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Bacteriemia/microbiologia , Ativação do Complemento , Infecções por Escherichia coli/microbiologia , Proteínas de Escherichia coli/metabolismo , Escherichia coli Extraintestinal Patogênica/fisiologia , Genes Bacterianos , Família Multigênica , Animais , Escherichia coli Extraintestinal Patogênica/genética , Camundongos , Camundongos Endogâmicos BALB CRESUMO
A carbon dioxide-dependent small-colony variant of Escherichia coli SH4888 was isolated from blood cultures of a patient with cholangitis. To date, little is known regarding the molecular mechanisms leading to formation of carbon dioxide-dependent phenotypes in clinical isolates, but abnormalities in the carbonic anhydrase are thought to cause carbon dioxide autotrophy. In this study DNA sequence analysis of the carbonic anhydrase-encoding can locus in the carbon dioxide-dependent E. coli SH4888 revealed that the isolate had a 325-bp deletion spanning from the 3'-terminal region of can to the 3'-terminal region of hpt, which encodes a hypoxanthine phosphoribosyltransferase. To confirm that the carbon dioxide-dependent SCV phenotype of E. coli SH4888 was due to the can mutation, we performed a complementation test with a plasmid carrying an intact can that restored the normal phenotype. However, E. coli SH4888 had increased virulence compared to the can-complemented E. coli SH4888 in a murine infection model. In conclusion, these data confirm that impaired carbonic anhydrase function can cause a carbon dioxide-dependent SCV phenotype in E. coli SH4888 and provides a fitness advantage in terms of infection.
Assuntos
Dióxido de Carbono/metabolismo , Anidrases Carbônicas/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Idoso , Animais , Hemocultura , Colangite/microbiologia , Farmacorresistência Bacteriana , Genes Bacterianos , Teste de Complementação Genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Viabilidade Microbiana/genética , Mutação , Análise de Sequência de DNARESUMO
BACKGROUND: Extraintestinal pathogenic E. coli (ExPEC) remains one of the most prevalent bacterial pathogens that cause extraintestinal infections, including neonatal meningitis, septicemia, and urinary tract (UT) infections (UTIs). Antibiotic therapy has been the conventional treatment for such infections, but its efficacy has decreased due to the emergence of antibiotic-resistant bacteria. Identification and characterization of bacterial factors that contribute to the severity of infection would facilitate the development of novel therapeutic strategies. The ExPEC periplasmic protease Prc contributes to the pathogen's ability to evade complement-mediated killing in the serum. Here, we further investigated the role of the Prc protease in ExPEC-induced UTIs and the underlying mechanism. METHODS: The uropathogenic role of Prc was determined in a mouse model of UTIs. Using global quantitative proteomic analyses, we revealed that the expression of FliC and other outer membrane-associated proteins was altered by Prc deficiency. Comparative transcriptome analyses identified that Prc deficiency affected expression of the flagellar regulon and genes that are regulated by five extracytoplasmic signaling systems. RESULTS: A mutant ExPEC with a prc deletion was attenuated in bladder and kidney colonization. Global quantitative proteomic analyses of the prc mutant and wild-type ExPEC strains revealed significantly reduced flagellum expression in the absence of Prc, consequently impairing bacterial motility. The prc deletion triggered downregulation of the flhDC operon encoding the master transcriptional regulator of flagellum biogenesis. Overexpressing flhDC restored the prc mutant's motility and ability to colonize the UT, suggesting that the impaired motility is responsible for attenuated UT colonization of the mutant. Further comparative transcriptome analyses revealed that Prc deficiency activated the σE and RcsCDB signaling pathways. These pathways were responsible for the diminished flhDC expression. Finally, the activation of the RcsCDB system was attributed to the intracellular accumulation of a known Prc substrate Spr in the prc mutant. Spr is a peptidoglycan hydrolase and its accumulation destabilizes the bacterial envelope. CONCLUSIONS: We demonstrated for the first time that Prc is essential for full ExPEC virulence in UTIs. Our results collectively support the idea that Prc is essential for bacterial envelope integrity, thus explaining how Prc deficiency results in an attenuated ExPEC.
Assuntos
Endopeptidases/genética , Infecções por Escherichia coli/genética , Proteínas de Escherichia coli/genética , Escherichia coli Extraintestinal Patogênica/genética , Flagelina/genética , Infecções Urinárias/genética , Animais , Farmacorresistência Bacteriana/genética , Infecções por Escherichia coli/microbiologia , Infecções por Escherichia coli/patologia , Escherichia coli Extraintestinal Patogênica/patogenicidade , Regulação Bacteriana da Expressão Gênica/genética , Humanos , Camundongos , Proteômica , Transdução de Sinais/genética , Infecções Urinárias/microbiologia , Infecções Urinárias/patologia , Escherichia coli Uropatogênica/genética , Escherichia coli Uropatogênica/patogenicidade , Fatores de Virulência/genéticaRESUMO
Uropathogenic E scherichia coli (UPEC) is the most common pathogen of urinary tract infections (UTIs). Antibiotic therapy is the conventional measure to manage such infections. However, the rapid emergence of antibiotic resistance has reduced the efficacy of antibiotic treatment. Given that the bacterial factors required for the full virulence of the pathogens are potential therapeutic targets, identifying such factors may facilitate the development of novel therapeutic strategies against UPEC UTIs. The peptidoglycan (PG) endopeptidase Spr (also named MepS) is required for PG biogenesis in E. coli. In the present study, we found that Spr deficiency attenuated the ability of UPEC to infect kidneys and induced a fitness defect during bladder colonization in a mouse model of UTI. Based on the liquid chromatography (LC)/mass spectrometry (MS)/MS analysis of the bacterial envelope, spr deletion changed the levels of some envelope-associated proteins, suggesting that Spr deficiency interfere with the components of the bacterial structure. Among the proteins, FliC was significantly downregulated in the spr mutant, which is resulted in reduced motility. Lack of Spr might hinder the function of the flagellar transcriptional factor FlhDC to decrease FliC expression. The motility downregulation contributed to the reduced fitness in urinary tract colonization. Additionally, spr deletion compromised the ability of UPEC to evade complement-mediated attack and to resist intracellular killing of phagocytes, consequently decreasing UPEC bloodstream survival. Spr deficiency also interfered with the UPEC morphological switch from bacillary to filamentous shapes during UTI. It is known that bacterial filamentation protects UPEC from phagocytosis by phagocytes. In conclusion, Spr deficiency was shown to compromise multiple virulence properties of UPEC, leading to attenuation of the pathogen in urinary tract colonization and bloodstream survival. These findings indicate that Spr is a potential antimicrobial target for further studies attempting to develop novel strategies in managing UPEC UTIs.
RESUMO
Prokaryotic CRISPR-Cas systems limit the acquisition of genetic elements and provide immunity against invasive bacteriophage. The characteristics of CRISPR-Cas systems in clinical Klebsiella pneumoniae isolates are still unknown. Here, 97 K. pneumoniae genomes retrieved from the Integrated Microbial Genomes & Microbiomes genome database and 176 clinical isolates obtained from patients with bloodstream (BSI, n = 87) or urinary tract infections (UTI, n = 89) in Taiwan, were used for analysis. Forty out of ninety-seven genomes (41.2%) had CRISPR-Cas systems identified by the combination of CRISPRFinder and cas1 gene sequence alignment. The phylogenetic trees revealed that CRISPR-Cas systems in K. pneumoniae were divided into two types (type I-E, 23; subtype I-E∗, 17) based on the sequences of Cas1 and Cas3 proteins and their location in the chromosome. The distribution of type I-E and I-E∗ CRISPR-Cas systems was associated with the multilocus sequence typing and the pulsed-field gel electrophoresis results. Importantly, no CRISPR-Cas system was identified in published genomes of clonal complex 258 isolates (ST11 and ST258), which comprise the largest multi-drug resistant K. pneumoniae clonal group worldwide. PCR with cas-specific primers showed that 30.7% (54/176) of the clinical isolates had a CRISPR-Cas system. Among clinical isolates, more type I-E CRISPR-Cas systems were found in UTI isolates (BSI, 5.7%; UTI, 11.2%), and subtype I-E∗ CRISPR-Cas systems were dominant in BSI isolates (BSI, 28.7%; UTI, 15.7%) (p = 0.042). Isolates which had subtype I-E∗ CRISPR-Cas system were more susceptible to ampicillin-sulbactam (p = 0.009), cefazolin (p = 0.016), cefuroxime (p = 0.039), and gentamicin (p = 0.012), compared to the CRISPR-negative isolates. The strains containing subtype I-E∗ CRISPR-Cas systems had decreased numbers of plasmids, prophage regions, and acquired antibiotic resistance genes in their published genomes. Here, we first revealed subtype I-E∗ CRISPR-Cas system in K. pneumoniae potentially interfering with the acquisition of phages and plasmids harboring antibiotic resistance determinants, and thus maintained these isolates susceptible to antibiotics.