Histidine Kinase Inhibitor Research Articles

Anti-quorum sensing activity of poly-amidoamine dendrimer generation 5 dendrimer loaded kinase inhibitor peptide against methicillin-resistant Staphylococcus aureus

Methicillin-resistant Staphylococcus aureus (MRSA) is a significant concern in both healthcare and community settings, as it causes numerous infections worldwide with high morbidity and mortality rates. One promising strategy is to target the quorum sensing (QS) system of MRSA using a dendrimer loaded with kinase inhibitor peptide. The present investigation has formulated a poly-amidoamine dendrimer (PAMAM) G5 dendrimer that is loaded with Quorum Quencher (QQ) peptide, which functions as a histidine kinase inhibitor. The particle average size of the formulated G5-QQ3 complex was determined to be 276 nm, and polydispersity index values of 0.33. The MIC50 for the formulated nanoparticles was 18 μM as demonstrated by a growth assay. Furthermore, the G5-QQ3 complex was able to inhibit the hemolysis activity of the MRSA with a concentration of 10 μM, and for Staphylococcus aureus was 3 μM. The G5-QQ3 complex possesses the ability to inhibit, penetrate, and eradicate biofilm in MRSA, Staphylococcus aureus, and different agr mutants with inhibition percentages ranging from 60 to 72%. Furthermore, live/dead viability assay confirmed the ability of the formulated nanoparticles to effectively kill all strains within the biofilm structure as evidenced by a confocal microscope, and the cytotoxicity of the G5-QQ3 complex was dose-dependent (p < 0.05). against RAW 264.7 cells. In general, the study confirmed that encapsulating QQ3 peptide within PAMAM G5 dendrimer results in a potent anti-virulence and anti-bacterial action and suggests a synergistic effect. The findings of this study have significant implications for the development of new treatments for MRSA infections, which are a major public health concern.

Identification of histidine kinase inhibitors through screening of natural compounds to combat mastitis caused by Streptococcus agalactiae in dairy cattle

BackgroundMastitis poses a major threat to dairy farms globally; it results in reduced milk production, increased treatment costs, untimely compromised genetic potential, animal deaths, and economic losses. Streptococcus agalactiae is a highly virulent bacteria that cause mastitis. The administration of antibiotics for the treatment of this infection is not advised due to concerns about the emergence of antibiotic resistance and potential adverse effects on human health. Thus, there is a critical need to identify new therapeutic approaches to combat mastitis. One promising target for the development of antibacterial therapies is the transmembrane histidine kinase of bacteria, which plays a key role in signal transduction pathways, secretion systems, virulence, and antibiotic resistance.ResultsIn this study, we aimed to identify novel natural compounds that can inhibit transmembrane histidine kinase. To achieve this goal, we conducted a virtual screening of 224,205 natural compounds, selecting the top ten based on their lowest binding energy and favorable protein–ligand interactions. Furthermore, molecular docking of eight selected antibiotics and five histidine kinase inhibitors with transmembrane histidine kinase was performed to evaluate the binding energy with respect to top-screened natural compounds. We also analyzed the ADMET properties of these compounds to assess their drug-likeness. The top two compounds (ZINC000085569031 and ZINC000257435291) and top-screened antibiotics (Tetracycline) that demonstrated a strong binding affinity were subjected to molecular dynamics simulations (100 ns), free energy landscape, and binding free energy calculations using the MM-PBSA method.ConclusionOur results suggest that the selected natural compounds have the potential to serve as effective inhibitors of transmembrane histidine kinase and can be utilized for the development of novel antibacterial veterinary medicine for mastitis after further validation through clinical studies.

Targeting multidrug resistant Staphylococcus infections with bacterial histidine kinase inhibitors.

The emergence of drug-resistant bacteria, such as methicillin-resistant Staphylococcus aureus (MRSA), which are not susceptible to current antibiotics has necessitated the development of novel approaches and targets to tackle this growing challenge. Bacterial two-component systems (TCSs) play a central role in the adaptative response of bacteria to their ever-changing environment. They are linked to antibiotic resistance and bacterial virulence making the proteins of the TCSs, histidine kinases and response regulators, attractive for the development of novel antibacterial drugs. Here, we developed a suite of maleimide-based compounds that we evaluated against a model histidine kinase, HK853, in vitro and in silico. The most potent leads were then assessed for their ability to decrease the pathogenicity and virulence of MRSA, resulting in the identification of a molecule that decreased the lesion size caused by a methicillin-resistant S. aureus skin infection by 65% in a murine model.

A simple protein histidine kinase activity assay for high-throughput inhibitor screening

Bacterial two-component systems (TCSs), which typically consist of a sensor histidine kinase (HK) and a response regulator (RR), have been investigated as attractive antibacterial drug targets. Unfortunately, current HK activity assays based on the quantification of autophosphorylated HKs are hampered by the instability of the phosphohistidine (pHis) product, rendering them ill-suited for high-throughput screenings. To address this challenge, we developed a simple HK activity assay using readily available reagents, which we have termed AUDECY (AUtophosphorylation-DEphosphorylation CYcle assay). Instead of trying to preserve the fragile pHis, we deliberately decomposed it with a pHis-specific phosphatase to constitute an ATPase-like cycle for convenient colorimetric measurements. This kinetic assay was successfully employed for the kinetic characterization of E. coli EnvZ and for high-throughput inhibitor screening of vancomycin-resistant Enterococcus faecium (VRE) VanS, of which histidine kinase activity was hardly detectable with conventional methods. Through the screening, we identified OSU-03012, a potent VanS HK inhibitor, which sensitized VRE toward vancomycin, highlighting the potential of AUDECY in HK inhibitor discovery.

Histidine kinase inhibitors impair shoot regeneration in Arabidopsis thaliana via cytokinin signaling and SAM patterning determinants.

Reversible protein phosphorylation is a post-translational modification involved in virtually all plant processes, as it mediates protein activity and signal transduction. Here, we probe dynamic protein phosphorylation during de novo shoot organogenesis in Arabidopsis thaliana. We find that application of three kinase inhibitors in various time intervals has different effects on root explants. Short exposures to the putative histidine (His) kinase inhibitor TCSA during the initial days on shoot induction medium (SIM) are detrimental for regeneration in seven natural accessions. Investigation of cytokinin signaling mutants, as well as reporter lines for hormone responses and shoot markers, suggests that TCSA impedes cytokinin signal transduction via AHK3, AHK4, AHP3, and AHP5. A mass spectrometry-based phosphoproteome analysis further reveals profound deregulation of Ser/Thr/Tyr phosphoproteins regulating protein modification, transcription, vesicle trafficking, organ morphogenesis, and cation transport. Among TCSA-responsive factors are prior candidates with a role in shoot apical meristem patterning, such as AGO1, BAM1, PLL5, FIP37, TOP1ALPHA, and RBR1, as well as proteins involved in polar auxin transport (e.g., PIN1) and brassinosteroid signaling (e.g., BIN2). Putative novel regeneration determinants regulated by TCSA include RD2, AT1G52780, PVA11, and AVT1C, while NAIP2, OPS, ARR1, QKY, and aquaporins exhibit differential phospholevels on control SIM. LC-MS/MS data are available via ProteomeXchange with identifier PXD030754.

High-level carbapenem tolerance requires antibiotic-induced outer membrane modifications.

Antibiotic tolerance is an understudied potential contributor to antibiotic treatment failure and the emergence of multidrug-resistant bacteria. The molecular mechanisms governing tolerance remain poorly understood. A prominent type of β-lactam tolerance relies on the formation of cell wall-deficient spheroplasts, which maintain structural integrity via their outer membrane (OM), an asymmetric lipid bilayer consisting of phospholipids on the inner leaflet and a lipid-linked polysaccharide (lipopolysaccharide, LPS) enriched in the outer monolayer on the cell surface. How a membrane structure like LPS, with its reliance on mere electrostatic interactions to maintain stability, is capable of countering internal turgor pressure is unknown. Here, we have uncovered a novel role for the PhoPQ two-component system in tolerance to the β-lactam antibiotic meropenem in Enterobacterales. We found that PhoPQ is induced by meropenem treatment and promotes an increase in 4-amino-4-deoxy-L-aminoarabinose [L-Ara4N] modification of lipid A, the membrane anchor of LPS. L-Ara4N modifications likely enhance structural integrity, and consequently tolerance to meropenem, in several Enterobacterales species. Importantly, mutational inactivation of the negative PhoPQ regulator mgrB (commonly selected for during clinical therapy with the last-resort antibiotic colistin, an antimicrobial peptide [AMP]) results in dramatically enhanced tolerance, suggesting that AMPs can collaterally select for meropenem tolerance via stable overactivation of PhoPQ. Lastly, we identify histidine kinase inhibitors (including an FDA-approved drug) that inhibit PhoPQ-dependent LPS modifications and consequently potentiate meropenem to enhance lysis of tolerant cells. In summary, our results suggest that PhoPQ-mediated LPS modifications play a significant role in stabilizing the OM, promoting survival when the primary integrity maintenance structure, the cell wall, is removed.

An immuno-dot blot assay for screening histidine kinase inhibitors

Two-component signal transduction systems (TCSs), consisting of a histidine kinase (HK) and its cognate response regulator, are ubiquitous among bacteria and are associated with the virulence of pathogens. TCSs are potential targets for alternative antibiotics and antivirulence agents. It is, thus, very important to determine HK activity in bacterial TCSs. Here, we describe an immuno-dot blot assay for the inhibition profiling of HKs using the anti-N3-phosphohistidine antibody. This simple method promises reliable detection of HK activity, and it is likely applicable in high-throughput screening of HK inhibitors.

Interaction of vitamin D, homocystein and dental diseases

The 1,2,4-triazole nucleus is resistant to metabolism and is an important pharmacophore; it can increase solubility and improve the pharmacokinetic and pharmacodynamic profile of drugs. Domestic scientists pay much attention to the heterocyclic 1,2,4-triazole system. A two-component system comprising “EC 2.7.13.3 Histidine kinase” and a variable response protein may be crucial for the virulence of some fungal strains, which are often the cause of candidiasis in immunocompromised individuals. Because of the fact, that humans lack this two-component system, it may be a good target for antimicrobials to treat candidiasis. The purpose of the study was to carry out a molecular docking and evaluate the bioavailability of S-alkyl derivatives of 5-(3-fluorophenyl)-, 5-(5-bromofuran-2-yl)- and 5‑((((3-(pyridin-4-yl)-1 H -1,2,4-triazole-5-yl)thio)methyl)-4-methyl-4H-1,2,4-triazole nucleus in silico by methods promising as objects of biological properties for inhibitors «EC 2.7.13.3 Histidine kinase». Materials and methods. The synthesis of compounds is carried out according to the well-known method. A virtual screening of compounds was performed using a computer software program PASS. Molecular docking was performed using Autodock 4.2.6. The screening was performed on the crystallographic structure of the enzyme “EC 2.7.13.3 Histidine kinase” (1A0B). Results. Analysis of the results of a computer prediction demonstrates the prospect of finding inhibitors of histidine kinase, cytidine deaminase, STAT transcription factor, alkaline phosphatase, CYP2C9, insulin, nootropic and diuretic action, antituberculous activity in a number of these compounds. Molecular docking has shown a high affinity of the selected compounds to the enzyme “EC 2.7.13.3 Histidine kinase” with the corresponding values. The interaction of the ligand with the active center of the enzyme is quite complex and is mainly provided by van der Waals and π-bonds with water molecules and amino acid residues of the enzyme. An important point for binding to the enzyme is the presence of Sulfur atoms in the molecules of the test compounds. Based on the above, it is the promising structural modification of the nucleus by the position of the Sulfur atom. Conclusions. Data from a computer prediction indicate the prospect of searching among the two areas of bioregulators that could be potential histidine kinase inhibitors and diuretic agents. Molecular docking has shown a high affinity of the selected compounds for the “EC 2.7.13.3 Histidine kinase” enzyme, which is provided with van der Waals and π‑bonds with water molecules and amino acid residues of the enzyme.

Synthesis of histidine kinase inhibitors and their biological properties.

Infections caused by multidrug-resistant bacteria represent a significant and ever-increasing cause of morbidity and mortality. There is thus an urgent need to develop efficient and well-tolerated antibacterials targeting unique cellular processes. Numerous studies have led to the identification of new biological targets to fight bacterial resistance. Two-component signal transduction systems are widely employed by bacteria to translate external and cellular signals into a cellular response. They are ubiquitous in bacteria, absent in the animal kingdom and are integrated into various virulence pathways. Several chemical series, including isothiazolidones, imidazolium salts, benzoxazines, salicylanilides, thiophenes, thiazolidiones, benzimidazoles, and other derivatives deduced by different approaches have been reported in the literature to have histidine kinase (HK) inhibitory activity. In this review, we report on the design and the synthesis of these HKs inhibitors and their potential to serve as antibacterial agents.

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes.

In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.

New natural products to meet the antibiotic crisis: a personal journey.

The use of natural products and their derivatives has evolved as a promising approach for the treatment of various infectious diseases, particularly to combat drug-resistant microbial strains. In addition, these natural products characterized by the presence of novel structures and mechanisms of action may provide guidance toward the development of potential new chemotherapies. In the present review, antimicrobial resistance (AMR) is briefly introduced and research focused on the identification and characterization of actinomycete metabolites for antimicrobial activity is discussed. Three compounds, i.e., walkmycin B, waldiomycin, and signamycin B, with novel mechanisms of action as histidine kinase inhibitors, were isolated from the metabolites of actinomycetes. New antituberculosis antibiotics, tuberlactomicin A and caprazamycins, were discovered, and amycolamicin was identified as an antimethicillin-resistant Staphylococus aureus antibiotic. The discovery of these compounds encourages the discovery and investigation of more natural products active against antimicrobial-resistant species, thus providing scaffold for the development of effective drugs against various AMR species.

Study of Influence of Free Radical Species on Antioxidant Activity of Selected 1,2,4‐Triazole‐3‐thiones

AbstractThe free radical scavenging potency of four 1,2,4‐triazole‐3‐thione compounds towards ten chosen radicals was investigated. The thermodynamic parameters, Gibbs free energies of reactions and reaction enthalpies, were used to determine the most probable mechanism of action. The results indicated that the favourable mechanism of antiradical action was dependent both on the polarity of solvents and a nature of free radical species. The results suggested that Sequential Proton Loss Electron Transfer (SPLET) is the most probable for reaction in benzene, while Hydrogen Atom Transfer (HAT) was favorized for reaction occurring in methanol. The constant competition between these mechanisms of antioxidative action was present. The molecular docking study, obtained for parent molecules of chosen 1,2,4‐triazole‐3‐thiones compounds and their corresponding anionic species, showed their inhibitory nature against histidine kinase inhibitor Sda. Except in the case of compound 4 a, all the anionic species (4 d‐4 h) showed the lower inhibition constants, indicating better inhibition potency of Sda, than corresponding parent molecules.

Screening serine/threonine and tyrosine kinase inhibitors for histidine kinase inhibition

Histidine kinases of bacterial two-component systems are promising antibacterial targets. Despite their varied, numerous roles, enzymes in the histidine kinase superfamily share a catalytic core that may be exploited to inhibit multiple histidine kinases simultaneously. Characterized by the Bergerat fold, the features of the histidine kinase ATP-binding domain are not found in serine/threonine and tyrosine kinases. However, because each kinase family binds the same ATP substrate, we sought to determine if published serine/threonine and tyrosine kinase inhibitors contained scaffolds that would also inhibit histidine kinases. Using select assays, 222 inhibitors from the Roche Published Kinase Set were screened for binding, deactivation, and aggregation of histidine kinases. Not only do the results of our screen support the distinctions between ATP-binding domains of different kinase families, but the lead molecule identified also presents inspiration for further histidine kinase inhibitor development.

Distribution of β-lactamase genes of Klebsiella pneumoniae isolates in Zhejiang province, China, and regulation of gene expression

Klebsiella pneumoniae is a common causative agent of nosocomial infections with a high level of resistance toward ?-lactam antibiotics. Our previous study showed that TEM-1 and SHV-11 are the predominant ?-lactamase-encoding genes of K. pneumoniae isolates in the Zhejiangarea, China. In this study, more clinical K. pneumoniae isolates were collected for detecting their ?-lactamase-encoding gene profiles by PCR and sequencing. qRT-PCR was then performed to determine the role of cefotaxime or penicillin in low concentrations to induce the ?-lactamase gene expression of K. pneumoniae isolates. Moreover, the K. pneumoniae isolates were pretreated with closantel (CLO), a histidine kinase inhibitor, before antibiotic treatment, and qRT-PCR and the ?-lactamase phenotype confirmatory test were then applied to determine the effect of CLO on the expression of the ?-lactamase genes. The results showed that, except for KPC-2, the 1/4 MIC cefotaxime or penicillin induced significant mRNA elevation of the TEM-1, CTX-M-14, SHV-11 and OXA-1?-lactamase genes, but this induction could be inhibited by CLO. After pretreatment withCLO,78.4~81.4%of the ?-lactam-resistant isolates became sensitive and the positive rate of the ?-lactamase production phenotype in the isolates was decreased from 100% to 27.1%. The data indicate thatTEM-1 (70.7%), SHV-11 (64.2%) and CTX-M-14 (40.5%) are the predominant ?-lactamase genes of the K. pneumoniae isolates in Zhejiang and sublethal dosage of ?-lactam antibiotics can induce the ?-lactamase gene expression of K. pneumoniae through histidine kinase-mediated two-component signaling systems.

A conserved dimorphism-regulating histidine kinase controls the dimorphic switching in Paracoccidioides brasiliensis.

Paracoccidioides brasiliensis and P. lutzii, thermally dimorphic fungi, are the causative agents of paracoccidioidomycosis (PCM). Paracoccidioides infection occurs when conidia or mycelium fragments are inhaled by the host, which causes the Paracoccidioides cells to transition to the yeast form. The development of disease requires conidia inside the host alveoli to differentiate into yeast cells in a temperature-dependent manner. We describe the presence of a two-component signal transduction system in P. brasiliensis, which we investigated by expression analysis of a hypothetical protein gene (PADG_07579) that showed high similarity with the dimorphism-regulating histidine kinase (DRK1) gene of Blastomyces dermatitidis and Histoplasma capsulatum This gene was sensitive to environmental redox changes, which was demonstrated by a dose-dependent decrease in transcript levels after peroxide stimulation and a subtler decrease in transcript levels after NO stimulation. Furthermore, the higher PbDRK1 levels after treatment with increasing NaCl concentrations suggest that this histidine kinase can play a role as osmosensing. In the mycelium-yeast (M→Y) transition, PbDRK1 mRNA expression increased 14-fold after 24 h incubation at 37°C, consistent with similar observations in other virulent fungi. These results demonstrate that the PbDRK1 gene is differentially expressed during the dimorphic M→Y transition. Finally, when P. brasiliensis mycelium cells were exposed to a histidine kinase inhibitor and incubated at 37°C, there was a delay in the dimorphic M→Y transition, suggesting that histidine kinases could be targets of interest for PCM therapy.

Putative histidine kinase inhibitors with antibacterial effect against multi-drug resistant clinical isolates identified by in vitro and in silico screens.

Novel antibacterials are urgently needed to address the growing problem of bacterial resistance to conventional antibiotics. Two-component systems (TCS) are widely used by bacteria to regulate gene expression in response to various environmental stimuli and physiological stress and have been previously proposed as promising antibacterial targets. TCS consist of a sensor histidine kinase (HK) and an effector response regulator. The HK component contains a highly conserved ATP-binding site that is considered to be a promising target for broad-spectrum antibacterial drugs. Here, we describe the identification of putative HK autophosphorylation inhibitors following two independent experimental approaches: in vitro fragment-based screen via differential scanning fluorimetry and in silico structure-based screening, each followed up by the exploration of analogue compounds as identified by ligand-based similarity searches. Nine of the tested compounds showed antibacterial effect against multi-drug resistant clinical isolates of bacterial pathogens and include three novel scaffolds, which have not been explored so far in other antibacterial compounds. Overall, putative HK autophosphorylation inhibitors were found that together provide a promising starting point for further optimization as antibacterials.

Rational design of potent histidine kinase inhibitors with antimicrobial activities

The excess use of antibiotics has resulted in the appearance of multidrug resistant pathogens, described as one of the most eminent threats to human health in the 21st century. To address this problem many unique fundamental processes for bacteria are being probed for the development of new antimicrobial drugs. In bacteria signaling via the two‐component system is a widespread mechanism, with bacteria typically featuring in excess of 10 such systems per genome. The two‐component system consists of a sensor histidine kinase and a response regulator. A conserved two‐component system (YycFG aka WalRK) essential for viability has been discovered in most gram‐positive pathogens suggesting that inhibition of these systems would lead to cell death. However, targeting a single system can be expected to quickly result in drug resistances. It would thus seem desirable to discover compounds with the ability to inhibit multiple two‐component systems simultaneously. Synthetic compounds have been described as potent competitive inhibitors of DNA gyrase and one such compound has entered phase 1 clinical trials. Gyrases share a common catalytic ATP‐binding domain with histidine kinases; redesign of these compounds to bind the catalytic domain of the latter thus seemed possible. We utilized available high‐resolution structures of the inhibitor/gyrase complex and of a histidine kinase catalytic domain to rationally design novel compounds and probe histidine kinase binding in silico. The most promising in silico hits were synthesized and tested for their ability to inhibit several histidine kinases of Bacillus subtilis. A series compounds were found to be potent inhibitors of all tested histidine kinases but did not inhibit the structuraly distinct ser/thr kinases in vitro. Several of these compounds showed antimicrobial activities against methicillin‐resistant Staphylococcus aureus (MRSA) and vancomycin‐resistant Enterococcus faecalis (VRE) and other Gram‐positive pathogens. Hemolytic assays on sheep erythrocytes ruled out that these compounds act by simply lysing cells, a problem encountered with many previously discovered histidine kinase inhibitors. In summary, the discovered compounds are promising leads for the development of new antibacterial drugs.Support or Funding InformationThis work was supported by grant GM106085 from the US National Institute of General Medical Sciences, National Institutes of Health

Identification of an Antimicrobial Agent Effective against Methicillin-Resistant Staphylococcus aureus Persisters Using a Fluorescence-Based Screening Strategy.

Persisters are a subpopulation of normal bacterial cells that show tolerance to conventional antibiotics. Persister cells are responsible for recalcitrant chronic infections and new antibiotics effective against persisters would be a major development in the treatment of these infections. Using the reporter dye SYTOX Green that only stains cells with permeabilized membranes, we developed a fluorescence-based screening assay in a 384-well format for identifying compounds that can kill methicillin-resistant Staphylococcus aureus (MRSA) persisters. The assay proved robust and suitable for high throughput screening (Z`-factor: >0.7). In screening a library of hits from a previous screen, which identified compounds that had the ability to block killing of the nematode Caenorhabditis by MRSA, we discovered that the low molecular weight compound NH125, a bacterial histidine kinase inhibitor, kills MRSA persisters by causing cell membrane permeabilization, and that 5 μg/mL of the compound can kill all cells to the limit of detection in a 108 CFU/mL culture of MRSA persisters within 3h. Furthermore, NH125 disrupts 50% of established MRSA biofilms at 20 μg/mL and completely eradicates biofilms at 160 μg/mL. Our results suggest that the SYTOX Green screening assay is suitable for large-scale projects to identify small molecules effective against MRSA persisters and should be easily adaptable to a broad range of pathogens that form persisters. Since NH125 has strong bactericidal properties against MRSA persisters and high selectivity to bacteria, we believe NH125 is a good anti-MRSA candidate drug that should be further evaluated.

Predominant β-lactamase genotypes of Escherichia coli isolates and induction and inhibition mechanisms of β-lactamase gene expression

To understand the predominant β-lactamase genotypes and their carrying modes of Escherichia coli isolates in Zhejiang province, and the effects of β-lactam antibiotics on inducing or histidine kinase inhibitor closantel (CLO) on inhibiting the expression of β-lactamase genes. Micro-dilution method and E-test were applied to measure the resistant rate and minimal inhibitory concentration (MIC) in E. coli isolates against β-lactam antibiotics. PCR and sequence analysis of PCR products were conducted to detect the β-lactamase genotypes and their carrying modes. Real-time fluorescent quantitative RT-PCR and β-lactamase confirmation test were performed to determine the influence of 1/4 MIC penicillin and cefotaxime, and CLO on the transcription and expression of β-lactamase genes in the resistant E. coli isolates. Among the 462 E. coli strains isolated in Zhejiang, 285 (61.7%) were resistant to penicillin, ampicillin, cefoxitin, cefotaxim and ceftazidime. In the 285 resistant isolates, the detection rate of TEM or CTX-M β-lactamase gene (83.2% or 75.1%) was significantly higher than that of KPC, SHV or OXA β-lactamase gene (1.4%-10.2%) (P<0.01) and the carrying rate of two or more β-lactamase genes (68.8%) was also significantly higher than that of single β-lactamase gene (31.2%) (P<0.01), and 61.4% of the resistant isolates carried TEM+CTX-M genes (P<0.01). Except KPC gene, 1/4 MIC of cefotaxim and penicillin induced a rapid increase of TEM-mRNA, CTX-M-mRNA, SHV-mRNA or OXA-mRNA levels (P<0.01), but 50-500 µg/ml CLO inhibited these levels (P<0.01). After pre-treatment with 100 µg/ml CLO, 82.8%-85.6% of the resistant isolates became sensitive to β-lactam antibiotics (P<0.01), while the detection rate of β-lactamases was also decreased from 95.1% to 16.1% (P<0.01). TEM and CTX-M are the predominant β-lactamase genotypes in E. coli isolates in Zhejiang and TEM+CTX-M is the predominant carrying mode of β-lactamase genes. Low concentrations of β-lactam antibiotics can up-regulate the expression levels of β-lactamase genes in E. coli through bacterial two-component signaling systems, but this effect can be inhibited by CLO, a histidine kinase inhibitor.

The expression of ESBLs genes in Escherichia coli isolates induced by β-lactam antibiotics and inhibited by histidine kinase inhibitors

Objective To investigate the genotypes of extended spectrum β-lactamases (ESBLs) and their carrying modes in Escherichia coli (E.coli) isolates,and to analyze the mechanism of protein phosphorylation and ESBLs gene expression induced by β-lactam antibiotics or inhibited by histidine kinase inhibitors.Methods The predominant genotypes of ESBLs (KPC,TEM,SHV and CTX-M) and their carrying modes were identified by PCR and sequencing analysis.E-test and micro-tube dilution method were applied to measure minimal inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs).Immobilized metal ion affinity chromatography,bacterial protein phosphorylation detection kit and real-time fluorescent quantitation RT-PCR were performed to analyze the enhancing effects of 1/4 MIC penicillin or cefotaxime or the inhibitory effects of histidine kinase inhibitors (closantel,bromized or iodized methylimidazol) on protein phosphorylation and the expression of ESBLs at mRNA level in E.coli isolates.Results In 183 β-lactam antibiotics-resistant E.coli isolates,TEM and CTX-M genes (83.1％ and 77.1％) were highly expressed than other two ESBLs genes with a prevalent carrying mode of coexisting (65.0％) (P＜0.05).Penicillin or cefotaxime at 1/4 MIC induced the protein phosphorylation and promoted the expression of TEM,SHV and CTX-M at mRNA level (P＜0.05).Closantel (200 μmol),bromized methylimidazol (2 or 10 μmol) or iodized methylimidazol (20 or 50 μmol) could neither kill E.coli isolates nor inhibit their growth,but could inhibit the protein phosphorylation induced by above mentioned antibiotics and enhance the expression of ESBLs at mRNA level (P＜0.05).Moreover,the susceptibility of antibioticresistant E.coli strains to penicillin and cefotaxime were increased (P＜0.05).Conclusion TEM and CTX-M were the predominant genotypes of ESBLs carried by β-lactam antibiotics-resistant E.coli strains isolated from Zhejiang province,which were mostly found in a TEM plus CTX-M carrying mode.Sublethal dose of β-lactam antibiotics could up-regulate the expression of ESBLs genes in E.coli isolates via TCSS,but it could be inhibited by histidine kinase inhibitors. Key words: Escherichia coli; Extended spectrum β-lactamases; β-lactam antibiotics; Protein phosphorylation; Histidine kinase