Project description:The emergence and spread of polymyxin resistance, especially among Klebsiella pneumoniae isolates threaten the effective management of infections. This study profiled for polymyxin resistance mechanisms and investigated the activity of polymyxins plus vancomycin against carbapenem- and polymyxin-resistant K. pneumoniae.
Project description:K. pneumoniae, a Gram-negative bacterium, is normally associated with pneumonia in patients with weakened immune systems. However, it is also a prevalent nosocomial infectious agent that can be found in infected surgical sites and combat wounds. Many of these clinical strains display multi-drug resistance. We have worked with a clinical strain of K. pneumoniae that was initially isolated from a wound of an injured soldier. This strain demonstrated resistance to many commonly used antibiotics, but sensitivity to carbapenems. This isolate was capable of forming biofilms in vitro, contributing to its increased antibiotic resistance and impaired clearance. We were interested in determining how sublethal concentrations of carbapenem treatment specifically affect K. pneumoniae biofilms both in morphology and genomic expression. Scanning electron microscopy showed striking morphological differences between untreated and treated biofilms, including rounding, blebbing, and dimpling of treated cells. Comparative transcriptome analysis using RNA sequencing technology identified a large number of open reading frames (ORFs) differentially regulated in response to carbapenem treatment at 2 and 24 hours. ORFs upregulated with carbapenem treatment included genes involved in resistance, antiporters, and autoinducers. ORFs downregulated included metal transporters, membrane biosynthesis proteins, and motility proteins. Quantitative real time PCR validated the general trend of some of these differentially regulated ORFs. Treating K. pneumoniae biofilms with sublethal concentrations of carbapenems induced a wide-range of phenotypic and gene expression changes. This study reveals some of the mechanisms underlying how sublethal amounts of carbapenems could affect the overall fitness and pathogenic potential of K. pneumoniae biofilm cells.
Project description:Carbapenem-resistant Klebsiella pneumoniae classified as multilocus sequence type 258 (ST258)are a problem in healthcare settings in many countries globally. ST258 isolates are resistant tomultiple classes of antibiotics and can cause life-threatening infections, such as pneumonia andsepsis, in susceptible individuals. Treatment strategies for such infections are limited. Hence,understanding the response of K. pneumoniae to host factors in the presence of antibiotics couldreveal mechanisms employed by the pathogen to evade killing in the susceptible host, as well asinform treatment of infections. Here, we investigated the ability of subinhibitory concentrationsof antibiotics to alter K. pneumoniae capsule polysaccharide (CPS) production and survival innormal human serum. Several antibiotics tested enhanced ST258 survival in normal humanserum. Unexpectedly, subinhibitory concentrations of mupirocin increased survival in 7 of 10clinical isolates tested, and caused up-regulated expression of CPS biosynthesis genes and CPSproduction in a selected ST258 clinical isolate (34446) compared with untreated controls.Additionally, mupirocin treatment caused a reduction in the deposition of the serum complementproteins C3b and C5b-9 on the surface of ST258. Transcriptome analyses with isolate 34446indicated that genes implicated in serum resistance, such as aroE, csrD, pyrB, pyrC and traT,were up-regulated following mupirocin treatment. In conclusion, mupirocin causes changes inthe K. pneumoniae transcriptome that likely contribute to the observed decrease in serumsusceptibility via a multifactorial process. Whether these responses are triggered by othercomponents of host defense or therapeutics that were not tested here merits further investigation.
Project description:The study aimed to characterize plasmids mediating carbepenem resistance in Klebsiella pneumoniae in Pretoria, South Africa. We analysed 56 K. pneumoniae isolates collected from academic hospital around Pretoria. Based on phenotypic and molecular results of these isolates, 6 representative isolates were chosen for further analysis using long reads sequencing platform. We observed multidrug resistant phenotype in all these isolates, including resistance to aminoglycosides, tetracycline, phenicol, fosfomycin, floroquinolones, and beta-lactams antibiotics. The blaOXA-48/181 and blaNDM-1/7 were manily the plasmid-mediated carbapenemases responsible for carbapenem resistance in the K. pneumoniae isolates in these academic hospitals. These carbapenemase genes were mainly associated with plasmid replicon groups IncF, IncL/M, IncA/C, and IncX3. This study showed plasmid-mediated carbapenemase spread of blaOXA and blaNDM genes mediated by conjugative plasmids in Pretoria hospitals.
Project description:Klebsiella pneumoniae is an arising threat to human health. However, host immune responses in response to this bacterium remain to be elucidated. The goal of this study was to identify the dominant host immune responses associated with Klebsiella pneumoniae pulmonary infection. Pulmonary mRNA profiles of 6-8-weeks-old BALB/c mice infected with/without Klebsiella pneumoniae were generated by deep sequencing using Illumina Novaseq 6000. qRT–PCR validation was performed using SYBR Green assays. Using KEGG (Kyoto Encyclopedia of Genes and Genomes) analysis, we identified several immune associated pathways, including complement and coagulation cascades, Toll-like receptor signaling pathway, Rap1 signaling pathway, chemokine signaling pathway, TNF signaling pathway, phagosome and NOD-like receptor signaling pathway, were involved in Klebsiella pneumoniae pulmonary infection. Using ICEPOP (Immune CEll POPulation) analysis, we found that several cell types were involved in the host immune response to Klebsiella pneumoniae pulmonary infection, including dendritic cells, macrophages, monocytes, NK (natural killer) cells, stromal cells. Further, IL-17 chemokines were significantly increased during Klebsiella pneumoniae infection. This study provided evidence for further studying the pathogenic mechanism of Klebsiella pneumoniae pneumonia infection.
Project description:Klebsiella pneumoniae is a bacterial pathogen that causes nosocomial infection in humans and is acquiring antibiotic resistance at an alarming rate. This study investigates the effect of zinc limitation on the phosphoproteome of K. pneumoniae using quantitative mass spectrometry to provide insight into the cell signaling methods used to respond to nutrient limited conditions like those experienced when colonizing a host.
Project description:Klebsiella pneumoniae is a human gut communal organism and notorious opportunistic pathogen. The relative high burden of asymptomatic colonization by K. pneumoniae is often compounded by multidrug resistance-a potential problem for individuals with significant comorbidities or other risk factors for infection. A carbapenem-resistant K. pneumoniae strain classified as multilocus sequence type 258 (ST258) is widespread in the United States and can be resistant to many classes of clinically useful antibiotics. Thus, treatment of ST258 infections is often difficult. Inasmuch as new preventive and/or therapeutic measures are needed for treatment of such infections, we developed an ST258 pneumonia model in cynomolgus macaques and tested the ability of an ST258 capsule polysaccharide type 2 (CPS2) vaccine to moderate disease severity. Compared with sham-vaccinated animals, those vaccinated with ST258 CPS2 had significantly less disease as assessed by radiograph 24 h after intrabronchial installation of 108 CFUs of ST258. All macaques vaccinated with CPS2 ultimately developed ST258-specific antibodies that significantly enhanced serum bactericidal activity and killing of ST258 by macaque neutrophils ex vivo. Consistent with a protective immune response to CPS2, transcripts encoding inflammatory mediators were increased in infected lung tissues obtained from CPS-vaccinated animals compared with control, sham-vaccinated macaques. Taken together, our data provide support to the idea that vaccination with ST258 CPS can be used to prevent or moderate infections caused by ST258. As with studies performed decades earlier, we propose that this approach can be extended to include multiple capsule types