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Since antimicrobial resistance, especially β-lactam resistance genes were common in clinical Escherichia coli strains, this study had designed and developed multiplex amplification platform for rapid and accurate detection of such resistance genes in 542 clinical E. coli isolates. The obtained specimens were subjected to bacteriological examination, antimicrobial susceptibility testing, and detection of β-lactamase genes and plasmid replicons. The major virulence genes were detected by 7 groups of multiplex PCR and eight groups of multiplex PCR were designed to detect 8 different plasmid replicons including parA-parB , iteron, repA , and RNAI . It was found that most MDR isolates were co-resistant to penicillins (AMP) and fluoroquindones (LVX, CIP) and distribution of LVX and CIP resistance was significantly higher among female than male gender. RNAI (AY234375) showed the highest detection rate, followed by the iteron (J01724) and repA (M26308), indicating the relatively higher carriage rate of corresponding plasmids. Bla OXA acquired the highest carriage rate, followed by group 2 bla CTX-M and bla SHV-1 , indicating their prevalence among clinical E. coli . Among the β-lactamase genes, bla OXA acquired the highest carriage rate, followed by group 2 bla CTX-M and bla SHV-1 , indicating their prevalence among clinical E. coli . The RNAI (AY234375) showed the highest detection rate, followed by the iteron (J01724) and repA (M26308), indicating the relatively higher carriage rate of the corresponding plasmids by clinical E. coli isolates. It is shown that the developed multiplex amplification methodology is applicable to AMR detection, and such identification of plasmid replicons and β-lactamase genes may aid in the understanding of clinical E. coli isolate epidemiology.

Persisters are a sub-population of genetically sensitive bacteria that survive antibiotic treatment by entering a dormant state. The emergence of persisters from dormancy after antibiotic withdrawal leads to recurrent infection. Indole is an aromatic molecule with diverse signalling roles, including a role in persister formation. Here we demonstrate that indole stimulates the formation of Escherichia coli persisters against quinolone antibiotics which target the GyrA subunit of DNA gyrase. However, indole has no effect on the formation of E. coli persisters against an aminocoumarin, novobiocin, which targets the GyrB subunit of DNA gyrase. Two modes of indole signalling have been described: persistent and pulse. The latter refers to the brief but intense elevation of intracellular indole during stationary phase entry. We show that the stimulation of quinolone persisters is due to indole pulse, rather than persistent, signalling. In silico docking of indole on DNA gyrase predicts that indole docks perfectly to the ATP binding site of the GyrB subunit. We propose that the inhibition of indole production offers a potential route to enhance the activity of quinolones against E. coli persisters.

Indole is a signalling molecule produced by many bacterial species and involved in intraspecies, interspecies, and interkingdom signalling. Despite the increasing volume of research published in this area, many aspects of indole signalling remain enigmatic. There is disagreement over the mechanism of indole import and export and no clearly defined target through which its effects are exerted. Progress is hindered further by the confused and sometimes contradictory body of indole research literature. We explore the reasons behind this lack of consistency and speculate whether the discovery of a new, pulse mode of indole signalling, together with a move away from the idea of a conventional protein target, might help to overcome these problems and enable the field to move forward., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

A culture of stationary phase Escherichia coli cells has been reported to produce copious indole when exposed to high temperature (50°C), and this response has been proposed to aid survival. We reinvestigated this phenomenon and found that indole production under these conditions is probably not a direct response to heat stress. Rather, E. coli produces indole when growth is prevented, irrespective of whether this is due to heat stress, antibiotic treatment or the removal of nutrients. Moreover, 300μM indole produced at 50°C does not improve the viability of heat stressed cells. Interestingly, a much lower concentration of indole (20 μM) improves the survival of an indole-negative strain (ΔtnaA) when heat stressed during exponential growth. In addition we have shown that the distribution of tryptophanase, the enzyme responsible for indole synthesis, is highly heterogeneous among cells in a population, except during the transition between exponential and stationary phases. The observation that, despite the presence of the tryptophanase, very little indole is produced during early exponential phase suggests that there is post-translational regulation of the enzyme.

Indole is a bicyclic signaling molecule with effects on both eukaryotic and prokaryotic cells. The majority of studies of indole action have been performed with bacteria cultured under aerobic conditions and little information is available about its effects under anaerobic conditions. Here the effect of the indole on anaerobic metabolism of Escherichia coli WDHL was studied. Indole in the range 0.5-8mM was added to the culture medium and cell growth, hydrogen and metabolite production were compared to cultures lacking indole. Results showed that while 8mM indole abolished growth completely, 4mM indole had a partial bacteriostatic effect and the maximum optical density of the culture decreased by 44% compared to the control cultures. In addition, 4mM indole had an important effect on anaerobic metabolism. Hydrogen production increased from 650±115 to 1137±343mL H 2 /L, and hydrogen yield increased from 0.45±0.1 to 0.94±0.34mol H 2 /mol glucose, compared to the control culture. Carbon flux was also affected and the composition of the final by-products changed. Lactate (41mM) was the main metabolite in the control cultures, whereas ethanol (56.2mM) and acetate (41.2mM) were the main metabolites in the cultures with 2mM indole. We conclude that the supplementation of E. coli cultures with exogenous indole is a simple and novel strategy to improve the production of hydrogen as well as other metabolites such as ethanol used as biofuels., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The anaerobic global regulator FNR from Escherichia coli is a [4Fe-4S](2+) cluster containing, O(2) labile dimer that plays an important role in adapting the bacterium to its anaerobic lifestyle. Although functional significance of this global regulator has been well established, its structural and biochemical characterizations have been hindered by the intrinsic O(2) lability of FNR protein. To obtain oxygen stable FNR variants for in vitro characterizations, in this study, we utilized the approach of covalently linking two fnr alleles in tandem to promote their in situ dimerization in the absence of the [4Fe-4S](2+) cluster under aerobic conditions. By covalently linking two alleles of fnrD154A mutant, which has reduced charge repulsions between two FNR monomers, an FNR variant of FNRD154A-Linker-FNRD154A (designated as (FNRD154A)(2)) was constructed. (FNRD154A)(2) displayed significantly enhanced DNA binding affinities and transcriptional regulatory activities to various FNR dependent promoters under aerobic conditions. Further in vivo and in vitro studies demonstrated that this variant retains oxygen sensing capability and maintains a similar secondary structure as that formed by native monomers of FNRD154A. We conclude that this novel variant of FNR can be widely used in various biochemical and structural studies of FNR in the presence of O(2)., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Escherichia coli O157:H7 is a major foodborne pathogen causing food poisoning. Standard detection method for foodborne E. coli is plate counting, which is time consuming and omits VBNC cells. PSR is an isothermal amplification technique identifying trace amount DNA. However, its effectiveness is influenced by residual DNA from dead cells. This study aimed at designing a PMA-PSR system for accurate determination of viable cells of E. coli O157:H7. Firstly, PSR was optimized in fluorescence indicator, betaine concentration and reaction time. The optimized system could successfully identify rfbE, stx1 and stx2 genes within 45 min at 65 °C. Secondly, sensitivity and specificity were tested in pure culture and different food samples. With 100% specificity, PSR could determine DNA amount as low as 1.12 pg/μL. Thirdly, a PMA-PSR system was designed for accurate determination of viable cells. Cells were treated with 5 μg/mL PMA to remove residual DNA from dead cells. Pretreated cells were adapted to PSR, thus viable cells were specifically detected. Fourthly, the system was applied in food samples with VBNC cells. Viable cells could be accurately determined in accordance with regular assay. In conclusion, the PMA-PSR system was an efficient tool for accurate determination of E. coli O157:H7 viable cells. • The optimized PSR system could determine E. coli O157:H7 at 65 °C within 45 min. • PSR results can be determined by naked eye observation on color change. • PMA-PSR acquires high specificity (100%) and sensitivity (1.12 pg/μL). • PMA-PSR detects viable cells avoiding the impact of dead cells and food component. [ABSTRACT FROM AUTHOR]

As major food-borne pathogens worldwide, Escherichia coli are capable of toxin production directly causing severe human disease. However, routine methods are incapable of detecting viable but non-culturable (VBNC) bacteria in food products and raw materials, leading to false-negative identification. In this study, VBNC E. coli O157 strains were acquired after cryopreservation at −20 °C, with and without freeze-thawing; morphology was observed to be of shorter rod-shape, and toxin expression remained at relatively high levels. PMA-PCR assay for VBNC detection was also validated. Therefore, these results suggest that VBNC E. coli O157 strains may represent a strong threat to public health and food safety. [ABSTRACT FROM AUTHOR]

Background Carbapenem-resistant Gram-negative bacilli (GNB) have become an important cause of nosocomial infections of hospitalized patients. Methods To investigate the microbial infection patterns and molecular epidemiology characteristics of the carbapenem-resistant GNB isolates from a long-term hospitalized patient, antimicrobial susceptibility testing, phenotypic screening test for carbapenemase production, PCR screening and DNA sequencing of carbapenemase genes, repetitive extragenic palindromic sequence-based PCR (REP-PCR), multilocus sequencing typing (MLST) and genetic environment analysis were performed. Results Twelve strains with carbapenemase genes were detected from 63 carbapenem-resistant isolates, including two bla IMP-25 -carrying Pseudomonas aeruginosa , one bla NDM-1 -carrying Citrobacter freundii , three bla NDM-1 -carrying Klebsiella pneumoniae and six bla KPC-2 -carrying K. pneumoniae . Only the bla NDM-1 genes were successfully transferred from three K. pneumoniae strains to Escherichia coli C600 by conjugation. Genetic environment of bla IMP-25 , bla NDM-1 and bla KPC-2 genes in our study were consistent with previous reports. Molecular typing of K. pneumoniae performed by MLST revealed that most of the isolates belonged to ST11. bla NDM-1 -carrying K. pneumoniae sequencing type 1416 was first reported in our study. Conclusions Carbapenem-resistant GNB are common pathogens during long-term hospitalization, and ST11 bla KPC-2 -carrying K. pneumoniae is the dominant bacterium in our study. Colonization and horizontal transmission of resistance by plasmids of carbapenem-resistant GNB have increased the risks of persistent infection and mortality of long-term hospitalized patients. [ABSTRACT FROM AUTHOR]