The Role Of Quorum Sensing Escherichia Coli Regulator C(QseC), A Bacterial Adrenergic Receptor, In Implant-Associated Infection Caused By Escherichia Coli

[Background and Objective]With the increasing numbers of devices that are being implanted, implant-associated infection (IAI) has already become the commonly formidable infection in hospital. According to a report, IAI accounts for50%infections in hospital. Escherichia coli is one of the most common microorganisms associated with device-related infections. Infections of prosthetic heart valves, prosthetic joints, and ventriculoperitoneal shunts are often caused by Escherichia coli.Cardiopulmonary bypass during cardiac surgery, controlled hypotension during neurosurgical or orthopedic procedure, and hemorrhagic shock result in the translocation of bacteria, leading to bacteremia. Bacterium in blood adhered to indwelling devices and then bacterial biofilm was formed. Bacterial biofilm has been shown to play an important role in implant-associated infections. In the biofilm state, microorganisms are relatively immune to antibodies and resistant to conventional antimicrobial agents. Regulation of the biofilm formation involves the qseC quorum-sensing system. The quorum sensing E.coli regulator C (QseC) is the histidine sensor kinase that detects autoinducer-3/epinephrine/norepinephrine in the planktonic state. However, the role of QseC in the implant-associated infection caused by Escherichia coli remains unclear.We investigated biofilm formation on the surface of medical-grade polyvinyl chloride (PVC) by using the E.coli K-12strain as well as corresponding qseC mutant. E.coli qseC mutant was constructed using lambda Red mutagenesis. LB with epinephrine and without epinephrine was used, and then the experiment of bacterial biofilm formation on PVC material was taken. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM). Additionally, a rat model of nonlethal hemorrhagic shock was used. Whether the ability of E.coli to sense epinephrine/norepinephrine by QseC would have the effect on the bacterial translocation to indwelling devices in a rat model of hemorrhagic shock was studied. These results serve to give a deeper understanding of how Escherichia coli biofilm on indwelling devices works. Our results are helpful for understanding and preventing biofilm formation, as well as helpful for combating implant-associated infections.[Methods]1. Construction of the qseC gene knockoutThe E.coli K-12MC1000qseC mutant was constructed by using the lambda Red mutagenesis strategy. The Cm resistance cassette from plasmid pKD3, including approximately50-bp extensions from the5’and3’ends of the predicted qseC gene was amplified. PCR products were electroporated into MC1000(pKD46) that had been induced to express the λRed recombinase. Cmr recombinants were selected. Mutations were confirmed by PCR. To remove the Cm cassette from the ΔqseC::Cm mutant, plasmid pCP20was introduced into the strain. The mutant, ΔqseC, was confirmed by PCR.2. The role of QseC in growth curves and motilityThe growth curves and motility of E. coli MC1000and MC1000ΔqseC were assayed.3. The role of QseC in the biofilm formation on the surface of polyethylene chlorideWe investigated the role of the QseC in the formation of biofilms on the surface of medical-grade poly vinyl chloride by using the E.coli K-12MC1000strain as well as a corresponding qseC mutant. LB with epinephrine and without epinephrine was used, and then the experiment of bacterial biofilm formation on PVC material was taken. The thickness of bacterial community and bacterial community quantity in the unit area on PVC materials were measured by confocal laser scanning microscope (CLSM), and the surface structure of biofilm formation was observed by scanning electron microscope (SEM).4. The role of QseC in the bacterial translocation to indwelling devicesE. coli MC1000and MC1000ΔqseC were used to track BT after gavage in rats. A rat model of nonlethal hemorrhagic shock was used. The rats were divided into6groups:controls (SS), rats that received a sham shock and MC1000(M-SS), rats that received a sham shock and MC1000ΔqseC (A-SS), rats that received a hemorrhagic shock and were not fed with E. coli (HS), rats that received a hemorrhagic shock and MC1000(M-HS), and rats that received a hemorrhagic shock and MC1000ΔqseC (Δ-HS). At sacrifice (24h), portal venous blood, the mesenteric lymph nodes, the midsection of the spleen, and segment IV of the liver were aseptically removed for bacteriological examination, and segments of the ileum were harvested for histopathology. The polyethylene chloride from peritoneal cavity was observed by scanning electron microscope (SEM).[Results]1. Deletion of qseC using the lambda Red systemThe qseC-deleted mutant of E. coli was confirmed by various PCR and DNA sequencing. Gene qseC was completely deleted.2. The role of QseC in growth curves and motility①There was no significant difference in growth ability between the qseC mutant strain and the wild-type strain MC1000in LB medium. Growth was not altered after qseC was deleted.②EPI and NE were used at concentrations of50μmol L-1. At these concentrations, no significant alterations in the growth of E.coli MC1000and MC1000ΔqseC were observed.③In the absence of EPI、NE, MC1000ΔqseC was less mobile than the isogenic wild-type strain.④MC1000motility increased upon the addition of EPI/NE. MC1000ΔqseC did not respond significantly to EPI/NE addition.3. The role of QseC in the biofilm formation on the surface of polyethylene chloride①E.coli biofilm formation on the surface of polyethylene chloride:Bacterium community quantity and thickness of bacterium biofilm on PVC have changed over time. After being cultured18-24h, bacterium community quantity reached peak. After being cultured24h, thickness of bacterium biofilm reached peak.②The effect of QseC on bacterium biofilm formation on PVC surface:Deletion of qseC decreased biofilm formation in LB. Deletion of qseC led to the reduction of bacterium community quantity and biofilm thickness.③Effect of EPI/NE on biofilm formation of the qseC-deleted mutant of Escherichia coli on biomaterial:Biofilm formation was stimulated significantly by EPI/NE in LB medium for E.coli MC1000. But that in E.coli MC1000ΔqseC, biofilm formation was not stimulated significantly by EPI/NE.4. The role of QseC in the bacterial translocation to indwelling devices ①The streptomycin-resistant bacteria isolated from the portal venous blood, MLNs, spleen, liver and PVC in the M-HS group produced a band at1488bp, whereas the streptomycin-resistant extraintestinal bacteria in the Δ-HS group produced a band at237bp. So these bacteria were the E. coli MC1000or MC1000ΔqseC administered to the rats by gavage.②Rats treated with MC1000ΔqseC showed reduced levels of bacterial translocation following hemorrhagic shock compared with rats treated with MC1000.③The number of streptomycin-resistant bacteria found in the PVC in the A-HS group was considerably reduced and significantly lower than that of the M-HS group.[Conclusions]1. E.coli AqseC motility decreased. E.coli motility increased upon the addition of catecholamines. Catecholamines increased Escherichia coli motility through QseC.2. The bacterium biofilm formation on the surface of implant is a dynamic process. Bacterium community quantity and thickness of bacterium biofilm has changed over time. It is possibly the important factor resulting in repetatus infection caused by the implant.3. The qseC gene plays an important role in regulation of biofilm formation of Escherichia coli on the surface of implant. In E.coli AqseC, biofilm formation was not stimulated significantly by catecholamines, which indicates that catecholamines stimulate E.coli biofilm formation on the surface of implant through QseC.4. Rats treated with E.coli AqseC, which has a weakened motility and does not respond to catecholamines, showed reduced levels of bacterial translocation following hemorrhagic shock. The blockade of the QseC receptor-mediated action may be useful to attenuate bacterial translocation to indwelling devices after hemorrhage.