Phloretin Attenuates Listeria monocytogenes Virulence Both In vitro and In vivo by Simultaneously Targeting Listeriolysin O and Sortase A

The critical roles of sortase A (SrtA) and listeriolysin O (LLO) in Listeria monocytogenes pathogenicity render these two virulence factors as ideal targets for the development of anti-virulence agents against L. monocytogenes infection. Additionally, the structures of SrtA and LLO are highly conserved among the members of sortase enzyme family and cholesterol dependent toxin family. Here, phloretin, a natural polyphenolic compound derived from apples and pears that has little anti-L. monocytogenes activity, was identified to simultaneously inhibit LLO expression and neutralize SrtA catalytic activity. Phloretin neutralized SrtA activity by causing a conformational change in the protein's active pocket, which prevented engagement with its substrate. Treatment with phloretin simultaneously reduced L. monocytogenes invasion into host cells and blocked the escape of vacuole-entrapped L. monocytogenes into cytoplasm. Further, L. monocytogenes-infected mice that received phloretin showed lower mortality, decreased bacterial burden and reduced pathological injury. Our results demonstrate that phloretin is a promising anti-infective therapeutic for infections caused by L. monocytogenes due to its simultaneous targeting of SrtA and LLO, which may result in fewer side effects than those caused by other antibiotics

A Wohlfahrtiimonas chitiniclastica with a novel type of blaVEB–1-carrying plasmid isolated from a zebra in China

BackgroundWohlfahrtiimonas chitiniclastica is an emerging fly-borne zoonotic pathogen, which causes infections in immunocompromised patients and some animals. Herein, we reported a W. chitiniclastica BM-Y from a dead zebra in China.MethodsThe complete genome sequencing of BM-Y showed that this isolate carried one chromosome and one novel type of blaVEB–1-carrying plasmid. Detailed genetic dissection was applied to this plasmid to display the genetic environment of blaVEB–1.ResultsThree novel insertion sequence (IS) elements, namely ISWoch1, ISWoch2, and ISWoch3, were found in this plasmid. aadB, aacA1, and gcuG were located downstream of blaVEB–1, composing a gene cassette array blaVEB–1–aadB–aacA1–gcuG bracketed by an intact ISWoch1 and a truncated one, which was named the blaVEB–1 region. The 5′-RACE experiments revealed that the transcription start site of the blaVEB–1 region was located in the intact ISWoch1 and this IS provided a strong promoter for the blaVEB–1 region.ConclusionThe spread of the blaVEB–1-carrying plasmid might enhance the ability of W. chitiniclastica to survive under drug selection pressure and aggravate the difficulty in treating infections caused by blaVEB–1-carrying W. chitiniclastica. To the best of our knowledge, this is the first report of the genetic characterization of a novel blaVEB–1-carrying plasmid with new ISs from W. chitiniclastica

A clinical Pseudomonas juntendi strain with blaIMP−1 carried by an integrative and conjugative element in China

ObjectiveTo precisely determine the species of a carbapenem-resistant Pseudomonas strain 1809276 isolated from the urine of a Chinese patient and analyze its integrative and conjugative element (ICE) 1276 formation mechanism.MethodsSingle-molecule real-time (SMRT) sequencing was carried out on strain 18091276 to obtain the complete chromosome and plasmid (pCN1276) sequences, and average nucleotide identity (ANI) was used for precise species identification. The ICEs in GenBank with the same integrase structure as ICE 1276 were aligned. At the same time, the transfer ability of blaIMP−1 and the antibiotic sensitivity of Pseudomonas juntendi 18091276 were tested.ResultsThis bacterium was P. juntendi, and its drug resistance mechanism is the capture of the accA4' gene cassette by the Tn402-like type 1 integron (IntI1-blaIMP−1) to form In1886 before its capture by the ΔTn4662a-carrying ICE 1276. The acquisition of blaIMP−1 confers carbapenem resistance to P. juntendi 18091276.ConclusionThe formation of blaIMP−1-carrying ICE 1276, its further integration into the chromosomes, and transposition and recombination of other elements promote bacterial gene accumulation and transmission

Chalcone attenuates Staphylococcus aureus virulence by targeting sortase A and alpha-hemolysin

Staphylococcus aureus (S.aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti-S. aureus activity, could significantly inhibit SrtA activity with an IC50 of 53.15 uM and Hla hemolysis activity with an IC50 of 17.63 uM using a fluorescence resonance energy transfer (FRET) assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA) display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla

Chalcone Attenuates Staphylococcus aureus Virulence by Targeting Sortase A and Alpha-Hemolysin

Staphylococcus aureus (S.aureus) resistance, considered a dilemma for the clinical treatment of this bacterial infection, is becoming increasingly intractable. Novel anti-virulence strategies will undoubtedly provide a path forward in combating these resistant bacterial infections. Sortase A (SrtA), an enzyme responsible for anchoring virulence-related surface proteins, and alpha-hemolysin (Hla), a pore-forming cytotoxin, have aroused great scientific interest, as they have been regarded as targets for promising agents against S. aureus infection. In this study, we discovered that chalcone, a natural small compound with little anti-S. aureus activity, could significantly inhibit SrtA activity with an IC50 of 53.15 μM and Hla hemolysis activity with an IC50 of 17.63 μM using a fluorescence resonance energy transfer (FRET) assay and a hemolysis assay, respectively. In addition, chalcone was proven to reduce protein A (SpA) display in intact bacteria, binding to fibronectin, formation of biofilm and S. aureus invasion. Chalcone could down-regulate the transcriptional levels of the hla gene and the agrA gene, thus leading to a reduction in the expression of Hla and significant protection against Hla-mediated A549 cell injury; more importantly, chalcone could also reduce mortality in infected mice. Additionally, molecular dynamics simulations and mutagenesis assays were used to identify the mechanism of chalcone against SrtA, which implied that the inhibitory activity lies in the bond between chalcone and SrtA residues Val168, Ile182, and Arg197. Taken together, the in vivo and in vitro experiments suggest that chalcone is a potential novel therapeutic compound for S. aureus infection via targeting SrtA and Hla

Curcumin Promotes the Clearance of Listeria monocytogenes both In Vitro and In Vivo by Reducing Listeriolysin O Oligomers

The pore-forming toxin listeriolysin O (LLO), an essential virulence factor that is secreted by Listeria monocytogenes (L. monocytogenes), is responsible for bacterial breaching at the phagosomal membranes and subsequent release into the cytoplasm; it cannot be recognized by the host immune system. The vital role that LLO plays in bacterial pathogenicity and evading host immune clearance makes this virulence a promising target for addressing L. monocytogenes infection. In this study, we hypothesized that curcumin, a polyphenol derived from turmeric that could effectively inhibit LLO pore-forming activity, might be useful in the prevention or treatment of L. monocytogenes infection. Thus, the in vitro protective effects of curcumin against L. monocytogenes infection by targeting LLO were assessed via hemolytic activity assays, cytotoxicity tests, intracellular growth assays, and confocal microscopy. Our results revealed that treating infected macrophages with curcumin can lead to a decrease in LLO-mediated bacteria phagosomal escape and limit the intracellular growth of L. monocytogenes. Moreover, results from animal experiments show that this natural compound effectively increases protection against bacterial infection and helps the host to clear the invading pathogen completely from an animal model, establishing it as a potent antagonist of L. monocytogenes. The results from our molecular modeling and mutational analysis demonstrated that curcumin directly engages with domains 2 and 4 of LLO, thereby decreasing the hemolytic activity of LLO by influencing its oligomerization. Taken together, these results suggest that, as an antitoxin agent, curcumin can be further developed into a novel therapy against L. monocytogenes infections by targeting LLO

Inhibition of sortase A by chalcone prevents Listeria monocytogenes infection

The critical role of sortase A in gram-positive bacterial pathogenicity makes this protein a good potential target for antimicrobial therapy. In this study, we report for the first time the crystal structure of Listeria monocytogenes sortase A and identify the active sites that mediate its transpeptidase activity. We also used a sortase A (SrtA) enzyme activity inhibition assay, simulation, and isothermal titration calorimetry analysis to discover that chalcone, an agent with little anti-L. monocytogenes activity, could significantly inhibit sortase A activity with an IC50 of 28.41 +/- 5.34 mu M by occupying the active site of SrtA. The addition of chalcone to a co-culture of L. monocytogenes and Caco-2 cells significantly inhibited bacterial entry into the cells and L. monocytogenes-mediated cytotoxicity. Additionally, chalcone treatment decreased the mortality of infected mice, the bacterial burden in target organs, and the pathological damage to L. monocytogenes-infected mice. In conclusion, these findings suggest that chalcone is a promising candidate for the development of treatment against L. monocytogenes infection. (C) 2016 Elsevier Inc. All rights reserved

Characteristics of Carbapenem-resistant Klebsiella pneumoniae in sewage from a tertiary hospital in Jilin Province, China.

Carbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a serious problem in hospitals worldwide. We monitored a tertiary hospital in Changchun, Jilin Province, China, and found that CRKP was the major species among the carbapenem-resistant isolates in sewage. Subsequently, we evaluated the drug susceptibility, resistance genes, virulence genes, outer pore membrane protein-related genes (OmpK35 & OmpK 36), multi-locus sequence typing and replicons, biofilm formation capabilities, and resistance to chlorine-containing disinfectants among KP isolates. Identification of drug sensitivity, multiple resistance profiles were observed including 77 (82.80%) multidrug resistant (MDR), 16 (17.20%) extensive drug resistant (XDR). Some antibiotic resistance genes were detected, the most prevalent carbapenemase gene was blaKPC, and 16 resistance genes were associated with other antibiotics. In addition, 3 (3.23%) CRKP isolates demonstrated loss of OmpK-35 and 2 (2.15%) demonstrated loss of OmpK-36. In the detection of multi-locus sequence typing (MLST), 11 ST11 isolates carried virulence genes. The most common replicon type was IncFII. Biofilm-forming capabilities were demonstrated by 68.8% of the isolates, all of which were resistant to chlorine-containing disinfectants. The results of the study showed that antibiotic-resistant isolates, especially CRKP, could resist disinfectants in hospital wastewater, and improper treatment of hospital wastewater may lead to the spread of drug-resistant bacteria and their genes. Thus, these bacteria must be eliminated before being discharged into the municipal sewage system

MIC of 93 <i>Klebsiella pneumoniae</i> isolates against imipenem.

Note. Rows represent imipenem concentration gradients, and columns represent percentages of isolates.</p

Replicon typing in CRKP isolates.

BackgroundCarbapenem-resistant Klebsiella pneumoniae (CRKP) infection is a serious problem in hospitals worldwide. We monitored a tertiary hospital in Changchun, Jilin Province, China, and found that CRKP was the major species among the carbapenem-resistant isolates in sewage. Subsequently, we evaluated the drug susceptibility, resistance genes, virulence genes, outer pore membrane protein-related genes (OmpK35 & OmpK 36), multi-locus sequence typing and replicons, biofilm formation capabilities, and resistance to chlorine-containing disinfectants among KP isolates. Identification of drug sensitivity, multiple resistance profiles were observed including 77 (82.80%) multidrug resistant (MDR), 16 (17.20%) extensive drug resistant (XDR). Some antibiotic resistance genes were detected, the most prevalent carbapenemase gene was blaKPC, and 16 resistance genes were associated with other antibiotics. In addition, 3 (3.23%) CRKP isolates demonstrated loss of OmpK-35 and 2 (2.15%) demonstrated loss of OmpK-36. In the detection of multi-locus sequence typing (MLST), 11 ST11 isolates carried virulence genes. The most common replicon type was IncFII. Biofilm-forming capabilities were demonstrated by 68.8% of the isolates, all of which were resistant to chlorine-containing disinfectants. The results of the study showed that antibiotic-resistant isolates, especially CRKP, could resist disinfectants in hospital wastewater, and improper treatment of hospital wastewater may lead to the spread of drug-resistant bacteria and their genes. Thus, these bacteria must be eliminated before being discharged into the municipal sewage system.</div